Oxidative Stress and Age-Related Neurodegeneration

Oxidative Stress and Age-Related Neurodegeneration

OXIDATIVE STRESS AND DISEASE Series Editors

LESTER PACKER, PH.D. ENRIQUE CADENAS, M.D., PH.D. University of Southern California School of Pharmacy Los Angeles, California

1. Oxidative Stress in Cancer, AIDS, and Neurodegenerative Diseases, edited by Luc Montagnier, René Olivier, and Catherine Pasquier 2. Understanding the Process of Aging: The Roles of Mitochondria, Free Radicals, and Antioxidants, edited by Enrique Cadenas and Lester Packer 3. Redox Regulation of Cell Signaling and Its Clinical Application, edited by Lester Packer and Junji Yodoi 4. Antioxidants in Diabetes Management, edited by Lester Packer, Peter Rösen, Hans J. Tritschler, George L. King, and Angelo Azzi 5. Free Radicals in Brain Pathophysiology, edited by Giuseppe Poli, Enrique Cadenas, and Lester Packer 6. Nutraceuticals in Health and Disease Prevention, edited by Klaus Krämer, Peter-Paul Hoppe, and Lester Packer 7. Environmental Stressors in Health and Disease, edited by Jürgen Fuchs and Lester Packer 8. Handbook of Antioxidants: Second Edition, Revised and Expanded, edited by Enrique Cadenas and Lester Packer 9. Flavonoids in Health and Disease: Second Edition, Revised and Expanded, edited by Catherine A. Rice-Evans and Lester Packer 10. Redox–Genome Interactions in Health and Disease, edited by Jürgen Fuchs, Maurizio Podda, and Lester Packer 11. Thiamine: Catalytic Mechanisms in Normal and Disease States, edited by Frank Jordan and Mulchand S. Patel 12. Phytochemicals in Health and Disease, edited by Yongping Bao and Roger Fenwick 13. Carotenoids in Health and Disease, edited by Norman I. Krinsky, Susan T. Mayne, and Helmut Sies

14. Herbal and Traditional Medicine: Molecular Aspects of Health, edited by Lester Packer, Choon Nam Ong, and Barry Halliwell 15. Nutrients and Cell Signaling, edited by Janos Zempleni and Krishnamurti Dakshinamurti 16. Mitochondria in Health and Disease, edited by Carolyn D. Berdanier 17. Nutrigenomics, edited by Gerald Rimbach, Jürgen Fuchs, and Lester Packer 18. Oxidative Stress, Inflammation, and Health, edited by Young-Joon Surh and Lester Packer 19. Nitric Oxide, Cell Signaling, and Gene Expression, edited by Santiago Lamas and Enrique Cadenas 20. Resveratrol in Health and Disease, edited by Bharat B. Aggarwal and Shishir Shishodia 21. Molecular Interventions in Lifestyle-Related Diseases, edited by Midori Hiramatsu, Toshikazu Yoshikawa, and Lester Packer 22. Oxidative Stress and Age-Related Neurodegeneration, edited by Yuan Luo and Lester Packer

Oxidative Stress and Age-Related Neurodegeneration

edited by

Yuan Luo Lester Packer

Boca Raton London New York

A CRC title, part of the Taylor & Francis imprint, a member of the Taylor & Francis Group, the academic division of T&F Informa plc.

Published in 2006 by CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 © 2006 by Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group No claim to original U.S. Government works Printed in the United States of America on acid-free paper 10 9 8 7 6 5 4 3 2 1 International Standard Book Number-10: 0-8493-3725-9 (Hardcover) International Standard Book Number-13: 978-0-8493-3725-3 (Hardcover) Library of Congress Card Number 2005053811 This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the consequences of their use. No part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright.com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC) 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe.

Library of Congress Cataloging-in-Publication Data Oxidative stress and age-related neurodegeneration / [edited by] Yuan Luo, Lester Packer. p. ; cm. -- (Oxidative stress and disease; 20) Includes bibliographical references and index. ISBN0-8493-3725-9 1. Alzheimer's disease--Pathophysiology. 2. Oxidative stress. I. Luo, Yuan, 1956- II. Packer, Lester. III. Series. [DNLM: 1. Alzheimer Disease. 2. Oxidative stress. 3. Aging. 4. Antioxidants--pharmacology. 5. Neurodegeneration Diseases. WT 155 O98 2005] RC523.O95 2005 616.8'3107--dc22

2005053811

Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com Taylor & Francis Group is the Academic Division of T&F Informa plc.

and the CRC Press Web site at http://www.crcpress.com

Series Introduction Oxygen is a dangerous friend. Through evolution, oxygen—itself a free radical— was chosen as the terminal electron acceptor for respiration. The two unpaired electrons of oxygen spin in the same direction; thus, oxygen is a biradical. Other oxygen-derived free radicals, such as superoxide anion or hydroxyl radicals, formed during metabolism or by ionizing radiation are stronger oxidants (i.e., endowed with a higher chemical reactivity). Oxygen-derived free radicals are generated during oxidative metabolism and energy production in the body, and are involved in regulation of signal transduction and gene expression; activation of receptors and nuclear transcription factors; oxidative damage to cell components; the anti-microbial and cytotoxic action of immune system cells, neutrophils, and macrophages; and in aging and age-related degenerative diseases. Overwhelming evidence indicates that oxidative stress can lead to cell and tissue injury; however, the same free radicals that are generated during oxidative stress are produced during normal metabolism and, as a corollary, are involved in both human health and disease. In addition to reactive oxygen species, research on reactive nitrogen species has been gathering momentum to develop an area of enormous importance in biology and medicine. Nitric oxide or nitrogen monoxide (NO) is a free radical generated by nitric oxide synthase (NOS). This enzyme modulates physiological responses in the circulation, such as vasodilation (eNOS) or signaling in the brain (nNOS). During inflammation, however, a third isoenzyme is induced—iNOS— resulting in the overproduction of NO and causing damage to targeted infectious organisms and to healthy tissues in the vicinity. More worrisome, however, is the fact that NO can react with superoxide anion to yield a strong oxidant—peroxynitrite. Oxidation of lipids, proteins, and DNA by peroxynitrite increases the likelihood of tissue injury. Both reactive oxygen and nitrogen species are involved in the redox regulation of cell functions. Oxidative stress is increasingly viewed as a major upstream component in the signaling cascade involved in inflammatory responses and stimulation of adhesion molecule and chemoattractant production. Hydrogen peroxide decomposes in the presence of transition metals to the highly reactive hydroxyl radical, which by two major reactions—hydrogen abstraction and addition—accounts for most of the oxidative damage to proteins, lipids, sugars, and nucleic acids. Hydrogen peroxide is also an important signaling molecule that, among others, can activate NF-KB, an important transcription factor involved in inflammatory responses. At low concentrations, hydrogen peroxide regulates cell signaling and stimulates cell proliferation; at higher concentrations, it triggers apoptosis and, at even higher levels, necrosis. Virtually all diseases thus far examined involve free radicals. In most cases, free radicals are secondary to the disease process, but in some instances, free radicals

are causal. Thus, a delicate balance exists between oxidants and antioxidants in health and disease. Their proper balance is essential for ensuring healthy aging. The term oxidative stress indicates that the antioxidant status of cells and tissues is altered by exposure to oxidants. The redox status is thus dependent on the degree to which cells’ components are in the oxidized state. In general, the reducing environment inside cells helps to prevent oxidative damage. In this reducing environment, disulfide bonds (S–S) do not spontaneously form because sulfhydryl groups are maintained in the reduced state (SH), thus preventing protein misfolding or aggregation. This reducing environment is maintained by oxidative metabolism and by the action of antioxidant enzymes and substances, such as glutathione, thioredoxin, vitamins E and C, and enzymes such as superoxide dismutases, catalase, and the selenium-dependent glutathione reductase, as well as glutathione and thioredoxin hydroperoxidases, which serve to remove reactive oxygen species (hydroperoxides). Changes in the redox status and depletion of antioxidants occur during oxidative stress. The thiol redox status is a useful index of oxidative stress mainly because metabolism and NADPH-dependent enzymes maintain cell glutathione (GSH) almost completely in its reduced state. Oxidized glutathione (glutathione disulfide [GSSG]) accumulates under conditions of oxidant exposure and this changes the ratio GSSG/GSH; an increased ratio is usually taken as indicating oxidative stress. Other oxidative stress indicators are ratios of redox couples such as NADPH/NADP, NADH/NAD, thioredoxinreduced/thioredoxinoxidized, dihydrolipoic acid/α-lipoic acid, and lactate/pyruvate. Changes in these ratios affect the energy status of the cell, largely determined by the ratio ATP/ADP ⫹ AMP. Many tissues contain large amounts of glutathione, 2–4 mM in erythrocytes or neural tissues, and up to 8 mM in hepatic tissues. Reactive oxygen and nitrogen species can oxidize glutathione, thus lowering the levels of the most abundant nonprotein thiol, sometimes designated as the cell’s primary preventative antioxidant. Current hypotheses favor the idea that lowering oxidative stress can have a health benefit. Free radicals can be overproduced or the natural antioxidant system defenses weakened, first resulting in oxidative stress, and then leading to oxidative injury and disease. Examples of this process include heart disease, cancer, and neurodegenerative disorders. Oxidation of human low-density lipoproteins is considered an early step in the progression and eventual development of atherosclerosis, thus leading to cardiovascular disease. Oxidative DNA damage may initiate carcinogenesis. Environmental sources of reactive oxygen species are also important in relation to oxidative stress and disease. A few examples include: UV radiation, ozone, cigarette smoke, and others are significant sources of oxidative stress. Compelling support for the involvement of free radicals in disease development originates from epidemiological studies demonstrating that an enhanced antioxidant status is associated with reduced risk of several diseases. Vitamins C and E, in the prevention of cardiovascular disease, are a notable example. Elevated antioxidant status is also associated with decreased incidence of cataracts, cancer, and neurodegenerative disorders. Some recent reports have suggested an inverse

correlation between antioxidant status and the occurrence of rheumatoid arthritis and diabetes mellitus. Indeed, the indications in which antioxidants may be useful in the prevention or the treatment of disease are increasing in number. Oxidative stress, instead of being the primary cause of disease, is more often a secondary complication in many disorders. Oxidative stress diseases include inflammatory bowel diseases, retinal ischemia, cardiovascular disease and restenosis, AIDS, adult respiratory distress syndrome, and neurodegenerative diseases such as stroke, Parkinson’s disease, and Alzheimer’s disease. Such indications may prove amenable to antioxidant treatment (in combination with conventional therapies) because a clear involvement of oxidative injury exists in these disorders. In this series of books, the importance of oxidative stress and disease associated with organ systems of the body is highlighted by exploring the scientific evidence and the medical applications of this knowledge. The series also highlights the major natural antioxidant enzymes and antioxidant substances such as vitamins E, A, and C, flavonoids, polyphenols, carotenoids, lipoic acid, co-enzyme Q10, carnitine, and other micronutrients present in food and beverages. Oxidative stress is an underlying factor in health and disease. More evidence indicates that a proper balance between oxidants and antioxidants is involved in maintaining health and longevity, and that altering this balance in favor of oxidants may result in patho-physiological responses that cause functional disorders and disease. This series is intended for researchers in the basic biomedical sciences and clinicians. The potential of such knowledge for healthy aging and disease prevention warrants further knowledge about how oxidants and antioxidants modulate cell and tissue function. Lester Packer Enrique Cadenas Series Editors

Preface Alzheimer’s disease, together with other age-related neurodegenerative disorders, is severely affecting our society. Substantial experimental evidence now supports a role of oxidative stress in this devastating human degenerative disorder. Research in this field is moving forward at a remarkable pace and there is unprecedented interest among the scientific community and the public. Some obvious questions are: ●

●

● ●

●

What are the similarities and differences in terms of susceptibility or site of action of oxidative stress between aging and neurodegeneration? What are the effects of oxidative stress in Alzheimer’s disease and on normal aging? How can oxidative stress or its effects be attenuated? Do antioxidants prevent or slow the progress of the disease? If yes, by what mechanism(s)? Is there any role for natural micronutrients in the attenuation of oxidative stress and prevention of age-related neurodegeneration?

These and similar questions prompted us to organize a book on this topic as part of the series on “Oxidative Stress and Disease.” This volume, entitled Oxidative Stress and Age-Related Neurodegeneration, will provide a timely collection of articles that cover many of the recent developments. The book will be of value to students and scholars as an orientation and introduction to this exciting field of biomedical research. Moreover, this book will serve as a valuable reference for researchers in academia, industry, and clinical medicine. The editors thank the scholars whose excellent chapters make this book an important contribution to the ongoing science. We also thank Miss Laura Causey for her editorial assistance. Yuan Luo Lester Packer

Editors Yuan Luo is currently an associate professor at the School of Pharmacy of the University of Maryland. A native of Beijing, China, she obtained her B.S. and M.S. degrees from Peking University. She then took a position at the Chinese Academy of Sciences. In 1993, Dr. Luo obtained her Ph.D. in pharmacology (Neuroscience Program) from the State University of New York, Upstate Medical University at Syracuse, New York. She then held two postdoctoral positions at the Massachusetts Institute of Technology and Harvard Medical School. Dr. Luo joined the faculty of the University of Southern Mississippi (USM) in 2000, where she was a director of the Cellular and Molecular Neuroscience Research Laboratory. Dr. Luo’s research program has been recognized internationally and her research has been supported by grants from the National Institutes of Health and IPSEN France. She has published numerous papers and has served as editor of several scientific journals. Her recent research focuses on neuroprotective mechanisms of the Ginkgo biloba extract in Alzheimer’s disease.

Lester Packer received his Ph.D. in microbiology and biochemistry from Yale University and was professor and senior researcher at the University of California at Berkeley for 40 years. Most recently, Dr. Packer joined the Department of Molecular Pharmacology & Toxicology within the School of Pharmacy at the University of Southern California to pursue studies related to the molecular, cellular, and physiological aspects of oxidants and antioxidants in biological systems. In addition to his membership in numerous professional research societies, Dr. Packer has held offices as president of the International Society of Free Radical Research, president of the Oxygen Club of California, and vice-president of UNESCO - Global Network on Molecular and Cell Biology. Dr. Packer is also the recipient of numerous scientific achievement awards including three honorary doctoral degrees. He serves on editorial advisory boards for scientific journals related to biochemistry, antioxidant metabolism, and nutrition. Dr. Packer has published over 800 scientific papers and more than 100 books on every aspect of antioxidants and health, including standard references such as

Vitamin E in Health and Disease, Vitamin C in Health and Disease, The Handbook of Natural Antioxidants, Understanding the Process of Aging: The Roles of Mitochondria, Free Radicals, and Antioxidants, Carotenoids and Retinoids: Molecular Aspects and Health Issues. The Antioxidant Miracle, published in 1999, is a book for nonscientists. Dr. Lester Packer has an unmatched scientific record on antioxidants and micronutrients, which are considered key components in achieving healthy aging.

Contributors Bruce N. Ames University of California Berkeley, California

Shigeru Chiba Asahikawa Medical College Asahikawa, Japan

Tamar Amit Technion Israel Institute of Technology Haifa, Israel

Yves Christen Ispen Institute Paris, France

Brian J. Bacskai Massachusetts General Hospital Charlestown, Massachusetts Stephen Barnes University of Alabama Birmingham, Alabama Stéphane Bastianetto Douglas Hospital Research Center Montreal, Canada

Carl W. Cotman University of California Irvine, California Jessy Deshane University of Alabama Birmingham, Alabama Sarah A. Dodwell Massachusetts General Hospital Charlestown, Massachusetts

Stig Berg Jönköping University Jönköping, Sweden

Tamagno Elena University of Turin Turin, Italy

Ashley I. Bush Harvard Medical School Boston, Massachusetts

Shannon Eliuk University of Alabama Birmingham, Alabama

Peter Butko University of Southern Mississippi Hattiesburg, Mississippi

Monica Garcia-Alloza Massachusetts General Hospital Charlestown, Massachusetts

D. Allan Butterfield University of Kentucky Lexington, Kentucky

Elizabeth Head University of California Irvine, California

Rudy J. Castellani Michigan State University East Lansing, Michigan

Ho Jin Heo Jeollanamdo Innovation Agency Suncheon, South Korea

Gregory Hook American Life Science Pharmaceuticals San Diego, California

Summer Lind Joseph Stokes Jr. Research Institute Philadelphia, Pennsylvania

Vivian Hook University of California La Jolla, California

Jiankang Liu Oakland Research Institute Oakland, California

Harry Ischiropoulos University of Pennsylvania Philadelphia, Pennsylvania

Yuan Luo University of Maryland Baltimore, Maryland

James A. Joseph Tufts University Boston, Massachusetts Jeffrey A. Kaye Oregon Health & Science University Portland, Oregon Dae-Ok Kim Kyung Hee University Yongin, South Korea Helen Kim University of Alabama Birmingham, Alabama Francis C. Lau Tufts University Boston, Massachusetts Chang Yong Lee Cornell University Geneva, New York Hyoung-gon Lee Case Western Reserve University Cleveland, Ohio Rena Li L.J. Roberts Center for Alzheimer’s Research Sun City, Arizona

Silvia A. Mandel Technion Israel Institute of Technology Haifa, Israel Tabaton Massimo University of Genoa Genoa, Italy Joseph R. Mazzulli Joseph Stokes Jr. Research Institute Philadelphia, Pennsylvania Sreelatha Meleth University of Alabama Birmingham, Alabama Thomas J. Montine University of Washington Seattle, Washington Paula I. Moreira Case Western Reserve University Cleveland, Ohio Jason D. Morrow Vanderbilt University Nashville, Tennessee

Jackob Moskovitz University of Kansas Lawrence, Kansas

Julie V. Smith William Carey College Hattiesburg, Massachusetts

Sven E. Nilsson Jönköping University Jönköping, Sweden

Mark A. Smith Case Western Reserve University Cleveland, Ohio

Akihiko Nunomura Asahikawa Medical College Asahikawa, Japan George Perry Case Western Reserve University Cleveland, Ohio Matthew J. Picklo, Sr. University of North Dakota Grand Forks, North Dakota H. Fai Poon University of Kentucky Lexington, Kentucky Joseph F. Quinn Oregon Health & Science University Portland, Oregon Terry Reisine American Life Science Pharmaceuticals San Diego, California Quirion Rémi Douglas Hospital Research Center Montreal, Canada Thomas B. Shea University of Massachusetts Lowell, Massachusetts Barbara Shukitt-Hale Tufts University Boston, Massachusetts

Rukhsana Sultana University of Kentucky Lexington, Kentucky Kazuki Tabata Asahikawa Medical College Asahikawa, Japan Flaubert Tchantchou University of Maryland Baltimore, Maryland Thomas Toneff University of California La Jolla, California Orly Weinreb Technion Israel Institute of Technology Haifa, Israel Han Ying-Shan Douglas Hospital Research Center Montreal, Canada Moussa B. H. Youdim Technion Israel Institute of Technology Haifa, Israel Yafei Zhang L.J. Roberts Center for Alzheimer’s Research Sun City, Arizona Baolu Zhao Institute of Biophysics Chinese Academy of Science Beijing, China

Hailin Zheng Technion Israel Institute of Technology Haifa, Israel

Xiongwei Zhu Case Western Reserve University Cleveland, Ohio

Table of Contents Chapter 1 Proteomics Identification of Oxidatively Modified Proteins in the Alzheimer’s Disease Brain and Models Thereof: Insights into Potential Mechanisms of Neurodegeneration ..................................1 D. Allan Butterfield, H. Fai Poon, and Rukhsana Sultana Chapter 2 Direct Evaluation of Alzheimer’s Disease-Specific Oxidative Stress with Multiphoton Microscopy.............................................27 Monica Garcia-Alloza, Sarah A. Dodwell, and Brian J. Bacskai Chapter 3 Ginkgo biloba Extract and Alzheimer’s Disease: Is the Neuroprotection Explained Merely by the Antioxidant Action? ...43 Yves Christen Chapter 4 Mitochondrial Nutrients: Reducing Mitochondrial Decay to Delay or Treat Cognitive Dysfunction, Alzheimer’s Disease, and Parkinson’s Disease.................................................................59 Jiankang Liu and Bruce N. Ames Chapter 5 Reactive Oxygen and Nitrogen Species: Weapons of Neuronal Destruction ...................................................................................107 Joseph R. Mazzulli, Summer Lind, and Harry Ischiropoulos Chapter 6 Amyloid-β and τ in Alzheimer’s Disease: What is the Neuropathology Trying to Tell Us? .............................................121 Rudy J. Castellani, Akihiko Nunomura, Hyoung-gon Lee, Xiongwei Zhu, George Perry, and Mark A. Smith Chapter 7 Antioxidant Therapies in the Prevention and Treatment of Alzheimer Disease .......................................................................131 Paula I. Moreira, Xiongwei Zhu, Akihiko Nunomura, Mark A. Smith, and George Perry Chapter 8 F2-Isoprostanes as Biomarkers of Late Onset Alzheimer’s Disease .........................................................................................147 Thomas J. Montine, Joseph F. Quinn, Jeffrey A. Kaye, and Jason D. Morrow

Chapter 9

Aspirin and Alzheimer’s Disease Protection ...............................159

Sven E. Nilsson and Stig Berg Chapter 10 The Disposition of Lipid-Derived Carbonyls in Alzheimer’s Disease .......................................................................................181 Matthew J. Picklo, Sr. Chapter 11 Methionine Sulfoxide Reductase System: Possible Roles in Protection against Neurodegenerative Diseases.........................199 Jackob Moskovitz and Ashley I. Bush Chapter 12 Nutritional Antioxidant Enrichment and Improved Cognitive Function in Canines ...................................................................213 Elizabeth Head and Carl W. Cotman Chapter 13 Green Tea and Resveratrol as Protective Agents against Neurotoxins ................................................................................225 Stéphane Bastianetto, Han Ying-Shan, and Quirion Rémi Chapter 14

Oxidative Stress and Aβ PP Processing .....................................235

Tabaton Massimo and Tamagno Elena Chapter 15 Apple Phenolics and Alzheimer’s Disease.................................247 Ho Jin Heo, Dae-Ok Kim, and Chang Yong Lee Chapter 16

Effects of Nicotine in Models of Alzheimer’s Disease..............265

Baolu Zhao Chapter 17 The Essentiality of Iron Chelation in Neuroprotection: A Potential Role of Green Tea Catechins ......................................277 Silvia A. Mandel, Tamar Amit, Hailin Zheng, Orly Weinreb, and Moussa B.H. Youdim Chapter 18 Ginkgo biloba Extract EGb 761 Extends Life Span and Attenuates H2O2 Levels in a Caenorhabditis elegans Model of Alzheimer’s Disease...............................................................301 Julie V. Smith and Yuan Luo

Chapter 19 Cysteine Proteases as β -Secretases for Aβ Production in the Major Regulated Secretory Pathway of Neurons: Implications for Therapeutic Strategies in Alzheimer’s Disease...................................................................327 Vivian Hook, Thomas Toneff, Gregory Hook, and Terry Reisine Chapter 20 The Molecular Mechanism of the Neuroprotective Action of Antioxidants Compared with the Anti-Parkinson Drug, Rasagiline ...................................................................................343 Orly Weinreb, Tamar Amit, Silvia A. Mandel, and Moussa B.H. Youdim Chapter 21 Temporal Primacy of Oxidative Stress in the Pathological Cascade of Alzheimer Disease...................................................365 Akihiko Nunomura, Kazuki Tabata, Shigeru Chiba, Mark A. Smith, and George Perry Chapter 22 Age-Related Neuronal and Behavioral Deficits are Improved by Polyphenol-Rich Blueberry Supplementation ...........................373 Francis C. Lau, Barbara Shukitt-Hale, and James A. Joseph Chapter 23 Amyloid-β Peptide, a Therapeutic Target for Alzheimer’s Disease?......................................................................................395 Yuan Luo and Peter Butko Chapter 24

Nutrition, Brain Aging, and Alzheimer’s Disease .....................409

Yafei Zhang and Rena Li Chapter 25 Interaction between Dietary and Genetic Deficiencies in the Modulation of Homocysteine Elimination.................................443 Flaubert Tchantchou and Thomas B. Shea Chapter 26 Nutriproteomics Approach to Understanding Dementia-Relevant Brain Protein Changes in Response to Grape Seed Extract, a Dietary Antioxidant....................................................................457 Helen Kim, Shannon Eliuk, Jessy Deshane, Stephen Barnes, and Sreelatha Meleth Index .................................................................................................................481

Identification 1 Proteomics of Oxidatively Modified Proteins in the Alzheimer’s Disease Brain and Models Thereof: Insights into Potential Mechanisms of Neurodegeneration D. Allan Butterfield, H. Fai Poon, and Rukhsana Sultana University of Kentucky Lexington, Kentucky

CONTENTS 1.1 1.2

1.3

Introduction..................................................................................................2 Methodology ................................................................................................3 1.2.1 Alzheimer’s Disease Brain Tissue ...................................................3 1.2.2 Cholinergic Animal Model ..............................................................4 1.2.3 Caenorhabditis Elegans...................................................................4 1.2.4 Senescence-Accelerated Mice Prone 8 (SAMP8) ...........................4 1.2.5 Two-Dimensional Polyacrylamide Gel Electrophoresis..................4 1.2.6 Mass Spectrometry and Database Searching...................................6 Proteomics Studies on Oxidatively Modified Proteins in the Alzheimer’s Disease Brain ..........................................................................8 1.3.1 Proteins Involved in Energy Metabolism ........................................8 1.3.2 Proteins Involved in Glutamate Reuptake or Conversion ...............9 1.3.3 Altered Synaptic Function ...............................................................9 1

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Oxidative Stress and Age-Related Neurodegeneration

1.3.4 Proteasomal Dysfunction.................................................................9 1.3.5 Lipid Abnormalities and Cholinergic Failure ................................10 1.3.6 Neuritic Abnormalities...................................................................10 1.3.7 Cell Cycle ......................................................................................10 1.3.8 pH Maintenance.............................................................................11 1.4 Oxidized Proteins in Alzheimer’s Disease Plasma....................................11 1.5 Proteomic Studies on Oxidatively Modified Proteins in Animal Models of Alzheimer’s Disease ....................................................11 1.5.1 Cholinergic Dysfunction-Related Animal Model..........................12 1.5.2 SAMP8 Mice .................................................................................13 1.5.3 Caenorhabditis Elegans.................................................................14 1.6 Future of Proteomics in Alzheimer’s Disease ...........................................16 Acknowledgments ...............................................................................................16 References ...........................................................................................................16

1.1 INTRODUCTION Alzheimer’s disease (AD) affects approximately 5 million persons in the United States, and in a few decades, more than 14 million individuals will be at risk if either the progression of the disease is not slowed down or a cure is not identified. The etiology of AD is largely unknown, but mutations of the genes for presenilin-1 (PS-1), presenilin-2 (PS-2),1 and amyloid precursor protein (APP)2 have been observed in inherited AD.3 In addition, there is an association between AD and allele 4 of the apolipoprotein E (APOE) gene,4,5 endothelial nitric oxide synthase-3 gene,6 and α-2-macroglobulin.7 Alzheimer’s disease is characterized by progressive loss of memory and, histopathologically, by intracellular neurofibrillary tangles (NFT), extracellular senile plagues (SP), and synaptic loss. NFT consists of paired helical filaments and related straight filaments, which are composed of aggregates of the hyperphosphorylated microtubule-associated protein tau.8 The main component of SP is amyloid β -peptide (Aβ ), a 40–42-amino-acid peptide derived from proteolytic cleavage of APP by the action of β - and γ -secretases. Aβ is considered to play a causal role in the development and progress of AD.9 Among the various neurotransmitter systems, cholinergic neurons in the nucleus basalis of Meynert (NBM) are lost early in the course of AD, and the dysfunction of cholinergic neurons is believed to be involved in cognitive deficits in this disease.10 Several lines of evidence suggest an important role for oxidative stress in the pathogenesis and progression of AD.11–15 This damage is characterized by oxidative modification of a number of cellular macromolecular targets, including proteins, lipids, carbohydrates, DNA, and RNA. Protein carbonyls, 4-hydroxy-2-nonenal (HNE), and 3-nitrotyrosine (3-NT) are among the early oxidative markers observed after the oxidative insult in a cell.13,16–20 Protein carbonyl levels were found to be elevated in the AD brain.21,22 In addition, in the AD brain, protein oxidation occurs in Aβ -rich regions such as the cortex and hippocampus, but is not observed in the cerebellum, where the Aβ was found to be negligible.23

Proteomics Identification of Oxidatively Modified Proteins

3

Protein carbonyl groups can be introduced into proteins by direct oxidation of certain amino acid side chains, peptide backbone scission, or by Michael addition reactions with products of lipid peroxidation or glycooxidation.13 In cell cultures, the use of vitamin E diminishes Aβ (1–42)-induced toxicity, further supporting the role of oxidative stress in AD pathology.24–26 Redox proteomics, which involves the coupling of two-dimensional polyacrylamide gel electrophoresis (2D–PAGE) separation of proteins with mass spectrometry (MS) techniques, is a valuable modality to determine oxidatively modified brain proteins.27 Proteomics has enabled us to identify a large number of oxidatively modified proteins that were previously undetected by other methods such as immunoprecipitation. There are several serious limitations to the use of immunoprecipitation to identify proteins; for example, the necessary availability of the antibody for the protein of interest, knowledge about the proteins, and the time-consuming and laborious nature of the process. Sometimes, a posttranslational modification can change the structure of proteins, thereby preventing the formation of the appropriate antigen–antibody complex. In this chapter, we discuss the identification and implications of oxidatively modified proteins in the pathology and biochemistry of AD and models thereof using proteomics techniques.

1.2 METHODOLOGY 1.2.1 ALZHEIMER’S DISEASE BRAIN TISSUE Human brain tissue samples, including inferior parietal lobule, hippocampus, and cerebellum used in the analyses, were taken at autopsy from the AD and control subjects, immediately frozen in liquid nitrogen, and stored at –80°C. The Rapid Autopsy Program of the University of Kentucky Alzheimer’s Disease Research Center (UK ADRC) resulted in extremely short postmortem intervals (PMIs). All AD subjects displayed progressive intellectual decline and met NINCDS-ADRDA Workgroup criteria for the clinical diagnosis of probable AD.28 Hematoxylin-eosin and modified Bielschowsky staining and 10-D-5, ubiquitin, and α-synuclein immunohistochemistry were used on multiple neocortical, hippocampal, entorhinal, amygdala, brainstem, and cerebellum sections for diagnosis. Some AD patients were also diagnosed with dementia with Lewy bodies, but the results were no different from AD patients with or without Lewy bodies. Control subjects underwent annual mental status testing as a part of the UK ADRC normal volunteer longitudinal aging study and did not have a history of dementia or other neurologic disorders. All control subjects had test scores in the normal range. Neuropathologic evaluation of control brains revealed only age-associated gross and histopathologic alterations. Brain tissues were minced and homogenized in 10 mM HEPES buffer (pH 7.4), containing 137 mM NaCl, 4.6 mM KCl, 1.1 mM KH2PO4, 0.6 mM MgSO4, and proteinase inhibitors — leupeptin (0.5 mg/mL), pepstatin (0.7 µg/mL), type IIS soybean trypsin inhibitor (0.5 µg/mL), and PMSF (40 µg/mL) — and were centrifuged at 14,000g for 10 min to remove debris.

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Oxidative Stress and Age-Related Neurodegeneration

Protein concentration in the supernatant was determined by the Pierce bicinchoninic acid (BCA) method.

1.2.2 CHOLINERGIC ANIMAL MODEL A cholinergic animal model was mimicked by injecting 1 µL of Aβ (1–42) (4 µg/µL) by Hamilton syringe into right-NBM, a main source for cholinergic innervations to the cerebral cortex,29,30 under sodium pentobarbital (45 mg/kg i.p.) anesthesia at the stereotaxic coordinates: AP ⫽ ⫺ 0.2, L ⫽ ⫺ 2.8 from Bregma, and H ⫽ 7 from the dura.31 Control rats were injected with 1 µL of saline solution. The experiments were carried out according to the guidelines of the European Community’s Council for Animal Experiments (86/609/EEC). On the seventh day postinjection, the rat brain regions were dissected out and stored at ⫺80°C. The entire right hippocampus was taken, and the right front cortex and NBM were dissected at the following approximate coordinates (from Bregma): frontal cortex, AP ⫽ ⫹ 2.2 to ⫹ 4.70 mm and L ⫽ 0 to ⫹ 2.5 mm; NBM, AP ⫽ ⫺ 0.4 to 1.80 mm and L ⫽ ⫹ 1.5 to 3.0 mm.

1.2.3 CAENORHABDITIS ELEGANS Transgenic Caenorhabditis elegans that express human Aβ (1–42) through a bodywall muscle myosin promoter and an Aβ minigene32 have been used as in vivo models to study Aβ toxicity and deposition.33,34 The temperature-inducible Aβ expression system in C. elegans is a good model for investigating the relationship between Aβ toxicity, fibril formation, and oxidative stress. C. elegans expressing Aβ (1–42) demonstrates increased protein oxidation prior to fibrillar deposition of the peptide, suggesting that small, soluble oligomers of the peptide are the toxic species.35

1.2.4 SENESCENCE-ACCELERATED MICE PRONE 8 (SAMP8) The SAMP8 mouse strain has undergone a natural mutation, which has resulted in age-dependent learning and memory deficits.36 SAMP8 mice have increased amounts of Aβ and Aβ -like protein immunoreactive granular structures in brain similar to those moieties observed in AD.37,38 Moreover, brains of SAMP8 mice show spheroidal axonal dystrophy in dorsal column nuclei, small neurons in gracile nucleus, and some well-defined or swollen axons.39 Age-related shrinkages of the cholinergic neurons of the laterodorsal tegmental nucleus are observed in aged SAMP8 mice brains.39

1.2.5 TWO-DIMENSIONAL POLYACRYLAMIDE GEL ELECTROPHORESIS Two-dimensional gel electrophoresis (2D-PAGE) is a sensitive technique that allows the separation of proteins based on two physicochemical properties, unlike sodium dodecyl sulfate (SDS)–PAGE, which separates proteins based only on molecular weight. The first step in 2D-PAGE involves separation of complex

Proteomics Identification of Oxidatively Modified Proteins

5

mixtures of proteins on the basis of their isoelectric points, i.e., isoelectric focusing (IEF) in which an inherent property of a protein is utilized, followed by the second dimension separation of proteins based on their relative mobility (Mr).40 The protein maps so obtained allow comparison of different sets of samples in terms of expression, posttranslational modification, etc., using a computerassisted program. Usually, a single spot on the 2D gel corresponds to a single protein.41 In addition, 2D-PAGE is used to catalog proteins and create databases.42 The 2D-PAGE method is sensitive and reliable, with high reproducibility, but many challenges still exist. The serious limitation of 2D-PAGE is the solubilization process for membrane proteins.43 The ionic detergents necessary are not compatible with IEF since a charge would be introduced to the protein, thereby interfering with its separation in first dimension. The second limitation is the inability to detect low-abundance proteins, and the third limitation is its insensitivity to proteins high in trypsin. Chaotropic agents, such as urea and thiourea, coupled with nonionic or zwitterionic detergents can be used to solubilize proteins and avoid protein precipitation during IEF and the SDS-PAGE gel.44 The use of immobilized pH IEF strips eliminates the typical cathodic drift associated with previously used tube gels and improves the protein map reproducibility between samples.45 In addition, the use of narrow-range IEF strips enables the investigator to separate proteins over a wide range of pH, with a unit pH difference of 1. However, the normally employed IEF strip pH range, i.e., 3 to 10, limits the identification of highly basic proteins. The identification of the low-abundance proteins in a given sample is, as noted above, a limitation, one that is important when a protein of this group is involved in the pathogenesis of a disease. Other separation methods, including 2D-HPLC and isotopically coded affinity tags (ICAT), are also used for protein separation. HPLC is often used to separate peptides produced by trypsin digestion of a protein mixture. This technique couples a strong cation-exchange column in tandem with a reverse-phase (RP) column46,47 that is connected to a mass spectrometer. Analysis of protein expression in two different sets of samples together can be achieved by labeling the samples with different isotopes that bind to cysteine residues using the ICAT method. The isotopic labels help in the evaluation of mass spectra and the differences in expression of a protein in the two samples.48 Of course, a limitation of the ICAT method is that cysteine-deficient proteins are refractive to this method. The redox proteomics techniques in our laboratory are used to identify oxidatively modified proteins in the AD brain and related models. In this method, we use a parallel analysis in which we couple 2D-PAGE with immunochemical detection of protein carbonyls derivatized by 2,4-dinitrophenylhydrazine (DNPH), nitrated proteins indexed by 3-NT, and protein adducts of HNE, followed by MS analysis, as shown in Figure 1.1. Proteins containing reactive carbonyl groups/3NT/HNE in AD and control brain samples are detected by 2D Western-blot analysis using specific antibodies. The 2D Western blots and 2D gel images are matched by computer-assisted image analysis, and the anti-DNP/nitrotyrosine/ HNE immunoreactivity of individual proteins is normalized to their content, obtained by measuring the intensity of colloidal Coomassie Blue staining or

6

Oxidative Stress and Age-Related Neurodegeneration Oxyblot

Sample

10 mM DNPH, 2 M HCl

Image analysis

2 M HCl

2D protein map

Protein identification

In-gel trypsin digestion

Database searching

Mass spectrometry

FIGURE 1.1 Protocol for the identification of the oxidized protein by proteomics.

SYPRO ruby stained spots. Such analysis allows comparison of levels of oxidatively modified brain proteins in AD vs. control subjects.

1.2.6 MASS SPECTROMETRY AND DATABASE SEARCHING The proteins of interest are excised, exposed to trypsin, eluted from the gel, and subjected to MS analysis. The method most commonly used is in-gel digestion of protein with a protease that not only cleaves the protein into small peptides, but also produces sequence-specific proteolysis. These mass fingerprints are characteristic of a particular protein and facilitate the identification of a particular protein using a suitable database (Table 1.1) that compares the experimental masses with theoretical masses of trypsin-generated protein sequences. Mass spectrometry is essential for the identification of the peptide masses and amino acid sequences for the proteins of interest. Early MS studies could not provide a precise peptide mass owing to the molecular fragmentation of the sensitive ion. However, the introduction of softer ionization techniques helped to overcome the fragmentation, thereby enabling better identification of proteins. The two most-used MS techniques are matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI). In MALDI analysis, the peptide sample is mixed with a matrix (usually α-cyano-4-hydroxycinnamic acid or 2,5-dihydroxybenzoic acid) and deposited on a plate that is subjected to laser radiation. The matrix absorbs the energy, which is then transferred to the peptides. The peptides then evaporate as detectable MH⫹ ions by an unknown mechanism related to the acidic nature of the matrix. However, in ESI, HPLC is coupled to MS and the liquid sample from the HPLC column enters directly into MS for characterization. Owing to the high potential difference between the capillary and the MS

Proteomics Identification of Oxidatively Modified Proteins

7

TABLE 1.1 Mass Spectrometry Search Engines for Peptide Mass Fingerprinting Search Engine Mascot MOWSE Profound MS-fit Peptident

URL http://www.matrixscience.com http://www.hgmp.mrc.ac.uk/Bioinformatics/Webapp/mowse http://prowl.rockefeller.edu/profound_bin/webProFound.exe http://prospector.ucsf.edu/ucsfhtml4.0/msfit.htm http://ca.expasy.org/tools/peptident.html

instrument, the inlet sample is dispersed as small droplets. These droplets undergo solvent evaporation until droplet fission occurs, owing to the high charge-to-surface tension ratio, finally leading to the formation of a single detectable ion per droplet. For better on-line pre-separation of peptides with HPLC and MS, a low salt concentration is important, since salt may interfere with ionization. In addition, reducing the flow time to nanoliters per minute can increase the time for analysis. Tandem MS/MS provides better isolation and fragmentation of a specific ion. This tandem MS/MS provides further information about the sequence of the protein.49 In MS/MS analysis, the isolation of a single ion is achieved by scanning all of the ions that were generated from a sample, followed by the application of a wide range of frequencies, except for the resonating frequency of the ion of interest. Fragmentation of the isolated ion, which provides additional information for protein identification or for evaluation of possible protein modification, is the final step in MS/MS. An additional proteomics tool in which MS is utilized is surface-enhanced laser desorption/ionization time of flight (SELDI-TOF), which couples the classical methods of chromatographic sample preparation with MS analysis. This method has potential applications in biomarker analysis, but is limited to proteins below a relatively low molecular weight. We did not use this method in the studies outlined in this chapter, but SELDI-TOF is described in detail in our recent review.50 After MS analysis, the correct identity of the protein is determined by on-line protein databases. The protein sequence database SwissProt is the most commonly used database for protein identification that is based on computer algorithms,51 and is available through the Internet. These databases are described in Table 1.1. These search engines provide a theoretical protease digestion of the proteins contained in the database, and the search engines compare the experimental masses obtained by MS and also account for several factors, such as the protein size and the probability of a single peptide occurring in the whole database. The search engine produces a probability score for each entry, which is calculated by a mathematical algorithm that is specific for each search engine. Any hit with a score higher than the one specific for the particular search engine is considered statistically significant and has an excellent and legitimate chance of being the protein cut from a given spot. In addition, the molecular weight and pI of the

8

Oxidative Stress and Age-Related Neurodegeneration

protein are calculated on the basis of the position in the 2D map to avoid any false identification. In many cases, validation of protein identification is achieved by immunochemical means.52–54

1.3 PROTEOMICS STUDIES ON OXIDATIVELY MODIFIED PROTEINS IN THE ALZHEIMER’S DISEASE BRAIN Protein oxidation levels were reported to be increased in the AD brain as indexed by increased protein carbonyls and 3-NT levels.17,19,21,23,55 Oxidative modification of proteins may render them less active, leading to loss of function.12,23,56 Identification of specifically oxidized proteins in the AD brain provides an insight into the role of oxidative stress in AD and also helps to unravel the mechanism(s) associated with AD pathology. Using proteomics, we identified in the inferior parietal and hippocampal regions of AD brains several such oxidatively modified proteins: creatine kinase BB (CK), glutamine synthase (GS), ubiquitin carboxyterminal hydrolase L-1 (UCH L-1), dihydropyrimidinase-related protein 2 (DRP-2), α-enolase, phosphoglycerate mutase 1 (PGM1), γ -soluble NSF attachment protein (SNAP), carbonic anhydrase II (CA-II), and peptidyl prolyl cis–trans isomerase (Pin 1). Similarly, proteomics has been used to identify 3-NT posttranslationally modified proteins in the AD brain: neuropolypeptide h3, triosephosphate isomerase and α-enolase, CA-II, and glyceraldehyde 3-phosphate dehydrogenase.17,55,57,58 All these proteins were assigned to different groups based on their functions and linked to the observed AD pathology (Table 1.2).

1.3.1 PROTEINS INVOLVED

IN

ENERGY METABOLISM

Creatine kinase (BB isoform), α-enolase, triosephosphate isomerase, glyceraldehyde 3-phosphate dehydrogenase, PGM1, and α ATPase are enzymes involved in

TABLE 1.2 Proteomic Identification of Specifically Oxidatively Modified Proteins in the AD Brain Energy-related enzymes Neurotransmitter-related proteins

Proteasome-related proteins Cholinergic system Structural proteins Cell cycle pH regulation protein

Creatine kinase; α-enolase; triosephosphate isomerase; phosphoglycerate mutase 1 Gamma-soluble N-ethylmaleimide-sensitive factor attachment protein; glutamine synthase; glutamate transporter EAAT2 Ubiquitin carboxy-terminal hydrolase L-1; heat shock protein Neuropolypeptide h3 Dihydropyrimidinase-related protein 2; β-actin Peptidyl prolyl cis–trans isomerase Carbonic anhydrase II

Proteomics Identification of Oxidatively Modified Proteins

9

ATP production and energy metabolism, either directly or indirectly. In the AD brain, CK activity is decreased, and the oxidation of this protein suggests the impairment of ATP synthesis.52,59 In addition, oxidation of other proteins involved in glucose metabolism, a main source of ATP production in brain, may lead to cellular dysfunction such as impaired ion-motive ATPase to maintain potential gradients, operate pumps, and maintain membrane lipids asymmetry. ATP diminution also induces hypothermia, leading to abnormal tau hyperphosphorylation through differential inhibition of kinase and phosphatase activities, ion pumps, electrochemical gradients, cell potential, and voltage-gated ion channels.60

1.3.2 PROTEINS INVOLVED

IN

GLUTAMATE REUPTAKE

OR

CONVERSION

In the AD brain, the glutamate transporter EAAT2 has been shown to be oxidatively modified by HNE, a lipid peroxidation product.12 Additionally, synaptosomes treated with Aβ (1–42) induced oxidative modification of EAAT2 by HNE.61 HNE has been shown to induce conformational changes in protein that could impair the function of protein.61 Therefore, oxidative modification of EAAT2 in the AD brain implies the loss of activity of this protein, resulting in accumulation of glutamate in the synaptic cleft, which, in turn, could lead to an influx of calcium into the cell via the N-methyl-D-aspartate (NMDA) and amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors and consequently induce cell death.62

1.3.3 ALTERED SYNAPTIC FUNCTION Studies have shown loss of synaptic circuitry in the AD brain that can be related to the observed cognitive deficits in AD individuals.63,64 γ -SNAP, a member of synaptosomal protein-like soluble N-ethylmaleimide-sensitive factor (NSF) attachment proteins (SNAPs), proteins that play an important role in vesicular transport in the constitutive secretory pathway as well as in neurotransmitter release, hormone secretion, and mitochondrial organization65,66 has been observed to be oxidized in the AD brain.65,66 The oxidation of SNAP can impair the learning and memory processes and alter neurotransmitter systems in the AD brain.

1.3.4 PROTEASOMAL DYSFUNCTION The proteasomal system is crucial for proteolytic degradation of damaged or aggregated proteins from a cell. Such proteins are ubiquintylated, forming a poly(ubiquitin) chain that serves as a signal for export to the 26S proteosome. UCH L-1, an enzyme that removes ubiquitin from the poly(ubiquitin) chain prior to insertion of the damaged protein into the core of the proteasome, i.e., a signal for protein degradation,67 helps to maintain the pool of ubiquitin in cells. UCH L-1 was found to be oxidized in the AD brain.52 Assuming similar findings for other oxidatively modified proteins, the oxidation of UCH L-1 would decrease the activity or inactivate the function of this protein, thereby depleting the free pool of ubiquitin or causing saturation of the proteasome with poly(ubiquitin) chains. In addition, such a scenario also leads to accumulation of the damaged protein and synaptic deterioration and degeneration. A recent in vitro study showed that HNE,

10

Oxidative Stress and Age-Related Neurodegeneration

a lipid peroxidation product, decreases hydrolase activity of recombinant UCH L-1.62,68 Both the cross-linked protein and HNE-bound protein can clog the pore of the proteasome.68 Inactivated UCH L-1 itself leads to oxidative stress in brain.69

1.3.5 LIPID ABNORMALITIES AND CHOLINERGIC FAILURE Neuropolypeptide h3, a phosphatidylethanolamine-binding protein (PEBP) or hippocampal cholinergic neurostimulating peptide (HNCP), has been identified as a specifically oxidized protein in the AD brain.17 PEBP conceivably plays an important role in maintaining phospholipid asymmetry, which is crucial to the structure and function of membranes.70 Moreover, loss of PEBP activity conceivably can lead to the exposure of phosphatidylserine to the outer bilayer leaflet of the membrane, a signal that triggers apoptosis and consequently, cell death. Consistent with the proposed role of Aβ (1–42) in oxidative stress and neurodegeneration in the AD brain,13–15 Aβ (1–42) added to synaptosomes leads to loss of phospholipid asymmetry,71 as does HNE,72 which in turn is formed by Aβ (1–42)-induced lipid peroxidation.12 Another function of this enzyme is to upregulate the level of choline acetyltransferase; the activity of this enzyme is reported to be decreased in the AD brain.73 In synaptosomes treated with Aβ (1–42), HNE is bound to cholineacetyltransferase,13 suggesting the possible role of oxidation of this protein in cognitive decline in the AD.

1.3.6 NEURITIC ABNORMALITIES Proteins such as β -actin and DRP-2 have been found to be expressed differentially or oxidized in the AD brain.17,55,74 Loss of actin activity could be related to the known loss of cytoskeletal network integrity and activation of cellular events that may lead to apoptosis. DRP-2 plays an important role in maintaining interneuronal communication and repair and also regulates the activity of collapsin, a protein that is involved in dendritic elongation and pathfinding. DRP-2 is normally expressed in the developing brain and is rarely found in adult brain. In the AD brain, oxidation of this protein has been observed, which conceivably could be related to the observed cognitive impairment in the AD.17,55,74 The brain is always involved in a constant process of developing new synaptic connections to incorporate newly experienced information, and the oxidation of this protein might lead to impaired memory due to shortening of dendrites and synapse loss in the AD brain.75

1.3.7 CELL CYCLE Recent studies showed that the cell cycle machinery is altered in the AD brain.76 Peptidyl-prolyl isomerases (PPIases) or Pin 1 are chaperone enzymes that catalyze the conversion of the cis to trans conformation, and vice versa, of proteins between given amino acids and a proline.77 PPIases have also been shown to be necessary for entry into mitosis, and we determined by proteomics that PPIase is oxidized in the AD brain.58 This modification can cause dramatic structural modifications, which can affect the properties of targeted proteins. Pin 1 possesses a WW domain, which specifically recognizes pSer-Pro and pThr-Pro motifs in which the first amino acid

Proteomics Identification of Oxidatively Modified Proteins

11

is phosphorylated. Pin 1 binds to many proteins implicated in cell cycle regulation (e.g., p53, Myt1, Wee1, and Cdc25C), and also targets tau, a protein-forming part of the neuronal cytoskeleton, which is hyperphosphorylated in patients suffering from AD.78 Recent studies show that Pin 1 is colocalized with phosphorylated tau and that an inverse relationship between expression of tau and Pin 1 in Alzheimer’s tautopathies exists.79–81 Pin 1 could, therefore, be involved in the pathogenesis of AD. The oxidation of Pin 1 may alter its isomerase activity, thereby increasing the accumulation of phophorylated tau protein in the AD brain. Lu et al.82 showed that Pin 1 could restore the function of tau protein in AD. Pin 1 is unique, and its oxidative alteration could be one of the initial events that trigger tangle formation, cell cyclerelated abnormalities, and oxidative damage.

1.3.8

PH

MAINTENANCE

The pH of the cells plays a crucial role in maintaining the activity of enzymes. − CA-II regulates cellular pH, CO2, and HCO 3 transport, and is involved in main83 taining water and electrolyte balance. CA-II also catalyzes the reversible hydration of CO2, a reaction fundamental to many cellular and systemic processes, including glycolysis and acid and fluid secretion. Catalytically active CA-II is confined to oligodendrocytes and subtypes of protoplasmic astrocytes in the central nervous system (CNS). The physiological functions of CA-II are regulation of cellular pH regulation, production of cerebrospinal fluid (CSF), and the synthesis of glucose and lipids.84 CA-II deficiency results in osteoporosis, renal tubular acidosis, and cerebral calcification, and also leads to cognitive defects varying from disabilities to severe mental retardation. Oxidative modification of CA-II as determined by redox proteomics57 probably explains the diminished enzyme activity that has been reported in the AD brain compared with age-matched control brain.85 Consequently, oxidized CA-II may not be able to balance both the extracellular and intracellular pH and may lead to a pH imbalance in the cell, mitochondrial alterations in oxidative phosphorylation, and impaired synthesis of glucose and lipids. Since pH plays such a crucial role in the functioning of enzymes, modification of CA-II may contribute to the progression of AD.

1.4 OXIDIZED PROTEINS IN ALZHEIMER’S DISEASE PLASMA In AD plasma, isoforms of fibrinogen γ -chain precursor protein and of α-1-antitrypsin precursor were found by proteomics to be oxidized86 and had been implicated in the AD pathology. Indeed, identification of oxidized proteins in plasma using proteomics is being touted as an important biomarker for AD.87

1.5 PROTEOMIC STUDIES ON OXIDATIVELY MODIFIED PROTEINS IN ANIMAL MODELS OF ALZHEIMER’S DISEASE The use of animal models of AD has been of principal importance for providing insight into the neurochemical and cellular changes associated with this disease.

12

Oxidative Stress and Age-Related Neurodegeneration

Several gene knock-in and knock-out models have been constructed to study the role of genetic mutations, for example, APP, PS-1, PS-1/APP double-mutant, tau, and tau/APP/PS-1 animal models containing mutated human genes that represent a means to familial AD or are involved in one or more of the pathological hallmarks of AD. In addition, human gene mutations in animals also facilitate study of the consequences of protein oxidation on cellular activities, as exemplified by the gracile axonal dystrophy (GAD) mouse model. The GAD mouse has a defective UCH L-1, a proteomics-identified oxidized protein in the AD brain,52,69 which showed oxidization of a number of proteins that were identified by proteomics.69

1.5.1 CHOLINERGIC DYSFUNCTION-RELATED ANIMAL MODEL Degeneration of the basal forebrain cholinergic neurons is pronounced in AD and is associated with cognitive deficits.88,89 A cholinergic model of AD has been mimicked by intracerebral injection of Aβ (1–42) into the nucleus basalis magnocellarius of rat brain.90 In this animal model, more extensive protein oxidation was observed in the hippocampus compared to cortex and nucleus basalis, although all three regions demonstrated oxidative damage.91 With the use proteomics, a large number of oxidized proteins were identified, including proteins involved in cellular structure (β -synuclein), signal transduction (14-3-3 zeta), and energy metabolism (PGM1, pyruvate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase), and stress responses (heat shock protein 60 [HSP60]).91 PGM1 was found to be one of the oxidized proteins in the AD hippocampus.57 14-3-3 Proteins are involved in a number of cellular functions, including signal transduction, protein trafficking, and metabolism,92,93 and the expression of these proteins was reported to be increased in the AD brain94 and CSF 95 14-3-3 Proteins are associated with NFT in the AD brain.96 Moreover, 14-3-3 zeta has been shown to act as an effector of tau protein phosphorylation,97 and many act as a scaffolding protein to promote the polymerization of tau protein.98 Recently, it has been shown that 14-3-3 zeta acts as a scaffolding protein simultaneously binding to tau and glycogen synthase kinase-3 β (GSK3β ) in a multiprotein tau phosphorylation complex.99 The oxidation of 14-3-3 zeta in this rat model following Aβ (1–42) injection into NBM could unite both the importance of Aβ (1–42) and the hyperphosphorylation of tau, as observed in AD. β -Synuclein is a presynaptic protein that normally plays a role in synaptic vesicle homeostasis. In rat brain, β -synuclein has been shown to play a role in catecholaminergic components of the CNS, while β -synuclein is associated with cholinergic components, particularly in the basal forebrain.100 If β -synuclein is involved in synapse formation in cholinergic regions of the brain, loss of function due to oxidation could result in loss of synapses and cholinergic deficits documented in AD.64,89,90 Recently, β -synuclein has been shown to increase Akt activity by direct interaction with Akt in neuroblastoma cells transfected with β -synuclein. The increase in Akt activity was shown to protect neurons against rotenone, suggesting that β -synuclein may play a protective role in the CNS.101 If this is the case, oxidation of β -synuclein could lead to a conformation change in the protein preventing its direct interaction with Akt and abolishing this protective effect.

Proteomics Identification of Oxidatively Modified Proteins

13

Heat shock proteins are critical to neuronal function.102,103 Chaperonin 60 (Cpn60), also called HSP60, is a mitochondrial chaperone protein that is involved in mediating the proper folding and assembly of mitochondrial proteins, especially in response to oxidative stress.104 Expression of HSP60 is significantly decreased in AD,105 and Aβ (25–35) has been shown to induce oxidation of HSP60 in fibroblasts derived from AD patients compared with age-matched controls.106 The loss of function of HSP60 could lead to increased protein misfolding and aggregation as well as an increased vulnerability to oxidative stress. This is particularly important owing to the lack of mechanisms to protect mitochondrial proteins from oxidative stress. The proximity of mitochondrial proteins to reactive oxygen species generated during normal oxidative phosphorylation and elevated oxidative stress associated with mitochondcial dysfunction increase risk for mitochondcial oxidative damage.

1.5.2 SAMP8 MICE The SAMP8 mouse strain shows accelerated senescence and develops deficits in learning and memory by 12 months of age and also shows increased oxidative stress in brain.107 The SAMP8 mouse shows Aβ deposition at ages 4 to 12 months at 7- to 20-fold excess compared with Aβ deposition observed in the normal aging mouse brain.108 This large increase in Aβ in the SAMP8 mouse brain is much closer to the estimated 50% increase in Aβ observed in AD brain109 than to that observed in APP transgenic mouse models. Hence, SAMP8 mouse may be an excellent model for studying Aβ toxicity. Previously oxidized proteins were identified in aged mice using a combination of MudPIT techniques with a hydrazine biotin–streptavidin isolation of carbonylated proteins.110 Recently, we identified α-enolase, γ -enolase, lactate dehydrogenase 2, α-spectrin, CK, and DRP-2 as oxidized proteins in SAMP8 mice brain.53 Among these proteins, α-enolase, γ -enolase, DRP-2, LDH, and CK are the oxidized proteins commonly observed in the human AD brain and SAMP8 mice by proteomics. The expression of α-spectrin and DRP-2 was reported to be altered in AD brain. As noted above, DRP-2 is one of the four members of the dihydropyrimidinase-related protein family (DRP-1, -2, -3, and -4) that is involved in axonal outgrowth and pathfinding through transmission and modulation of extracellular signals.111–114 One of the identified extracellular signals is mediated by the proteins of the collapsin–semaphorin family in collaboration with their receptor, neuropilion.115,116 Collapsin contributes to axonal pathfinding by inducing growth cone collapse, which repels the outgrowing axon.117 It was reported that DRP-2 can induce growth cone collapse112,118 by ρ -kinase phosphorylation,119 and binding to tubulin heterodimers and bundled microtubule as carriers to promote microtubules assembly and dynamics.120,121 Many neurodegenerative diseases are associated with DRP-2. It was suggested that incorporation of DRP-2 in the NFT decreases cytosolic DRP-2 and leads to abnormal neuritic and axonal growth, thus accelerating the neurotic degeneration in AD.122 Decreased expression of DRP-2 protein was observed in AD, adult Down’s syndrome (DS),74 fetal DS,123 schizophrenia, and affective disorders.124 Thus, oxidation of DRP-2 in both SAMP8 and AD could conceivably contribute to the cognitive dysfunction observed in each case.

14

Oxidative Stress and Age-Related Neurodegeneration

The spectrins are a family of widely distributed filamentous proteins. α-Spectrin is a membrane-associated cytoskeletal protein that forms a supporting and organized scaffold for intracellular cohesion with the association of actins.125 α-Spectrin breakdown products are used as markers of apoptosis.126 Aβ can also induce these α-spectrin breakdown products in cultured rat cortical neurons by activating caspases.127 Consistent with these studies, our proteomics results demonstrated a decreased level of α-spectrin in aged SAMP8 mouse brain as well as an increased specific carbonyl level, suggesting that the proteolytic mechanism in apoptosis involves oxidative modification and degradation of α-spectrin.53 This suggests that loss of α-spectrin by oxidation or degradation would disrupt the cytoskeleton and the structure of cells in brain, thereby affecting intercellular and intracellular communications and, consequently, contributing to the learning and memory deficits observed in SAMP8 mice. Consistent with the role of oxidative damage in SAMP8 mice,128,129 α-lipoic acid given to SAMP8 mice leads to modulation of the oxidatively modified proteins, as demonstrated by proteomics.130 Moreover, the learning and behavior of aged SAMP8 mice observed following α-lipoic acid treatment suggest that oxidative stress underlies the cognitive impairment of accelerated aging. Consistent with this notion, antisense oligonucleotide directed at the Aβ region of the APP gene not only improved the learning and behavior of aged mice, but also lowered Aβ levels, decreased oxidative stress, and led to modulation of specifically oxidized brain proteins in aged SAMP8 mice.53,130

1.5.3 CAENORHABDITIS ELEGANS Transgenic C. elegans serve as an in vivo model of Aβ -associated toxicity and deposition. Human Aβ (1–42) is expressed via a body-wall muscle myosin promoter and an Aβ minigene.34,35 Recently, we showed that C. elegans expressing Aβ (1–42) exhibited increased oxidative stress and also suggested association of methionine 35 of Aβ (1–42) in oxidative damage.34 The temperature-inducible Aβ expression system in the C. elegans is an excellent model for studying the relationship between Aβ toxicity, fibril formation, and oxidative stress by proteomics.131 In this animal model, 16 proteins were identified to be oxidatively modified. These proteins were involved in a variety of cellular functions, including energy metabolism, protein degradation, cytoskeletal integrity, the antioxidant system, signal transduction, and lipid metabolism. Protein oxidation has been shown to alter protein conformation, leading to loss of function.12,23,55,57,61 Thus, it is likely that oxidation of the proteins identified in this study also leads to loss of function. The proteins involved in energy metabolism and cell signaling that were found to be oxidized in C. elegans include acyl-CoA dehydrogenase, transketolase, guanine nucleotide-binding proteins, adenine kinase, arginine kinase (analogous to CK in humans), and malate dehydrogenase. Although, until this study, there was no evidence that these proteins were oxidized in the AD brain, by analogy, these proteins were reported to have altered activity and expression.132,133 Future studies will help us to understand the mechanism of Aβ -induced oxidative stress and associated toxicity.

Proteomics Identification of Oxidatively Modified Proteins

15

Glutathione S-transferases (GSTs), antioxidant enzymes that catalyze the reaction of reactive alkenals like HNE, with glutathione belong to a class of major antioxidants that are abundant in the brain.134–137 Recently, we reported excessive binding of HNE to GST in AD brain that could be associated with the observed decreased GST activity.54 The observation that this protein is one of the oxidized proteins in AD brain is consistent with the view that oxidative stress plays a role in the progression of AD.13–15,54,138,139 Similar to the AD brain, C. elegans also showed an increase in oxidation of actin.21,64,117,138 In the AD brain, proteasomal activity is inhibited by oxidation of proteosomal UCH L-1, whereas in C. elegans we observed an oxidation of proteasome α subunit 4, suggesting that cytoskeletal alterations may play a role in proteasome inhibition.140–143 In addition, C. elegans expressing human Aβ (1–42) also showed an increased oxidation of myosin light chain 1, suggesting that the oxidation of myosin is in response to Aβ (1–42) and likely a direct effect of the proximity of the protein to Aβ -mediated ROS (reactive oxygen species). Another protein that is found to be oxidized is lipid-binding protein 6 (LBP-6), a fatty-acid-binding protein that plays roles in lipid metabolism and fatty acid transport. Oxidation of LBP-6 was found to beinduced by overexpressing Aβ (1–42) in C. elegans, suggesting Aβ (1–42)-induced alteration in lipid metabolism. This finding is consistent with the observation of elevated lipid peroxidation in AD12,138 and perhaps altered cholesterol homeostasis and decreased membrane fluidity in AD. Moreover, cholesterol modulates the toxicity of Aβ (1–42) on neuronal membranes.144 However, in brain from APP/PS-1 doublemutant mice raised on a high-cholesterol diet, the elevated oxidative stress caused by the mutations (presumably due to excess Aβ (1–42)) was not increased further by the high-cholestrol diet.71

AD Glutamine synthase, UCHL-1, -SNAP, carbonic anhydrase II, Pin 1, neuropolypeptide h3, triosephosphate isomerase

SAMP 8 mice, Deposits A
(1– 42) Lactate dehydrogenase 2, -Spectrin

-Enolase, DRP-2, -Enolase CK

Glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate mutase 1 GST


-Synuclein, 14-3-3 zeta, pyruvate dehydrogenase, HSP 60

A
(1–42) mediated-cholinergic deficit animal model

Acyl CoA dehydrogenase, transketolase, guanine nucleotide-binding protein, Adenine kinase, Proteasome  subunit 4, myosin light chain 1, lipid-binding protein-6

C. elegans, expressing human A
(1–42)

FIGURE 1.2 Comparison of oxidatively modified proteins in AD brain and different models of this disorder.

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1.6 FUTURE OF PROTEOMICS IN ALZHEIMER’S DISEASE A number of common proteins were found to be oxidatively modified in all the models that are in common with the AD brain (Figure 1.2). The presence of these oxidized proteins in various animal models as well as the AD brain suggests the role of Aβ (1–42) in the induction of oxidative stress and related altered AD biochemistry and pathology. Hence, proteomics approaches, together with these animal models, provide insight into the mechanisms of neurodegeneration in AD and could also help in the development of therapeutic approaches to prevent or delay the onset of this devastating disease. With the improvement in proteomics technology, other kinds of protein posttranslational modifications can be studied. Continued proteomics analysis of AD and models thereof are in progress in our laboratory.

ACKNOWLEDGMENTS This work was supported, in part, by NIH grants (AG-05119; AG-10836). We thank Professors John. B. Klein, University of Louisville, and Bert Lynn, University of Kentucky, for help and discussions about proteomics applications.

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124. Johnston-Wilson NL, Sims CD, Hofmann JP, Anderson L, Shore AD, Torrey EF, Yolken RH. Disease-specific alterations in frontal cortex brain proteins in schizophrenia, bipolar disorder, and major depressive disorder. The Stanley Neuropathology Consortium. Mol Psychiatry, 2000; 5:142–149. 125. Leto TL, Fortugno-Erikson D, Barton D, Yang-Feng TL, Francke U, Harris AS, Morrow JS, Marchesi VT, Benz EJ, Jr. Comparison of nonerythroid alpha-spectrin genes reveals strict homology among diverse species. Mol Cell Biol, 1988; 8:1–9. 126. Vanderklish PW, Bahr BA. The pathogenic activation of calpain: a marker and mediator of cellular toxicity and disease states. Int J Exp Pathol, 2000; 81:323–339. 127. Harada J, Sugimoto M. Activation of caspase-3 in beta-amyloid-induced apoptosis of cultured rat cortical neurons. Brain Res, 1999; 842:311–323. 128. Farr SA, Poon HF, Dogrukol-Ak D, Drake J, Banks WA, Eyerman E, Butterfield DA, Morley JE. The antioxidants alpha-lipoic acid and N-acetylcysteine reverse memory impairment and brain oxidative stress in aged SAMP8 mice. J Neurochem, 2003; 84:1173–1183. 129. Butterfield DA, Hensley K, Cole P, Subramaniam R, Aksenov M, Aksenova M, Bummer PM, Haley BE, Carney JM. Oxidatively induced structural alteration of glutamine synthetase assessed by analysis of spin label incorporation kinetics: relevance to Alzheimer’s disease. J Neurochem, 1997; 68:2451–2457. 130. Poon H, Farr S, Thongboonkerd V, Lynn BC, Banks WA, Morley JE, Klein JB, DA B. Proteomic analysis of specific brain proteins in aged SAMP8 mice treated with alpha-lipoic acid: implications for aging and age-related neurodegenerative disorders. Neurochem Int, 2005; 46:159–168. 131. Boyd-Kimball D, Poon HF, Lynn BC, Cai J, Pierce WM, Klein JB, Ferguson J, Link CD, Butterfield DA. Proteomic identification of proteins specifically oxidized in Caenorhabditis elegans expressing human Ab(1-42): implications for Alzheimer’s disease. Neurobiol Aging, 2005 (in press). 132. Gibson GE, Sheu KF, Blass JP, Baker A, Carlson KC, Harding B, Perrino P. Reduced activities of thiamine-dependent enzymes in the brains and peripheral tissues of patients with Alzheimer’s disease. Arch Neurol, 1988; 45:836–840. 133. Aksenov M, Aksenova M, Butterfield DA, Markesbery WR. Oxidative modification of creatine kinase BB in Alzheimer’s disease brain. J Neurochem, 2000; 74:2520–2527. 134. Xie C, Lovell MA, Markesbery WR. Glutathione transferase protects neuronal cultures against four hydroxynonenal toxicity. Free Radic Biol Med, 1998; 25:979–988. 135. Leiers B, Kampkotter A, Grevelding CG, Link CD, Johnson TE, Henkle-Duhrsen K. A stress-responsive glutathione S-transferase confers resistance to oxidative stress in Caenorhabditis elegans. Free Radic Biol Med, 2003; 34:1405–1415. 136. Mark RJ, Lovell MA, Markesbery WR, Uchida K, Mattson MP. A role for 4-hydroxynonenal, an aldehydic product of lipid peroxidation, in disruption of ion homeostasis and neuronal death induced by amyloid beta-peptide. J Neurochem, 1997; 68:255–264. 137. Lauderback CM, Kanski J, Hackett JM, Maeda N, Kindy MS, Butterfield DA. Apolipoprotein E modulates Alzheimer’s Abeta(1-42)-induced oxidative damage to synaptosomes in an allele-specific manner. Brain Res, 2002; 924:90–97. 138. Markesbery WR, Lovell MA. Four-hydroxynonenal, a product of lipid peroxidation, is increased in the brain in Alzheimer’s disease. Neurobiol Aging, 1998; 19:33–36.

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139. Lovell MA, Xie C, Markesbery WR. Decreased glutathione transferase activity in brain and ventricular fluid in Alzheimer’s disease. Neurology, 1998; 51:1562–1566. 140. Zheng WH, Bastianetto S, Mennicken F, Ma W, Kar S. Amyloid beta peptide induces tau phosphorylation and loss of cholinergic neurons in rat primary septal cultures. Neuroscience, 2002; 115:201–211. 141. Sullivan PG, Dragicevic NB, Deng JH, Bai Y, Dimayuga E, Ding Q, Chen Q, Bruce-Keller AJ, Keller JN. Proteasome inhibition alters neural mitochondrial homeostasis and mitochondria turnover. J Biol Chem, 2004; 279:20699–20707. 142. Keller JN, Hanni KB, Markesbery WR. Impaired proteasome function in Alzheimer’s disease. J Neurochem, 2000; 75:436–439. 143. Jayarapu K, Griffin TA. Protein-protein interactions among human 20S proteasome subunits and proteassemblin. Biochem Biophys Res Commun, 2004; 314:523–528. 144. Eckert GP, Kirsch C, Leutz S, Wood WG, Muller WE. Cholesterol modulates amyloid beta-peptide’s membrane interactions. Pharmacopsychiatry, 2003; 36 (Suppl 2):S136–S143.

Evaluation of 2 Direct Alzheimer’s DiseaseSpecific Oxidative Stress with Multiphoton Microscopy Monica Garcia-Alloza, Sarah A. Dodwell, and Brian J. Bacskai Massachusetts General Hospital Charlestown, Massachusetts

CONTENTS 2.1 2.2

Introduction................................................................................................27 Mechanisms of Oxidative Stress-related Toxicity in Alzheimer's Disease...................................................................................28 2.2.1 Reactive Oxygen Species in Alzheimers’s Disease.......................29 2.2.2 Transition Metals in Alzheimer’s Disease .....................................29 2.3 Natural Antioxidants ..................................................................................30 2.4 Oxidative Stress Markers...........................................................................31 2.5 Multiphoton Microscopy ...........................................................................31 2.5.1 Imaging Reactive Oxygen Species using Multiphoton Microscopy ...............................................................32 2.5.2 Effect of Antioxidants on Reactive Oxygen Species associated with Senile Plaques using Multiphoton Microscopy.....................33 Acknowledgments ...............................................................................................36 References ...........................................................................................................37

2.1 INTRODUCTION Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by a progressive decline in memory, impairment of cognitive function and behavioral deterioration. Senile plaques are a major hallmark of the disease, and are mainly 27

28

Oxidative Stress and Age-Related Neurodegeneration

composed of the 39–43-amino-acid peptide amyloid-β (Aβ )1 derived from the proteolytic processing of the transmembrane glycoprotein amyloid precursor protein (APP). APP can be secreted or cleaved by α, β and γ secretases.2 Several genes have been described that link Aβ with the disease, including the APP gene located on chromosome 21, presenilin genes 1 and 2 located on chromosomes 14 and 1, and the apolipoprotein E gene located on chromosome 19. Even though the ultimate neurotoxic mechanisms of Aβ are unknown, free radical hypotheses can contribute to the development of AD as both genetic and nongenetic causes are involved.3 In this context, the high energy requirements, high oxygen consumption, and relatively low antioxidant defenses make the central nervous system extremely vulnerable to oxidative stress,4 supporting the implication of oxidation-mediated damage in AD. There is no successful treatment to significantly delay or prevent the progression of AD. At present only palliative treatment is available and is mostly circumscribed to acetylcholinesterase inhibitors, although new approaches include N-methyl-D-aspartate (NMDA) receptor inhibitors, anti-inflammatory drugs, or antiamyloid agents.5 Therefore, treating dementia has become a challenging obstacle for physicians, and antioxidant therapy is a promising approach for delaying or preventing the progression of the illness. Most current work on oxidative stress in AD utilizes indirect markers of oxidative stress to check protein, lipid, or DNA oxidation. While extremely valuable, these methods only provide surrogate markers of oxidative damage. A more direct approach, using multiphoton microscopy (MPM), provides an advantage over these methods as it allows imaging of oxidative stress both in vitro in brain sections, and in vivo in the intact animal. By this means it is possible to image the oxidative stress associated with the senile plaques of DA. Using MPM, the protective ability of several known antioxidants including natural polyphenols, vitamin E, Ginkgo biloba extract, and the well-characterized spin trap N-tert-butyl-αphenylnitrone (PBN) were characterized in vitro. Several of these compounds were then tested for protective activity from oxidative stress resulting from senile plaques in living, transgenic mice. Using these tools, MPM opens the door for systematically screening new antioxidants, in vitro to in vivo, helping in the search for rational therapies for the prevention or reduction of oxidative stress in AD.

2.2 MECHANISMS OF OXIDATIVE STRESS-RELATED TOXICITY IN ALZHEIMER’S DISEASE Alzheimer’s is a multifactorial disease, and even though the neurotoxic mechanisms of Aβ have not been completely elucidated (probably owing to the complexity of the concomitant alterations) oxidative stress seems to play an important role. There is abundant evidence of the damaging role of free radicals as they provoke membrane lipid peroxidation, protein and DNA oxidation, or lead to the production of advanced glycation end products, not only in AD but also in other diseases, such as ischemia, Parkinson’s disease, or amyotrophic lateral sclerosis.6 Moreover, the low content of glutathione in neurons and the high requirement of oxygen for brain metabolism increase the vulnerability of the central nervous system. The most striking risk factor

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29

for AD, i.e. aging, supports the implication of oxidative stress in the illness as the effect of free radicals accumulate over the years.7 Although some studies have shown that Aβ has a neuroprotective role,8 the major body of research supports the toxic effect of Aβ .9–11 Aβ can induce the production of H2O212 and it is capable of causing protein peroxidation.13 Aβ peptides are also responsible for the formation of free radicals in cell culture, causing membrane lipid peroxidation and leading to cell death in vitro.14,15 On the other hand Pratico et al.16 have described that lipid peroxidation precedes Aβ deposition, suggesting that oxidative stress is a cause and not a consequence of Aβ deposition. Indirect markers of oxidative stress have also detected some compensating mechanisms in postmortem brain tissue from AD patients, as increased levels of defensive enzymes such as superoxide dismutase (SOD), catalase or glutathione peroxidase,17 increased lipid peroxidation,18 increased protein oxidation,19 or DNA oxidation.20 Other studies have also detected reduced levels of antioxidants such as vitamins A, C, or E in plasma and cerebrospinal fluid from AD patients.21–23 Aβ interacts with the receptor for advanced glycation end products (RAGE) and directly increases the production of intracellular reactive oxygen species (ROS) and indirectly induces the activation of microglia.24 Microglial cells present near senile plaques can also produce and release free radicals.25 All these data come to confirm the importance of oxidative stress and the related neurotoxic effects in AD.

2.2.1 REACTIVE OXYGEN SPECIES

IN

ALZHEIMERS’S DISEASE

Reactive oxygen species are normal products of the cell cycle with activities including host defense to neuronal signal transduction. However, when their potentially damaging role is not counterbalanced by detoxification, oxidative stress appears. Many free radicals are probably involved, including nitric oxide, peroxinitrite, hydroxyl radicals, superoxide or hydrogen peroxide. Supporting evidence demonstrates that Aβ toxicity is mediated through ROS. Peak values of nitric oxide and reactive nitrogen species are found in Tg2576 mice at 9 months, just when Aβ plaque deposition begins, suggesting that ROS may have a potentially predisposing role in AD,26 and Aβ 1-40 also leads to a significant increase in macrophage nitric oxide production.27 Aβ increases hydrogen peroxide levels in cell culture10 and hydroxyl radicals seem to be initiators of lipid peroxidation in Aβ stimulated PC12 cells.14 Moreover, amyloid seems to drive superoxide-mediated endothelial damage28 and, it also activates NADPH oxidase in microglia by increasing superoxide levels.29,30 Tabner et al.30 have shown that solutions of Aβ peptides liberate hydroxyl radicals upon incubation in vitro followed by the addition of small amounts of Fe(II), suggesting that the direct production of hydrogen peroxide during the formation of the abnormal protein aggregates may be responsible for cell death in AD.

2.2.2 TRANSITION METALS

IN

ALZHEIMER’S DISEASE

The transition metals are of special relevance in oxidative stress (for review see [3,6,31]) as they have been found in the core and in the surroundings of plaques.

30

Oxidative Stress and Age-Related Neurodegeneration

Transition metals are both a direct source of ROS and accelerators of Aβ aggregation.6,32 The conversion of superoxide radicals and water peroxide into hydroxyl radicals33 can only occur in the presence of catalytic amounts of transition metals. Following this idea, iron has been shown to be a free radical source, and the distribution of iron in the brain of AD patients seems to overlap with the distribution of senile plaques and tangles.34 Aluminum has been described as a contributor to the pathogenesis of AD,35 although this point has not been confirmed in other studies.36 Copper has also been implicated in AD: copper is a transition metal necessary in enzyme activities37 that can catalyze the production of ROS. APP possesses a copper binding site,38,39 and negative correlations between copper serum levels and cognitive function have also been detected.35 On the other, hand selenium, chromium, or cobalt seem to have some protective effect on cognitive function.35 It has also been pointed out that metal chelation may interfere with Aβ fibrillization40 and that the copper/zinc chelator clioquinol reduces Aβ accumulation in Tg2576 mice,41 supporting the potential utility of metal chelator therapies in the treatment of the illness.

2.3 NATURAL ANTIOXIDANTS Owing to the limited success of the pharmacological treatments to combat AD, and because of the implications of oxidative stress in the disorder, many efforts have been directed toward studying the potential activity of antioxidants to prevent or reduce oxidative stress in AD. The protective effect of diets rich in antioxidants is widely accepted in disorders such as cardiovascular disease or cancer, and similar assertions have been made in relation to AD.42 Among these substances, natural antioxidants such as vitamins, proanthocyanidins, or bioflavonoids have received special attention because of their high concentration in fruits, vegetables, seeds, nuts, and leaves, as well as because of their wide range of biological, pharmacological, and therapeutic activities against free radicals and oxidative stress.43,44 Some of these compounds have shown a positive role in one or more aspects related to AD, including cell culture experiments,45–47 animal models of AD48,49 or even in studies with AD patients.50,51 Natural polyphenols can act as reducing agents, hydrogen-donating antioxidants, or singlet oxygen quenchers.44 Individually assessed polyphenols, such as quercetin, chrysin, or rutin, have been shown to reduce protein glycation,52 lipid peroxidation,53 or even to inhibit the formation and extension of Aβ as well as to disrupt preformed Aβ fibrils.43,47,54 A wine related polyphenol, resveratrol, has been shown to protect hippocampal cells against nitric oxide toxicity55 and epidemiological studies have shown that moderate red wine consumption has a protective effect on the occurrence of AD.56 Moreover, plant extracts, rich in different polyphenols, such as tea extract, grape seed extract, or more commonly Ginkgo biloba extract (see chapter 3 by Christen, chapter 18 by Smith and Luo and chapter 23 by Luo and Butko for further information) have also shown antioxidant and neuroprotective effects.43,54 Ginkgo biloba extract

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31

protects hippocampal cells against ROS and Aβ -induced toxicity57,58 and it also improves cognitive function in patients suffering from dementia.51,59 On the other hand the effect of vitamins A, C, or E, as natural antioxidants highly abundant in fruits and vegetables, has been widely assessed in different paradigms associated with AD.47,60,61 Epidemiological studies have shown significant reductions in vitamins A, C, or E in plasma or cerebrospinal fluid from AD patients,21,62 although these data have not been reproduced in other studies or seem to depend on the brain area under study.63,64 It is still not clear whether these alterations in vitamin levels are part of the etiology of the illness or just a consequence of it.65 At this point it seems clear that these natural antioxidants are responsible for some beneficial effects on oxidative stress and neuroprotection in one or more aspects related to AD.

2.4 OXIDATIVE STRESS MARKERS Although there is no doubt about the implication of oxidative stress in AD, the extent of the damage has been difficult to assess directly. Most of the studies have been performed on AD postmortem brain tissue using surrogate markers or histopathologic evidence of oxidative stress.6 Similar approaches have been used in the case of animal models or cell culture studies. Different markers ofprotein peroxidation have been analyzed in AD brains, showing increased levels of protein carbonyls.13,66 Related enzymes such as carbonyl reductase, alcohol dehydrogenase,67 or advanced glycation end products68 have also been assessed in the illness. DNA oxidation has been mostly assessed by determination of the DNA adduct 8hydroxy-2⬘-deoxyguanosine; in this case whereas mitochondrial DNA seems to be affected in the early stages of the illness, nucleic DNA does not seem to be damaged.20,69 Increased lipid peroxidation has been assessed by measuring malondialdehyde levels with the thiobarbituric acid-reacting substrates (TBARS) in brains from AD patients,18 although these results seem to be determined by the brain region under study.66 Moreover, this test is nonspecific, and it is generally poor when applied to biological samples.70 These techniques provide extremely valuable information about the oxidation status in animal models as well as in AD brains, and the disturbances observed seem to affect primarily the most severely affected brain areas in AD, such as the hippocampus and temporal cortex. However, as surrogate markers they cannot establish a direct relation between the observed effects and amyloid plaques. Therefore, an approach that allows direct monitoring of ADspecific oxidative stress would be very informative. An in vivo approach would be ideal, allowing direct monitoring of oxidative metabolism in the mouse models.

2.5 MULTIPHOTON MICROSCOPY MPM is a technique that allows real-time imaging of fluorescence in thick, living tissue with confocal-like resolution. MPM has been used for in vivo imaging of

32

Oxidative Stress and Age-Related Neurodegeneration

fluorescence in mice, rats, and even in cats.71 Minimally invasive MPM offers a new approach to image brain structures in intact animals, providing about two orders of magnitude higher spatial resolution than classical brain imaging technologies, such as magnetic resonance imaging (MRI), positron emission tomography (PET), or single-photon emission tomography (SPECT). MPM spatial resolution, in the scale of micrometers, allows the possibility of assessing cellular and subcellular structures in vivo.72 The number of synthetic contrast agents available for fluorescence imaging is enormous and is continuously increasing, both for structural and functional readouts, providing new approaches to investigate biological and pathological processes. Moreover, a single-excitation wavelength can excite different fluorophors simultaneously allowing multiplexed measurements. The imaging resolution of MPM, ~1 µm, allows cellular and subcellular imaging of the metabolic state of cells, spine morphology, signaling mechanisms, and individual synapses; structures and features too small to be detected with classical neuroimaging technologies. Brain MPM imaging is mostly conducted through cranial windows,73–75 however it is also possible to image the brain through a thinned skull of living animals.76 This approach allows imaging the brain time and again, making this technique particularly useful for the study of chronic diseases or for pharmacological treatments. Experimental studies using MPM in living animals have been successfully applied to transgenic mouse models of AD73,74 to study the dynamic process of Aβ deposition in the living brain as well as novel therapeutic approaches. In this sense, MPM has been used with fluorescent structural markers to monitor the dynamics of senile plaques in transgenic mice. It is also possible to use MPM to monitor functional fluorescent probes in the living brain.

2.5.1 IMAGING REACTIVE OXYGEN SPECIES MICROSCOPY

USING

MULTIPHOTON

In order to measure oxidative stress directly related to AD, we have recently developed a technique that allows the study of oxidative stress in association with Aβ plaques in vivo and in vitro.77 With this approach, MPM can provide real-time imaging of fluorescent oxidation reporter molecules: 2⬘,7⬘-Dichlorodihydrofluorescein (H2DCF) that after oxidation by ROS forms the green fluorescent 2⬘,7⬘-dichlorofluorescein (DCF),78 or Amplex red (AR) reagent (10-acetyl-3,7-dihydroxyphenoxazine) that oxidizes to the red fluorescent resorufin in the presence of hydrogen peroxide.79 In this case, MPM is especially useful because the UV or blue-green light used to detect H2DCF in confocal microscopy rapidly oxidizes the agent, whereas the near-infrared light used for multiphoton imaging does not lead to H2DCF or AR activation, even after prolonged exposures of more than 15 min.77 Moreover MPM permits the simultaneous detection of a histochemical marker of dense-core senile plaques and the fluorescent reporter molecule. By this means, it is possible to detect the fluorescent reporter AR in one optical channel in combination with thioflavin S in a second optical channel. In the same way it is possible to work with the reporter

Direct Evaluation of Alzheimer’s Disease-Specific Oxidative Stress

33

molecule H2DCF in combination with thiazine red. Using either AR or H2DCF, MPM provides a reliable, quick, and easy method to test the effectiveness of therapies targeting Aβ and its capacity to generate free radicals in the brain of AD tissue samples as well as in different transgenic models77 of AD, including PDAPP,80 Tg2576,81 or B6C3.82 McLellan et al.77 demonstrated the activation of both H2DCF and AR by dense-core thioflavin S-positive amyloid plaques, but not diffuse deposits. This suggests that free radical oxidation occurs in close physical proximity to densecore senile plaques (Figure 2.1). However, this does not preclude the possibility that diffuse amyloid deposits or even soluble, oligomeric forms of Aβ could be a low-level source of oxidative stress. No cellular response was necessary for the detected oxidative stress, as similar results were observed in fixed tissues sections. Moreover,neither AR nor H2DCF crosses cell membranes, suggesting that the observed fluorescence is a consequence of extracellular oxidation or free radicals. These in vitro studies demonstrated that quantitative screening of AD-specific antioxidative agents was possible with a “high throughput” approach. It is also possible to generate quantitative dose–response curves for each compound or complimentary antioxidant mixture, in order to determine the necessary amount for effective prevention of Aβ -derived oxidative stress. Moreover, the utility of the method to examine free radical oxidation was used not only in ex vivo studies, but also in living transgenic mice. This approach provides a distinct advantage over other ex vivo studies using surrogate markers of oxidative stress, as by this means it is possible to check not only the current status of oxidative processes, but the chronic evolution of the illness or the effectiveness of pharmacological treatments. Moreover, as ROS seem to be implicated in Parkinson’s disease, Down syndrome, ischemia, amyotrophic lateral sclerosis or head trauma,83 MPM may provide a useful approach in the treatment of other diseases.

2.5.2 EFFECT

OF

ANTIOXIDANTS ON REACTIVE OXYGEN SPECIES SENILE PLAQUES USING MULTIPHOTON

ASSOCIATED WITH

MICROSCOPY The MPM technique, to study oxidative processes in relation to senile plaques, opens the door to check the efficacy of antioxidants using reporting agents sensitive to free radicals. Some antioxidants may be fluorescent, and would interfere with the assay reagents, although it is unlikely that a compound would interfere with both green DCF as well as AR fluorescence. Owing to the well-characterized antioxidant activity of PBN it was possible to test the ability of the spin-trap molecule to reduce both AR and H2DCF oxidation PBN is a nitrone compound that interacts not only with oxygen radicals but also with carbon- or nitrogen-centered radicals, such as nitric oxide. PBN has been shown to reduce the lesion size in ischemic models,84 probably by removing free radicals produced in the ischemic process. Other studies have shown the neuroprotective effect of PBN in vitro, using the colorimetric MTT reduction assay,85 as well as its capacity to lower oxidant production measured by H2DCF.86

34

Oxidative Stress and Age-Related Neurodegeneration Dense-core plaques

Diffuse amyloid (B)

(C)

(D)

(E)

(F)

Anti-amyloid-


Thioflavine S

Amplex red

(A)

FIGURE 2.1 Dense-core plaques produce free radicals ex vivo in human AD tissue. Dense-core plaques oxidize Amplex Red (A), but diffuse amyloid-β (Aβ) does not (B). Thioflavin S staining (C,D) and anti-amyloid-β immunohistochemistry (E, F), in the same tissue sections confirm that Amplex Red reports oxidative stress specifically from thioflavin S-positive dense-core plaques, but not diffuse amyloid-β alone. Scale bars: A, C, E ⫽ 50 µm; B, D, F ⫽ 100 µm. (From McLellan ME, Kajdasz ST, Hyman BT, Bacskai BJ, J Neurosci, 2003; 23:2212–2217.Copyright 2003, The Society for Neuroscience.)

It has also been shown that PBN can effectively reduce brain cell membrane lipid peroxidation.87 McLellan et al.,77 using MPM, demonstrated that the deleterious oxidative effects resulting from senile plaques in the brain can be significantly reduced with PBN. When assessed ex vivo, in brain slices from Tg2576 or PDAPP mice, PBN

Direct Evaluation of Alzheimer’s Disease-Specific Oxidative Stress

35

was capable of reducing extracellular free radical oxidation of H2DCF associated with senile plaques. Ex vivo dose–response studies also show a statistically significant reduction in AR intensity, associated with senile plaques in Tg2576 mice, when PBN doses between 1 mM and 10 µM are used (Figure 2.2A). After proving the efficacy of PBN ex vivo in the same study McLellan et al.77 used MPM to asses PBN antioxidant activity in vivo. PBN (300 mg/Kg) was administered i.p. 20 h and 15 to 20 min before brain surgery, as well as locally (100 µM) on the brain surface before imaging with H2DCF and thioflavin S. PBN was capable of reducing H2DCF fluorescence in vivo by ~40%, supporting the protective role of this compound in oxidation processes associated with senile plaques in AD (Figure 2.2B). We have also assessed the effect of some natural antioxidants including vitamin E, natural polyphenols such as hesperitin, or natural extracts such as Ginkgo biloba extract ex vivo with MPM. In this way we have detected that there is a significant effect of these compounds in reducing AR intensity at the highest doses under study (1 mM for hesperitin and vitamin E, and 1 mg/Kg for Ginkgo biloba extract) when compared with control values; ~40% in case of vitamin E and Ginkgo biloba, and ~25% in case of hesperitin. Table 2.1 shows the effect of these compounds on AR oxidation associated with senile plaques ex vivo.

(A)

(B)

Ex vivo †

120 Plaque intensity (% control)

In vivo

† 100 80 60

∗

∗

∗

40 20 0

Control 1mM 100µm 10µm

1µm 100nm 10nm

Control PBN

PBN

FIGURE 2.2 Quantitative inhibition of oxidative stress from senile plaques. A) In vitro: tissue sections from Tg2576 mouse brain after treatment with PBN (1 mM-10 nM) using Amplex Red (AR) as oxidation reporting molecule. Significant differences were determined by one-way ANOVA followed by Tukey-b test (* P⬍0.05 vs. Control: † P⬍0.05 vs. PBN 1 mM, 100 µM and 10 µM). (B) In vivo: Tg2576 intact mice after treatment with PBN (300 mg/Kg ip and100 µM locally) using 2⬘,7⬘-dichlorodihydrofluorescein (H2DCF) as oxidation reporting molecule. Significant differences were determined by Student’s t-test (*P⬍0.001). Fluorescence intensity of AR and H2DCF resulting from plaque-mediated oxidation was normalized to thioflavin S intensity. Data are expressed as percentage of control values. Data are representative of 12 to 144 plaques from 2 to 3 animals.

36

Oxidative Stress and Age-Related Neurodegeneration

TABLE 2.1 Quantitative Reduction of Oxidative Stress from Senile Plaques in Tissue Sections from Mouse Brain. Treatment

Class of Compound

Reduction in Plaque-Derived Oxidative Stress (% of Control)

Hesperitin (100 µM) Vitamin E (100 µM) Ginkgo Biloba extract (1 mg/mL)

Natural antioxidant Vitamin Complex plant extract

31.12 ⫾ 13.27* 55.50 ⫾ 6.97* 46.95 ⫾ 4.18*

Note: Fluorescence intensity of Amplex Red, resulting from plaque-mediated oxidation is expressed as percentage of reduction of control values. Data are representative of 12–96 plaques. Significant differences were determined by one-way ANOVA followed by Tukey-B test (*P⬍0.05 vs. Control Group).

Because of the clinical studies that indicate a beneficial effect of antioxidant supplementation on AD progression50,51,88 and the above described results demonstrated with direct imaging of oxidative stress surrounding senile plaques,77 it is possible to speculate that antioxidant efficacy may be at least in part due to a reduction in free radical damage associated with senile plaques. Moreover, these data also set a basis to systematically screen the effect of potential antioxidants that differ structurally, and come from variable drug classes, but share the ability to protect against free radical-mediated cellular injury. In this sense there is an extensive bibliography supporting the antiamyloidogenic, antioxidant and neuroprotective role of natural polyphenols, plant extracts or vitamins as discussed above. MPM offers an incomparable opportunity to directly assess the effect of these compounds on senile plaques, helping to elucidate the possible neuroprotective effect of widely studied antioxidants such as Ginkgo biloba extract, hesperitin, or vitamin E. In conclusion, MPM provides a useful tool to directly screen libraries of antioxidants with the potential capacity to reduce oxidative stress associated with senile plaques in AD. Moreover MPM offers the possibility of assessing the effect of antioxidants in vivo by chronically imaging the effect of antioxidants in the intact animal. This technique supports the search for rational therapies for the prevention or reduction of oxidative stress in AD as well as in other central nervous system disorders where oxidative damage has been observed.

ACKNOWLEDGMENTS Supported by NIH AG020570, EB00768 (BJB) and a scholarship from the Secretaria de Estado de Educacion y Universidades, cofinanced by the Fondo Social Europeo (EX2004-0250, MG-A). Additional support provided by the IPSEN.

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Oxidative Stress and Age-Related Neurodegeneration 19. Hensley K, Hall N, Subramaniam R, Cole P, Harris M, Aksenov M, Aksenova M, Gabbita SP, Wu JF, Carney JM, Lovell M, Markesberg WR, Bulterfield DA. Brain regional correspondence between Alzheimer’s disease histopathology and biomarkers of protein oxidation. J Neurochem, 1995; 65:2146–2156. 20. Mecocci P, MacGarvey U, Beal MF. Oxidative damage to mitochondrial DNA is increased in Alzheimer’s disease. Ann Neurol, 1994; 36:747–751. 21. Jimenez-Jimenez FJ, de Bustos F, Molina JA, Benito-Leon J, Tallon-Barranco A, Gasalla T, Orti-Pareja M, Guillamon F, Rubio JC, Arenas J, Enriquez-deSalamanca R. Cerebrospinal fluid levels of alpha-tocopherol (vitamin E) in Alzheimer’s disease. J Neural Transm, 1997; 104:703–710. 22. Bourdel-Marchasson I, Delmas-Beauvieux MC, Peuchant E, Richard-Harston S, Decamps A, Reignier B, Emeriau JP, Rainfray M. Antioxidant defences and oxidative stress markers in erythrocytes and plasma from normally nourished elderly Alzheimer patients. Age Ageing, 2001; 30:235–241. 23. Rinaldi P, Polidori MC, Metastasio A, Mariani E, Mattioli P, Cherubini A, Catani M, Cecchetti R, Senin U, Mecocci P. Plasma antioxidants are similarly depleted in mild cognitive impairment and in Alzheimer’s disease. Neurobiol Aging, 2003; 24:915–919. 24. Yan SD, Chen X, Fu J, Chen M, Zhu H, Roher A, Slattery T, Zhao L, Nagashima M, Morser J, Migheli A, Nawroth P, Stern D, Schmidt AM. RAGE and amyloidbeta peptide neurotoxicity in Alzheimer’s disease. Nature, 1996; 382:685–691. 25. Kiprianova I, Schwab S, Fandrey J, Spranger M. Suppression of the oxidative burst in murine microglia by nitric oxide. Neurosci Lett, 1997; 226:75–78. 26. Apelt J, Bigl M, Wunderlich P, Schliebs R. Aging-related increase in oxidative stress correlates with developmental pattern of beta-secretase activity and betaamyloid plaque formation in transgenic Tg2576 mice with Alzheimer-like pathology. Int J Dev Neurosci, 2004; 22:475–484. 27. Klegeris A, Walker DG, McGeer PL. Activation of macrophages by Alzheimer beta amyloid peptide. Biochem Biophys Res Commun, 1994; 199:984–991. 28. Thomas T, Thomas G, McLendon C, Sutton T, Mullan M. beta-Amyloid-mediated svasoactivity and vascular endothelial damage. Nature, 1996; 380:168–171. 29. Bianca VD, Dusi S, Bianchini E, Dal Pra I, Rossi F. beta-amyloid activates the O-2 forming NADPH oxidase in microglia, monocytes, and neutrophils. A possible inflammatory mechanism of neuronal damage in Alzheimer’s disease. J Biol Chem, 1999; 274:15493–15499. 30. Tabner BJ, Turnbull S, El-Agnaf OM, Allsop D. Formation of hydrogen peroxide and hydroxyl radicals from A(beta) and alpha-synuclein as a possible mechanism of cell death in Alzheimer’s disease and Parkinson’s disease. Free Radic Biol Med, 2002; 32:1076–1083. 31. Miranda S, Opazo C, Larrondo LF, Munoz FJ, Ruiz F, Leighton F, Inestrosa NC. The role of oxidative stress in the toxicity induced by amyloid beta-peptide in Alzheimer’s disease. Prog Neurobiol, 2000; 62:633–648. 32. Dyrks T, Dyrks E, Hartmann T, Masters C, Beyreuther K. Amyloidogenicity of beta A4 and beta A4-bearing amyloid protein precursor fragments by metal-catalyzed oxidation. J Biol Chem, 1992; 267:18210–18217. 33. Halliwell B, Gutteridge JM. Free radicals, lipid peroxidation, and cell damage. Lancet 1984; 2:1095. 34. Smith MA, Harris PL, Sayre LM, Perry G. Iron accumulation in Alzheimer disease is a source of redox-generated free radicals. Proc Natl Acad Sci USA, 1997; 94:9866–9868.

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35. Smorgon C, Mari E, Atti AR, Dalla Nora E, Zamboni PF, Calzoni F, Passaro A, Fellin R. Trace elements and cognitive impairment: an elderly cohort study. Arch Gerontol Geriatr Suppl, 2004; 393–402. 36. Bjertness E, Candy JM, Torvik A, Ince P, McArthur F, Taylor GA, Johansen SW, Alexander J, Gronnesby JK, Bakketeig LS, Edwardson JA. Content of brain aluminum is not elevated in Alzheimer disease. Alzheimer Dis Assoc Disord, 1996; 10:171–174. 37. Linder MC, Hazegh-Azam M. Copper biochemistry and molecular biology. Am J Clin Nutr, 1996; 63:797S–811S. 38. Multhaup G, Scheuermann S, Schlicksupp A, Simons A, Strauss M, Kemmling A, Oehler C, Cappai R, Pipkorn R, Bayer TA. Possible mechanisms of APP-mediated oxidative stress in Alzheimer’s disease. Free Radic Biol Med, 2002; 33:45–51. 39. Multhaup G, Ruppert T, Schlicksupp A, Hesse L, Bill E, Pipkorn R, Masters CL, Beyreuther K. Copper-binding amyloid precursor protein undergoes a site-specific fragmentation in the reduction of hydrogen peroxide. Biochemistry, 1998; 37:7224–7230. 40. House E, Collingwood J, Khan A, Korchazkina O, Berthon G, Exley C. Aluminium, iron, zinc and copper influence the in vitro formation of amyloid fibrils of Abeta42 in a manner which may have consequences for metal chelation therapy in Alzheimer’s disease. J Alzheimers Dis, 2004; 6:291–301. 41. Cherny RA, Atwood CS, Xilinas ME, Gray DN, Jones WD, McLean CA, Barnham KJ, Volitakis I, Fraser FW, Kim Y, Huang X, Goldstein LE, Moir RD, Lim JT, Beyreuther K, Zheng H, Tanzi RE, Masters CL, Bush AI. Treatment with a copper–zinc chelator markedly and rapidly inhibits beta-amyloid accumulation in Alzheimer’s disease transgenic mice. Neuron, 2001; 30:665–676. 42. Engelhart MJ, Geerlings MI, Ruitenberg A, van Swieten JC, Hofman A, Witteman JC, Breteler MM. Dietary intake of antioxidants and risk of Alzheimer disease. JAMA, 2002; 287:3223–3229. 43. Bagchi D, Bagchi M, Stohs SJ, Das DK, Ray SD, Kuszynski CA, Joshi SS, Pruess HG. Free radicals and grape seed proanthocyanidin extract: importance in human health and disease prevention. Toxicology, 2000; 148:187–197. 44. Rice-Evans CA, Miller NJ, Paganga G. Structure-antioxidant activity relationships of flavonoids and phenolic acids. Free Radic Biol Med, 1996; 20:933–956. 45. Savaskan E, Olivieri G, Meier F, Seifritz E, Wirz-Justice A, Muller-Spahn F. Red wine ingredient resveratrol protects from beta-amyloid neurotoxicity. Gerontology, 2003; 49:380–383. 46. Montilla-Lopez P, Munoz-Agueda MC, Feijoo Lopez M, Munoz-Castaneda JR, Bujalance-Arenas I, Tunez-Finana I. Comparison of melatonin versus vitamin C on oxidative stress and antioxidant enzyme activity in Alzheimer’s disease induced by okadaic acid in neuroblastoma cells. Eur J Pharmacol, 2002; 451:237–243. 47. Ono K,Yoshiike Y, Takashima A, Hasegawa K, Naiki H,Yamada M. Potent anti-amyloidogenic and fibril-destabilizing effects of polyphenols in vitro: implications for the prevention and therapeutics of Alzheimer’s disease. J Neurochem, 2003; 87:172–181. 48. Stackman RW, Eckenstein F, Frei B, Kulhanek D, Nowlin J, Quinn JF. Prevention of age-related spatial memory deficits in a transgenic mouse model of Alzheimer’s disease by chronic Ginkgo biloba treatment. Exp Neurol, 2003; 184:510–520. 49. Yao Y, Chinnici C, Tang H, Trojanowski JQ, Lee VM, Pratico D. Brain inflammation and oxidative stress in a transgenic mouse model of Alzheimer-like brain amyloidosis. J Neuroinflammation, 2004; 1:21.

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Oxidative Stress and Age-Related Neurodegeneration 50. Klatte ET, Scharre DW, Nagaraja HN, Davis RA, Beversdorf DQ. Combination therapy of donepezil and vitamin E in Alzheimer disease. Alzheimer Dis Assoc Disord, 2003; 17:113–116. 51. Kanowski S, Hoerr R. Ginkgo biloba extract EGb 761 in dementia: intent-to-treat analyses of a 24-week, multi-center, double-blind, placebo-controlled, randomized trial. Pharmacopsychiatry, 2003; 36:297–303. 52. Asgary S, Naderi GA, Sarraf Zadegan N, Vakili R. The inhibitory effects of pure flavonoids on in vitro protein glycosylation. J Herb Pharmcother, 2002; 2:47–55. 53. Drummen GP, Makkinje M, Verkleij AJ, Op den Kamp JA, Post JA. Attenuation of lipid peroxidation by antioxidants in rat-1 fibroblasts: comparison of the lipid peroxidation reporter molecules cis-parinaric acid and C11-BODIPY(581/591) in a biological setting. Biochim Biophys Acta, 2004; 1636:136–150. 54. Smith JV, Luo Y. Elevation of oxidative free radicals in Alzheimer’s disease models can be attenuated by Ginkgo biloba extract EGb 761. J Alzheimers Dis, 2003; 5:287–300. 55. Bastianetto S, Zheng WH, Quirion R. Neuroprotective abilities of resveratrol and other red wine constituents against nitric oxide-related toxicity in cultured hippocampal neurons. Br J Pharmacol, 2000; 131:711–720. 56. Orgogozo JM, Dartigues JF, Lafont S, Letenneur L, Commenges D, Salamon R, Renaud S, Breteler MB. Wine consumption and dementia in the elderly: a prospective community study in the Bordeaux area. Rev Neurol (Paris), 1997; 153:185–192. 57. Bastianetto S, Quirion R. Natural extracts as possible protective agents of brain aging. Neurobiol Aging, 2002; 23:891–897. 58. Bastianetto S, Quirion R. EGb 761 is a neuroprotective agent against beta-amyloid toxicity. Cell Mol Biol (Noisy-le-grand), 2002; 48:693–697. 59. Le Bars PL, Velasco FM, Ferguson JM, Dessain EC, Kieser M, Hoerr R. Influence of the severity of cognitive impairment on the effect of the Gnkgo biloba extract EGb 761 in Alzheimer’s disease. Neuropsychobiology, 2002; 45:19–26. 60. El-Demerdash FM. Antioxidant effect of vitamin E and selenium on lipid peroxidation, enzyme activities and biochemical parameters in rats exposed to aluminium. J Trace Elem Med Biol, 2004; 18:113–121. 61. Werman MJ, Ben-Amotz A, Mokady S. Availability and antiperoxidative effects of beta-carotene from Dunaliella bardawil in alcohol-drinking rats. J Nutr Biochem, 1999; 10:449–454. 62. Bourdel-Marchasson I, Vincent S, Germain C, Salles N, Jenn J, Rasoamanarivo E, Emeriau JP, Rainfray M, Richard-Harston S. Delirium symptoms and low dietary intake in older inpatients are independent predictors of institutionalization: a 1-year prospective population-based study. J Gerontol A Biol Sci Med Sci, 2004; 59:350–354. 63. Schippling S, Kontush A, Arlt S, Buhmann C, Sturenburg HJ, Mann U, MullerThomsen T, Beisiegel U. Increased lipoprotein oxidation in Alzheimer’s disease. Free Radic Biol Med, 2000; 28:351–360. 64. Sinclair AJ, Bayer AJ, Johnston J, Warner C, Maxwell SR. Altered plasma antioxidant status in subjects with Alzheimer’s disease and vascular dementia. Int J Geriatr Psychiatry, 1998; 13:840–845. 65. Youdim KA, Joseph JA. A possible emerging role of phytochemicals in improving age-related neurological dysfunctions: a multiplicity of effects. Free Radic Biol Med, 2001; 30:583–594.

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66. McIntosh LJ, Trush MA, Troncoso JC. Increased susceptibility of Alzheimer’s disease temporal cortex to oxygen free radical-mediated processes. Free Radic Biol Med, 1997; 23:183–190. 67. Balcz B, Kirchner L, Cairns N, Fountoulakis M, Lubec G. Increased brain protein levels of carbonyl reductase and alcohol dehydrogenase in Down syndrome and Alzheimer’s disease. J Neural Transm Suppl, 2001; 61:193–201. 68. Kaufmann E, Boehm BO, Sussmuth SD, Kientsch-Engel R, Sperfeld A, Ludolph AC, Tumani H. The advanced glycation end-product N(varepsilon)-(carboxymethyl)lysine level is elevated in cerebrospinal fluid of patients with amyotrophic lateral sclerosis. Neurosci Lett, 2004; 371:226–229. 69. Te Koppele JM, Lucassen PJ, Sakkee AN, Van Asten JG, Ravid R, Swaab DF, Van Bezooijen CF. 8OHdG levels in brain do not indicate oxidative DNA damage in Alzheimer’s disease. Neurobiol Aging, 1996; 17:819–826. 70. Moore K, Roberts LJ, 2nd. Measurement of lipid peroxidation. Free Radic Res, 1998; 28:659–671. 71. Denk W, Strickler JH, Webb WW. Two-photon laser scanning fluorescence microscopy. Science, 1990; 248:73–76. 72. Levene MJ, Dombeck DA, Kasischke KA, Molloy RP, Webb WW. In vivo multiphoton microscopy of deep brain tissue. J Neurophysiol, 2004; 91:1908–1912. 73. Bacskai BJ, Hickey GA, Skoch J, Kajdasz ST, Wang Y, Huang GF, Mathis CA, Klunk WE, Hyman BT. Four-dimensional multiphoton imaging of brain entry, amyloid binding, and clearance of an amyloid-beta ligand in transgenic mice. Proc Natl Acad Sci USA, 2003; 100:12462–12467. 74. Bacskai BJ, Kajdasz ST, McLellan ME, Games D, Seubert P, Schenk D, Hyman BT. Non-Fc-mediated mechanisms are involved in clearance of amyloid-beta in vivo by immunotherapy. J Neurosci, 2002; 22:7873–7878. 75. Bacskai BJ, Klunk WE, Mathis CA, Hyman BT. Imaging amyloid-beta deposits in vivo. J Cereb Blood Flow Metab, 2002; 22:1035–1041. 76. Christie R, Yamada M, Moskowitz M, Hyman B. Structural and functional disruption of vascular smooth muscle cells in a transgenic mouse model of amyloid angiopathy. Am J Pathol, 2001; 158:1065–1071. 77. McLellan ME, Kajdasz ST, Hyman BT, Bacskai BJ. In vivo imaging of reactive oxygen species specifically associated with thioflavine S-positive amyloid plaques by multiphoton microscopy. J Neurosci, 2003; 23:2212–2217. 78. LeBel CP, Ischiropoulos H, Bondy SC. Evaluation of the probe 2⬘,7⬘-dichlorofluorescin as an indicator of reactive oxygen species formation and oxidative stress. Chem Res Toxicol, 1992; 5:227–231. 79. Zhou M, Diwu Z, Panchuk-Voloshina N, Haugland RP. A stable nonfluorescent derivative of resorufin for the fluorometric determination of trace hydrogen peroxide: applications in detecting the activity of phagocyte NADPH oxidase and other oxidases. Anal Biochem, 1997; 253:162–168. 80. Games D, Adams D, Alessandrini R, Barbour R, Berthelette P, Blackwell C, Carr T, Clemens J, Donaldson T, Gillespie F, Guido T, Hagopian S, Johnson-Wood K, Khan K, Mikelee, Leibowitz P, Lieberburg I, Little S, Masliah E, McConlogue L, Montoya-Zavala M, Macke L, Paganini L, Penniman E, Power M, Schenk D, Seubert P, Snyder B, Soriano F, Tan H, Vitale J, Wadsworth S, Wolozin B, Zhao J. Alzheimer-type neuropathology in transgenic mice overexpressing V717F betaamyloid precursor protein. Nature, 1995; 373:523–527.

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Oxidative Stress and Age-Related Neurodegeneration 81. Hsiao K, Chapman P, Nilsen S, Eckman C, Harigaya Y, Younkin S, Yang F, Cole G. Correlative memory deficits, Abeta elevation, and amyloid plaques in transgenic mice. Science, 1996; 274:99–102. 82. Jankowsky JL, Slunt HH, Gonzales V, Jenkins NA, Copeland NG, Borchelt DR. APP processing and amyloid deposition in mice haplo-insufficient for presenilin 1. Neurobiol Aging, 2004; 25:885–892. 83. Grundman M, Delaney P. Antioxidant strategies for Alzheimer’s disease. Proc Nutr Soc, 2002; 61:191–202. 84. Cao X, Phillis JW. alpha-Phenyl-tert-butyl-nitrone reduces cortical infarct and edema in rats subjected to focal ischemia. Brain Res, 1994; 644:267–272. 85. Koenig ML, Meyerhoff JL. In vitro neuroprotection against oxidative stress by pre-treatment with a combination of dihydrolipoic acid and phenyl-butyl nitrones. Neurotox Res, 2003; 5:265–272. 86. Hagen TM, Wehr CM, Ames BN. Mitochondrial decay in aging. Reversal through supplementation of acetyl-L-carnitine and N-tert-butyl-alpha-phenyl-nitrone. Ann N Y Acad Sci, 1998; 854:214–223. 87. Choi CW, Hwang JH, Chang YS, Shin SM, Park WS, Lee M. Effects of alphaphenyl-N-tert-butyl nitrone (PBN)on brain cell membrane function and energy metabolism during transient global cerebral hypoxia-ischemia and reoxygenationreperfusion in newborn piglets. J Korean Med Sci, 2004; 19:413–418. 88. Sano M, Ernesto C, Thomas RG, Klauber MR, Schafer K, Grundman M, Woodbury P, Growdon J, Cotman CW, Pfeiffer E, Schneider LS, Thal LJ. A controlled trial of selegiline, alpha-tocopherol, or both as treatment for Alzheimer’s disease. The Alzheimer’s Disease Cooperative Study. N Engl J Med, 1997; 336:1216–1222.

Ginkgo biloba Extract 3 and Alzheimer’s Disease: Is the Neuroprotection Explained Merely by the Antioxidant Action? Yves Christen Ipsen Institute Paris, France

CONTENTS Abstract................................................................................................................43 3.1 Introduction................................................................................................44 3.2 Why Antioxidants Such as EGb 761 are Important in the Fight against Alzheimer’s Disease? ....................................................................44 3.3 Ginkgo biloba Extracts ..............................................................................46 3.4 Effects of EGb 761 on Human Subjects....................................................47 3.4.1 Effects on Cognitive Symptoms in Alzheimer’s Disease ..............47 3.4.2 Effect on Prevention of Alzheimer’s Disease ................................47 3.4.3 Other Cognitive Effects .................................................................48 3.5 EGb 761 as a Free-radical Scavenger........................................................48 3.6 Effects of EGb 761 on Amyloid-β.............................................................48 3.7 Effect of EGb 761 on Gene Expression ....................................................49 3.8 Which Additional Effects of EGb 761 could be Linked to the Antioxidant Action?...................................................................................50 3.9 Why Advance a Hypothesis Whereby There is an Action Over and above a Traditional Antioxidant and Neuroprotective Effect? ...........50 References ...........................................................................................................52

ABSTRACT Ginkgo biloba extract (EGb 761) has been found to have many biological effects in experimental models and in human subjects. In the case of Alzheimer’s disease 43

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(AD), the extract not only acts on the symptoms, but it could also — and this is perhaps the essential benefit — contribute to preventing the onset of the disease. Extensive therapeutic trials are currently being conducted with a view to demonstrate this. These actions may be explained by a wide variety of experimental data. It should, in particular, be noted that EGb 761 is a powerful free-radical scavenger with proven effectiveness in various experimental models, notably in vivo and even in human subjects. There are many other explanations for the action in pathologies such as AD, including the reduction in the production, oligomerization, and toxicity of amyloid-β. EGb 761 also has a major effect on gene expression. Although seemingly distinct, many of these effects may be interrelated. In this respect, we form the hypothesis that free radicals (and free-radical scavengers) play their biological roles not only through conventional mechanisms directly related to their effect on toxicity (protection being linked to the direct suppression of this toxicity), but also by means of an action regulating cellular functions, notably with respect to gene expression.

3.1 INTRODUCTION Ginkgo biloba extract EGb 761, first marketed in France and Germany 30 years ago, is an ethical drug in many countries (including most European countries), and is considered a food supplement in the United States. It was initially indicated for various vascular, cerebral, and neurosensory deficits, but more and more data now offer evidence of its potential for treating neurodegenerative disorders. In fact, the knowledge of this product evolved as new scientific and medical discoveries were made.1 In the 1960s, brain disorders in old people were mainly thought to be linked to circulatory deficits. The emphasis placed on Alzheimer’s disease (AD) altered this perspective, and the real physiological target became the neuron rather than the blood vessels in the brain. Hundreds of publications have now been dedicated to Ginkgo biloba extracts, and mainly to EGb 761. These cover many properties, including biological effects and trials involving healthy or ill human subjects and, notably, AD sufferers. Concerning AD, published and ongoing trials concern both action on cognitive symptoms and preventive effects.

3.2 WHY ANTIOXIDANTS SUCH AS EGb 761 ARE IMPORTANT IN THE FIGHT AGAINST ALZHEIMER’S DISEASE? Alzheimer’s disease represents a considerable challenge.2 Demographic projections and epidemiological figures point out the severity of the public health concern. Sloane et al.3 evaluated the impact of treatment advances, both for delaying disease onset and for treating existing disease, and concluded that AD care is likely to remain a major public health problem during the coming decades. This explains the pharmaceutical sector’s keen interest in this disease. Some medicines have been approved for use in the treatment of symptoms, and many others are considered as having potential for delaying disease onset. Many new compounds are also

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being tested currently. While at present it is impossible to predict future developments, it would appear reasonable to summarize the following three approaches: 1. Cholinesterase inhibitors approved for the treatment of Alzheimer’s symptoms have only a moderate effect. The recent AD2000 trial, which was conducted under the sponsorship of the British National Health Service, concluded that, despite the slight effect on cognition (0.8 unit difference in the Mini-mental State Examination), Donepezil (the most commonly prescribed of these medicines) “is not cost effective, with benefits below minimally relevant thresholds. More effective treatments than cholinesterase inhibitors are needed for Alzheimer’s disease”.4 This trial has given rise to controversy. However, according to Schneider,5 the trial does not completely contradict the findings of previous work. Most authors agree that the effect is moderate, which is generally regarded as preferable to having no effect at all. They also agree that this category of medicine — like the recently marketed memantine — is far from constituting an ideal solution.6 2. In the past few years, many classes of drugs have been considered likely to have a preventive effect, either due to the results of epidemiological studies, or due to the drugs’ modes of action. The potential implications of these treatments are important since projections indicate that only therapies that delay disease onset will markedly reduce the overall disease prevalence.3 These mainly include estrogens, traditional nonsteroid anti-inflammatory drugs, cyclooxygenase-2-inhibiting anti-inflammatory drugs, hypocholesterolemic agent from the statin family, and antioxidants. This hope led to a large number of clinical trials, including a number of ambitious prevention trials focusing on estrogens, the two categories of anti-inflammatory drugs, and the two main antioxidants (vitamin E and EGb 761). It was shown that neither of the two categories of anti-inflammatory drugs (represented by Naproxen and Rofecoxib) resulted in any beneficial effect on symptoms,7,8 and that the cognitive functions were even adversely affected by the estrogens.9–11 The fact that other adverse events were noted (especially cardiovascular disease with respect to the anti-inflammatory drugs) prompted the suspension of the ongoing prevention trials (estrogens in early 2004, and Naproxen and Rofecoxib at the end of 2004). These medicines are therefore not as safe as was thought, and the same goes for certain statins, which have been shown to have a negative effect on muscles. Today, therefore, only the antioxidants are being tested for preventive properties, a fact that serves to heighten interest in this category, notably since a number of epidemiological studies appear to indicate that dietary intake of antioxidants from food (fruit, vegetables, and wine) has preventive properties.12–16 3. The new compounds being tested belong to a variety of categories: vaccines or antibodies for use in immunization; β - or γ -secretase inhibitors used to inhibit peptide amyloid-β (Aβ ) formation; β -sheet breaker

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peptides to block Aβ deposits; acyl-coenzyme A:cholesterol acyltransferase (ACAT) inhibitors; inhibitors of neurofibrillary degeneration; metal chelators; and many others.6 So far, and despite the significant amount of research in basic science carried out to discover the causes and pathogenic features of AD, progress toward effective treatments has been remarkably less.17 In particular, while the study of the amyloid cascade as a crucial pathogenic element has enabled the development of a large number of theoretical therapeutic approaches, no real medicine has yet emerged.18 There are various explanations for this. While certain researchers believe that it is too early to expect as viable treatment for the disease, others believe that the amyloid theory is simply false. More reasonably, it is possible that disease modification strategies would be most effective for prevention and not when pathology is established. Another difficulty could be that a number of mechanisms involved in the formation of Aβ — and notably those involving β - and γ -secretases — play a key biological role, and therefore their inhibition would have highly toxic results. Whatever the hypothesis of choice, the fact remains that despite the scientific interest of the remarkable research work that has been conducted, the therapeutic results obtained have been meager, particularly when compared with those achieved over the same period with respect to AIDS and cancers. The only candidate preventive treatments currently being tested, and of which the efficacy can therefore be demonstrated over the next 5 years, are certain antioxidants: EGb 761, which is used in two prevention trials, and vitamin E, which is currently used in another trial. This means that these substances represent the only serious hope of identifying a proven preventive treatment within a reasonable time frame, since, even if other preventive medicines now existed, the length of any clinical trials would mean that there would be little hope of proving their efficacy within less than 7 or 8 years (taking the time needed for the inclusion of several thousand patients, for monitoring, and for the analysis of results as 1 year, 5 years, and several months, respectively). The attractiveness of antioxidant medicines, and particularly of EGb 761, is enhanced by the fact that side effects are extremely limited. For this reason, these substances seem increasingly worthy of interest, despite the length of time for which they have been known, and given the relative ineffectiveness of the other approaches.19

3.3 GINKGO BILOBA EXTRACTS Most of the scientific literature on Ginkgo biloba extracts concerns EGb 761, which is the best known and the unique Ginkgo biloba extract approved for treatment of mild to moderate AD. It is standardized to contain 24% flavonoids and 6% terpene lactones (3.1% ginkgolides and 2.9% bilobalide). Nevertheless, other extracts have also been marketed, notably in the United States. It is essential to be aware of the fact that these are not identical to EGb 761 and may sometimes differ considerably with regard to their biological effects.20,21 In a paper for the

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Natural Health Products Directorate of Canada, McCutcheon 21 claims that only German- and French-manufactured products (EGb 761) met their label claims. In many other samples, neither terpenes nor levels of bilobalide were detectable, and only one out of three Ginkgo products produced significant cognitive activation. Even the antioxidant activity varied considerably among preparations from different suppliers.22

3.4 EFFECTS OF EGb 761 ON HUMAN SUBJECTS 3.4.1 EFFECTS

ON

COGNITIVE SYMPTOMS

IN

ALZHEIMER’S DISEASE

Several reports show a beneficial effect on cognitive symptoms of AD,23–25 although this was not confirmed by van Dongen et al. 26,27. On the basis of the meta-analyses in the Cochrane collaboration database, a recent review of the published data on the efficacy of Ginkgo biloba for the treatment of patients with dementia or cognitive decline concludes that “there is promising evidence of improvement in cognition and function”.28 The odds ratio for global improvement is exactly the same for EGb 761 (2.16) and for Donepezil (2.18), according to the Cochrane meta-analysis for this product.29 According to the data of an American study (partly published 30) and to a meta-analysis of the data currently available,31 the effect of EGb 761 on cognitive functions could be particularly evident in AD patients with behavioral and psychological symptoms of dementia (BPSD).

3.4.2 EFFECT

ON

PREVENTION

OF

ALZHEIMER’S DISEASE

EGb 761 also seems to have a preventive action according to the data on the Toulouse cohort (n⫽1462) of the EPIDOS study.32 The odds ratios were 0.78, 0.51, and 0.38 for at least one exposure, two consecutive exposures, and at least three consecutive exposures, respectively. EGb 761 is currently the focus of two major interventional studies — the GEM (Ginkgo Evaluation of Memory) study in the United States and the GuidAge trial in France — aimed at evaluating its effect in the prevention of AD. The GEM study, which is being conducted with the support of the NIH and coordinated by S. DeKosky (University of Pittsburgh), aims to evaluate the potential of EGb 761 to prevent Alzheimer’s dementia in a group of 3000 subjects aged over 75 and receiving 240 mg of EGb 761 per day or a placebo, over a 5-year period. The GuidAge study, which is being conducted in France and coordinated by B. Vellas (University of Toulouse), aims to evaluate the potential of EGb 761, administered at a dose of 240 mg/day, to prevent Alzheimer’s dementia in patients aged over 70 years who are presenting a risk because they spontaneously consulted their general practitioners (GPs) for memory complaint (an indicator of AD risk according to Palmer et al. 33). The results of this study, which concerns 2,800 patients, are expected in 2009–2010. It will be very interesting to compare the results of these two studies, which are relatively similar, apart from the fact that

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GuidAge includes patients who may be considered at risk insofar as they have spontaneously consulted for memory problems. A third study, conducted with the support of the NIH in Oregon, concerns 200 subjects aged over 85. It aims to evaluate the level of prevention of mild cognitive impairment (MCI) obtained for subjects receiving 240 mg of EGb 761 daily or a placebo, over a 3-year period.

3.4.3 OTHER COGNITIVE EFFECTS In cerebral and cognitive conditions, clinical studies have demonstrated an improvement in short-term memory,34 an action that is enhanced by memory training;35 in working memory, psychomotor performance, and executive processes;36,37 in cognitive deficits associated with aging;38,39 and in cognitive aspects of multiple sclerosis.40 Although the data about the action of EGb 761 in healthy subjects vary, most observations conclude that the effect is beneficial;37,41–45 however, Solomon et al.46 did not observe any benefits.

3.5 EGb 761 AS A FREE-RADICAL SCAVENGER The free-radical scavenging effect of EGb 761 has been shown in numerous models in vitro,47,48 but also directly in the brain, where Pierre et al.49 showed the reduction of lipid peroxidation in cerebral ischemia experiments, and even in humans, in open-heart surgery.50 Oxidative processes seem to be involved in AD51 and even in mild cognitive impairment,52,53 and they very probably participate in neuronal death. Aβ can produce reactive oxygen species,54 but lipid peroxidation also leads to Aβ deposition.55 Consequently, oxidative stress could be both a cause and a consequence of Aβ damage. Antioxidants are, therefore, good candidates as therapeutic agents, and notably with regard to AD prevention.56 It was, therefore, particularly important to study the free-radical scavenger effect of EGb 761 in the pathological situation of AD or in corresponding pathological models.57 Ramassamy et al.58,59 found that EGb 761 protects the frontal cortices of AD patients. Using multiphoton microscopy, Bacskai et al.60 found that EGb 761 reduces the oxidative stress resulting from the senile plaques of AD.

3.6 EFFECTS OF EGb 761 ON AMYLOID-β There is a great amount of experimental data suggesting that EGb 761 has an effect on several mechanisms linked to the pathogenesis of AD. In particular, EGb 761 affords protection against neuronal death due to apoptosis caused by Aβ 61–63 or several other neurotoxic agents such as glutamate and nitric oxide.64,65 The protective effect of Aβ is linked to the inhibition of the activation of caspase-3 (an enzyme involved in apoptosis) and to the formation of Aβ fibrils,63 an essential mechanism in the pathogenesis of AD. EGb 761 also limits the formation of Aβ by inducing α-secretase 66 and lowering free cholesterol levels.67 It also modulates Aβ oligomerization in transgenic Caenorhabditis elegans with constitutive

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expression of human Aβ 1–42 by producing more nontoxic Aβ dimers and monomers, which leads to reduced Aβ deposition (in contrast, Congo red that was bound to Aβ fibrils did not reverse them into the nontoxic forms of Aβ and did not protect against toxicity 68).

3.7 EFFECT OF EGb 761 ON GENE EXPRESSION Pioneering studies have shown an effect on down-regulation of the transcription factor AP-169 and of the peripheral-type benzodiazepine receptor (PBR; an effect responsible for the inhibition of glucocorticoid production and specifically linked to ginkgolide B, a particular component of EGb 76170,71): stimulation of the expression of mitochondrial DNA encoded cytochrome oxidase subunit III;72 an increase in the transcription of protector genes such as those of hemeoxygenase-1,73,74 of mitochondrial superoxide dismutase and of the regulatory subunit ofγ -glutamyl-cysteinyl synthetase (the rate-controlling enzyme of glutathione synthesis 75); an increase in p65 and in HSP70, as well as a decrease in glutathione reductase and glutathione S-transferase;76 stimulation of the expression of NADH dehydrogenase subunit I 77. While most of these effects appear to be linked to the flavonoid fraction of EGb 761,75 terpenoids also play a role, with ginkgolide B acting on PBR and bilobalide on mitochondrial cytochrome oxidase subunit III.78 More recent and exhaustive work, using high-density oligonucleotide microarrays, has shown changes in the expression of several hundreds of genes.75,79,80 These observations reveal an adaptation of the transcription aimed at increasing the antioxidant status of the cell and inhibiting the risk of DNA damage.79 They have also pointed, unexpectedly, to an interesting antineoplastic action. In particular, in human bladder cancer cell line T-24, which contains an activated c-Has-ras oncogene, Gohil et al.79 demonstrated a beneficial effect of EGb 761 on the regulation of various signaling systems involved in oncogenesis, and, in particular, a down-regulation of hepatocyte growth factor receptor dose and time dependency. This discovery was confirmed by Papadopoulos et al.,80 who observed a down-regulation of 37 genes, particularly of the PBR coding gene the expression and localization of which are correlated with the proliferation of breast cancer cells. Watanabe et al.81 have demonstrated that treatment of mice with EGb 761 in vivo activates several relevant genes in the cortex and hippocampus: transthyretin (which probably exerts a neuroprotective role through sequestration of Aβ ), growth hormone, prolactin, NfX1 protein, purinergic region binding protein α, chloride channel protein 3, calcium channel GluRB (AMPA-2), neuronal tyrosine/threonine phosphatase, and microtubule-associated tau. This may be associated with the antioxidant action of the drug, as free-radical scavengers modify gene expression, probably because cells activate protective genes in response to the presence of pools of free radicals. By protecting the cells from free radicals, the scavengers make this activation less useful. However, the action of EGb 761 seems to be more complex, because it generally induces rather than diminishes

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protective cellular mechanisms, such as superoxide dismutase, glutathione, the chaperone protein hsp 70, and heme-oxygenase 1. The protective action of freeradical scavenging is thus coupled with another beneficial effect that may not be present in synthetic antioxidant molecules. EGb 761, as a natural product shaped by evolutionary natural selection, may therefore be better adapted to the complexity of cellular processes than synthetic products.1

3.8 WHICH ADDITIONAL EFFECTS OF EGb 761 COULD BE LINKED TO THE ANTIOXIDANT ACTION? Many of the effects of EGb 761 are due, either directly or indirectly, to its freeradical scavenger action. One of the main reasons for attributing the benefits to an antioxidant effect is the fact that EGb 761 has been proven effective in a variety of neurodegenerative diseases, and not solely with respect to AD. Examples include animal models of cerebral ischemia and stroke,82–84 mouse transgenic model of amyotrophic lateral sclerosis,85 N-methyl-4-phenyl 1,2,3,6-tetrahydropyridine (MPTP) model of Parkinson’s disease,86,87 Tat transgenic mouse model of brain-targeted HIV infection,88 etc. All of these models and diseases probably involve free-radical injury in addition to the specific lesion characteristic of the disease. In the case of AD, even the characteristic lesions may be linked to oxidative stress, hence the protective effect associated with an antioxidant action. This is the case with inhibition of Aβ toxicity. Vitamin E, the benchmark antioxidant, also affords protection against Aβ toxicity. However, it has been observed that the protection provided by EGb 761 is not identical, since it is not merely preventive. Unlike vitamin E, EGb 761 has an effect even when introduced after Aβ .61 It is also likely that the modulation of Aβ oligomerization 68 is partly due to the free-radical scavenger effect, since, in this model, it limits the intracellular hydrogen peroxide level in a dose-dependent manner. It is, however, interesting to note that, in this model too, EGb 761 behaves differently from the other antioxidant used in this experiment – L-ascorbate – which does not protect against paralysis caused by Aβ. This suggests that the action of EGb 761 is due to at least two distinct mechanisms – one directly involving inhibition of oligomerization, and the other acting specifically on the toxicity. Notwithstanding this, even trials directly concerning the effect on oxidative stress show differences between antioxidants. Backsai et al. found that acute treatments with vitamin E (trolox) or EGb 761 showed statistically significant reductions of oxidative stress in senile plaques in vivo, while only EGb 761 was significantly efficient in vivo in oral chronic treatment (personal communication).

3.9 WHY ADVANCE A HYPOTHESIS WHEREBY THERE IS AN ACTION OVER AND ABOVE A TRADITIONAL ANTIOXIDANT AND NEUROPROTECTIVE EFFECT? Since free radicals are most probably involved in the neurodegenerative process and in neuronal death, it appears logical that free-radical scavengers should have

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a neuroprotective effect.56 However, this effect is manifested only after chronic treatment or in situations involving rapid neuronal death. EGb 761 has a number of effects in both chronic and acute treatment, and even in situations where there is no reason to suspect a neuronal death process. Examples include the stimulation of memory in human subjects 35 and an even more salient effect in tests such as dual coding.89 There are several possible explanations for these acute effects: action on membrane receptors, on energy metabolism, and on gene expression. These three actions correspond to elements of the mode of action of EGb 761.1,90 The effects on the energy metabolism and on gene expression may prove crucially important, since they are capable of modifying numerous cellular targets simultaneously. Such a process is more in line with the modern vision of cellular and molecular biology, according to which intracellular molecules are seen no longer as isolated elements but as elements functioning in association with others in the form of clusters. Knowledge of these processes leads us to the era of complexity. In this context, natural extracts, which are, themselves, made up of many components and are therefore complex, could prove particularly compatible with this new therapeutic vision.1,91 Although actions affecting the energy metabolism or gene expression are, theoretically, distinct from the antioxidant effect, it is not clear that this distinction is complete. The cell is sensitive to the presence of a pool of free radicals. Thus, when the pool size increases, transcription factors such as activator protein-1 (AP-1) and nuclear factor κ B (NFκ B) are activated. Free-radical scavengers, therefore, often have the effect of limiting the expression of these transcription factors. All of this suggests that, over and above their directly deleterious action, which leads to neuronal death, free radicals have a more immediate effect on cellular functions. For the same reason, free-radical scavengers can also act immediately by simply reducing the pool of free radicals (and consequently gene expression) in the cell. This suggests that the action of free radicals and that of free-radical scavengers are more complex than is ordinarily believed, and that these elements play a role across the entire spectrum of metabolic regulation. Clearly, the role of EGb 761 is even more complex than that of better defined antioxidants. Several of the components in this extract have different effects on cells. The flavonoids – and perhaps the terpenes – are antioxidants, and also have other effects. In addition to this, the other components of EGb 761 have a wide variety of effects, and there is a unique interdependency between actions of the various constituents of the extract, which appears to be responsible for its therapeutic activity.92 We wish to view the data presented in this chapter within a hypothesis of integration of oxidative mechanisms across the whole of cellular metabolism. It is probable that the formation of reactive oxygen species does not result from a simple error in cellular functioning, since, in such an event, natural selection would have automatically selected the mechanisms enabling this phenomenon to be effectively combated. We think that free radicals not only have a deleterious effect, but they also play a role in the activation of protective mechanisms that are

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sensitive to their production. In practice, this hypothesis suggests that free-radical scavengers are useful in combating excessive production of free radicals, but that the most effective medicines will be those that are not only capable of scavenging free radicals or inhibiting lipid peroxidation, but also act in such a way as to take into account cellular complexity. This supposes, in particular, that these substances do not inhibit the activation of protective mechanisms by free radicals. EGb 761 would appear to be an excellent candidate medicine, not only as a free-radical scavenger, but also as a cellular-function regulator in accordance with adaptive procedures. This particularity is, doubtless, due to the fact that it is a natural extract that has come about through evolution and natural selection, which are by themselves sources of adaptation.

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74. Zhuang H, Pin S, Christen Y, Dore S. Induction of heme oxygenase 1 by Ginkgo biloba in neuronal cultures and potential implications in ischemia. Cell Mol Biol, 2002; 48:647–653. 75. Gohil K, Maguire JJ, Packer L. Ginkgo biloba extract, EGb 761, activates antioxidant response and genes for intracellular transport: an in vitro study with GeneChips and a human cancer cell line. In: Christen Y, ed. Ginkgo biloba Extract (EGb 761) as a neuroprotective agent: from basic studies to clinical trials. Marseille: Solal, 2001:13–31. 76. Soulié C, Nicolle A, Christen Y, Ceballos-Picot I. The Ginkgo biloba extract EGb 761 increases viability of hNT human neurons in culture and affects the expression of genes implicated in the stress response. Cell Mol Biol, 2002; 48:641–646. 77. Tendi EA, Bosetti F, DasGupta SF, Giuffrida Stella AM, Drieu, Rapoport S. Ginkgo biloba extracts EGb 761 and bilobalide increase NADH dehydrogenase mRNA level and mitochondrial respiratory control ratio in PC12 cells. Neurochem Res, 2002; 27:319–323. 78. DeFeudis FV. Effects of Ginkgo biloba Extract (EGb 761) on gene expression: possible relevance to neurological disorders and age-associated cognitive impairment. Drug Dev Res, 2002; 57; 214–215. 79. Gohil K, Roy RK, Farzin S, Maguire JJ, Packer L. mRNA expression profile of a human cancer cell line in response to Ginkgo biloba extract: induction of antioxidant response and the Golgi system. Free Rad Res, 2000; 33:831–849. 80. Papadopoulos V, Kapsis A, Li H, Amri H, Hardwick M, Culty M, Kasprzyk PG, Carlson M, Moreau JP, Drieu K. Drug-induced inhibition of the peripheral-type benzodiazepine receptor expression and cell proliferation in human breast cancer cells. Anticancer Res, 2000; 20:2835–2847. 81. Watanabe CMH, Wolffram S, Ader P, Rimbach G, Packer L, Maguire JJ, Schultz PG, Gohil K. The in vivo neuromodulatory effects of the herbal medicine ginkgo biloba. Proc Natl Acad Sci USA, 2001; 98:6577–6580. 82. Clark WM, Rinker LG, Lessov NS, Lowery SL, Cipolla MJ. Efficacy of antioxidant therapies in transient focal ischemia in mice. Stroke 2001; 32:1000–1004. 83. Spinnewyn B. Ginkgo biloba Extract (EGb 761) protects against delayed neuronal deth in gerbils. In: Costentin J, Christen Y, Lacour M, eds. Effects of Gingko biloba extract EGb 761 on the central nervous system. Paris: Elsevier, 1992:113–118. 84. Lee EJ, Chen HY, Wu TS, Chen TY, Ayoub IA, Maynard KI. Acute administration of Ginkgo biloba extract (EGb 761) affords neuroprotection against permanent and transient focal cerebral ischemia in Sprague-Dawley rats. J Neurosci Res, 2002; 68:636–645. 85. Ferrante RJ, Klein AM, Dedeoglu A, Beal MF. Therapeutic efficacy of EGb 761 (Ginkgo biloba Extract) in a transgenic mouse model of amyotrophic lateral sclerosis. J Mol Neurosci, 2001; 17:89–96. 86. Ramassamy C, Clostre F, Christen Y, Costentin J. In vivo Ginkgo biloba extract (EGb 761) protects against neurotoxic effects induced by MPTP: investigations into its mechanism(s) of action. In: Christen Y, Costentin J, Lacours M, eds. Effects of Ginkgo biloba Extract (EGb 761) on the Central Nervous System. Paris: Elsevier, 1992:27–36. 87. Rojas P, Garduno B, Rojas C, Vigueras RM, Rojas-Castaneda J, Rios C, SerranoGarcia N. EGb 761 blocks MPP+-induced lipid peroxidation in mouse copus striatum. Neurochem Res, 2001; 26:1245–1251.

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Oxidative Stress and Age-Related Neurodegeneration 88. He J, Kim BO, Liu Y. Neuroprotective function of Ginkgo bioba extract (EGb 761) in the brain of doxycycline inducible, brain-targeted human immunodeficiency virus type 1 TAT transgenic mice. In: Ginkgo biloba Extract. From traditional medicine to a medicine of the future; or molecular biology attending a medicine of complexity. Meeting Berlin, October 11–13, 2002. Abstract. 89. Allain H, Raoul P, Lieury A, LeCoz F, Gandon JM, d’Arbigny P. Effect of two doses of Ginkgo biloba extract (EGb 761) on the dual-coding test in elderly subjects. Clin Ther, 1993; 15:549–558. 90. Christen Y. Ginkgo biloba and neurodegenerative disorders. Frontiers Biosci, 2004; 9:3091–3104. 91. Christen Y, Olano-Martin E, Packer L. EGb 761 in the post genomic era; new tools from molecular biology for the study of complex products such as Ginkgo biloba extract. Cell Mol Biol, 2002; 48:593–600. 92. DeFeudis FV. Ginkgo biloba extract (EGb 761). From Chemistry to the Clinic. Wiesbaden: Ullstein Medical, 1998.

Nutrients: 4 Mitochondrial Reducing Mitochondrial Decay to Delay or Treat Cognitive Dysfunction, Alzheimer’s Disease, and Parkinson’s Disease Jiankang Liu

Oakland Research Institute Oakland, California

Bruce N. Ames

University of California Berkeley, California

CONTENTS Abstract................................................................................................................60 4.1 Introduction................................................................................................61 4.2 Mitochondrial Decay with Age..................................................................61 4.3 The Km concept and metabolism ...............................................................62 4.4 Possible underlying mechanisms for altered enzyme Km ..........................63 4.4.1 Protein Oxidation and Adduction by Aldehydes ...........................63 4.4.2 Protein Nitration and Chlorination ................................................64 4.4.3 Protein Glycation and Cross-Linking ............................................64 4.4.4 Protein Degradation/Turnover .......................................................64 4.4.5 Aging-Associated Protein/Enzyme Oxidation...............................65 4.5 Mt-nutrients and mitochondrial decay.......................................................66 4.6 Mt-nutrient deficiency and cognitive dysfunction.....................................70 4.6.1 Iron Deficiency ..............................................................................71 4.6.2 Zinc Deficiency..............................................................................74 4.6.3 Copper Deficiency .........................................................................74 4.6.4 Pantothenic Acid Deficiency..........................................................74 4.6.5 Vitamin B6 Deficiency ...................................................................75 59

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4.6.6 4.6.7 4.6.8 4.6.9 4.6.10 4.6.11

Biotin Deficiency ...........................................................................75 Thiamine Deficiency......................................................................75 Riboflavin Deficiency ....................................................................76 Niacin Deficiency ..........................................................................76 Folate Deficiency ...........................................................................76 Docosahexaenoic Acid and Eicosapentaenoic Acid (EPA) Deficiency ......................................................................................76 4.6.12 Choline Deficiency ........................................................................77 4.6.13 Acetyl-Carnitine, R-α-Lipoic Acid, Coenzyme Q10, and Creatine Decline ............................................................................78 4.7 Mt-nutrient Supplementation for the prevention and amelioration of AD and PD ............................................................................................78 4.7.1 Mitochondrial Decay Due to Oxidative Damage Is a Key Contributor to Aging and Age-Associated Neurodegenerative Diseases, AD and PD.....................................................................78 4.7.2 Thiamine Supplementation ............................................................80 4.7.3 Riboflavin Supplementation ..........................................................81 4.7.4 Niacin/NADH Supplementation ....................................................81 4.7.5 Folic Acid, B6, and B12 Supplementation ......................................82 4.7.6 Docosahexaenoic Acid Supplementation ......................................82 4.7.7 Choline Supplementation...............................................................83 4.7.8 Acetyl-L-Carnitine/L-Carnitine Supplementation ..........................83 4.7.9 α-Lipoic Acid /Dihydrolipoic Acid Supplementation ...................84 4.7.10 Coenzyme Q10 Supplementation ..................................................86 4.7.11 Creatine Supplementation..............................................................86 4.8 Effectiveness of combinations of mt-nutrient supplementation on AD and PD prevention and treatment........................................................87 4.9 Conclusions and Perspectives ....................................................................89 Acknowledgments ...............................................................................................89 References ...........................................................................................................90

ABSTRACT Mitochondria are the source of energy and also the source and target of oxidants. Mitochondrial decay due to oxidative damage and nutrient deficiencies is a major contributor to brain aging and age-related neurodegenerative diseases, such as Alzheimer’s disease (AD) and Parkinson’s disease (PD). Therefore, protecting mitochondria from oxidative damage is essential for delaying brain aging and preventing/treating age-related neurodegenerative diseases. We define mitochondrial nutrients (mt-nutrients) as those which protect mitochondria from oxidative damage and improve mitochondrial function, including those that can (1) inhibit or prevent oxidant production in mitochondria; (2) scavenge and inactivate free radicals and reactive oxygen species; (3) repair mitochondrial damage or induce phase-2 enzymes to enhance antioxidant defenses, and (4) act as cofactors/substrates to protect mitochondrial enzymes and/or stimulate enzyme activity. The cofactor/substrate

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protection is based on the idea that oxidatively modified enzymes lose activity and have reduced binding affinity for substrates and cofactors, and the loss of binding affinity may be overcome by providing high doses of enzyme substrates and cofactors similar to that of high dose B vitamins remedy for some genetic diseases. We discuss the relationships among mt-nutrient deficiency, mitochondrial decay, and cognitive dysfunction, and summarize available evidence suggesting an effect of mt-nutrient supplementation on AD and PD. It appears that longer term administration of combinations of a number of mt-nutrients is more effective in delaying and protecting mitochondrial decay. Thus, optimal doses of combinations of mt-nutrients to delay and repair mitochondrial decay could be a strategy for delaying and treating neurodegenerative diseases, including AD and PD.

4.1 INTRODUCTION Neurodegenerative diseases, including Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis, and Huntington’s disease, are ageassociated. Mitochondrial decay may be a principal underlying event in aging1–7 and may also be associated with the onset and development of neurodegenerative diseases.8–15 Thus, neurodegenerative diseases, such as AD and PD may be delayed or ameliorated by treatment with mitochondrial nutrients (mt-nutrients), which can delay or repair mitochondrial damage, thus improving mitochondrial function. We define mt-nutrients as those that can (1) elevate substrates and coenzymes of mitochondrial enzymes, (2) induce phase-2 enzymes to enhance cellular antioxidant defense, (3) scavenge free radicals and prevent oxidant production, and (4) repair mitochondrial membranes. In the present chapter, we survey the published literature to identify some mt-nutrients, including L-carnitine/acetyl-L-carnitine (ALCAR), α-lipoic acid (LA)/dihydrolipoic acid (DHLA)/ lipoamide, coenzyme Q10 (CoQ), creatine, choline, phospholipids, glutathione (GSH)/N-acetylcysteine, pyruvate, and various vitamins (e.g., A, B, C, and E). Then, we discuss the relationships among mt-nutrient deficiency, mitochondrial decay, and cognitive dysfunction, and also summarize available evidence of mt-nutrient supplementation on ameliorating AD and PD.

4.2 MITOCHONDRIAL DECAY WITH AGE Mitochondria provide energy for basic metabolic processes, produce oxidants as inevitable by-products, and decay with age, impairing cellular metabolism and leading to cellular decline. Mitochondrial membrane potential, respiratory control ratios, and cellular oxygen consumption decline with age, and oxidant production increases.4,16,17 Oxidative damage to DNA, RNA, proteins, and lipid membranes in mitochondria may be involved. Mutations in mitochondrial genes compromise mitochondria by altering components of the electron-transport chain, resulting in inefficient electron transport and increased superoxide production.4,18 The resulting oxidative damage to mitochondria may compromise their ability to meet cellular energy demands. Oxidized proteins accumulate with age19

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and cause mitochondrial inefficiencies, leading to more oxidant formation. Mitochondrial membrane fluidity also declines with age;20,21 this may lead to deformation of membrane proteins and cause mitochondrial dysfunction. The significant age-related loss of cardiolipin, a phospholipid that occurs primarily in the mitochondrial inner membrane, may occur in part because of greater oxidative damage or reduced biosynthesis. Loss of cardiolipin, coupled with oxidation of critical thiol groups in key proteins, adversely affects transport of substrates and cytochrome c oxidase activity22 necessary for mitochondrial function. These changes could directly affect the ability of mitochondria to maintain their membrane potential.

4.3 THE Km CONCEPT AND METABOLISM As many as one third of all mutations in a gene result in the corresponding enzyme having a decreased binding affinity for its coenzyme, i.e., an increased Michaelis constant (Km).23 Clinical symptoms associated with about 50 human genetic diseases can be remedied or ameliorated by the administration of high doses of the B-vitamin component of the corresponding coenzyme, which was shown to markedly raise levels of the coenzyme and partially restore enzymatic activity.23 Five single-nucleotide polymorphisms were also discussed in the review,23 in which the variant amino acid reduces coenzyme binding and enzymatic activity, and the enzyme dysfunction may be reversible by increasing cellular concentrations of the cofactor through high-dose vitamin therapy. Among these 50 genetic diseases, 14 are with defective mitochondrial enzymes, suggesting that vitamin supplementation results in increased coenzyme levels in mitochondria as well as the rest of the cell. The example of enzymes requiring B-vitamin-derived coenzymes likely represent only a small fraction of the total number of defective enzymes that would be responsive to therapeutic mt-nutrients. We suggest this loss in activity may be often recoverable by high doses of enzyme substrates and cofactors in the same way that high-dose vitamins have shown to remedy genetic diseases. For example, with age, the mitochondrial complexes III and IV show a significant increase in Km value for substrate (complex III for ubiquinol and complex IV for reduced cytochrome c) and decrease in Vmax.24 Feuers also showed that dietary restriction, which has been shown to reduce oxidative stress, effectively reversed these decreases in activity of the complexes and substrate affinity. Carnitine acetyltransferase, which transfers an acetyl group between CoA and carnitine, loses activity with age owing to a poorer binding affinity for CoA and acetyl carnitine.25 Feeding the substrate ALCAR together with LA, a mitochondrial antioxidant, restores the velocity of the reaction, the Km value of carnitine acetyltransferase for ALCAR and CoA, and mitochondrial function.25 In addition, a recent clinical study shows that high doses of riboflavin (a precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) and elimination of dietary red meat promoted the recovery of some motor functions in PD patients.26 Mitochondrial complex I, which uses FMN and FAD as cofactors, is defective in PD.27 Therefore, administration of a

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high-dose mt-nutrient to target mitochondrial decay may be a potential remedy for many degenerative diseases including AD and PD. Besides restoring the efficiency of enzymes damaged by oxidative decay, the high concentration of substrates and cofactors may also protect enzymes, because the presence of a high concentration of substrate or coenzyme may prevent further oxidative inactivation of the active sites. Thus, clinical tests of a high-dose mt-nutrient pill, or high-dose individual mt-nutrients, as a reasonably safe and potentially helpful prevention and therapy strategy for AD and PD, may be desirable.

4.4 POSSIBLE UNDERLYING MECHANISMS FOR ALTERED ENZYME Km 4.4.1 PROTEIN OXIDATION AND ADDUCTION

BY

ALDEHYDES

Oxidants and oxidative products are known to modify proteins, altering enzyme activity and affecting cellular functions, receptor-mediated processes, and protein turnover. The modification is due at least in part to the oxidation of amino acid residues in proteins. Free amino acids can be reversibly or irreversibly modified or destroyed by oxidation,28–31 and thus quenching of oxidants by free amino acids may play an important role in antioxidant defense against protein and tissue damage.32 Oxidative modification of amino acids in proteins can cause deformation and inactivation of enzymes. Oxidation of aliphatic amino acids causes deamination and yields mainly carboxylic acids and aldehydes, while oxidation of aromatic amino acids targets the indole ring and aromatic group with minor deamination. Mild oxidation in vitro, which mimics the situation in vivo, can modify a number of different amino acid residues in proteins without cleavage of peptide bonds.19,33 Thus, the effect of amino acid modifications on a protein may vary from slight structure modification to large denaturation accompanied by fragmentation. The most significant modifications are those of histidine, arginine, lysine, proline, methionine, and cysteine. Mixed-function oxidation reactions inactivate a number of key metabolic enzymes, such as glutamine synthetase, pyruvate kinase, lactate dehydrogenase, and phosphoglycerate kinase.34 Enzyme inactivation can be protected either by antioxidants and metal chelators or by high concentrations of substrates.34 Aldehyde products derived from lipid peroxidation of membranes react with amino and sulfhydryl groups,35 thus also potentially inactivating proteins.36,37 Malondialdehyde (MDA) and 4-hydroxy-2-nonenal (HNE) are two of the many known active aldehydes formed from lipid peroxidation that contribute significantly to enzyme inactivation. For example, in vitro experiments showed that MDA and HNE cause loss of activity and decrease in binding affinity (increased Km) to substrates of carnitine acetyltransferase and pyruvate dehydrogenase.25 The enzyme dysfunction induced by lipid peroxidation products such as MDA and HNE may be a common mechanism of age-associated dysfunction of enzymes with amino and sulfhydryl groups at or near their active sites. Specifically oxidized proteins have been identified in brain tissue from AD patients using proteomics-based techniques.38

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4.4.2 PROTEIN NITRATION AND CHLORINATION Nitric oxide reacts with superoxide anion to form peroxynitrite, which, along with other reactive oxygen species, can attack proteins to form nitrotyrosine. In addition, HOCl and NO2Cl may also attack proteins leading to oxidation of thiol groups and methionine residues, or chlorination of aromatic rings of tyrosines and –NH2 groups.39 Using proteomic techniques, six targets of protein nitration have been identified in brain tissue from AD patients, establishing a link between reactive nitrogen species-related protein modification and neurodegeneration.40 The nucleophilic form of vitamin E, γ -tocopherol, has been shown to protect against protein nitration41 and inflammation.42

4.4.3 PROTEIN GLYCATION AND CROSS-LINKING Protein amino groups can condense nonenzymatically with glucose to form Schiff bases, which form Amadori products by rearrangement. The Amadori product degrades into α-ketoaldehydes such as 1- and 3-deoxyglucosones, which can react with proteins to form cross-links as well as chromo/fluorophoric adducts called Maillard products or advanced glycation endproducts (AGE). Transition metal overload, with a concomitant increased oxidation rate and lipid peroxidation, may contribute to the production of Amadori products and AGE.43

4.4.4 PROTEIN DEGRADATION/TURNOVER Normally, proteins are degraded by proteolytic enzymes in proteasomes and lysosomes, but after cross-linking the proteins may become resistant to degradation.44 Cell aging may depend, in part, on a deleterious accumulation of insoluble inert protein that has escaped physiological proteolytic degradation. Mechanisms that may result in oligomerization of proteins include: (1) oxidants may create crosslinking by forming disulfide bridges and by the formation of ketogroups capable of reacting with free amino groups of neighboring proteins; and (2) carbonyl groups of glucose and related substances may initiate cross-linking between two amino groups to form AGE.44 Proteolytic systems consist of the ubiquitin–proteasome system, calpains, and lysosomes, which decline with age.45 The proteasome may play a key role in antioxidant defenses that delay aging and age-associated degenerative disease by minimizing protein aggregation and cross-linking, and by removing potentially toxic protein fragments.46,47 However, proteasomes can also be oxidatively modified. An age-related accumulation of oxidized proteins could, therefore, be a result of declining activity of the proteasome. As a consequence of this declining activity, oxidized proteins may begin to accumulate rapidly and thus contribute to cellular dysfunction and senescence.46,47 The accumulation of oxidized proteins and decline in protein turnover and activity of the proteasomal system are not only associated with postmitotic aging, but also are known to occur during proliferative senescence, resulting in an increased half-life of oxidized proteins.48–50

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Accumulation of insoluble protein deposits and their cross-linking is a feature of aging and neurodegeneration. For example, two types of fibrillar protein aggregates are present in AD brain: extracellular deposits (plaques) consisting primarily of β -amyloid peptide and intracellular deposits (tangles) composed predominantly of the microtubule-associated protein tau. Both plaques and tangles have AGE modifications, which occur primarily at lysine and arginine residues.51

4.4.5 AGING-ASSOCIATED PROTEIN/ENZYME OXIDATION With age, antioxidant systems decrease and oxidants increase. The accumulation of oxidized proteins is a characteristic feature of aging cells. An increase in the amount of oxidized proteins has been reported in many experimental aging models and in neurodegenerative diseases, as measured by the level of intracellular protein carbonyls or dityrosine, or by the accumulation of protein-containing pigments such as lipofuscin and ceroid bodies.19,33,46,47 Increased oxidative stress from nitrogen oxides is suggested by an increase in neuronal and inducible nitric oxide synthase (nNOS and iNOS) expression in the aged rat brain.52 Nitrotyrosine has been detected in neurofibrillary tangles of AD and also in lesions associated with other major neurodegenerative diseases of aging, such as PD and amyotrophic lateral sclerosis.53 The accumulation of Amadori products and AGE has been shown in aging54–56 and age-associated diseases such as diabetes.43 Mitochondrial enzymes are especially susceptible to inactivation by superoxide and hydroxyl radicals, as these oxidants are generated in mitochondria. Mitochondrial enzymes such as NADH dehydrogenase, NADH oxidase, succinate dehydrogenase, succinate oxidase, and ATPase are rapidly inactivated by hydroxyl radical or superoxide.57 Some of the enzyme damage is accompanied with an increase in Km value (a decrease in binding affinity) for their substrates or cofactors, including some receptor proteins, such as β -adrenergic receptor, transforming growth factor-β (TGF) receptor, dopamine receptor, acetylcholine receptor, adenylate cyclase, insulin receptor, serotonin receptor, steroid hormone receptor, retinoic acid receptor, inositol 1,4,5-trisphosphate receptor, and calmodulin, The Km value for delta-6desaturation of linoleic acid increased proportionally to the animal age, while the Vmax did not change until 25 months of age in liver microsomes of rats.58 Feuers24 studied the complexes of the electron transport system in gastrocnemius of young and old B6C3F1 female mice, and found that the Vmax of complex III and complex IV were significantly decreased with age and paralleled with a decrease in substrate affinity. He also showed that dietary restriction, which has been shown to reduce oxidative stress, effectively reversed these decreases in the activity of the complexes and substrate affinity. Hossain et al.59 showed that the apparent Km of the high-affinity form of GTP-cyclohydrolase was increased and the Vmax value of the low-affinity form in cerebellumon of 25-month-old rats decreased compared with that of the young animals. Cytosol proteins also display an age-associated decline of affinity to their substrates or cofactors, such as glyceraldehyde-3-phosphate dehydrogenase and tryptophan hydroxylase, which has an age-associated increased Km and changed

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activity in the midbrain or pons of rat brain.60 A combined effect of inefficient phosphorylation and oxidative damage of tryptophan hydroxylase may be responsible for lower tryptophan hydroxylase activity and binding affinity in aging brain. Aging causes a decrease in anabolic enzymes and an increase in catabolic enzymes for amine neurotransmitters in the central nervous system (CNS).61 Whether it is a general phenomenon and whether Km changes are correlated with changes in the function of anabolic and catabolic enzymes require further study. Nevertheless, we propose that feeding high levels of substrates and cofactors of defective enzymes in mitochondria may protect enzyme from oxidation and reverse some mitochondrial enzyme dysfunction due to aging and age-related degenerative diseases. As discussed above, oxidation or modification causes a structural deformation of enzymes, often altering binding affinity (Km) for the enzyme substrate. Such a modification can result in irreversible or reversible inhibition of enzyme activity. For example, an irreversible inhibitor, such as MDA, forms stable crosslinks with ε -amino groups of lysine residues in proteins.35 Some inhibitions can be reversed if substrate or coenzyme binding sites are affected, by increasing substrate or coenzyme levels. Reversible protein modification can be considered an antioxidant defense. A prime example is the oxidation of methionine residues of proteins to methionine sulfoxide, which is readily reversed by the action of methionine sulfoxide reductase.62,63

4.5 MT-NUTRIENTS AND MITOCHONDRIAL DECAY Clinical trials to determine whether micronutrients will improve memory in the elderly or will prevent or treat AD and PD are vigorously undertaken, and promising results (discussed below) suggest that such clinical trials are well justified.64,65 A few reviews have summarized the effects of different nutrients and antioxidants on neurological diseases including PD and AD.65–72 We have drawn on data summarized in these reviews and have also conducted an independent literature search to focus on a group of micronutrients, i.e., mt-nutrients which either are mitochondrial components or or their metabolites have an influence on mitochondrial structure and function. Table 4.1 summarizes mt-nutrients including CoQ, ALCAR, LA, creatine, phospholipids/fatty acids, and various vitamins that may improve mitochondrial function and their possible effects or function on mitochondria. Table 4.2 gives the dietary reference intakes (DRIs), tolerable upper levels (ULs), and mega-doses of mt-nutrients in this review. It should be emphasized that these mt-nutrients also play different roles in other parts of the cells. We propose that an inadequate supply of these mt-nutrients to mitochondria may result in acceleration of aging and neurodegenerative diseases, such as AD and PD. Figure 4.1 illustrates the possible relationships among mt-nutrients, mitochondrial decay, and brain aging/neurodegenerative diseases (AD and PD). Figure 4.2 illustrates some possible protective mechanisms of mt-nutrients in mitochondria. We will focus on a few mt-nutrients for targeting mitochondrial

B-vitamins

Mt-Nutrients

Biotin (B7)

Pyridoxine (B6)

Pantothenate (B5)

Niacin (B3)

Riboflavin (B2)

Thiamine (B1)

Precursor of coenzyme thiamine pyrophosphate, which is required for the key reactions catalyzed by the mitochondrial α-ketoacid dehydrogenase (1.2.4.4), pyruvate decarboxylase (4.1.1.1), α-ketoglutarate dehydrogenase (1.2.4.2), etc Precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), both of which are involved in a variety of redox reactions essential for energy production and cellular respiration. Examples of mitochondrial enzymes that use an FAD or FMN (riboflavin) cofactor include protoporphrinogen oxidase (1.3.3.4), electron-transferringflavoprotein, and electron-transferring-flavoprotein ubiquinone oxidoreductase (1.5.5.1), short-, medium-, and long-chain acyl-CoA dehydrogenases (1.3.99.2, 1.3.99.3, and 1.3.99.13), and mitochondrial complex I (1.6.5.3) Precursor for nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP), which are substrates/cofactors for complex I. Examples of mitochondrial enzymes that use an NAD or NADP (niacin) cofactor include aldehyde dehydrogenase (NAD+) (1.2.1.3), complex I (1.6.5.3), and long-chain-3-hydroxyacyl-CoA dehydrogenase. NAD is also a substrate of poly(ADP-ribose) polymerase, which is involved in the nuclear DNA base excision-repair mechanism by producing a repeating poly(ADP-ribose) polymer sequence for integration into the repaired DNA. NAD may also work in the same way in mitochondrial DNA repair mechanism. NDAH and NADPH are also mitochondrial antioxidants A component of coenzyme A and the phosphopantetheine moiety of fatty acid synthase and thus is required for the metabolism of all fat, protein, and carbohydrate via the citric acid cycle and for fatty acid and cholesterol synthesis. More than 70 enzymes utilize coenzyme A or derivatives Precursor of pyridoxal phosphate, which is essential for energy production from amino acids. Examples of mitochondrial enzymes that use pyridoxal phosphate cofactors include ornithine aminotransferase (2.6.1.13), and erythroid (housekeeping)-specific δ -aminolevulinic acid synthase (2.3.1.37) Examples of mitochondrial enzymes that use biotin cofactors include pyruvate carboxylase (for synthesis of oxaloacetate or gluconeogenesis and replenishment of citric acid cycle), acetyl-CoA carboxylase (for fatty acid biosynthesis), propionyl-CoA carboxylase (for metabolism of amino acids, cholesterol and odd-chain fatty acids) methionine, leucine, methylcrotonyl-CoA, and valine metabolism), and holocarboxylase synthetase (Continued)

Relation to and Possible Functions in Mitochondria

TABLE 4.1 Mt-Nutrients and Their Possible Functions in Mitochondria

Mitochondrial Nutrients 67

Coenzyme of α-ketoglutarate dehydrogenase and pyruvate dehydrogenase; multifunctional mitochondrial antioxidant that can scavenge free radicals, recycle other antioxidants (including GSH, ascorbic acid, CoQ and thioredoxin, all of which can recycle vitamin E), and chelate catalyzing metals to prevent free radical generation; induces phase-2 enzymes Cell-permeant antioxidant and precursor of GSH, which is also a mitochondrial antioxidant Transfers long-chain fatty acids across the mitochondrial membrane, increases cardiolipin level, increases respiration, and also considered a secondary antioxidant

Increases phosphocreatine stores to prevent ATP depletion The substrate of mitochondrial enzyme pyruvate dehydrogenase, which decreases in aged brain and in AD, Down’s syndrome, and Huntington’s disease. Pyruvate is also a cell-permeant antioxidant that quenches intracellular cytosolic oxidants that may be inaccessible to the lipophilic antioxidant vitamin E

Coenzyme Q (CoQ)

R-α-lipoic acid (LA)/DHLA (reduced form of LA) N-acetylcysteine Carnitine/ ALCAR

creatine pyruvate

Antioxidants

Energy enhancers and others

Tocopherols are important naturally occurring lipophilic antioxidants, which accumulate in circulating lipoproteins, cellular membranes, and fat deposits, where they react very rapidly with oxidants, particularly abundant in mitochondrial inner membranes β -carotene, lycopene, and some other carotenoids are effective antioxidants. Dietary β -carotene has a highest distribution in mitochondria, increases vitamin E and A levels; β -carotene protects cells from photoaging-associated mt-DNA mutation, and protects cytochrome b in oxygenic photosynthetic membranes again oxidative toxicity Cofactor of mitochondrial complexes I, II, and III, and mitochondrial antioxidant

Hematopoietic vitamin. Various one-carbon tetrahydrofolate derivatives are used in biosynthetic reactions such as the synthesis of choline, serine, glycine, purines and dTMP. The most pronounced effect of folate deficiency is inhibition of nuclear DNA or mt-DNA synthesis due to decreased availability of purines and dTMP. Mitochondria have higher levels of several folate coenzymes such as 5-methyltetrahydrofolate, 5-, and 10-formyltetrahydrofolate, and tetrahydrofolate than cytosol and have many tetrahydrofolate-synthesizing enzymes Functions in reduction and hydroxylation reactions, and is a first-line antioxidant in cytosol and mitochondria

Ascorbic acid (vitamin C) α-, γ tocopherols (vitamin E) vitamin A/ carotenoids

Folic acid

Relation to and Possible Functions in Mitochondria

Other vitamins

Mt-Nutrients

TABLE 4.1 (Continued)

68 Oxidative Stress and Age-Related Neurodegeneration

Trace elements

Manganese

Zinc

Copper

Iron

Docosahex-aenoic acid (DHA) and eicosapen-taenoic acid (EPA),

Choline/ cytidine 5⬘diphosphoc holine

Precursor for synthesis of the phospholipids phosphatidylcholine and sphingomyelin, which are important for biological membrane function, intracellular signaling, and hepatic export of very low density lipoproteins; also required for synthesis and release of acetylcholine, an important neurotransmitter involved in memory storage, motor control, and other functions; choline acetyltransferase, acetylcholinesterase and receptor sites are more enriched in brain mitochondria. Mitochondria contain choline phosphotransferase (EC 2.7.8.2) and have the intrinsic capacity to synthesize phosphatidylcholine. Exogenous phosphatidylcholine is evidently incorporated into and turned over most rapidly in mitochondrial membranes and sphingomyelin is preferably incorporated into microsomal and plasma membranes. Cytidine 5⬘-diphosphocholine is able to restore the activity of mitochondrial ATPase and of membrane Na+/K+-ATPase in experimental cerebral injury models. A dithiothreitol-insensitive cytidine 5⬘-diphosphocholine:cholinephospho-transferase is localized in the inner mitochondrial membrane The long-chain polyunsaturated fatty acids (LCPUFA), docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), owing to their high degree of unsaturation, can incorporate into mitochondrial membranes, therefore, they are essential for mitochondrial function and proper brain development. EPA and DHA have different effects on mitochondrial biogenesis, and are metabolized via β -oxidation in mitochondria. EPA lowers plasma triacylglycerol by increased mitochondrial fatty acid oxidation and decreased availability of fatty acids for triacylglycerol synthesis Iron accumulation causes generation of oxidants to damage mitochondrial lipids, proteins, and DNA, leading to mitochondrial dysfunction and contributing to aging, and neurodegeneration. Similarly, iron deficiency consequently causes heme deficiency, which selectively interrupts assembly of mitochondrial complex IV and causes mitochondrial DNA damage Copper acts as a cofactor in many enzymes playing key roles in cell metabolism, including the mitochondrial cytochrome c oxidase, which is one of the hallmarks of mitochondrial decay in neurodegenerative disorders. Both copper accumulation and deficiency are associated with mitochondrial dysfunction and take place through the same oxidative-stress mechanism Zinc-metallopeptidase, an insulin-degrading enzyme, regulates both cerebral amyloid β -peptide and plasma insulin levels in vivo and is also present in mitochondria; Zn2+ acts as an inhibitor of ATP depletion-induced apoptosis induced by blocking Bax activation, apoptosome function, caspase activation, and cytochrome c release from mitochondria; Zn2+ can be released from metallothioneins and mitochondria to cause mitochondrial and extramitochondrial generation of oxidants Manganese is an essential component of several enzymes, including the mitochondrial superoxide scavenger, manganese superoxide dismutase (MnSOD). MnSOD is one of the most important antioxidant defenses of our body

Mitochondrial Nutrients 69

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TABLE 4.2 Dietary Reference Intakes (DRIs), Tolerable Upper Levels (ULs), and Megadoses of mt-Nutrients in this Chapter

B-vitamins

Other vitamins

Antioxidants

Energy enhancers and others Trace elements

Thiamine (B1) Riboflavin (B2) Niacin (B3) Pantothenate (B5) Pyridoxine (B6) Biotin (B7) Folic acid Ascorbic acid (Vitamin C) Alpha-, γ -Tocopherols (Vitamin E) Vitamin A (retinol) Coenzyme Q (CoQ) R-α-lipoic acid (LA)/ DHLA(reduced form of LA) N-Acetyl-cysteine Carnitine/ALCAR Creatine Iron Copper Zinc Manganese

DRI (Men/Women)

UL

Megadose

1.2/1.1 mg 1.3/1.1 mg 16/14 mg 5/5 mg 1.3/1.3 mg 30/30 µg 400/400 µg 90/75 mg

— — 35 mg — 100 mg — 1000 µg 2000 mg

1000 mg 400 mg 2,000 mg 150 mg 1,000 mg 100,000 µg 40,000 µg 10,000 mg

15/15 mg

—

800 mg

900/700 µg — —

— — —

10,000 µg 1,200 mg 300–1000 mg

— —

— —

3,000 mg 2,000 mg

— 8/8 mga 900/900 µg 11/8 mg 2.3/1.8 mg

— 40 mg 10 mg 40 mg 11 mg

5–20 g — — — —

a

The RDA for men and post-menopausal women is 8 mg/day but 18 mg/day for premenopausal women, and 27 mg/day for pregnant women.

dysfunction and discuss: (1) deficiency causes cognitive dysfunction and (2) supplementation prevents and ameliorates cognitive dysfunction in AD and PD.

4.6 MT-NUTRIENT DEFICIENCY AND COGNITIVE DYSFUNCTION Significantly lower dietary intake of vegetables, fruits, vitamin A, thiamin, riboflavin, and niacin has been associated with lower scores of cognitive function.73 Also, higher intake of vitamins E, A, B6, and B12 has been related to better performance on visuospatial recall and abstraction tests.74 Considerable evidence suggests that diets deficient in various micronutrients can accelerate mitochondrial

Mitochondrial Nutrients

71 Mitochondrial nutrients Sufficient and supraphysiological dosages

Deficient

Increased

Decre ased

Dam age

e Damag

Oxidative stress

Antioxidant defense Accelerating Increasing Exacerbating

Aging and Neurodegeneration Risk of onset of AD and PD Progress of AD and PD

Delaying Reducing Ameliorating

FIGURE 4.1 Schematic illustration of the relationships among mitochondrial nutrients, mitochondrial decay, and brain aging/neurodegenerative diseases (AD and PD). Antioxidant defense includes antioxidants (vitamin E, vitamin C, carotenoids, bilirubin, carnosine, ubiquinol GSH, uric acid, LA, etc.), and antioxidant enzymes (superoxide dismutase, catalase, GSH oxidase, GSH reductase, phase-2 antioxidant enzymes, etc.). Oxidative stress includes increased oxidants (hydrogen peroxide, lipid hydroperoxide, lipid alkoxyl and peroxyl radicals, α, β -unsaturated aldehydes, singlet oxygen, superoxide, hydroxyl radical, nitric oxide, peroxynitrate etc.) and decreased enzyme cofactors/ substrates and oxidative phosphorylation.

decay, and this could contribute to neurodegeneration. Below, we discuss mt-nutrient deficiencies that have been linked to cognitive dysfunction, focusing on those known to cause accelerated mitochondrial decay. These micronutrients include iron, zinc, copper, and the B vitamins.

4.6.1 IRON DEFICIENCY Approximately 2 billion people, mainly children and menstruating women, are iron deficient. The main cause of iron deficiency is malnutrition, i.e., insufficient intake of iron, especially insufficient consumption of meat in their diet. The effects of iron deficiency and supplementation on rats were studied.75 Iron deficiency in children is associated with difficulties in performing cognitive tasks and retardation in the development of the CNS.76,77 Mitochondrial functional parameters and mitochondrial DNA (mtDNA) damage were assayed in iron-deficient (ⱕ5 µg/day) and iron-normal (800 µg/day) rats and in both groups after daily high-iron supplementation (8000 µg/day) for 34 days. This high dose is equivalent to the daily dose

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Oxidative Stress and Age-Related Neurodegeneration

B2

Cytosol

Fatty acids Carnitine

Creatine

CoQ

Vitamin E

Outer membrane

Creatine kinase Inner membrane

I

NADH NAD +

FADH 2

IV

III

II

CoQ FAD

Niacin

O2

e

O2-

Cardio lipin

V

Cyt C e

H2O2

e

.OH e

H2O CAT

LA

ADP+ ATP Pi

BC-KADH

FAD

Biotin

Citric acid cycle

Citrate synthase

Folate

THF-related enzymes

Matrix

B1


-KGDH

PDH

B12 Methyl malonyl CoA

Carboxylases

B6 Succinyl CoA

CoA Asp

B5

Oxaloacetate

FIGURE 4.2 Some possible protective mechanisms of mitochondrial nutrients in mitochondria. The possible mitochondrial protective mechanisms may include: (1) preventing mitochondria from oxidative stress such as antioxidants and metal chelators, such as LA, vitamin E, and CoQ; (2) repairing mitochondrial membranes, such as carnitine and vitamin E; (3) functionally repairing and preventing mitochondrial damage such as substrates or coenzymes or precursors of mitochondrial enzymes, such as LA, niacin, pantothenic acid, riboflavinthiamine, biotin, and carnitine; and (4) inducing phase-2 antioxidant defense enzymes in cytosol as an indirect protection to mitochondria (not shown), such as LA. Abbreviations: B1, thiamine, B2, riboflavin; B3, niacin; B5, pantothenic acid; B6, pyridoxine; B12, cobalamin; CoQ, coenzyme Q10; Cyt C, cytochrome c, LA, α -LA; PDH, pyruvate dehydrogenase; α -KGDH, α-ketoglutarate dehydrogenase; BC-KADH, branched-chain ketoacid dehydrogenase, CAT, carnitine acyltransferases. Carboxylases includes pyruvate carboxylase, acetyl-CoA carboxylase, and propionyl-CoA carboxylase; and THF-related enzymes include methionine synthase and methylene tetrahydrofolate reductase.

commonly given to iron-deficient humans. Iron-deficient rats had lower liver mitochondrial respiratory control ratios and increased levels of oxidants in polymorphonuclear leukocytes, as assayed by the fluorescent dye 2⬘7⬘-dichlorodihydrofluorescein diacetate (DCFH) (P ⬍ 0.05). Rhodamine 123 fluorescence of polymorphonuclear leukocytes also increased (P ⬍ 0.05). Lowered respiratory control ratios were found in daily high-iron-supplemented rats regardless of the previous iron status (P ⬍ 0.05). mtDNA damage was observed in both iron-deficient rats and rats receiving daily high-iron supplementation, compared with ironnormal rats (P ⬍ 0.05). Both inadequate and excessive iron (10 ⫻ nutritional need) cause significant mitochondrial malfunction. Although excess iron has been

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known to cause oxidative damage, oxidant-induced damage to mitochondria from iron deficiency has not been observed earlier. Untreated iron deficiency as well as excessive-iron supplementation are deleterious and emphasize the importance of maintaining optimal iron intake.75 A large literature links iron deficiency to cognitive impairment.78–80 A major consequence of iron deficiency is heme deficiency. Heme deficiency selectively interrupts assembly of mitochondrial complex IV.81,82 Heme deficiency was studied in young and old normal human fibroblasts (IMR90). Regardless of age, heme deficiency increased the steady-state level of oxidants and lipid peroxidation and sensitized the cells to fluctuations in intracellular Ca2+. Heme deficiency selectively decreased the activity and protein content of mitochondrial complex IV (cytochrome c oxidase) by 95%, indicating a decrease in successful assembly. Complexes I–III and catalase remained intact under conditions of heme deficiency, whereas ferrochelatase was up-regulated. Complex IV is the only heme protein in the cell that contains heme-a, which may account for its susceptibility. The rate of removal and assembly of complex IV declines with age. The role of heme and iron-sulfur clusters in mitochondrial biogenesis, maintenance, and decay with age has been reviewed.83 Mitochondria decay with age owing to oxidative damage and loss of protective mechanisms. Resistance, repair, and replacement mechanisms are essential for mitochondrial preservation and maintenance. Iron plays an essential role in the maintenance of mitochondria through its two major functional forms: heme and iron-sulfur clusters. Both these iron-based cofactors are formed and utilized in the mitochondria and then distributed throughout the cell. This is an important function of mitochondria not directly related to the production of ATP. Heme and iron–sulfur clusters are important for normal assembly and for optimal activity of the electron transfer complexes. Loss of mitochondrial cytochrome c oxidase, integrity of mtDNA, and function can result from abnormal homeostasis of iron. Common causes of heme deficiency include aging, deficiency of iron and vitamin B6, and exposure to toxic metals such as aluminum.82 Iron and B6 deficiencies are especially important because they are widespread. Heme deficiency may be a factor in the mitochondrial and neuronal decay of aging.82,84 Heme, a major functional form of iron in the cell, is synthesized in the mitochondria by ferrochelatase, which inserts ferrous iron into protoporphyrin IX. Heme deficiency was induced with N-methylprotoporphyrin IX, a selective inhibitor of ferrochelatase, in two human brain cell lines, SHSY5Y (neuroblastoma) and U373 (astrocytoma), as well as in rat primary hippocampal neurons. Heme deficiency in brain cells decreases mitochondrial complex IV, activates nitric oxide synthase, alters amyloid precursor protein, and corrupts iron and zinc homeostasis. The metabolic consequences resulting from heme deficiency seem similar to dysfunctional neurons in patients with AD. Heme-deficient SHSY5Y or U373 cells die when induced to differentiate or to proliferate, respectively. The role of heme in these observations could result from its interaction with heme regulatory motifs in specific proteins or secondary to the compromised mitochondria. Since iron ron and B6 deficiencies are

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preventable with supplementation, heme deficiency or dysregulation may be a preventable component of the cognitive dysfunction.84

4.6.2 ZINC DEFICIENCY Zinc deficiency induces oxidant accumulation and oxidative DNA damage, disrupts p53, nuclear factor κ B (NFκ B), and activator protein 1 (AP1) DNA-binding, and affects DNA repair in a rat glioma cell line.85,86 Approximately 10% of the U.S. population ingests ⬍50% of the current recommended daily allowance for zinc. We investigated the effect of zinc deficiency on DNA damage, expression of DNA-repair enzymes, and downstream signaling events in a cell-culture model. Low zinc inhibited cell growth of rat glioma C6 cells and increased oxidative stress. Low intracellular zinc increased DNA single-strand breaks (comet assay). Zinc-deficient C6 cells also exhibited an increase in the expression of the zinc-containing DNA-repair proteins p53 and apurinic endonuclease (APE). Repletion with zinc restored cell growth and reversed DNA damage. APE is a multifunctional protein that not only repairs DNA, but also controls DNA-binding activity of many transcription factors that may be involved in cancer progression. The ability of the transcription factors p53, nuclear factor κ B, and activator protein 1 (AP1) to bind to consensus DNA sequences was decreased markedly with zinc deficiency. Thus, low intracellular zinc status causes oxidative DNA damage and induces DNA-repair protein expression, but binding of p53 and important downstream signals leading to proper DNA repair are lost without zinc.85,86 Mitochondrial decay due to heme deficiency is a plausible explanation for the oxidative stress.85,86 δ -Aminolevulinate dehydratase (δ -ALA-D) contains eight zinc atoms and catalyzes the asymmetric condensation of two δ -Aminolevulinic acids (δ -ALA) molecules to yield porphobilinogen in the cytoplasm, an intermediate in heme biosynthesis.82 Zinc deficiency has been linked to cognitive defects in humans.71,87,88

4.6.3 COPPER DEFICIENCY Copper deficiency also alters heme metabolism.82 Copper deficiency causes a selective decrease in mitochondrial complex IV, likely by decreasing its assembly89 since copper is a prosthetic group in complex IV.90 It has been suggested that deficiency of copper also impairs the insertion of heme-a into complex IV.89 Copper is essential for efficient heme synthesis;91 therefore copper deficiency may affect complex IV by decreasing heme content in the cell and affecting the synthesis of heme-a.92 A rat model for Wilson’s disease, a disorder of copper transport, exhibits abnormal heme metabolism,93 supporting the connection between copper and heme synthesis.

4.6.4 PANTOTHENIC ACID DEFICIENCY Pantothenic acid is the precursor of coenzyme A (CoA), and thus is important for the production of acetyl-CoA. A deficiency of pantothenic acid depresses heme

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synthesis in monkeys, and causes anemia.94 Pantothenic acid deficiency in Neurospora crassa decreases complex IV95 and its iron content.95 It seems likely that the level of heme and heme-a were decreased as a result of deficiency in pantothenic acid, which led to loss of complex IV.82 Clinical pantothenic acid deficiency in humans is very rare, although the frequency of marginal deficiency or age-related deficiency has not been investigated. Both LA and pantothenic acid are micronutrients essential for the normal supply of succinyl-CoA, the precursor for heme biosynthesis; a deficiency of either may reduce heme synthesis through a mechanism similar to that produced by a biotin deficiency.

4.6.5 VITAMIN B6 DEFICIENCY Vitamin B6 is converted into the coenzyme pyridoxal 5⬘-phosphate (PLP), which is directly involved in heme synthesis, as a cofactor for δ -Aminolevulinic acid synthetase (δ -ALAS),96 and result in a shortage of heme. Approximately 10% of the U.S. population consumes less than half of the RDA of Vitamin B6.97

4.6.6 BIOTIN DEFICIENCY Biotin deficiency is quite common, especially during pregnancy.98,99 Biotin is a prosthetic group in four biotin-dependent carboxylases,100 three of which are present only in mitochondria. All of these three enzymes catalyze anaplerotic reactions (i.e., reactions that replenish an intermediate) in the Krebs cycle. The Krebs cycle is the donor for succinyl-CoA, the precursor for heme. A biotin deficiency decreases the activity of these enzymes. In addition, during biotin deficiency, methylcrotonyl-CoA accumulates in the mitochondria and reacts with glycine,100 which may result in depletion of this amino acid from the mitochondrial matrix. Thus, biotin deficiency may cause a decrease in both mitochondrial succinyl-CoA and glycine, and thus heme deficiency,82 which is associated with cognitive dysfunction as discussed in iron deficiency.

4.6.7 THIAMINE DEFICIENCY Thiamin occurs in the human body as free thiamin and its phosphorylated forms: thiamin monophosphate (TMP), thiamin triphosphate (TTP), and thiamin pyrophosphate (TPP). TPP is a required coenzyme for the mitochondrial enzyme pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, and branched-chain ketoacid (BCKA) dehydrogenase. These enzymes catalyze the decarboxylation of pyruvate, α-ketoglutarate, and branched-chain amino acids to form acetylcoenzyme A, succinyl-coenzyme A, and derivatives of branched-chain amino acids, respectively, all of which play critical roles in the production of energy from food. Thiamin deficiency affects the cardiovascular, nervous, muscular, and gastrointestinal systems. Wernike-Korsakoff syndrome, a profound memory disorder, is caused by thiamin deficiency, possibly, owing to the dysfunction of the TPP-dependent enzymes, α-ketoglutarate dehydrogenase and transketolase, in the brains of patients. Thiamine deficiency appears to be related to AD and

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Oxidative Stress and Age-Related Neurodegeneration

other forms of dementia.101 Impaired thiamine pyrophosphate synthesis may contribute to AD because the activity of α-ketoglutarate dehydrogenase and transketolase, the thiamine pyrophosphate-dependent enzymes, decrease in AD brain. This decrease is accompanied by a decrease in brain levels of thiamine pyrophosphate.102,103

4.6.8 RIBOFLAVIN DEFICIENCY Riboflavin is a precursor of the mitochondrial coenzyme FMN and FAD. Riboflavin also plays a role in the activity of complex I, pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, and BCKA dehydrogenase. FAD is the coenzyme of GSH reductase (which produces reduced GSH), xanthine oxidase (which produces uric acid), methylene tetrahydrofolate reductase (which catalyzes homocysteine to methionine), and tetrahydrofolate-related enzymes. High levels of homocysteine104–106 and low levels of reduced GSH107 and uric acid108 are correlated to age or cognitive dysfunction. Therefore, it is possible that riboflavin deficiency indirectly affects cognition by affecting the electron-transport chain, antioxidant enzymes and homocysteine metabolism.

4.6.9 NIACIN DEFICIENCY Niacin is the precursor of the ubiquitous mitochondrial oxidation–reduction coenzyme NAD⫹ and NADP⫹. Niacin also plays important role in the activity of pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, and BCKA dehydrogenase. Dementia can be caused by severe niacin insufficiency.109 Pellagra, the late stage of severe niacin deficiency, is clinically manifested by a photosensitive dermatitis, diarrhea, and dementia. The neurologic symptoms of pellagra include headache, apathy, fatigue, depression, disorentation, and memory loss.101

4.6.10 FOLATE DEFICIENCY Folate plays an important role in nucleic acid synthesis and methylation reactions, which are essential for normal brain function. Mitochondria have higher levels of several folate coenzymes such as 5-methyltetrahydrofolate, 5-, and 10-formyltetrahydrofolate, and tetrahydrofolate than the cytosol and are the site of many tetrahydrofolate-synthesizing enzymes.110 The mitochondrial distribution of folate coenzymes in the brain (which have longer polyglutamate chains) is different from that in the liver.111 Decreased folate levels or high homocysteine levels are associated with cognitive impairment, PD, AD, and other types of dementia.112–116

4.6.11 DOCOSAHEXAENOIC ACID AND EICOSAPENTAENOIC ACID (EPA) DEFICIENCY The long-chain polyunsaturated fatty acids (LCPUFA), docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), are present in mitochondrial membranes

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and are essential for mitochondrial function. For instance, both EPA and DHA have effects on mitochondrial biogenesis, although their effects are different.117 In vitro experiments with mitochondrial fraction show that EPA and DHA are metabolized via β -oxidation.118 It has also been shown that EPA, but not DHA, lowers plasma triacylglycerol by increased mitochondrial fatty acid oxidation and decreased availability of fatty acids for triacylglycerol synthesis and that DHA is oxidized in peroxisomes. The different effects of EPA and DHA on cellular organelles are suggested to be related to their substrate preference.119 Docosahexaenoic acid is highly concentrated in cell membranes of retina and brain,120,121 accounting for roughly 35% of phosphatidyl serine phospholipids of brain gray matter.122 Although infants have some capacity to synthesize DHA from its precursor, the essential fatty acid α-linolenic acid,123,124 DHA is also supplied directly to the fetus through the placenta and to the postnatal infant in breast milk.125–127 DHA cannot be excluded as a factor contributing to increased performance of children fed breast milk compared to formula on some intelligence tests,128–130 but breast-feeding studies are known to be seriously confounded, and positive results must be considered uncertain.125,131,132 A number of animal studies have shown that rats whose diets were chronically and severely restricted in the EFA precursor of DHA, α-linolenic acid, exhibit behavioral changes that are not observed in restricted animals supplemented with DHA.133 Some studies in animals have also correlated lower levels of DHA in the brain and poor performance on learning tests with biochemical indicators of brain function, pointing to possible mechanisms by which DHA could affect learning.134–136 Evidence in nonhuman primates also suggests that animals subjected to similar dietary restriction exhibit poorer visual acuity and other behavioral abnormalities.137,138 In adulthood, LCPUFA deficiency has been associated with cognitive impairment, AD, and PD.139–141 One study has also reported an association of DHA deficiency with attention-deficit hyperactivity disorder in children.142 Fatty fish, a good source of DHA,143 is not a major component of the American diet, which tends to be quite low in DHA.144

4.6.12 CHOLINE DEFICIENCY Choline is an essential nutrient for humans and is an essential component of membrane phospholipids. Phospholipids serve not only as structural components in cellular membranes, but also as cofactors for activating enzymes. For example, β -hydroxybutyrate dehydrogenase requires phosphatidylcholine for activity. Choline is a precursor for the biosynthesis of the neurotransmitter acetylcholine and also is an important source of labile methyl groups.145–147 Choline acetyltransferase, acetylcholinesterase, and receptor sites are found to be more enriched in the mitochondrial fraction of fetal human brain.148 Rat lung mitochondria contain choline phosphotransferase (EC 2.7.8.2) and have the intrinsic capacity to synthesize phosphatidylcholine.149 An in vitro study with cultured neuroblastoma cells demonstrates that exogenous phosphatidylcholine is evidently incorporated

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Oxidative Stress and Age-Related Neurodegeneration

into and turned over most rapidly in mitochondrial membranes and that sphingomyelin is preferably incorporated into microsomal and plasma membranes.150 Choline deficiency in cell culture and fetal brain causes cell death by apoptosis,145,151 possibly owing to a decrease in membrane phosphatidylcholine and an accompanied increase in ceramide.145,152 Choline deficiency during development has been reported to affect memory and attention in rat offspring,153 and also to alter several biochemical, electrophysiological, and morphological properties in some brain regions.154–156 Evidence also suggests that choline supplementation during development enhances memory in offspring throughout their life span.145,153,157

4.6.13 ACETYL-CARNITINE, R-α -LIPOIC ACID, COENZYME Q10, AND CREATINE DECLINE Acetyl-carnitine, LA, CoQ, and creatine can be synthesized in animals and humans, thus they are not vitamins. The age-associated decline of these mt-nutrients may be associated with mitochondrial decay and cognitive dysfunction, and supplementation of these mt-nutrients may ameliorate mitochondrial decay and neurological diseases. The relationships among these mt-nutrients, mitochondrial decay, and cognition will be discussed in the following section.

4.7 MT-NUTRIENT SUPPLEMENTATION FOR THE PREVENTION AND AMELIORATION OF AD AND PD 4.7.1 MITOCHONDRIAL DECAY DUE TO OXIDATIVE DAMAGE IS A KEY CONTRIBUTOR TO AGING AND AGE-ASSOCIATED NEURODEGENERATIVE DISEASES, AD AND PD Increasing evidence suggests that AD and memory loss in AD and other dementia may involve a mitochondrial disorder.7,9,11,15,158–163 Defects in mitochondrial oxidative phosphorylation are frequently associated with AD, and both inherited and somatic mtDNA mutations are reported in certain AD cases, such as the recently found somatic mtDNA control-region mutations in AD brain that suppress mitochondrial transcription and replication.164 AD brains show increased oxidative damage, including increased lipid peroxidation, protein oxidation, DNA/RNA oxidation, and redox-active metals; decreased antioxidant defense; and increased vulnerability to oxidative stress. Oxidative stress-induced injury may involve the selective modification of different intracellular proteins, including key enzymes and structural proteins, which precedes and may lead to the neurofibrillary degeneration of neurons in the AD brain.165 AD is the third most costly disease after heart disease and cancer. Only a few drugs are available for treating less severely affected (mild to moderate) patients. Although the US Food and Drug Administration of recently approved the first drug (Menantine, with a brand name of Namenda, that can have some beneficial effect in moderate to severe AD patients, but still cannot modify the underlying

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pathology of AD (http://www.fda.gov/bbs/topics/NEWS/2003/NEW00961.html), medications that will stop or reverse the disease seem still far away. Therefore, an effective prevention would be a promising approach to slow or halt the development of AD. We propose that using mt-nutrients may be an appropriate approach to prevent AD by reducing protein modification and mitochondrial decay. Increasing evidence has shown that PD, caused either by the toxin 1-methyl4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), or by genetic defects and sporadic, is associated with oxidative damage.166–169 Attempts to attenuate oxidative damage and protect striatal dopaminergic neurons in PD have included blocking the dopamine transporter by mazindol, blocking the N-methyl-D-aspartate (NMDA) receptors by dizocilpin maleate, enhancing the survival of neurons by giving brain-derived neurotrophic factors, or inhibiting monoamine oxidase B by selegiline.166,167 Most of the current treatments for PD only relieve the symptoms without slowing down the disease process, and also cause significant side effects. Mitochondrial decay due to oxidative damage is a key contributor to aging4,5,170 and also to PD.27 The striatum of patients with PD displays a defect in complex I of the mitochondrial electron-transport chain, i.e., a reduction in the activity of NADH:CoQ reductase;27,169,171 MPTP, which causes a PD-like syndrome in humans and rodents, acts via inhibition of mitochondrial complex I.27,172 Similar to MPTP, rotenone and 6-hydroxydopamine (6-OHDA) cause a PD-like syndrome in animals by inhibiting the activity of complex I and increasing oxidative damage.166,173 More recently, the major physiologically and pathologically relevant reactive-oxygen species-generating site in mitochondria has been limited to the FMN group of complex I, not at the ubiquinone of complex III as commonly believed, which further emphasizes the importance of ameliorating complex I defect in PD.174 Antioxidants are the most commonly administered nutrients to AD patients.175 Antioxidants may protect the aging brain against oxidative damage associated with pathological changes of AD. The Cache County Study demonstrated that the use of vitamin Es and C supplements in combination with antioxidants is associated with reduced prevalence and incidence of AD.176 Prospective epidemiological studies have indicated that dietary intake of several exogenous antioxidants is associated with a lower risk for AD, suggesting that people at risk for developing AD or in the early phases of this disease may benefit from intervention with exogenous antioxidants.177 However, further clinical studies, based on larger cohorts over a longer term, are greatly needed. It has been suggested that balanced up-regulation of both exogenous and endogenous antioxidants should be considered as one of the best strategies for preventing or at least slowing down the progression of AD.177 As indicated in the previous section, deficiencies of many vitamins and minerals cause oxidant leakage from mitochondria; antioxidant intervention offers an effective means of relieving these deficiencies. A decrease in the level of mt-nutrients may impair effectiveness of the neuron’s antioxidant defense mechanisms, resulting in neurodegenerative diseases such as AD. Some evidence is available that administration of individual or combined nutritional cofactors is effective in increasing mitochondrial ATP production

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Oxidative Stress and Age-Related Neurodegeneration

and slowing or arresting the progression of clinical symptoms of mitochondrial disorders.69 Among the mt-nutrients, ALCAR, LA, CoQ, vitamins E and C, and some B vitamins are those most commonly studied. Deficits in cholinergic function contribute to the clinical manifestation of AD, affecting cognition, behavior, and activities of daily living. Both choline acetyltransferase (for the synthesis of acetylcholine) and acetylcholinesterase (for destruction of acetylcholine) are found to be affected by aging and AD. Choline acetyltransferase showed an age-dependent decrease in activity and increase in Km.61 Levels of acetylcholine are diminished in cholinergic neurons. In severe AD, levels of choline acetyltransferase and acetylcholinesterase are decreased by as much as 90%, while levels of butyrylcholinesterase increase, compared with normal.178 Different approaches have been proposed or tested for replacement of cholinergic function, including intervention with acetylcholine precursors, stimulation of acetylcholine release, use of muscarinic or nicotinic receptor agonists, and cholinesterase inhibition. The drugs for AD treatment at present are mainly cholinesterase inhibitors, which block the acetylcholine destruction and enhance the acetylcholine neurotransmission in the remaining cholinergic neurons.179 Choline supplementation has been shown to enhance synthesis and release of acetylcholine.180 Administering substrates and cofactors, such as choline, its precursors, and ALCAR (a precursor of acetyl group of acetylcholine (White HL and Scates PW, 1990 #1353)) might improve cholinergic dysfunction in AD. The use of mt-nutrients could be effective in reducing PD-related damage, by preventing and repairing mitochondrial damage. We believe that the use of mt-nutrients to treat people at risk for PD could provide an inexpensive way to prevent the onset and delay the progression of the disease, and lead to an effective therapy, as well as yield a better understanding of the underlying mechanism of the disease. A combination of mt-nutrients might be more effective than single nutrients, because PD is a complicated mitochondrial disease. These mitochondrial antioxidants/metabolites play different and complementary roles, and thus may be synergistic in attenuating mitochondrial decay. Some evidence is available that administration of individual or combinations of mt-nutrients is effective in relieving the symptoms of PD.

4.7.2 THIAMINE SUPPLEMENTATION Thiamine is phosphorylated to form thiamine pyrophosphate, a cofactor used by many enzymes, including mitochondrial enzymes such as α-ketoacid dehydrogenase, pyruvate decarboxylase, and α-ketoglutarate dehydrogenase. Thiamine is important in the metabolism of acetylcholine and its release from the presynaptic neuron. Reduced activities of thiamine-dependent enzymes, including mitochondrial thiamine-dependent enzymes, pyruvate dehydrogenase, and α-ketoglutarate dehydrogenase in the brains and peripheral tissues of patients with AD, have been reported.181–183 Blass et al.184 reported that Alzheimer’s patients treated with 3 g/day thiamine show statistically significant improvement in cognition. Meador et al.185 found that thiamine at pharmacologic dosages (3 to 8 g/day) may have a

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mild beneficial effect in dementia of Alzheimer’s type. Fursultiamine, a derivative of thiamine, at an oral dose of 100 mg/day had a mild beneficial effect in patients with AD in a 12-week open trial.186 However, one review of the literature concludes that evidence for thiamine as a useful treatment for the symptoms of AD is weak and thus not recommended.187 A plausible explanation for a thiamine effect in AD could be that mitochondrial decay leads to oxidized proteins in the mitochondria, and some of this damage affects the Km for thiamine pyrophosphate. A high dose of thiamine, which increases thiamine pyrophosphate in the mitochondria,23 could thus ameliorate enzyme damage, and in turn improve AD. As discussed previously,23 genetic defects in three mitochondrial enzymes (two of which, pyruvate dehydrogenase and α-ketoglutarate dehydrogenase, decrease during human aging and lead to brain dysfunction) are shown to affect the Km for thiamine pyrophosphate, and are remedied by high dose of thiamine administration. Thus, brain dysfunction characteristic of normal and pathological aging, of which AD is a common extreme case, might be ameliorated by a high dose of thiamine.

4.7.3

RIBOFLAVIN SUPPLEMENTATION

Since complex I appears to be deficient in PD, therapy with high riboflavin can be considered. As discussed previously, a recent study showed that high dose of riboflavin (30 mg orally every 8 h) (and the elimination of dietary red meat) promotes the recovery of some motor functions in PD patients.26 The authors suggested that riboflavin-sensitive mechanisms involved in PD, such as GSH depletion, cumulative mitochondrial DNA mutations, disturbed mitochondrial protein complexes, and abnormal iron metabolism, may be improved by this treatment. We suggest that riboflavin may stimulate mitochondrial enzymes with decreased activity and altered Km for FMN/FAD.

4.7.4 NIACIN/NADH SUPPLEMENTATION Niacin refers to both nicotinic acid and nicotinamide. Nicotinic acid is the precursor of NAD and NADP. High doses of nicotinic acid can raise NAD/NADP levels in both mitochondria and cytoplasm.23 Since nicotinamide can be deamidated to some extent in the intestine to nicotinic acid, it can also be a precursor of NAD and NADP. Nicotinamide is also used in nutritional supplements and in food fortification.101 High dose of nicotinic acid is a known anticholesterol treatment, while nicotinamide is sometimes used in antiaging studies, and is used in clinical trials as a therapy for diabetes and cancer. Nicotinic acid intake from foods is found to be inversely associated with AD, and higher food intake of nicotinic acid is associated with a slower annual rate of cognitive decline, suggesting that dietary nicotinic acid may protect against AD and age-related cognitive decline.109 NADH is a substrate for mitochondrial complex I. NADH and NADPH may act as endogenous antioxidants.188 In a preliminary trial of NADH therapy for

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patients with Alzheimer’s dementia, improvement was seen in cognitive function of all 17 patients tested.189 However, another study observed no cognitive improvement in patients with either AD or vascular and fronto-temporal dementia treated with NADH.190 PD patients have a significant lower intake of nicotinic acid.191 Nicotinamide, unlike nicotinic acid, is a poly(ADP-ribose) polymerase inhibitor, protects against neurotoxicity in MPTP-treated mice and poly(ADP-ribose) polymerase knockout mice, and attenuates neuronal injury and ATP depletion produced by focal ischemia, malonate, and MPTP.66 NADH may stimulate endogenous dopamine synthesis and alleviate motoric impairment and cognitive dysfunction of PD patients.192,193

4.7.5 FOLIC ACID, B6, AND B12 SUPPLEMENTATION Various one-carbon tetrahydrofolate derivatives are used in biosynthetic reactions such as the synthesis of choline, serine, glycine, purines, and dTMP. The most pronounced effect of folate deficiency is inhibition of DNA synthesis due to decreased availability of purines and dTMP. Kruman et al.105 showed that folic acid deficiency and homocysteine impair DNA repair in hippocampal neurons and sensitize neurons to amyloid-induced oxidative damage in APP mutant transgenic mice, an animal AD model. Hyperhomocysteinemia has also been shown in clinical study as an independent risk factor for development of dementia and AD.104 Since the hyperhomocysteinemia is caused by inadequate intake or decreased utilization of folate and vitamins B6 and B12, the hypothesis that folate, B6, and B12 supplementation may lower the risk AD is plausible.114,194 Elevated plasma homocysteine levels are found in a PD animal model106 and also in patients treated with L-dopa,195 and the homocysteine levels correlate inversely with folate, B12, and PLP levels.196 Thus, folate, B6, and B12 supplements may be warranted for PD patients on L-dopa therapy because the B-vitamin requirements necessary to maintain normal plasma homocysteine concentrations are higher in L-dopa-treated patients than in those who are not on L-dopa therapy.196

4.7.6 DOCOSAHEXAENOIC ACID SUPPLEMENTATION Vertebrates are unable to synthesize linoleic and α-linolenic acids, two essential polyunsaturated fatty acids and the only dietary precursors for LCPUFA. DHA, a (22:n–3) fatty acid, comprises about 30% of the fatty acids in the neurons. Intakes of dietary DHA ethyl ester and egg phosphatidylcholine improved maze-learning ability in young and old mice.197 DHA-rich phospholipids improve learning ability and visual function, and reverse biochemical modifications in old mice fed with n–3 polyunsaturated fatty acid-deficient diet; they also improve visual function in old mice fed with a balanced diet.198 Dietary supplementation with phospholipids and DHA improves age-related cognitive impairment.199 High DHA consumption is associated with reduced AD risk, and reduction of dietary n–3 PFA in an AD mouse model results in losses of the p85α subunit of

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phosphatidylinositol 3-kinase and the postsynaptic actin-regulating protein drebrin, as in AD brain.200 Treatment of n⫺3 PFA-restricted mice with DHA protects against loss of postsynaptic proteins, oxidation, and behavioral deficits, and increases antiapoptotic Bcl-xL/Bcl-2-Associated Death Promoter (BAD) phosphorylation suggesting that DHA protects from dendritic pathology in an AD mouse model.200 Concentrations of essential fatty acids are lower in the plasma and red blood cell phospholipids of AD patients compared with age-matched controls; decreased DHA in phosphatidylethanolamine was observed in AD patient brain, and low-serum DHA may be a significant risk factor in the development of early AD patients, while an essential fatty acid preparation improves the quality of life of AD patients.198

4.7.7 CHOLINE SUPPLEMENTATION Clinical use of choline in Alzheimer senile dementia was tried early in 1970s.201 The first long-term double-blind placebo-controlled trial of high-dose lecithin (a precursor of choline) in senile dementia of the Alzheimer type was reported by Little et al., in 1985 with a conclusion that there may be a “therapeutic window” for the effects of lecithin in the condition, and that this may be more evident in older patients.202 Besides choline, other cholinergic precursors, which are used to enhance ACh availability or release, include phosphatidylcholine (lecithin), cytidine 5⬘-diphosphocholine (citicoline or CDP-choline),203–205 choline alphoscerate,206 and phosphatidylserine. Some of these precursors have provided improvement of cognitive dysfunction in AD, especially cytidine 5⬘-diphosphocholine207 and choline alphoscerate.208 Soybean lecithin transphosphatidylated phosphatidylserine, prepared from soybean lecithin and L-serine by a transphosphatidylation reaction and fed to aged rats, significantly improved performance in the water maze escape and increased acetylcholine release and the Na(+), K(+)ATPase activity of the synaptosome in aged rats.209 Cytidine 5⬘-diphosphocholine is an intermediate in the biosynthetic pathway of the structural phospholipids of cell membranes, especially in that of phosphatidylcholine. Once absorbed after administration, the cytidine and choline disperse widely throughout the organism, cross the blood–brain barrier, and reach the CNS, where they are incorporated into the phospholipid fraction of the membrane and microsomes. Cytidine 5⬘-diphosphocholine can restore the activity of mitochondrial ATPase and membranal Na+/K+ ATPase, inhibit the activation of phospholipase A2, and accelerate the reabsorption of cerebral edema in various experimental models. The important clinical use of cytidine 5⬘-diphosphocholine is for improvement of senile cognitive impairment of AD and chronic cerebral vascular disease. Cytidine 5⬘-diphosphocholine is also effective as cotherapy for PD,207 although the FDA has not approved it as a drug for PD treatment.

4.7.8

ACETYL-L-CARNITINE/L-CARNITINE SUPPLEMENTATION

Acetyl-L-carnitine is the acetyl derivative of L-carnitine, which transports longchain fatty acids into mitochondria for fuel. ALCAR is better absorbed than L-carnitine and crosses the blood–brain barrier more efficiently.210 Tissue levels

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of carnitine in animals, including humans, decrease with age;211–213 this reduces the integrity of the mitochondrial membrane. Studies on rats, mice, and dogs showed that ALCAR supplementation improves age-associated cognitive dysfunction and nerve regeneration, protects neurons from the toxicity of mitochondrial uncouplers or inhibitors, attenuates neurological damage following brain ischemia and reperfusion, elevates levels of GSH and GABA in the brain, increases cardiolipin content, elevates activities of mitochondrial enzymes, and improves mitochondrial function17,67,213 ALCAR might improve the symptoms or slow disease progression in clinical trials with AD patients or people suffering from dementia-associated cognitive dysfunction. Calvani et al.214 reviewed the neuroprotective effects of ALCAR in more than 500 patients with AD or other age-related dementias, and concluded that oral ALCAR administration might slow the progression of neurodegeneration by improving mitochondrial energetics, increasing antioxidant activity, and stabilizing intracellular membranes and cholinergic neurotransmission. A small study with seven AD patients showed improvement in both clinical and CNS measurements in a double-blind placebo-controlled trial over a 1-year period.215 Another study showed significant improvements in all cognitive, behavioral, and emotional measurements except anxiety in a 40-day, double-blind, placebo-controlled study of 40 AD patients.216 A study of 6 months’ duration in outpatients showed mild improvements in tasks of attention and timing.217,218 A meta-analysis of 21 double-blind, placebo-controlled, prospective clinical trials of 3 to 12 months’ duration on the efficacy of ALCAR in mild cognitive impairment and early AD concluded that ALCAR had a significant beneficial response, compared with placebo.219 Beneficial effects of ALCAR supplementation were seen on both the clinical scales and the psychometric tests at the time of the first assessment at 3 months, and the benefits increased over time.219 Another analysis of 11 clinical trials of AD patients found that there is evidence for benefit of ALCAR on clinical global impression, but there was no evidence using objective assessments in any other area of outcome.220 The significant effect on early AD and mild impairment and the less-significant effect on AD patients may suggest that ALCAR is more effective in preventing and slowing the progression of AD than in treating severe AD. ALCAR was proposed as a therapeutic agent for protecting against MPTPinduced toxicity in neuroblastoma cells221 and in monkeys.222 However, no clinical trials of ALCAR on PD have been reported.

4.7.9

α -LIPOIC ACID /DIHYDROLIPOIC ACID SUPPLEMENTATION

LA is a coenzyme involved in mitochondrial metabolism. The reduced form of LA, DHLA, is a powerful mitochondrial antioxidant.223–226 It recycles other cellular antioxidants, including CoQ, vitamins C and E, GSH, and chelates iron and copper.223–226 LA readily crosses the blood–brain barrier and is accepted by human cells as a substrate, where it is reduced to DHLA by NADH-dependent mitochondrial dihydrolipoamide dehydrogenase.223

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Substantial evidence suggests that one mechanism by which β -amyloid peptides contribute to the etiology of AD is by stimulating the formation of oxidants. Thus, the antioxidant LA, which is able to cross the blood–brain barrier, would seem an appropriate nutrient in treating AD. LA has been used to treat or prevent diabetic peripheral and cardiac autonomic neuropathy,227,228 as well as other mitochondrial diseases (reviewed in Liu et al,213 2002). LA has also been studied as a treatment for Alzheimer-type dementia. A dose of 600 mg LA, given daily to nine patients with AD and related dementias for about 1 year, led to a stabilization of cognitive functions in the study group, as shown by constant scores in two neuropsychological tests.229 Although the study was small and not randomized, it suggests that treatment with LA is a possible neuroprotective therapy option for AD and related dementias. Treatment with LA protected cortical neurons against cytotoxicity induced by β -amyloid or hydrogen peroxide, and induced an increase in the level of Akt, an effector immediately downstream of phosphatidylinositol kinase, suggesting that the neuroprotective effects of the antioxidant LA are partly mediated through activation of the PKB/Akt signaling pathway.230 The effects of the different forms of LA (oxidized or reduced forms as well as the enantiomers of the natural R-α-LA and the synthetic S-α-LA) may be different under different systems.213,231 For example, Lovell et al.232 showed that DHLA, the reduced form of LA, in primary cortical and hippocampal neuronal cultures significantly protects against both β -amyloid- and Fe/H2O2-mediated toxicity, and that concomitant exposure to LA and Fe/H2O2 significantly potentiates the oxidative stress. They suggest that the oxidation state of LA is critical to its function, and that in the absence of studies of LA/DHLA equilibria in human brain, the use of LA as an antioxidant in disorders where there is increased Fe such as AD is of questionable efficacy.232 This might not be a serious problem if the in vivo reducing power of NADH and NADPH is functioning normally, because exogenously supplied LA can be readily reduced by NADH and NADPH when it is taken up by mitochondria.225 LA, as with other antioxidants such as vitamin E or flavonoids, could indirectly strengthen the antiglycation and antioxidant defense systems when GSH is limiting under conditions of oxidative stress and inflammation in AD. Excess production of reactive carbonyl compounds and subsequent AGE formation can contribute to cross-linking of protein fibrils and to pathological proinflammatory signaling, which contributes to pathological changes and dementia progression in AD. The human brain has developed the glyoxalase system, an effective defense system to scavenge small dicarbonyl compounds such as glyoxal and methylglyoxal; GSH is a rate-limiting cofactor for this defense system.51 Another mechanism of the protective effect of LA, like the thiol-reactive compound sulforaphane,233 is mediated through induction of the phase-2 enzyme response through the transcriptive factor Nrf2. LA was shown to induce Nrf2, Nrf2 binding to antioxidant response element, and consequently, higher glutamylcysteine ligase activity.234 Phase-2 enzymes (e.g., GSH transferases, NAD(P)H:quinone reductase) and GSH synthesis are part of the elaborate system

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for protection against the toxicity of reactive oxygen and nitrogen species and electrophiles, which are constant threats to the integrity of mammalian DNA.233 Induction of phase-2 enzymes, which neutralize reactive electrophiles and act as indirect antioxidants, appears to be an effective means for achieving protection against a variety of carcinogens and other oxidative damage in animals and humans.235 This mechanism appears to be an indirect mitochondria protection, because the induced phase-2 enzymes reduce cytosolic oxidative stress and enhance the cellular antioxidant defense, thus indirectly relieving oxidative stress to mitochondria. LA has not been tested in PD. However, several observations suggest that it might be effective. Enhanced expression of tumor necrosis factor α (TNF-α) is associated with the earliest stages of damage in the MPTP model of dopaminergic neurotoxicity. TNF-α receptor deficiency protects dopaminergic neurons, suggesting that this pro-inflammatory cytokine is an early effector in the neurodegenerative processes underlying PD.236 LA inhibits TNF-α-induced NF-κ B activation.237 LA can prevent the GSH depletion-induced decrease in mitochondrial complex I activity and increase cellular GSH; however, both decrease in PD.238

4.7.10 COENZYME Q10 SUPPLEMENTATION Coenzyme Q10, which is an electron carrier in the inner mitochondrial membrane, stabilizes respiratory chain components and acts as a mitochondrial antioxidant.27,239,240 The levels of CoQ decline with age.240 Supplementation of CoQ improves mitochondrial function and age-associated decline in cognitive function and neurodegeneration. There have been several reviews on the effects of ALCAR, LA, and CoQ on mitochondrial decay and cognitive dysfunction.65–67,69,213,241 Coenzyme Q10 has a beneficial effect on clinical outcomes and biochemical parameters in a variety of mitochondrial disorders including PD, Huntington’s disease, and Friedreich’s ataxia.27,242,243 It, however, has not been used in clinical AD studies. CoQ was found not to change in the plasma244 and serum245 of AD and dementia patients. But given the importance of CoQ in mitochondria, and the effect of CoQ in treating other mitochondrial diseases, CoQ might also be effective in treating AD. In MPTP-treated mice CoQ, attenuates MPTP neurotoxicity, elevates striatal dopamine levels, and increases the number of striatal mitochondria, the synthesis of ATP, and the activity of striatal complex I.27,66,167 CoQ attenuates MPTPinduced generation of oxidants and MPTP-induced dopamine depletion in the striatum.27,242 A recent clinical study demonstrated that CoQ administration to PD patients at high doses attenuates disability development, is safe, and is well tolerated at dosages of up to 1200 mg/day. Thus, it appears that CoQ slows the progressive deterioration of function in PD.243

4.7.11 CREATINE SUPPLEMENTATION Creatine increases creatine/phosphocreatine (PCr) and inhibits the opening of the mitochondrial permeability transition pore.66 The PCr system functions as a

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spatial energy buffer between the cytosol and mitochondria, using a unique mitochondrial creatine kinase (CK) isoform. A double-blind, placebo-controlled, cross-over trial with 45 young adult, vegetarian subjects showed that oral creatine supplementation (5 g/day for 6 weeks) had a significant positive effect on both working memory and intelligence, both tasks that require speed of processing.246 Creatine has also been used in several neurological diseases, including PD and Huntington’s disease, but has not been used for treating AD.

4.8 EFFECTIVENESS OF COMBINATIONS OF MT-NUTRIENT SUPPLEMENTATION ON AD AND PD PREVENTION AND TREATMENT On the basis of epidemiologic, laboratory, and limited clinical studies, it is suggested that a combination of nonsteroidal anti-inflammatory drugs (NSAIDs) and appropriate levels and types of micronutrients might be more effective than the individual agents in the prevention and in the treatment of AD.247 The concept of using NSAIDs and nutrients, combined with using mt-nutrients to target mitochondrial dysfunction, may provide a more effective strategy in preventing and treating AD. Combinations of a number of nutritional cofactors have been tested in different mitochondrial disorders for additive or synergistic effects, including riboflavin plus carnitine to improve muscle weakness and exercise capacity in complex I-deficient myopathy; riboflavin plus nicotinamide to improve encephalopathic symptoms and nerve conduction; vitamin K3 plus ascorbate to clinically improve exercise capacity in patients with complex III defect; CoQ plus vitamin K3, ascorbate, thiamin, riboflavin, and niacin to reduce mortality in mitochondrial myopathy and encephalomyopathies;69 and carnitine plus choline and caffeine to reduce body fat and serum leptin concentrations.248 We observed that ALCAR and LA are more effective in combination than when used individually to ameliorate the decay of mitochondria in old rats because they play different roles in restoring mitochondrial function, including the complementary effect of LA on ALCAR by inhibiting oxidative stress.25,249,250 Feeding a diet with vitamin E, Ginkgo biloba, pycnogenol, and ascorbyl palmitate to ApoE-deficient mice resulted in a significant increase in life span and a marked reduction of inclusion body histopathology in the hippocampus, and a significant reduction in the levels of DNA fragmentation.251 A mixture (vitamin E, phosphatidyl choline, and pyruvate) is shown to be more effective than single antioxidants in preventing β -amyloid toxicity in cell culture,252 and on buffering neuronal degeneration and oxidative stress in cultured cortical neurons and in central nervous tissue of apolipoprotein E-deficient mice.253 A clinical study suggested that vitamin E and C supplements may protect against vascular dementia and may improve cognitive function later in life.254 The population-based, prospective cohort study conducted in the Netherlands also showed that high dietary intake of vitamins C and E may lower the risk of AD,255 although another study showed that men using vitamins E and C (either for long

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or short term) were not at reduced risk for AD and for AD with vascular dementia or cerebrovascular disease.256 Another approach for AD treatment is to combine mt-nutrients with other AD drugs. An open study to evaluate the effect of ALCAR (2 g/day orally for 3 months) in association with donepezil or rivastigmine in patients with mild AD who had not responded to treatment with acetylcholinesterase inhibitors (AchE-I) showed that the response rate (by assessing cognitive functions, functional status and behavioral symptoms) is further improved after the addition of ALCAR. This result indicates that the combination of these two drugs may be a useful therapeutic option in AD patients.257 Cholinergic therapies can potentially cause unwanted side effects. Combinations of drugs may prove to be the most productive means of treating patients with AD.178 Tetrahydroaminoacridine (generic name: Tacrine), one of the cholinesterase inhibitors, has beneficial effects on cognitive status in patients with AD. Tetrahydroaminoacridine, given together with highly concentrated lecithin (a source of choline from its key component phosphatidylcholine), appears to improve cognition and functional autonomy.203 Lecithin seems to have a small additional benefit independent of Tacrine.258 Thus, we propose that combination of drugs or drug with mt-nutrients may prove to be a more effective strategy for AD treatment. LA may be more active and effective when combined with CoQ because DHLA (the reduced form of LA), which is soluble in the aqueous compartment, has been found to reduce CoQ to ubiquinol by the transfer of a pair of electrons, thereby increasing the antioxidant capacity in biomembranes.259 Packer et al.225 have suggested that LA can recycle most of the antioxidants, including CoQ, ascorbic acid, GSH, and thioredoxin. It may be useful to add LA to other antioxidants to recycle the oxidized antioxidants in vivo. ApoE4 is associated with sporadic and late-onset familial AD. Gene dose was shown to have an effect on the risk of developing AD, age of onset, accumulation of senile plaques in the brain, and reduction of choline acetyltransferase activity in the hippocampus of AD subjects.260 The human ApoE4 transgenic mouse shows cognitive dysfunction and is considered a good animal AD model.261 One mechanism underlying increased risk for AD due to the presence of apoE4 is that apoE4 is less effective in transporting cholesterol than apoE3 when neurons respond to neuronal stress, including β -amyloid toxicity, oxygen deficiency, oxidative damage, and head trauma, all of which require neuronal repair.262 LA has anticholesterol activity.263–265 ALCAR reduces age-associated increases in cholesterol in rat brain and blood.266,267 Therefore, both LA and ALCAR may slow AD progression by lowering cholesterol in apoE4 mice. In addition, the cholesterol-lowering drugs, such as Lovastatin, may lower tissue concentrations of CoQ, and supplementation with CoQ can return CoQ level to normal.268 Therefore, ALCAR, LA, and CoQ seem a rational combination in reducing apoE4-associated cholesterol metabolic abnormalities. Few studies have examined the effects of combinations of mt-nutrients in PD. However, considering the synergistic or additive effects of these mt-nutrients in other diseases such as in AD (discussed above), more promising effects, compared with single compounds, might be obtained in PD prevention and treatment.

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Beal’s group has shown that in the Huntington’s model transgenic mice, ramacemide and CoQ showed additive effects on survival (from 15–20% to 33%), behavior and weight loss; and a combination of four different agents (a transglutaminase inhibitor, an NOS inhibitor, ramacemide, and CoQ) showed better protective effects with increases in survival up to 46%.66 A combination of mt-nutrients thus might work similarly better for PD prevention and amelioration than the combination of mt-nutrients, similar to the cocktail therapy currently favored in treating HIV/AIDS.269–271

4.9 CONCLUSIONS AND PERSPECTIVES Although further investigations are needed for studying the molecular and cellular rationale and the clinical effects of mitochondrial nutrient supplementations, increasing evidence has demonstrated that the mitochondrial nutrients, including the B vitamins, ascorbic acid, α-tocopherol, β -carotene, coenzyme Q, α-LA, N-acetylcystein, ALCAR, creatine, choline, DHA, and trace elements, have beneficial effects and show promising applications for delaying, preventing, and repairing mitochondrial decay, thus delaying aging and preventing/ameliorating age-related neurodegenerative diseases. Therefore, the prevention of neurodegenerative disease through dietary intervention, either by the recommendation of specific diets or by the use of dietary supplements, has great potential for improving human health.23,66,67,69,213,241,272–274 A metabolic tune-up with mt-nutrients for preventing and ameliorating neurodegenerative diseases is likely to have the most health benefits for those with inadequate diets, such as many of the poor and the elderly, who need improvement the most. The issues discussed here highlight the need to educate the public about the crucial importance of optimal nutrition and the potential benefits of something as simple and affordable as a daily multivitamin/mineral supplement. A variety of PD models, both in vitro and in vivo, have been developed recently166–169 in addition to the well-known MPTP, paraquat, 6-OHDA, reserpine, and methamphetamine-induced acute animal models. Similarly, in vitro and in vivo AD models275,276 have been developed, such as APP transgenic mice,277 ApoE transgenic mice,278,279 Caenorhabditis elegans,280 tauopathy Drosophila,281 and also APP⫹ApoE mice.282 All of these models, especially the cell systems and small animals (Drosophila and nematode), have made the highly complicated studies with combinations of several mt-nutrients easier and more feasible, because more nutrients and their combinations can be screened in cell systems of Drosophila and nematode models than in mice and humans. The promising indications of success with individual mt-nutrients and combinations gives hope that combination therapies may have an important future role.

ACKNOWLEDGMENTS This chapter is adapted from a review published in Nutritional Neuroscience (Liu J and Ames BN, 2005, page 67–89). We are grateful for the helpful comments

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of Elizabeth Head at the Institute for Brain Aging and Dementia, Department of Neurology, University of California at Irvine; Lester Packer at the Department of Molecular Pharmacology and Toxicology, University of Southern California; Joyce McCann and Hani Atamna at the Children’s Hospital Oakland Research Institute; and editorial assistance from Teresa Klask and Ludmila Voloboueva. This study was supported by The National Center for Minority Health and Health Disparities Grant P60 MD00222 (B.N.A.), The National Center for Complementary and Alternative Medicine Research Scientist Award K05 AT001323 (B.N.A) and grant R21 AT001918 (B.N.A. and J.L.), and the National Institute of Aging grant AG02326501 (B.N.A and J.L.).

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Oxygen and 5 Reactive Nitrogen Species: Weapons of Neuronal Destruction Joseph R. Mazzulli, Summer Lind, and Harry Ischiropoulos The University of Pennsylvania Philadelphia, Pennsylvania

CONTENTS 5.1 5.2 5.3

Introduction..............................................................................................107 Acute Neural Injury .................................................................................108 Chronic Neurodegenerative Diseases ......................................................109 5.3.1 Alzheimer’s Disease ....................................................................109 5.3.2 Parkinson’s Disease .....................................................................110 5.4 Concluding Remarks................................................................................113 References .........................................................................................................113

5.1 INTRODUCTION Recent advancements in understanding the biochemical actions of reactive oxygen and nitrogen species (RONS) have identified many biological targets that reliably serve as indices of oxidative and nitrative stress. Oxidative stress occurs when uncontrolled and excessive amounts of oxidants are produced, resulting in the formation of oxidized macromolecules, whereas nitrative stress occurs when excessive reactive nitrogen species are produced resulting in the formation of nitrated macromolecules. The most frequently detected oxidant-modified biological targets include: 1) the polyunsaturated fatty acid oxidation products, isoprostanes and the electrophiles 4-hydroxy-2(E)nonenal (4-HNE) and malondialdehyde (MDA), 2) 8-hydroxy-2⬘-deoxyguanosine, the most studied product of DNA oxidation, and 3) oxidatively modified proteins including advanced glycation endproducts (AGEs) and proteins with reactive carbonyl side groups. Modification by 107

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reactive nitrogen species (reactive species derived from nitric oxide (NO) or metabolites of NO such as dinitrogen trioxide, nitrous acid, nitrogen dioxide, and peroxynitrite) lead to the formation of nitrated macromolecules by the addition of a nitro(–NO2) group. Biological targets modified by nitration include: 1) tyrosine and tryptophan residues in proteins, 2) polyunsaturated fatty acids, 3) DNA bases, and 4) sugars. All of these oxidative modifications can be readily detected in biological samples, providing molecular footprints to track the generation and actions of RONS. Moreover, the identification and quantification of these biomarkers may provide a reliable assessment of disease risk and severity. Despite the plethora of molecular markers available, detection of these processes alone does not directly demonstrate a causal relationship between RONS and disease mechanisms. The use of in vivo experimental models including knock-out and transgenic mice has proven valuable in ascertaining a causal role for RONS in pathological mechanisms that lead to neurodegeneration. Additional supporting evidence is also derived by in vitro analysis that has revealed specific consequences of oxidant-induced modifications on protein structure and function at a more basic level. In this chapter, associations, causes, and consequences of oxidative and nitrative damage in both acute neural injury and chronic neurodegenerative disorders will be discussed.

5.2 ACUTE NEURAL INJURY A substantial amount of evidence linking oxidative and nitrative damage to acute neural injury comes from a combination of pharmacological and genetic manipulations of enzymes involved in the production or removal of RONS. For example, overexpression of mitochondrial superoxide dismutase (SOD2), an enzyme that converts superoxide radical into hydrogen peroxide, results in protective effects in mice after ischemic brain injury, shown by decreased neuronal death, protein tyrosine nitration, and lipid peroxidation products.1 The importance of SOD2 has been confirmed through knock-out mice, which die within 2 to 3 weeks as a result of cardiac failure and neurodegeneration of the basal ganglia and brainstem, presumably due to mitochondrial oxidative damage.2,3 Manipulation of the expression levels of the cytosolic form of SOD (Cu, Zn SOD1) has shown similar results in cell culture and ischemic mouse models.4,5 These studies have been corroborated by pharmacological augmentation of neuronal antioxidant capacity. For example, administration of the SOD mimetic M40401 prior to hypoxic-ischemic challenge results in a drastic decrease in brain lesion volume and in improved cognitive function in neonatal rats.6,7 Flavonoid polyphenols present in plants, such as epigallocatechin gallate, also have the ability to protect neurons against ischemic damage through putative antioxidant properties and other cellular signaling events.8,9 Vitamin E has been shown to attenuate brain lipid peroxidation and to improve cognitive function following traumatic brain injury in a mouse model of Alzheimer’s disease (AD).10 These studies provide evidence that RONS are produced in the central nervous system and are capable of causing damage resulting in neuronal cell death.

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Additional evidence supporting the involvement of reactive nitrogen species in stroke and acute glutamate neurotoxicity is derived from studies employing mice deficient in neuronal nitric oxide synthase (nNOS). For example, nNOS ⫺/⫺ mice-derived cortical cells exposed to the toxic stimulus N-methyl-D-aspartate (NMDA) are protected from cell death.11 Sensitivity to NMDA toxicity can be restored by the addition of NO donors, underscoring a key role of NO or NO metabolites in the initiation of this process.11 Other studies using nNOS ⫺/⫺ mice have shown partial resistance to ischemia revealed by decreased infarct size after 24 to 72 h of middle cerebral artery occlusion.12 Selectivity of nitrative damage has been shown in similar ischemic models, demonstrated by increases in tyrosine-nitrated protein immunoreactivity only in brain regions damaged by the occlusion, suggesting that the likely culprits responsible for neuronal cell death are NO-derived reactive nitrogen species.13 Furthermore, in a mouse model of head injury it was found that injury induces the upregulation of the inducible isoform of nitric oxide synthase (NOS) accompanied by a localized increase in tyrosine-nitrated proteins.14 Overall, inappropriate or untimely production of NO by either the neuronal or the inducible isoforms of NOS result in the formation of reactive nitrogen species. These species induce modification of proteins and other macromolecules that could induce neuronal dysfunction and ultimately cell death.

5.3 CHRONIC NEURODEGENERATIVE DISEASES 5.3.1 ALZHEIMER’S DISEASE Alzheimer’s disease (AD) is characterized by progressive brain neurodegeneration associated with deteriorations in executive, cognitive, and behavioral functions. Pathologically, AD brains contain proteinaceous lesions in certain susceptible regions consisting of extracellular amyloid plaques made of aggregated amyloid beta (A β) peptides and intracellular neurofibrillary tangles composed of aggregated tau, a microtubule-associated protein. A major risk factor in the development of AD is age, as prevalence can reach 30% of the population over 80 years of age.15 Owing to this association, it is hypothesized that in some cases of AD, a pathogenic phenotype arises gradually over many years in individuals with compromised antioxidant defenses or repair mechanisms, causing a slow buildup of toxic oxidized and nitrated biomolecules. Many therapies for AD have therefore focused on restoration or augmentation of antioxidant function in an effort to delay the onset of disease. This section will outline the association of oxidative damage with AD and review existing evidence supporting a causal role of RONS in disease processes. The use of molecular markers has established an association of oxidative stress with AD pathology. Increased detection of protein carbonyls within specific brain areas more prominently affected by the disease such as the hippocampus, have been reported.16 AGE-modified proteins have been identified in neurofibrillary tangles17 and in isolated tau aggregates called paired helical filaments (PHF-tau).18

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Furthermore, an AGE modification of tau occurs in the tubulin-binding domain resulting in reduced binding to tubulin in vitro.18,19 This presents a possible in vivo situation, where decreased microtubule interactions increase the pool of unbound tau, thus increasing the possibility of forming PHF-tau. Other in vitro studies have demonstrated that AGE-modified tau aggregates more rapidly than unmodified tau18 suggesting a close association of oxidative damage and formation of neurofibrillary tangles. Lipid peroxidation has been shown in AD brains by the detection of elevated 4-HNE in ventricular fluid20 and by histological detection in neurofibrillary tangles and amyloid plaques.21,22 In vitro studies have shown that addition of 4-HNE can induce aggregation of phosphorylated tau.23 Elevated levels of isoprostanes, another index of in vivo lipid peroxidation,24 have been shown in postmortem AD brains.25 In addition, analysis of a transgenic mouse model of AD has revealed that lipid peroxidation occurs prior to the formation of Aβ aggregates, implying that oxidative events occur early in the disease process.26 Detection of protein tyrosine nitration (either by immunohistochemistry using anti-nitrotyrosine antibodies or by analytical methodologies) appears to be one of the earliest markers of oxidative stress in AD brains.27,28 This association has been shown by postmortem analysis, demonstrating that nitrated tau is a component of neurofibrillary tangles and of isolated PHF-tau.29 Cell culture studies have shown that tau is a selective target for nitration upon exposure to peroxynitrite.29 Additional experiments using purified recombinant tau protein have shown that peroxynitrite and H2O2 can induce tau aggregation through disulfide linkages, resulting in decreased microtubule stability.30 Further evidence substantiating a role of oxidative and nitrative stress in AD is demonstrated by manipulation of antioxidant defense mechanisms such as reactive species scavengers and metal chelators. For example, administration of a copper/zinc chelator (iodochlorhydroxyquin, or clioquinol) was shown to effectively delay the onset of Aβ pathology observed in a mouse model of AD.31 Cell culture studies have supported a role for redox copper in AD, demonstrating a potentiation of Aβ -induced toxicity by the addition of copper to the media.32 Other antioxidants such as α-tocopherol have been found to alter neurodegenerative processes in both mouse models and human cases of neurodegeneration. Mice deficient in the α-tocopherol transport protein develop delayed-onset ataxia,33 and administration of large doses of vitamin E (2000 IU/day) has shown to delay the progression of AD in humans.34 Tau pathology in a mouse model of AD can also be delayed by the administration of α-tocopherol35 demonstrating that oxidative processes may have a causal role in chronic progression of AD.

5.3.2 PARKINSON’S DISEASE Parkinson’s disease (PD) is a motor disorder characterized clinically by resting tremor, cogwheel rigidity, and postural instability. Pathological examination of PD brains shows an extensive loss of dopaminergic neurons of the nigrostriatal pathway accompanied by the presence of intracellular proteinaceous inclusions known

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as Lewy bodies, primarily found in the remaining neurons of the substatia nigra pars compacta. Although Lewy bodies contain a plethora of proteins, a major component is the protein α-synuclein.36,37 This was discovered subsequent to genetic analysis, which revealed an autosomal dominant mutation in the α-synuclein gene, resulting in conversion of alanine 53 to threonine (A53T).38 Other genetic analysis revealed a mutation converting alanine 30 to proline (A30P),39 demonstrating a clear link between α-synuclein and PD. Although the association of α-synuclein in PD pathology is apparent, the exact pathological mechanisms have not been delineated. This is in part due to the unclear physiological function of α-synuclein. However, several putative functions have been proposed that may explain the role of α-synuclein in PD pathogenesis. Studies utilizing α-synuclein knock-out mice have shown deficits in neurotransmitter release in the nigrostriatal pathway and reserve pool depletion in hippocampal cultures.40,41 Other possible physiological functions include negative regulation of phospholipase D2, an enzyme responsible for regulating vesicular transport,42 regulation of intracellular dopamine levels by inhibiting the rate-limiting synthesis enzyme tyrosine hydroxylase,43 and inhibition of the dopamine transporter.44 Sequence homology with 143-3 protein suggests it may function as a chaperone.45 Additionally, α-synuclein has been shown to inhibit aggregation of thermally or chemically denatured proteins, further supporting a role as a chaperone-like protein.46 In pathological conditions, α-synuclein is thought to mediate toxicity through a change in secondary structure to form β -sheet rich fibrils and insoluble aggregates. Owing to the presence of these fibrils in Lewy bodies of PD brain, the process of structure change is considered an important pathological event. Thus, major efforts have currently been devoted to understanding the process of α-synuclein aggregation in vitro. Treatments of α-synuclein by oxidizing agents such as redox active iron, copper, and hydrogen peroxide can promote aggregation,47,48 and it has recently been proposed that oxidative dimer formation through o-o⬘ dityrosine crosslinks is the critical step in fibril formation.49 Dopamine oxidation is also known to affect α-synuclein fibril formation in vitro by inhibiting the formation of mature fibrils while stabilizing an oligomeric intermediate termed the protofibril.50 This oxidative modification may have particular importance in the mechanisms of PD pathology, since dopamine neurons are predominantly affected in this disease. While the pathological consequence of a putative in vivo dopamine–α-synuclein interaction is not fully understood, it has been proposed that α-synuclein protofibrils may exhibit toxicity through the formation of pore-like structures that can permeate synthetic vesicles,51,52 while α-synuclein fibrils represent inert species that reflect secondary effects of the disease process. However, owing to the lack of in vivo evidence for a dopamine–α-synuclein interaction or a kinetically stable protofibril species, the process of fibril formation remains the more likely hypothesis to explain the neuronal cell death that occurs in PD. Nitrative modifications have also been implicated in the fibril forming process. Antibodies that preferentially recognize a nitrated form of α-synuclein have specifically localized nitrated α-synuclein within the lesions in human PD brain and other synucleinopathies, thus providing a potential direct link to pathogenesis.53 Nitrative

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modifications to tyrosine residues of α-synuclein have beenshown to promote stabilization of oligomeric species.54,55 Furthermore, addition of isolated nitrated monomers and dimers to unmodified α-synuclein has the ability to accelerate the fibril formation process.56 Nitrated α-synuclein exhibits decreased binding to phospholipids, which represents an alteration in putative physiological functions, and is degraded at a slower rate compared to unmodified α-synuclein.56 Both of these processes may result in an increase in the critical concentration of cytosolic α-synuclein, promoting aggregation conditions in vivo. Taken together, these studies suggest that modification of α-synuclein by tyrosine nitration may diminish putative physiological functions while aiding in the formation of fibrils. In addition to α-synuclein mediated toxicity, recent studies have shown that mitochondrial deficits leading to an increase in the production of reactive oxygen species may contribute to PD pathogenesis. This is best described by investigations utilizing environmental toxins that function as complex I inhibitors. The pesticides rotenone and paraquat, which inhibit the transfer of electrons by complex I, are both known to cause neuronal injury and can reproduce features of PD in rodents and primates.57–60 A well-established experimental animal model of PD utilizes the dopaminergic-selective neurotoxin, 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) which among other functions is a complex I inhibitor.61 MPTP-induced parkinsonism has been shown to reproduce many phenotypic and biochemical features of the naturally occurring human disease, including motor abnormalities and α-synuclein-positive proteinaceous inclusions.62,63 It is hypothesized that interruptions in the electron flow mediated by complex I in the respiratory chain contribute to the generation of RONS leading to dopaminergic cell death. Although the pathogenic mechanisms of MPTP are not fully understood, studies from mouse models have established a role of NO-derived reactive species in this process. For example, mice deficient in the neuronal or inducible isoforms of NOS are partially protected from MPTP-induced toxicity,64,65 and these studies have been supported by pharmacological inhibition of neuronal NOS by 7-nitroindazole.66,64 Tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis, is a selective target of reactive nitrogen species generated through MPTP exposure. Oxidation of cysteine residues67,68 or nitration of critical tyrosine residues in a loop above the catalytic cleft decrease the activity of the enzyme.69,70 Overall, these studies demonstrate that RONS are generated in human PD brains and in experimental models, providing a direct link to the pathogenesis of PD and other neurodegenerative disorders. Genetic factors are known to play a role in the pathogenesis of PD, and may act synergistically with other factors to produce mitochondrial dysfunction and oxidative stress. In addition to the disease-associated A30P and A53T α-synuclein mutations, genetic analysis has linked locus triplication and thus overexpression of α-synuclein to PD.71 Interestingly, overexpressing α-synuclein in cultured cells results in compromised mitochondrial function accompanied by oxidative stress.72 Mutations in PTEN-induced kinase 1 (PINK1), a putative mitochondrial protein kinase, have been implicated in PD, possibly linking aberrant phosphorylation to mitochondrial dysfunction.73 Other mutations affecting the

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cellular proteolytic pathway including the E3 ubiquitin ligase parkin74 and ubiquitin C-terminal hydrolase (UCH-L1) have been found,75 and may explain the accumulation of aggregated α-synuclein in Lewy bodies. Mice deficient in parkin display abnormal metabolism, reductions in mitochondrial cellular respiration, and elevated protein carbonyls and 4-HNE immunostaining, suggesting that parkin may also act to regulate mitochondrial function and oxidative stress.76 Additionally, mutations that reduce protein stability and function in DJ-1, a putative antioxidant protein, have been identified in a familial case of PD.77,78,79 Collectively, these data suggest that PD associated mutations may lead to increased steady-state levels of α-synuclein, altered mitochondrial function, and elevated oxidative stress.

5.4 CONCLUDING REMARKS Oxidative stress has been implicated in the physiological changes associated with aging as well as pathological mechanisms of chronic neurodegenerative disorders such as AD, PD, and acute neuronal injuries derived from stroke or trauma. Obtaining evidence for oxidative/nitrative damage in human pathogenesis relies primarily upon the detection of oxidized or nitrated biological targets of reactive species, such as lipids, nucleic acids, and proteins. Detection of these targets does not directly demonstrate a causal relationship between oxidative stress and disease. However, direct evidence can be derived using molecular or pharmacological manipulations in experimental models, which have suggested that reactive oxygen and nitrogen species are capable of initiating and propagating degenerative processes that occur in the central nervous system.

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48. Paik, S.R., Shin, H.J., and Lee, J.H. (2000) Metal-catalyzed oxidation of alphasynuclein in the presence of Copper(II) and hydrogen peroxide. Arch. Biochem. Biophys., 378:269–277. 49. Krishnan, S., Chi, E.Y., Wood, S.J., Kendrick, B.S., Li, C., Garzon-Rodriguez, W., Wypych, J., Randolph, T.W., Narhi, L.O., Biere, A.L., Citron, M., and Carpenter, J.F. (2003). Oxidative dimer formation is the critical rate-limiting step for Parkinson’s Disease alpha-Synuclein fibrillogenesis. Biochemistry, 42:829–837. 50. Conway, K.A., Rochet, J.C., Bieganski, R.M., and Lansbury, P.T. (2001). Kinetic stabilization of the alpha-synuclein protofibril by a dopamine–alpha-synuclein adduct. Science, 294:1346–1349. 51. Lashuel, H.A., Hartley, D., Petre, B.M., Walz, T., and Lansbury, P.T. (2002). Neurodegenerative disease: amyloid pores from pathogenic mutations. Nature, 418:291. 52. Volles, M.J., Lee, S.J., Rochet, J.C., Shtilerman, M.D., Ding, T.T., Kessler, J.C., and Lansbury, P.T. (2001) Vesicle permeabilization by protofibrillar alpha-synuclein: implications for the pathogenesis and treatment of Parkinson’s disease. Biochemistry, 40:7812–7819. 53. Giasson, B.I., Duda, J.E., Murray, I.V., Chen, Q., Souza, J.M., Hurtig, H.I., Ischiropoulos, H., Trojanowski, J.Q., and Lee, V.M.-Y. (2000b). Oxidative damage linked to neurodegeneration by selective alpha-synuclein nitration in synucleinopathy lesions. Science, 290:985–989. 54. Souza, J.M., Giasson, B.I., Chen, Q., Lee, V.M.-Y., and Ischiropoulos, H. (2000b). Dityrosine cross-linking promotes formation of stable alpha-synuclein polymers. Implication of nitrative and oxidative stress in the pathogenesis of neurodegenerative synucleinopathies. J. Biol. Chem., 275:18344–18349. 55. Norris, E.H., Giasson, B.I., Ischiropoulos, H., and Lee,V.M.-Y. (2003). Effects of oxidative and nitrative challenges on alpha-synuclein fibrillogenesis involve distinct mechanisms of protein modifications. J. Biol. Chem., 278: 27230–27240. 56. Hodara, R., Norris, E.H., Giasson, B.I., Mishizen-Eberz, A.J., Lynch, D.R., Lee, V.M., and Ischiropoulos, H. (2004) Functional consequences of alpha-synuclein tyrosine nitration: diminished binding to lipid vesicles and increased fibril formation. J. Biol. Chem., 279:47746–47753. 57. Manning-Bog, A.B., McCormack, A.L., Li, J., Uversky, V.N., Fink, A.L., and DiMonte, D.A. (2002) The herbicide paraquat causes up-regulation and aggregation of alpha-synuclein in mice: paraquat and alpha-synuclein. J. Biol. Chem., 277:1641–1644. 58. Betarbet, R., Sherer, T.B., MacKenzie, G., Garcia-Osuna, M., Panov, A.V., and Greenamyre, J.T. (2000). Chronic systemic pesticide exposure reproduces features of Parkinson’s disease. Nat. Neurosci., 3:1301–1306. 59. Sherer, T.B., Betarbet, R., Stout, A.K., Lund, S., Baptista, M., Panov, A.V., Cookson, M.R., and Greenamyre, J.T. (2002). An in vitro model of Parkinson’s disease: linking mitochondrial impairment to altered alpha-synuclein metabolism and oxidative damage. J. Neurosci., 22:7006–7015. 60. Lee, H.J., Shin, S.Y., Choi, C., Lee, Y.H., and Lee, S.J. (2002) Formation and removal of alpha-synuclein aggregates in cells exposed to mitochondrial inhibitors. J. Biol. Chem., 277:5411–5417. 61. Vila, M. and Przedborski, S. (2003) Targeting programmed cell death in neurodegenerative diseases. Nat. Rev. Neurosci., 4:365–375.

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62. Forno, L.S., Langston, J.W., DeLanney, L.E., and Irwin, I. (1988) An electron microscopic study of MPTP-induced inclusion bodies in an old monkey. Brain Res., 448:150–157. 63. Kowall, N.W., Hantraye, P., Brouillet, E., Beal, M.F., McKee, A.C., and Ferrante, R.J. (2000) MPTP induces alpha-synuclein aggregation in the substatia nigra of baboons. Neuroreport, 11:211–213. 64. Przedborski, S., Jackson-Lewis, V., Yokoyama, R., Shibata, T., Dawson, V.L., and Dawson, T.M. (1996) Role of neuronal nitric oxide in 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine (MPTP)-induced dopaminergic neurotoxicity. Proc. Natl. Acad. Sci. USA, 93:4565–4571. 65. Liberatore, G.T., Jackson-Lewis, V., Vukosavic, S., Mandir, A.S., Vila, M., McAuliffe, W.G., Dawson, V.L., Dawson, and T.M., Przedborski, S. (1999) Inducible nitric oxide synthase stimulates dopaminergic neurodegeneration in the MPTP model of Parkinson disease. Nat. Med., 5:1403–1409. 66. Schulz, J.B., Matthews, R.T., Muqit, M.M., Browne, S.E., and Beal, M.F. (1995) Inhibition of neuronal nitric oxide synthase by 7-nitroindazole protects against MPTP-induced neurotoxicity in mice. J. Neurochem., 64:936–939. 67. Kuhn, D.M., Aretha, C.W., and Geddes, T.J. (1999) Peroxynitrite inactivation of tyrosine hydroxylase: mediation by sulfhydryl oxidation, not tyrosine nitration. J. Neurosci., 19:10289–10294. 68. Kuhn, D.M., Sadidi, M., Liu, X., Kreipke, C., Geddes, T., Borges, C., and Watson, J.T. (2002) Peroxynitrite-induced nitration of tyrosine hydroxylase: identification of tyrosines 423, 428, and 432 as sites of modification by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and tyrosine-scanning mutagenesis. J. Biol. Chem., 277:14336–14342. 69. Ara, J., Przedborski, S., Naini, A.B., Jackson-Lewis, V., Trifiletti, R.R., Horwitz, J., and Ischiropoulos, H. (1998) Inactivation of tyrosine hydroxylase by nitration following exposure to peroxynitrite and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Proc. Natl. Acad. Sci. USA, 95:7659–7663. 70. Blanchard-Fillion, B., Souza, J.M., Friel, T., Jiang, G.C., Vrana, K., Sharov, V., Barron, L., Schoneich, C., Quijano, C., Alvarez, B., Radi, R., Przedborski, S., Fernando, G.S., Horwitz, J., and Ischiropoulos, H. (2001) Nitration and inactivation of tyrosine hydroxylase by peroxynitrite. J. Biol. Chem., 276:46017–46023. 71. Singleton, A.B., Farrer, M., Johnson, J., Singleton, A., Hague, S., Kachergus, J., Hulihan, M., Peuralinna, T., Dutra, A., Nussbaum, R., Lincoln, S., Crawley, A., Hanson, M., Maraganore, D., Adler, C., Cookson, M.R., Muenter, M., Baptista, M., Miller, D., Blancato, J., Hardy, J., and Gwinn-Hardy, K. (2003) alphaSynuclein locus triplication causes Parkinson’s disease. Science, 302:841. 72. Hsu, L.J., Sagara, Y., Arroyo, A., Rockenstein, E., Sisk, A., Mallory, M., Wong, J., Takenouchi, T., Hashimoto, M., and Masliah, E. (2000) alpha-synuclein promotes mitochondrial deficit and oxidative stress. Am. J. Pathol., 157:401–410. 73. Valente, E.M., Abou-Sleiman, P.M., Caputo, V., Muqit, M.M., Harvey, K., Gispert, S., Ali, Z., Del Turco, D., Bentivoglio, A.R., Healy, D.G., Albanese, A., Nussbaum, R., Gonzalez-Maldonado, R., Deller, T., Salvi, S., Cortelli, P., Gilks, W.P., Latchman, D.S., Harvey, R.J., Dallapiccola, B., Auburger, G., and Wood, N.W. (2004) Hereditary early-onset Parkinson’s disease caused by mutations in PINK1. Science, 304:1158–1160. 74. Kitada, T., Asakawa, S., Hattori, N., Matsumine, H., Yamamura, Y., Minoshima, S., Yokochi, M., Mizuno, Y., and Shimizu, N. (1998) Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism. Nature, 392:605–608.

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Amyloid-β and τ in 6 Alzheimer’s Disease: What is the Neuropathology Trying to Tell Us? Rudy J. Castellani

Michigan State University East Lansing, Michigan

Akihiko Nunomura

Asahikawa Medical College Asahikawa, Japan

Hyoung-gon Lee, Xiongwei Zhu, George Perry, and Mark A. Smith Case Western Reserve University Cleveland, Ohio

CONTENTS Abstract..............................................................................................................121 6.1 Introduction..............................................................................................122 6.2 Amyloid-β ................................................................................................122 6.3 Tau............................................................................................................124 6.4 Summary ..................................................................................................125 Acknowledgments .............................................................................................125 References .........................................................................................................126

ABSTRACT For nearly a century, the pathological hallmarks of Alzheimer’s disease (AD), namely, senile plaques and neurofibrillary tangles (NFT), have provided a reasonable basis to conclude that their major components, amyloid-β and tau, respectively, are central mediators of disease pathogenesis. Therefore, not surprisingly, efforts to 121

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understand disease mechanisms have concentrated on the biochemistry of amyloidβ deposition as senile plaques, or the phosphorylation and aggregation of tau as NFT. Unfortunately, it now appears that this focus on pathology as a central contributor to disease may be misguided. In fact, rather than being initiators of disease pathogenesis, recent evidence shows that the lesions not only occur consequent to oxidative stress but, importantly, that they may function as a primary line of antioxidant defense. In this paradigm, it is not surprising that the increased sensitivity to oxidative stress in the aged brain, even in control individuals, is marked by the appearance of both amyloid-β and tau. Moreover in AD, where chronic oxidative stress persists and is superimposed upon an age-related vulnerable environment, an increased lesion load is predictable. That amyloid-β and tau accumulations serve as age-related physiological reactions to chronic stress calls into question the rationale of current therapeutic efforts targeted toward lesion removal. Moreover, if this concept holds true for pathology in other neurodegenerative diseases, we may need to structure a paradigm shift before significant progress can be made in terms of therapeutic intervention.

6.1 INTRODUCTION Amyloid-β and tau protein are among the best studied proteins in all of neurobiology and figure centrally into much of the research dedicated to Alzheimer disease (AD). While this is not surprising since the pathological diagnosis of AD is dependent upon the quantity of amyloid-β and tau deposits within cortical gray matter,1,2 we suggest that this strict linkage of diagnostic and mechanistic views is misleading, particularly in the case of neurodegenerative disease. It is important to recognize that, unlike the pathological diagnosis of most other processes, the pathological diagnosis of AD brains merely represents an association of presumed pathological findings together with a given clinical disease. Therefore, since AD-type pathological changes in cognitively-intact elderly patients3 or, conversely, AD-like dementia in the absence of AD-type pathology is well known,4 the diagnosis of AD requires positive data for both clinical and pathological aspects. Nevertheless, since amyloid-β and tau are crucial proteins that are exploited for diagnostic purposes, this focus has led many to conclude that these same proteins, corresponding to stereotypical AD-type lesions, are signatures of an aberration that speaks directly to disease etiology. Unfortunately, it now appears that such an inductive leap is not warranted upon objective review of available data. Rather, we believe the mechanistic importance of senile plaques/amyloid-β and neurofibrillary tangles (NFT)/tau has far less to do with their consequences than with the factors that led to their formation. In this chapter, we present an alternative hypothesis for the role of amyloid-β and tau deposition in AD that may herald a dramatic shift in our view of neurodegenerative diseases.

6.2 AMYLOID-β The scientific literature as well as popular media accounts are replete with the fundamental concept that amyloid-β causes disease.5 Genetic data are often suggested

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as a priori evidence of this “fact” since, for example, amyloid β protein precursor (Aβ PP) mutations lead to familial, early-onset AD (autsomal-dominant), and patients with Down’s syndrome, who carry an extra copy of the Aβ PP gene, consistently develop AD changes with prolonged survival. Clinicopathological data may also be cited, as amyloid-β deposits are increased in the AD brain.6 On the other hand, AD kindreds with Aβ PP mutations are exceedingly rare and it remains to be determined whether these kindreds are only tangentially representative of sporadic AD.7 Indeed, it is notable that oxidative stress precedes amyloid-β deposition by decades in Down’s syndrome, sporadic AD, in familial AD.7–11 Similarly, the genetic aberration in Down’s syndrome clearly leads to a “cascade” of pathophysiology over and above amyloid-β deposits in sporadic AD.7 Moreover, the notion of amyloid-β deposits per se as primary neurotoxic lesions in AD may be called into question simply by the early appearance of oxidative stress sequelae relative to amyloid-β deposits in sporadic7,8,12 and genetic cases11 of disease. It is now known that neurons respond to oxidative stress by increasing amyloid-β production13 and that this increased amyloid-β is associated with a consequent reduction in oxidative stress.7,8 Similarly, we recently demonstrated that amyloid-β is a genuine antioxidant that can act as a potent superoxide dismutase.14 By this logic, therefore, AD kindreds with Aβ PP mutations lose effective antioxidant capacity (owing to mutation-driven protein dysfunction), while the extensive amyloid-β deposits themselves are signatures not of neurotoxicity per se but of oxidative imbalance and an oxidative stress response. This is consistent with the data that virtually everyone over the age of 40 exhibit detectable amyloid-β deposits,8 and is manifestly more logical than the alternative view that everyone at midlife is on the verge of developing AD, a view also directly contradicted by the fact that a large percentage of cognitively-intact, aged individuals contain amyloid-β loads equivalent to patients with AD.3 Fibrillar or aggregated forms of amyloid-β, such as in senile plaque cores, in a cell culture environment are toxic to cultured neurons in vitro.15,16 However, in vivo, the presence and density of amyloid-β correlates weakly with the onset and severity of AD,17,18 while recent data suggest that the presence of the soluble form of amyloid-β in the brain may be a better predictor of the disease.19 Specifically, sodium dodecyl sulfate (SDS)-stable oligomers, and not monomers, of this form of amyloid-β seem to play an important role, as shown by the augmented presence of these oligomers during the expression of mutations in Aβ PP or presenilin,20 as well as by their capacity to inhibit neuronal plasticity parameters long-term potentiation (LTP) in vivo when micro-injected into the brains of rodents.21 Conversely, amyloid-β is not always present in the brains of cognitively normal elderly individuals. Whether this indicates that some individuals have efficient endogenous antioxidant defense systems and thus age more effectively, or whether such individuals may have supplemented their diets with antioxidants throughout their lifespan, compensating for age-related declines in antioxidant defenses, remains to be elucidated.22–24 If amyloid-β deposition possesses antioxidant function, this process will be recruited during times when oxidative stress is high and the endogenous antioxidant defenses are compromised. On the other

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hand, if this system is efficient and is supported by exogenous antioxidant supplementation, the antioxidant effects of amyloid-β may not be necessary. Some stereological studies have suggested that there may be little or no neuronal loss during “normal” aging despite, as pointed out above, the presence of an increasing number of plaques.25 Interestingly, even the hyperphysiologic levels of amyloid-β in mouse models of AD26 only lead to senile plaque formation in middle-aged mice and, like their human counterparts, these mice show evidence of oxidative stress that precedes the amyloid-β deposits.27–30 Taken together, these findings indicate that amyloid-β is a consequence of the pathogenesis that serves an antioxidant function. The idea that amyloid-β is protective should not necessarily be surprising. Neuronal degeneration is associated with a number of responses including the induction of heat shock proteins31,32 that, like amyloid-β, show a relationship with cognitive decline. Yet only amyloid-β is considered pathogenic since amyloid-β is neurotoxic in vitro and is weakly associated with neuronal loss in vivo.17,18 On the other hand, as alluded to above, neurotoxicity in cultured cells may be an artifact of in vitro conditions,16 since neither isolated senile plaques nor immobilized amyloid-β elicit neurotoxity in vivo or in vitro.33–35 Thus, the capacity of amyloid-β to induce oxidative stress remains controversial36 but may be akin to the known pro-oxidant effect of all antioxidants, which is dependent on environmental conditions. The few reports demonstrating neuronal loss in some transgenic AD models 37 argue that amyloid-β is a bioactive substance, but do not provide a compelling analogy to sporadic AD in humans. In addition, there is little evidence demonstrating behavioral deficits in mice transgenic for only Aβ PP mutations, while the most consistent deficits have been shown in mice transgenic for more than one mutation, e.g., Aβ PP/PS,138,39 superimposed upon an aged environment.

6.3 TAU Tau accumulation in the form of neurofibrillary pathology may also represent oxidative imbalance.8 According to recent studies, quantitative analysis of the extent of oxidative damage is reduced in those neurons with the most cytopathology.8 Further studies suggest that most neuronal loss in AD occurs prior to NFT deposition,40,41 a period associated with high levels of oxidative stress, while subsequent deposition of NFT decreases these levels.10 The physiological modification of tau and neurofilament proteins by lipid peroxidation products and carbonyls is consistent with this view.42,43 Indeed, oxidative stress and attendant modification of tau by-products of oxidative stress including 4-hydroxy-2-nonenal (HNE)44 and other cytotoxic carbonyls,45 enables such neurons to survive for decades.46 Interestingly, although tau and neurofilaments, being cytoskeletal proteins, have a long half-life, the extent of carbonyl modification is comparable throughout the aging spectrum, as well as along the length of the axon.47 A logical explanation for this finding is that the oxidative modification of cytoskeletal proteins is under tight regulation.

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A high content of lysine-serine-proline (KSP) domains on both tau and neurofilament protein suggests that they are uniquely adapted to oxidative attack. Exposure of these domains on the protein surface is effected by extensive phosphorylation of serine residues, resulting in an oxidative “sponge” of surfacemodifiable lysine residues.47 Since phosphorylation plays this pivotal role in redox balance, it is neither surprising that oxidative stress, through activation of MAP kinase pathways, leads to phosphorylation,48–50 nor that conditions associated with chronic oxidant stress, such as AD, are associated with extensive phosphorylation of cytoskeletal elements. Indeed, other neurological conditions where phosphorylated tau and neurofilament protein accumulations occur also show evidence of oxidative adducts, e.g., progressive supranuclear palsy,51 corticobasal degeneration,52 and frontal temporal dementia.53 Given this protective role of tau phosphorylation, it is not surprising that embryonic neurons that survive treatment with oxidants have more phospho-tau immunoreactivity relative to those that die.54 Further, since heme oxygenase induction and tau expression are opposing,44,55 reduced oxidative damage in neurons with tau accumulation may be a part of the antioxidant function of phosphorylated tau. The concept that intracellular inclusions are manifestations of cell survival has recently found support in a Huntington’s disease model.56 In this neuronal model, cell death was mutant-huntingtin dose and polyglutamine-dependent; however, huntingtin inclusion formation correlated with cell survival. Thus, in this model, as in AD, inclusion formation represents adaptation, or a productive, beneficial response to the otherwise neurodegenerative process. Taken together with our studies, this represents a fundamental and necessary change in which pathological manifestations of neurodegenerative disease are interpreted.

6.4 SUMMARY The long-held notion that pathological lesions in neurodegenerative diseases provide direct insight into etiology may be a fundamental misconception. The observed decrease in oxidative damage with amyloid-β and tau accumulation suggests, rather, that senile plaques and neurofibrillary pathology are empirical manifestations of cellular adaptation. Efforts aimed solely at eliminating amyloidβ or tau may therefore be directed against a biochemical process that is more physiological than pathological and therefore unlikely to produce the desired results. We further suggest that the classical notion of neurodegenerative disease pathology as signifying disease per se be reorganized into a modern framework that recognizes the difference between cause and effect.57 Only through such an effort will the greatest potential for continued diagnostic and therapeutic advances be realized.

ACKNOWLEDGMENTS Work in the authors’ laboratories is supported by the John Douglas French Alzheimer’s Foundation.

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30. Drake J, Link CD, Butterfield DA. Oxidative stress precedes fibrillar deposition of Alzheimer’s disease amyloid beta-peptide (1-42) in a transgenic Caenorhabditis elegans model. Neurobiol Aging, 2003; 24:415–420. 31. Anthony SG, Schipper HM, Tavares R, Hovanesian V, Cortez SC, Stopa EG, Johanson CE. Stress protein expression in the Alzheimer-diseased choroid plexus. J Alzheimers Dis, 2003; 5:171–177. 32. Smith MA, Kutty RK, Richey PL, Yan SD, Stern D, Chader GJ, Wiggert B, Petersen RB, Perry G. Heme oxygenase-1 is associated with the neurofibrillary pathology of Alzheimer’s disease. Am J Pathol, 1994; 145:42–47. 33. Frautschy SA, Cole GM, Baird A. Phagocytosis and deposition of vascular betaamyloid in rat brains injected with Alzheimer beta-amyloid. Am J Pathol, 1992; 140:1389–1399. 34. Canning DR, McKeon RJ, DeWitt DA, Perry G, Wujek JR, Frederickson RC, Silver J. beta-Amyloid of Alzheimer’s disease induces reactive gliosis that inhibits axonal outgrowth. Exp Neurol, 1993; 124:289–298. 35. DeWitt DA, Perry G, Cohen M, Doller C, Silver J. Astrocytes regulate microglial phagocytosis of senile plaque cores of Alzheimer’s disease. Exp Neurol, 1998; 149:329–340. 36. Walter MF, Mason PE, Mason RP. Alzheimer’s disease amyloid beta peptide 25–35 inhibits lipid peroxidation as a result of its membrane interactions. Biochem Biophys Res Commun, 1997; 233:760–764. 37. Calhoun ME, Wiederhold KH, Abramowski D, Phinney AL, Probst A, SturchlerPierrat C, Staufenbiel M, Sommer B, Jucker M. Neuron loss in APP transgenic mice. Nature, 1998; 395:755–756. 38. Joseph J, Shukitt-Hale B, Denisova NA, Martin A, Perry G, Smith MA. Copernicus revisited: amyloid beta in Alzheimer’s disease. Neurobiol Aging, 2001; 22:131–146. 39. Gordon MN, King DL, Diamond DM, Jantzen PT, Boyett KV, Hope CE, Hatcher JM, DiCarlo G, Gottschall WP, Morgan D, Arendash GW. Correlation between cognitive deficits and Abeta deposits in transgenic APP+PS1 mice. Neurobiol Aging, 2001; 22:377–385. 40. Gomez-Isla T, Hollister R, West H, Mui S, Growdon JH, Petersen RC, Parisi JE, Hyman BT. Neuronal loss correlates with but exceeds neurofibrillary tangles in Alzheimer’s disease. Ann Neurol, 1997; 41:17–24. 41. Kril JJ, Patel S, Harding AJ, Halliday GM. Neuron loss from the hippocampus of Alzheimer’s disease exceeds extracellular neurofibrillary tangle formation. Acta Neuropathol (Berl), 2002; 103:370–376. 42. Smith MA, Rudnicka-Nawrot M, Richey PL, Praprotnik D, Mulvihill P, Miller CA, Sayre LM, Perry G. Carbonyl-related posttranslational modification of neurofilament protein in the neurofibrillary pathology of Alzheimer’s disease. J Neurochem, 1995; 64:2660–2666. 43. Sayre LM, Zelasko DA, Harris PL, Perry G, Salomon RG, Smith MA. 4-Hydroxynonenal-derived advanced lipid peroxidation end products are increased in Alzheimer’s disease. J Neurochem, 1997; 68:2092–2097. 44. Takeda A, Smith MA, Avila J, Nunomura A, Siedlak SL, Zhu X, Perry G, Sayre LM. In Alzheimer’s disease, heme oxygenase is coincident with Alz50, an epitope of tau induced by 4-hydroxy-2-nonenal modification. J Neurochem, 2000; 75:1234–1241. 45. Calingasan NY, Uchida K, Gibson GE. Protein-bound acrolein: a novel marker of oxidative stress in Alzheimer’s disease. J Neurochem, 1999; 72:751–756.

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46. Morsch R, Simon W, Coleman PD. Neurons may live for decades with neurofibrillary tangles. J Neuropathol Exp Neurol, 1999; 58:188–197. 47. Wataya T, Nunomura A, Smith MA, Siedlak SL, Harris PL, Shimohama S, Szweda LI, Kaminski MA, Avila J, Price DL, Cleveland DW, Sayre LM, Perry G. High molecular weight neurofilament proteins are physiological substrates of adduction by the lipid peroxidation product hydroxynonenal. J Biol Chem, 2002; 277:4644–4648. 48. Zhu X, Rottkamp CA, Boux H, Takeda A, Perry G, Smith MA. Activation of p38 kinase links tau phosphorylation, oxidative stress, and cell cycle-related events in Alzheimer disease. J Neuropathol Exp Neurol, 2000; 59:880–888. 49. Zhu X, Castellani RJ, Takeda A, Nunomura A, Atwood CS, Perry G, Smith MA. Differential activation of neuronal ERK, JNK/SAPK and p38 in Alzheimer disease: the ‘two hit’ hypothesis. Mech Ageing Dev, 2001; 123:39–46. 50. Zhu X, Raina AK, Rottkamp CA, Aliev G, Perry G, Boux H, Smith MA. Activation and redistribution of c-jun N-terminal kinase/stress activated protein kinase in degenerating neurons in Alzheimer’s disease. J Neurochem, 2001; 76:435–441. 51. Odetti P, Garibaldi S, Norese R, Angelini G, Marinelli L, Valentini S, Menini S, Traverso N, Zaccheo D, Siedlak S, Perry G, Smith MA, Tabaton M. Lipoperoxidation is selectively involved in progressive supranuclear palsy. J Neuropathol Exp Neurol, 2000; 59:393–397. 52. Castellani R, Smith MA, Richey PL, Kalaria R, Gambetti P, Perry G. Evidence for oxidative stress in Pick disease and corticobasal degeneration. Brain Res, 1995; 696:268–271. 53. Gerst JL, Siedlak SL, Nunomura A, Castellani R, Perry G, Smith MA. Role of oxidative stress in frontotemporal dementia. Dement Geriatr Cogn Disord, 1999; 10 (Suppl 1):85–87. 54. Ekinci FJ, Shea TB. Phosphorylation of tau alters its association with the plasma membrane. Cell Mol Neurobiol, 2000; 20:497–508. 55. Takeda A, Perry G, Abraham NG, Dwyer BE, Kutty RK, Laitinen JT, Petersen RB, Smith MA. Overexpression of heme oxygenase in neuronal cells, the possible interaction with Tau. J Biol Chem, 2000; 275:5395–5399. 56. Arrasate M, Mitra S, Schweitzer ES, Segal MR, Finkbeiner S. Inclusion body formation reduces levels of mutant huntingtin and the risk of neuronal death. Nature, 2004; 431:805–810. 57. Lee HG, Petersen RB, Zhu X, Honda K, Aliev G, Smith MA, Perry G. Will preventing protein aggregates live up to its promise as prophylaxis against neurodegenerative diseases? Brain Pathol, 2003; 13:630–638.

Therapies in 7 Antioxidant the Prevention and Treatment of Alzheimer Disease Paula I. Moreira, Xiongwei Zhu, Mark A. Smith, and George Perry Case Western Reserve University Cleveland, Ohio

Akihiko Nunomura

Asahikawa Medical College Asahikawa, Japan

CONTENTS Abstract..............................................................................................................132 7.1 Introduction..............................................................................................132 7.2 Oxidative Stress .......................................................................................132 7.3 Mitochondria: Source And Target of Oxidative Stress............................133 7.4 Antioxidant Therapies..............................................................................135 7.5 Metabolic Antioxidants............................................................................135 7.6 Direct Antioxidants ..................................................................................137 7.7 Indirect Antioxidants................................................................................139 7.8 Conclusion ...............................................................................................141 Acknowledgments .............................................................................................141 References .........................................................................................................141

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ABSTRACT Oxidative stress is a key factor involved in the development and progression of Alzheimer disease (AD), and it is well documented that free-radical oxidative damage is extensive in the brains of AD patients. Consequently, antioxidants that prevent the detrimental consequences of oxidative stress are considered to offer a promising approach to neuroprotection and clinical benefits. Epidemiological and clinical studies present the benefits of increased antioxidant status for reduction of onset and controversial results of intervention studies. Antioxidants constitute a major part of the panel of clinical and experimental drugs that are currently considered for AD prevention and therapy. This chapter focuses mainly on three distinct classes of antioxidants: metabolic (α-lipoic acid, acetyl-L-carnitine, and idebenone), direct (vitamin E, estrogen, flavonoids, and terpenoids), and indirect (desferrioxamine and clioquinol).

7.1 INTRODUCTION Alzheimer disease (AD) is a progressive neurodegenerative disorder affecting the elderly and is characterized by a slow insidious decline in learning ability, memory, and behavior, with a deadly outcome. The prevalence of the disease is expected to quadruple by the year 2047 owing to increased survival of the aged to other causes of death.1 The disorder is a growing public health concern with potentially devastating effects. There are no known cures or preventions for AD; however, delaying its onset by only 5 years would decrease its prevalence by halting consequent burden on families and the public health systems.1 Early clinical diagnosis of AD is currently a major topic of investigation. Classification is mainly dependent on measures of global and cognitive function that reveal a condition, termed mild cognitive impairment (MCI), that does not always resolve benign senile forgetfulness from early AD. Autopsies of cases of AD show the histological features characterized by Alois Alzheimer2 in the brain cortex, extracellular deposits of amyloid-β (Aβ ) plaques, and intraneuronal neurofibrillary tangles (NFT). Another constant observation in postmortem AD brain is the loss of cholinergic markers such as acetylcholine and choline acetyltransferase. Furthermore, it has also been demonstrated that in AD, the brain shows signs of increased generation of reactive oxygen species (ROS) and oxidative stress. Accumulating evidence indicates that oxidative stress occurs prior to the onset of symptoms in AD, and that such oxidative changes are pervasive throughout the body, detectable peripherally as well as in association with vulnerable regions of the brain affected in disease (for a review, see [3,4]). If oxidative damage is a key player in the initiation and progression of AD, then antioxidant therapies envisaging the reduction of oxidative damage and the increase of endogenous antioxidant defenses should prevent, delay, or ameliorate the disease symptoms.

7.2 OXIDATIVE STRESS Oxidative stress is highly relevant in the brain5 for several reasons since the brain (1) is a postmitotic tissue with a high-energy demand; (2) is exposed to high oxygen

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concentrations, utilizing about one fifth of basal oxygen consumption; (3) contains relatively poor concentrations of the classic antioxidants and related enzymes; (4) is rich in polyunsaturated fatty acids, which are prone to oxidation; and (5) is enriched in iron, which accumulates in the brain as a function of age and can be a potent catalyst for oxidative species formation. It is widely considered that the major portion (about 95–98%) of the total ROS produced during aerobic metabolism comes as a by-product of the electron-transport chain of mitochondria.6 Under normal conditions, cells are capable of counteracting the oxidant insults by regulating their homeostatic balance. However, during the progression of neurodegenerative conditions, the capacity of cells to maintain redox balance puts major demands on cellular defenses and leads to the accumulation of oxidized macromolecules, mitochondrial dysfunction, and neuronal injury. Excessive lipid peroxidation, protein oxidation, DNA and RNA oxidation, and glycooxidation have all been documented in AD.7

7.3 MITOCHONDRIA: SOURCE AND TARGET OF OXIDATIVE STRESS Extensive evidence indicates that cerebral metabolism is reduced in AD,8 making mitochondria the key players in this scenario.9 Mitochondria are essential organelles for neuronal function because the limited glycolytic capacity of these cells makes them highly dependent on aerobic oxidative phosphorylation for their demanding energy needs. However, oxidative phosphorylation is a major source of endogenous, toxic-free radicals, including hydrogen peroxide (H2O2), hydroxyl radical (•OH), and superoxide (O2⫺•), which are products of normal cellular respiration10 (Figure 7.1). With inhibition of the electron-transport chain, electrons accumulate in complex I and coenzyme Q, where they can be donated directly to molecular oxygen to give O⫺2 •, which can be detoxified by the mitochondrial manganese superoxide dismutase (MnSOD) to give H2O2 which, in turn, can be converted into H2O by glutathione peroxidase (GPx) or catalase. However, O⫺2 • in the presence of nitric oxide (NO•) formed during the conversion of arginine into citrulline by nitric oxide synthase (NOS) can yield peroxynitrite (ONOO⫺). Furthermore, H2O2 in the presence of reduced transition metals can be converted to toxic •OH via Fenton or Haber–Weiss reactions, a process that we have specifically localized to neurofibrillary pathology and cytosol in AD11,12 (Figure 7.1). Inevitably, if the amount of free radical species overwhelms the capacity of neurons to counteract these harmful species or leads to major homeostatic responds compromising cell viability, oxidative stress occurs, followed by mitochondrial dysfunction and neuronal damage. Reactive species generated by mitochondria have several cellular targets including mitochondrial components themselves (lipids, proteins, and DNA/RNA) (Figure 7.1). The lack of histones in mitochondrial DNA (mtDNA) and diminished capacity for DNA repair render mitochondria an easy target of oxidative stress events. By releasing high levels of H2O2, dysfunctional mitochondria propagate a series of interactions between redox metals and oxidative response elements.13 The role of mitochondria in AD was studied in our laboratory using in situ hybridization to mtDNA, immunocytochemistry of cytochrome oxidase, and morphometry of electron micrographs of biopsy specimens to determine

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O2– • MnSOD

NO• ONOO–

•OH Haber–Weiss or Fenton reaction(s)

H2O2

Free radicals Targets: O2

Lipids Proteins DNA/RNA

ATP H+

FIGURE 7.1 Besides the fundamental role of mitochondria in the generation of energy (ATP), these organelles are also the main producers of reactive species. If the defense mechanisms are debilitated, these reactive species initiate a cascade of deleterious events within the cell. •OH, hydroxyl radical; H2O2, hydrogen peroxide; MnSOD, manganese ⫺ superoxide dismutase; O⫺ 2 •, superoxide; ONOO , peroxynitrite.

whether there were mitochondrial abnormalities in AD.9,14 We found that the neurons showing increased oxidative damage in AD also possess a striking and significant increase in mtDNA and cytochrome oxidase. Surprisingly, much of the mtDNA and cytochrome oxidase is found in the neuronal cytoplasm and in the case of mtDNA, the autophagic vacuoles associated with lipofuscin, whereas morphometric analysis showed that mitochondria are significantly reduced in AD. We also observed a major overall reduction in microtubules in AD compared with controls.15 Altogether, these data indicate that the abnormal mitochondrial turnover, as indicated by increased perikaryal mtDNA and mitochondrial protein accumulation in the face of reduced numbers of mitochondria, could be due to a defective microtubule metabolism resulting in deficient mitochondrial transport in the axon. This may, in turn, set up a pathological cascade of events in the perikaryal mitochondria. Accumulating evidence indicates that heme-oxygenase-1 (HO-1) is induced in AD brains.16,17 HO-1 catalyzes the conversion of heme to biliverdin and iron. Biliverdin, in turn, is reduced to bilirubin, an antioxidant. Since HO-1 is induced in proportion to the level of heme,18 the induction of HO-1 suggests that there may be an abnormal increase in turnover of heme in AD. This idea is consistent with the mitochondrial abnormalities associated with AD, since it is well known that many heme-containing enzymes are found in mitochondria. As previously discussed, our ultrastructural studies concerning mitochondria suggest a high rate of mitochondrial turnover and redox activity in the residual body of lipofuscin.9 In turn, the increase in heme induces synthesis of more HO-1, suggesting that mitochondrial turnover promotes oxidative stress via increase of redox-active iron.19

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Furthermore, we observed that oxidized nucleic acids are commonly observed in the cytoplasm of the neurons that are particularly vulnerable to degeneration in AD.20 8-hydroxyguanosine(8OHG), a marker of nucleic acid oxidation, is likely to form at the site of •OH production, a process dependent on redox-active metal-catalyzed reduction of H2O2 together with cellular reductants such as ascorbate or O⫺ 2 . Interestingly, the levels of 8OHG are inversely related to the extent of Aβ deposits inside and outside neurons ([20] and unpublished) suggesting a complex interplay between Aβ and redox metal activity that may be critical to metal dynamics within the neuronal cytoplasm. A possible key element of these dynamics is mitochondria in the neuronal cell body.

7.4 ANTIOXIDANT THERAPIES The role of oxidative stress in the etiology of AD has been hypothesized, described, and supported by a variety of experimental and clinical studies (for a review, see [3,21]). The possibility that oxidative stress is a primary event in AD has led to research exploring how antioxidants in foods and supplements can prevent and delay onset of AD. There are various types of antioxidant compounds: direct, indirect, and metabolic. Here, we will focus on antioxidants whose therapeutic efficacy was analyzed in epidemiological/clinical trials.

7.5

METABOLIC ANTIOXIDANTS

Metabolic antioxidants are involved in cellular energy production and act as cofactors of several metabolic enzymes. α-Lipoic acid is a coenzyme for mitochondrial pyruvate and α-ketoglutarate dehydrogenases. It is a powerful antioxidant and can recycle other antioxidants such as vitamin C, vitamin E, and glutathione.22 It was reported that old rats supplemented with (R)-α-lipoic acid showed an improvement in mitochondrial function, decreased oxidative damage, and increased metabolic rate.23 Accordingly, Suh and colleagues24 reported that old rats injected with (R)-αlipoic acid presented an improvement in glutathione (GSH) redox status of both cerebral and myocardial tissues when compared with control rats. Hager and collaborators25 reported that the administration of 600 mg α-lipoic acid/day to nine patients with AD for an average of 337 days promoted the stabilization of cognitive measures. However, Frolich et al.26 reported that (R)-α-lipoic acid stimulates deficient brain pyruvate dehydrogenase complex in vascular dementia but not in AD. In vitro studies showed that the pretreatment of dissociated primary hippocampal cultures with lipoic acid promotes significant protection against Aβ and iron/H2O2 toxicity. In contrast, concomitant treatment of cultures with lipoic acid and iron/H2O2 significantly potentiated the toxicity.27 Acetyl-L-carnitine (ALC) is a compound that acts as an intracellular carrier of acetyl groups across inner mitochondrial membranes. It also appears to have neuroprotective properties and has recently been shown to reduce attention deficits in

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patients with AD after long-term treatment. Bianchetti et al.28 performed an open study to evaluate the effect of ALC (2 g/day orally for 3 months) in association with donepezil or rivastigmine in 23 patients with mild AD who had not responded to treatment with acetylcholinesterase inhibitors (AChE-I). Clinical effects were evaluated by assessing cognitive functions, functional status, and behavioral symptoms. The response rate, which was 38% after AChE-I treatment, increased to 50% after the addition of ALC, indicating that the combination of these two drugs may be a useful therapeutic option for AD patients. These data do not permit a conclusion as to the possible mechanism of action of the association of the two treatments. Furthermore, the efficacy of ALC in MCI and mild (early) AD was investigated with a meta-analysis of double-blind, placebo-controlled, prospective, parallel group comparison studies of at least 3-months duration.29 The authors reported beneficial effects on both the clinical scales and the psychometric tests. To establish whether ALC is clinically effective in the treatment of people with dementia, Hudson and Tabet30 analyzed and compared 11 double-blind, randomized trials involving people with dementia in which treatment with ALC was compared with a placebo group. The analysis indicates a benefit of ALC on clinical global impression, but there was no evidence using objective assessments in any other area of outcome. Furthermore, the authors emphasized that given the large number of comparisons made, the statistically significant result may be due to chance. Idebenone is structurally similar to ubiquinone, which is normally synthesized in cells as part of the mitochondrial oxidative phosphorylation system. Ubiquinol, the reduced form of ubiquinone, can act as a very active antioxidant and can be absorbed through the diet. In a double-blind, placebo-controlled, multicenter, human study, 450 patients were given either placebo for 12 months followed by idebenone 90 mg three times per day for another 12 months, 90 mg three times per day for 24 months, or 120 mg three times per day for 24 months. Significantly, dose-dependent improvements were seen in measurements of clinical status and in neuropsychiatric tests compared with placebo. These improvements continued over the 2-year study.31 In another study,32 300 patients with mild to moderate AD were randomized to receive either placebo, idebenone 30 mg three times per day, or 90 mg three times per day for 6 months. Statistically significant improvement was noted in the Alzheimer Disease Assessment Scale (ADAS) total and in one cognitive parameter (ADAS-cog). An analysis of therapy responders revealed significant improvement in three outcome measures (clinical global response, ADAScog, and noncognitive scores) in the idebenone 90 mg three times per day group, compared with placebo. The study performed by Gutzmann et al.33 evaluated the safety and efficacy of idebenone vs. tacrine in a prospective, randomized, doubleblind, parallel-group, multicenter study in patients suffering from dementia of the Alzheimer type (DAT) of mild to moderate degree. Patients randomized to idebenone showed a higher benefit from treatment than patients randomized to tacrine. The authors concluded that the benefit–risk ratio is favorable for idebenone compared with tacrine, and furthermore, that this ratio is likely to be similar when comparing idebenone with other cholinesterase inhibitors. Thal et al.34 performed a 1-year, multicenter, double-blind, placebo-controlled, randomized

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trial including subjects aged 50 years with a diagnosis of probable AD and had Mini-Mental State Examination (MMSE) scores between 12 and 25. Subjects were treated with idebenone 120, 240, or 360 mg tid, each of which was compared with placebo. The authors observed that idebenone failed to slow cognitive decline in AD that was of sufficient magnitude to be clinically significant.

7.6 DIRECT ANTIOXIDANTS Direct antioxidants have the potential to interact directly with ROS. Their activity is not dependent on endogenous cellular macromolecules (e.g., enzymes) to exert their primary action, but they can by themselves chemically react with the damaging free radicals at the molecular level. Nevertheless, to some extent, there is also interaction with intracellular enzymes since some of these compounds are recycled either by endogenous oxyreductases directly, or indirectly via intracellular reducing shuttles such as ascorbate or some thiols. This class of antioxidants includes, among others, vitamins, 17β -estradiol, flavonoids, and terpenoids. Gingko biloba contains a variety of components, including flavonoids and terpenoids, that have free-radical scavenging ability. Recently, Stackman and colleagues35 tested the ability of Gingko biloba to antagonize the age-related behavioral impairment and neuropathology exhibited by Tg2576, a transgenic mouse model for AD. They observed that transgenic mice treated with Gingko biloba exhibited spatial memory retention comparable with that of wild-type mice. However, there were no differences in soluble Aβ and hippocampal Aβ plaque burden between treated and untreated TG2576 mice. Paradoxically, the levels of protein carbonyls were increased in Gingko biloba-treated mice. In a randomized controlled trial, EGb 716, an extract of Gingko biloba, was examined in 327 patients, 45 years or older, with mild to severe dementia resulting from either AD or vascular dementia.36 In this study, 150 AD patients were included and half of them received placebo for 1 year. Patients in the active treatment group had a significantly higher score on ADAS-cog, a measure of cognitive impairment, and in improved Geriatric Evaluation by Relative’s Rating Instrument (GERRI) score, a measure of daily living and social behavior. However, there was no difference between groups in the Clinical Global Impression of Change, an interview-based global rating that quantifies the clinician’s judgment of the amount of change in overall impairment compared with baseline. They also did not present any difference concerning the number or severity of adverse events between patients given extract or placebo. However, these findings suggest that Gingko biloba may be beneficial in patients with AD and vascular dementia. Vitamin E (a chain-breaking, lipid-soluble antioxidant) and selegiline (a selective monoamine oxidase type b inhibitor with antioxidant properties) appeared to be beneficial in patients with moderately severe AD by delaying the time of progression to severe dementia, loss of ability to perform activities of daily living, institutionalization, or death.37 However, it was observed that both treatments given together had no additional benefit over either alone. Other studies on the effect of vitamin E supplementation in AD subjects have yielded interesting

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results.37,38 Those subjects supplemented with 2000 IU/day experienced delay before they became institutionalized, but supplementation had no effect on loss of cognitive performance. Two studies39,40 published in the Journal of the American Medical Association suggest that higher dietary levels of vitamin E and vitamin C can help protect against the onset of symptoms associated with AD. Both studies were based on a large number of elderly people who were followed over 3–6 years, and the development of dementia and cognitive decline associated with AD were monitored. Both studies reported a significant decrease in the development of AD in subjects with high dietary intake of vitamins E and C. Recently, a crosssectional and prospective study indicated that the use of vitamin E and vitamin C supplements in combination is associated with reduced prevalence and incidence of AD.41 However, contradictory results were obtained in other studies. Luchsinger et al.42 investigated the relationship between AD and the intake of carotenes, vitamin C, and vitamin E in 980 elderly subjects in the Washington Heights-Inwood Columbia Aging Project who were free of dementia at baseline and were followed for a mean time of 4 years. The authors observed that the intake of carotenes and vitamin C or vitamin E in supplemental or dietary (nonsupplemental) form or in both forms, was not related to a decreased risk of AD. Recently, Laurin et al.43 examined the association of midlife dietary intake of antioxidants with late-life dementia and its subtypes. Data were obtained from the Honolulu-Asia Aging Study, a prospective community-based study of Japanese-American men who were aged 45–68 years in 1965–1968, when a 24-h dietary recall was administered. The authors concluded that intakes of β -carotene, flavonoids, and vitamins E and C were not associated with the risk of dementia or its subtypes, suggesting that midlife dietary intake of antioxidants does not modify the risk of late-life dementia or its most prevalent subtypes. Estrogen (ovarian hormone) has long been used to treat the physical symptoms of menopause and to aid in the prevention of osteoporosis in postmenopausal women. Cumulative evidence from basic science and clinical research suggests that estrogen also plays a significant neuromodulatory and neuroprotective role. The numerous estrogenic effects in the brain include the modulation of synaptogenesis, increased cerebral blood flow, mediation of important neurotransmitters and hormones, protection against apoptosis, antiinflammatory actions, and antioxidant properties. These multiple actions in the central nervous system support estrogen as a potential treatment for the cognitive decline associated with AD, the most common form of dementia (for a review, see [44]). Twenty postmenopausal women with AD were randomized to receive either 0.10 mg/day of 17β -estradiol by skin patch or a placebo patch for 8 weeks. Subjects were evaluated at baseline, at weeks 3, 5, and 8 during treatment, and again 8 weeks after treatment termination. During each visit, cognition was assessed with a battery of neuropsychological tests, and blood samples were collected to measure plasma estradiol as well as several other neuroendocrine markers of interest.45 The results indicate that the administration of a higher dose of estrogen may enhance attention and memory for postmenopausal women with AD. In another placebo-controlled, double-blind, parallel-group design study, 20

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women were randomized to receive either 0.10 mg/day of transdermal 17β -estradiol or a placebo for 8 weeks and were retrospectively evaluated as to whether basal levels of Aβ 40 were affected by prestudy use of hormone replacement therapy (HRT).46 Blood samples were collected and cognitive tests were administered at baseline; at weeks 3, 5, and 8 during treatment; and again 8 weeks after treatment termination. The results support an effect of estradiol on Aβ -processing for women with AD who are HRT-naive. This finding suggests that the hormone may serve as an Aβ -lowering agent for HRT-naive women with AD, which may, in turn, have ultimate ramifications for the progression of AD pathology.46 Although these findings provide clinical evidence to support a cognitive benefit of estrogen for women with AD, more recent studies based on larger sample sizes and for longer treatment durations do not support the efficacy of estrogen on AD. Geerlings and colleagues47 determined whether higher endogenous estradiol levels were associated with lower risk of dementia in older men and women not using HRT, using a case-cohort design within the Rotterdam Study, a populationbased follow-up study on chronic diseases, including dementia, in 7983 subjects aged 55 years or older, and ongoing since 1990. After adjustments for potential confounders, the authors observed that in men, no clear association was observed between estradiol levels and risk of dementia or its subtypes. The findings do not support the hypothesis that higher levels of endogenous estradiol reduce risk of dementia, neither in women nor in men. In the same line, Thal et al.48 studied 120 postmenopausal women who underwent hysterectomy and who were treated for AD with Premarin (equine estrogen) for 1 year. Plasma estradiol and estrone levels were determined at multiple points during the 1-year treatment trial. The change from baseline level at 2 and 12 months was associated with the change score on seven different assessments of cognitive functioning. The data obtained indicate that although Premarin elevated estradiol and estrone levels, there was no association between hormone levels and cognitive functioning after either 2 or 12 months of treatment. Many other compounds have been tested in vitro and in animal models of AD with some degree of success: curcumin,49–51 resveratrol,52,53 and ferulic acid.54,55 However, more clinical studies are needed to validate the efficacy of those antioxidants.

7.7 INDIRECT ANTIOXIDANTS Indirect antioxidants are compounds that do not have antioxidant capacity per se but help to prevent or reduce free radical formation. Among these, we can find metal-chelating agents such as desferrioxamine (DFO) and clioquinol. It has been shown that dyshomeostasis of the redox-active biometals (copper and iron) and oxidative stress are intimately connected, contributing to the neuropathology of AD. Using an in situ detection system, we showed that NFT and senile plaques are major sites for catalytic redox reactivity. Pretreatment with DFO or diethylenetriaminepentaacetic acid abolished the ability of the lesions to catalyze the H2O2-dependent oxidation of 3,3⬘-diaminobenzidine (DAB), strongly suggesting

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the involvement of associated transition metal ions.11,56 Indeed, following chelated removal of metals, incubation with iron or copper salts reestablished lesion-dependent catalytic redox reactivity. Our findings suggest that NFT and senile plaques contain redox-active transition metals and may thereby exert prooxidant or possibly antioxidant activities, depending on the balance among cellular reductants and oxidants in the local microenvironment. Savory et al.57 performed a study aimed at determining an optimal DFO treatment protocol in an animal model exhibiting AD-like intraneuronal protein aggregates. New Zealand white rabbits were injected intracisternally with either aluminum maltolate (aluminum has many similarities to iron but lacks redox activity) or with saline on day 0. Intramuscular injections of DFO were given to selected rabbits for 2 days prior to sacrifice on days 4, 6, or 8. Bielschowsky’s silver impregnation demonstrated widespread neurofibrillary degeneration (NFD) in neuronal cell bodies and neurites of brain and spinal cord from aluminum-treated rabbits. The authors observed that DFO treatment was capable of reversing tau aggregation after 2 days of treatment. McLachlan and collaborators58,59 performed a 2-year, singleblind study aimed at investigating whether the progression of dementia could be slowed by the trivalent ion chelator, DFO. In this study, 48 patients with probable AD were randomly assigned to receive DFO (125 mg intramuscularly twice daily, 5 days per week, for 24 months), oral placebo (lecithin), or no treatment. Analysis showed that the treatment and no-treatment groups were closely matched at entry into the trial but that the rate of decline, as measured over 2 years of observation, was twice as rapid in the no-treatment group compared with the DFO-treated group. Furthermore, trace-metal analysis of autopsied brain confirmed that extended treatment with DFO lowered neocortical brain aluminum concentrations to near-control concentrations. The results obtained suggest that the administration of DFO may slow the clinical progression of dementia associated with AD, and this chelator could be an useful palliative treatment for AD. Clioquinol, a 8-hydroxyquinoline derivative, has shown very encouraging results in the treatment of AD. Its biological effects are most likely ascribable to complexation of specific metal ions, such as copper(II) and zinc(II), critically associated with protein aggregation and degeneration processes in the brain. Cherny and collaborators60 reported a 49% decrease in brain Aβ deposition (⫺375 µg/g wet weight, p⫽0.0001) in a blinded study of Aβ PP2576 transgenic mice treated orally for 9 weeks with clioquinol. This was accompanied by a modest increase in soluble Aβ, Aβ PP, while synaptophysin and GFAP levels remained unaffected. Recently, Ritchie et al.61 developed a pilot phase-2 clinical trial where 36 patients with moderately severe AD were treated with clioquinol. The effect of treatment was significant in the more severely affected group owing to a substantial worsening of scores in those taking placebo compared with minimal deterioration for the clioquinol group. Plasma Aβ 42 levels declined in the clioquinol group and increased in the placebo group. Plasma zinc levels rose in the clioquinol-treated group. However, further clinical studies based on larger sample sizes and longer study periods should be performed.

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O2– •

Metabolic

Direct SOD

H2O2

SOD mimetics

FeII/CuI

HO•

Metal chelation

FIGURE 7.2 Diagrammatic representation of the sites of action of various classes of antioxidants. Cu, copper; Fe: iron, •OH, hydroxyl radical; H2O2, hydrogen peroxide; O⫺ 2 •, superoxide; SOD, superoxide dismutase.

7.8 CONCLUSION Antioxidants represent a promising therapeutic against AD (Figure 7.2). However, the broad occurrence of the disease (almost 50% by the age of 85), the limited regenerative nature of the central nervous system, and the fact that diagnosis often does not occur until late in disease progression, suggest that the ideal antioxidant should be a prophylactic given to most of the aged population. Owing to their low toxicity, low cost, and their ability to target the earliest sources of oxidative stress in AD, antioxidant therapies are particularly attractive for this prophylactic role.

ACKNOWLEDGMENTS This research was supported by the National Institutes of Health, Alzheimer’s Association, Philip Morris USA Inc., and Philip Morris International. Mark A. Smith and George Perry are consultants and own equity in Voyager Pharmaceuticals.

REFERENCES 1. Brookmeyer R, Gray S, Kawas C. Projections of Alzheimer’s disease in the United States and the public health impact of delaying disease onset. Am J Public Health, 1998; 88:1337–1342. 2. Alzheimer A. Uber einen eigenartigen schweren Krankheitsprozess der Hirnrinde. Zentralbl Nervenkrankheiten, 1906; 25:1134. 3. Moreira PI, Smith MA, Zhu X, Santos MS, Oliveira CR, Perry G. Therapeutic potential of oxidant mechanisms in Alzheimer disease. Expert Rev Neurotherapeutics, 2004; 4:995–1004. 4. Smith MA, Nunomura A, Lee HG, Zhu X, Moreira PI, Avila J, Perry G. Chronological primacy of oxidative stress in Alzheimer disease. Neurobiol Aging, 2005; 26:579–580; discussion 587–595. 5. Halliwell B. Protection against tissue damage in vivo by desferrioxamine: what is its mechanism of action? Free Radic Biol Med, 1989; 7:645–651.

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6. Skulachev VP. Mitochondrial physiology and pathology; concepts of programmed death of organelles, cells and organisms. Mol Aspects Med, 1999; 20:139–184. 7. Perry G, Nunomura A, Hirai K, Zhu X, Perez M, Avila J, Castellani RJ, Atwood CS, Aliev G, Sayre LM, Takeda A, Smith MA. Is oxidative damage the fundamental pathogenic mechanism of Alzheimer’s and other neurodegenerative diseases? Free Radic Biol Med, 2002; 33:1475–1479. 8. Kalaria RN, Gravina SA, Schmidley JW, Perry G, Harik SI. The glucose transporter of the human brain and blood-brain barrier. Ann Neurol, 1988; 24:757–764. 9. Hirai K, Aliev G, Nunomura A, Fujioka H, Russell RL, Atwood CS, Johnson AB, Kress Y, Vinters HV, Tabaton M, Shimohama S, Cash AD, Siedlak SL, Harris PL, Jones PK, Petersen RB, Perry G, Smith MA. Mitochondrial abnormalities in Alzheimer’s disease. J Neurosci, 2001; 21:3017–3023. 10. Wallace DC. Mitochondrial diseases in man and mouse. Science, 1999; 283:1482–1488. 11. Smith MA, Harris PL, Sayre LM, Perry G. Iron accumulation in Alzheimer disease is a source of redox-generated free radicals. Proc Natl Acad Sci USA, 1997; 94:9866–9868. 12. Honda K, Smith MA, Zhu X, Baus D, Merrick WC, Tartakoff AM, Hattier T, Harris PL, Siedlak SL, Fujioka H, Liu Q, Moreira PI, Miller FP, Nunomura A, Shimohama S, Perry G. Ribosomal RNA in Alzheimer disease is oxidized by bound redox-active iron. J Biol Chem, 2005; 280: 20978–20986. 13. Zhu X, Raina AK, Lee HG, Casadesus G, Smith MA, Perry G. Oxidative stress signalling in Alzheimer’s disease. Brain Res, 2004; 1000: 32–39. 14. Castellani R, Hirai K, Aliev G, Drew KL, Nunomura A, Takeda A, Cash AD, Obrenovich ME, Perry G, Smith MA. Role of mitochondrial dysfunction in Alzheimer’s disease. J Neurosci Res, 2002; 70: 357–360. 15. Cash AD, Aliev G, Siedlak SL, Nunomura A, Fujioka H, Zhu X, Raina AK, Vinters HV, Tabaton M, Johnson AB, Paula-Barbosa M, Avila J, Jones PK, Castellani RJ, Smith MA, Perry G. Microtubule reduction in Alzheimer’s disease and aging is independent of tau filament formation. Am J Pathol, 2003; 162:1623–1627. 16. Smith MA, Kutty RK, Richey PL, Yan SD, Stern D, Chader GJ, Wiggert B, Petersen RB, Perry G. Heme oxygenase-1 is associated with the neurofibrillary pathology of Alzheimer’s disease. Am J Pathol, 1994; 145:42–47. 17. Premkumar DR, Smith MA, Richey PL, Petersen RB, Castellani R, Kutty RK, Wiggert B, Perry G, Kalaria RN. Induction of heme oxygenase-1 mRNA and protein in neocortex and cerebral vessels in Alzheimer’s disease. J Neurochem, 1995; 65:1399–1402. 18. Keyse SM, Tyrrell RM. Heme oxygenase is the major 32-kDa stress protein induced in human skin fibroblasts by UVA radiation, hydrogen peroxide, and sodium arsenite. Proc Natl Acad Sci USA, 1989; 86:99–103. 19. Honda K, Casadesus G, Petersen RB, Perry G, Smith MA. Oxidative stress and redox-active iron in Alzheimer’s disease. Ann NY Acad Sci, 2004; 1012:179–182. 20. Nunomura A, Perry G, Pappolla MA, Wade R, Hirai K, Chiba S, Smith MA. RNA oxidation is a prominent feature of vulnerable neurons in Alzheimer’s disease. J Neurosci, 1999; 19: 1959–1964. 21. Christen Y. Oxidative stress and Alzheimer disease. Am J Clin Nutr, 2000; 71:621S–629S.

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-Isoprostanes as 8 FBiomarkers of Late Onset 2

Alzheimer’s Disease Thomas J. Montine

University of Washington Seattle, Washington

Joseph F. Quinn and Jeffrey A. Kaye Oregon Health & Science University Portland, Oregon

Jason D. Morrow Vanderbilt University Nashville, Tennessee

CONTENTS 8.1 Alzheimer’s Syndrome, Disease, and Dementia .....................................147 8.2 Free Radical-Mediated Damage in Alzheimer’s Disease ........................149 8.3 Assays for F2-IsoPs..................................................................................149 8.4 F2-IsoPs in the Brain................................................................................150 8.5 F2-IsoPs in Ventricular Cerebrospinal Fluid ............................................151 8.6 F2-IsoPs in Lumbar Cerebrospinal Fluid .................................................151 8.7 F2-IsoPs in Plasma and Urine ..................................................................152 8.8 Summary ..................................................................................................153 Acknowledgments .............................................................................................154 References .........................................................................................................154

8.1 ALZHEIMER’S SYNDROME, DISEASE, AND DEMENTIA What is commonly referred to as Alzheimer’s disease (AD) is really a syndrome, a common clinicopathologic entity that has multiple causes. Unusual forms of this syndrome are caused by highly penetrant autosomal-dominant mutations in one of three different genes: amyloid precursor protein (APP) gene, presenilin (PS) 1 gene, or PS 2 gene.1 Apparently, the same pathologic processes very commonly afflict adults with trisomy 21 or Down’s syndrome. However, it is late onset AD (LOAD) that represents a significant and growing public health burden, a “silent epidemic,” currently affecting between 2.5 and 4 million people in the United 147

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States, and more than 10 million people worldwide.2,3 The causes of LOAD are not yet clarified, but several environmental and genetic risk factors have been identified, the most potent of these being inheritance of the ε 4 allele of the apolipoprotein (apo) E gene.4 It is estimated that LOAD will grow to staggering prevalence in the next generation with an estimated 8 to 12 million patients by the year 2050 in the United States alone. In addition to the untold suffering it causes patients and their families, AD is the third most costly medical condition in the United States.5–7 As the number of patients afflicted continues to mount, the need for safe and effective therapy to delay or avert LOAD will become imperative.8 It is now well recognized that the pathologic processes of AD precede clinically diagnosed dementia by as much as two or three decades. Indeed, as early as 1976, Katzman applied the chronic disease model to AD and proposed the existence of a latent stage where some structural damage accrues but where there are no functional or behavioral changes, followed by a prodromal stage during which more structural damage accrues and mild functional and behavioral changes occur, and ultimately by a clinical stage with substantial irreversible damage and behavioral abnormalities.9 AD latency and prodrome are receiving increasing attention because it is here that interventions would have the greatest public health impact and because of the growing realization that treatment strategies at these earlier stages may differ from treatment of established dementia.10 Several clinical, neuroimaging, and clinicopathologic studies have investigated the prodromal stage of AD (reviewed in [11]). In addition, clinicopathologic studies have shown that AD-type neurodegenerative changes, viz. neuritic plaques (NPs) and neurofibrillary tangles (NFTs), are commonly present in older individuals rigorously demonstrated to be cognitively normal.12–24 Broadly summarizing these many studies, of cognitively normal individuals ⬎65 years old, approximately 80% or more will have NFTs in entorhinal cortex and ⬎50% will have NPs in isocortex as identified by standard silver-stain techniques. These data from cognitively normal older individuals, along with elegant clinical and neuroimaging studies, form the basis for the widely held view that AD has latent and prodromal phases during which damage occurs, although the person does not meet clinical criteria for dementia. Progression to Alzheimer’s dementia is marked by increased density and more extensive distribution of these histopathologic features. A very large number of pathological and experimental studies have proposed that formation of NPs and NFTs lies at the distal end of a complex molecular cascade that remains to be fully characterized.25 Important elements in this cascade include increased oxidative damage, loss of synapses, and the formation of nonsoluble protein aggregates. Combined with the results of the above-mentioned autopsy studies, these data strongly suggest that a substantial subset of older individuals who do not meet clinical criteria for dementia are experiencing not only histopathologic but also other structural and biochemical features of AD-type neurodegeneration. Importantly, this concept of prodromal AD implies that the opportunity may exist to intervene before the onset of symptoms to prevent clinically apparent dementia. Indeed, one interpretation of the apparent discordance between epidemiologic studies that associate protection from subsequent

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dementia by a particular drug and clinical trials of the same drug that show no therapeutic effect in patients with established dementia is that these drugs may be efficacious only during AD latency and prodrome but not during dementia.10 While LOAD is thought to contribute to ~70% of cases of dementia in the elderly, the situation is often complicated by coexisting pathologic changes in about one third to one half of these demented patients. One such coexisting condition is Lewy body (LB) accumulation in limbic and isocortical brain regions; this condition is referred to as Dementia with Lewy bodies (DLB) or the Lewy body variant of AD.26 Similarly, a substantial subset of demented patients with AD-type neurodegeneration also display significant vascular damage in the form of microvascular lesions (MVLs), an area under active investigation by several groups.27,28 In further analogy to the histopathologic lesions of AD, low levels of LBs and MVLs are found in subsets of aged individuals who are not demented, perhaps indicating latency and prodromal stages for these processes as well.15,27 Leaving aside the most obvious limitations of so-called mouse models of AD, there are no models of dementia that even come close to approximating the complexity of dementia in the elderly, thus underscoring the importance of studying brains obtained from patients that have been carefully evaluated both clinically and pathologically.

8.2 FREE RADICAL-MEDIATED DAMAGE IN ALZHEIMER’S DISEASE Abundant in vitro and in vivo data have strongly implicated free radical-mediated injury to diseased regions of brain as a pathogenic mechanism in AD.29 Several potentially overlapping sources for this AD-related increased free radical damage exist, including oligomers or higher order aggregates of amyloid β (Aβ ) peptides, mitochondrial dysfunction, innate immune activation, excitotoxicity, and others.25 Owing to the high concentration of polyunsaturated fatty acids in brain relative to other organs, lipid peroxidation is one of the major outcomes of free radical-mediated injury to brain.30 A critically important aspect of lipid peroxidation is that it is self-propagating and will proceed until the substrate is consumed or termination occurs. In this way, lipid peroxidation is fundamentally different from other forms of free radical injury in that it is a self-sustaining process capable of extensive tissue damage.31 There are many products generated by lipid peroxidation; one group of molecules, the F2-isoprostanes (F2-IsoPs) have received considerable attention because of their chemical stability and minimal metabolism in situ, making them ideal candidates for quantitative biomarkers of lipid peroxidation.32

8.3 ASSAYS FOR F2-ISOPS F2-IsoPs are formed in vivo by free radical-mediated attack on arachidonic acid followed by oxygen insertion and cyclization. F2-IsoPs are a complex mixture of 64 enantiomers contained within four regioisomeric families. In the study of AD,

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F2-IsoPs have been quantified by one of four different methods: commercially available enzyme-linked immunosorbent assays (ELISAs),33 two different gas chromatography- (GC) mass spectrometry (MS) stable isotope dilution methods that we refer to as the original method32 and modified method,34 respectively, and most recently by liquid chromatography- (LC) MS.35 The two GC-MS methods are similar and quantify subsets of F2-IsoPs that coelute with the deuterated internal standard used; this will be key to comparing studies presented in detail below. The original GC-MS method uses a commercially available deuterated F2-IsoP, 8-iso-PGF2α (also known as iPF2α-III), as internal standard and quantifies those F2-IsoPs in the peak that comigrates with this molecule.36 Morrow and colleagues36 have shown by reverse-phase HPLC and electrospray ionization MS that this peak not only contains 8-iso-PGF2α but also additional as yet uncharacterized F2-IsoPs; it is for this reason that the subset quantified by the original GCMS method is conservatively referred to as “F2-IsoPs.” The modified GC-MS method uses a different GC protocol and additional internal standards: iPF2α-III, iPF2α-VI, or 8-,12-iso-iPF2α-VI.34,37 This assay quantifies the peak that comigrates with each deuterated standard and refers to what is quantified as iPF2α-III, iPF2α-VI, or 8-,12-iso-iPF2α-VI. Recently, we showed that iPF2α-VI comigrates with 8-iso-PGF2α in the original GC-MS method, proving that iPF2α-VI is included in the peak we call F2-IsoPs, along with 8-isoPGF2α (iPF2α-III) and other as yet uncharacterized F2-IsoPs. In contrast, we found that another isomer, deuterated iPF2α-IV, does not comigrate with the F2-IsoP peak quantified by the original GC-MS method. 8-,12-iso-iPF2α-VI is not available for analysis.

8.4 F2-ISOPS IN THE BRAIN The first step in investigating lipid peroxidation in AD was to establish relevance in human postmortem tissue. The main advantage of this approach is that autopsy classification remains the gold standard for diagnosing not only LOAD but also common comorbid conditions such as LB disease and significant MVLs. A disadvantage is that almost all patients have advanced disease at the time of death. This point is particularly important because it means that finding changes postmortem does not inform about whether these processes occur early in the disease process, making them potential therapeutic targets, or are late-stage consequences of disease. Two groups have measured F2-IsoPs in brain of patients who died with advanced AD. Pratico et al.38 demonstrated elevated iPF2a-III and iPF2a-VI levels in frontal and temporal lobes of AD patients compared with controls using the modified GC-MS method. We expanded these findings by measuring F2-IsoPs (that contain iPF2a-III and iPF2a-VI among others) in the temporal and parietal cortices, hippocampus, and cerebellum of AD patients and age-matched controls, all with short postmortem intervals using the original GC-MS method.39 Our data also showed greater lipid peroxidation in diseased regions of AD brain but not in the cerebellum, a region relatively spared by AD.

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TABLE 8.1 Comparison of results from different methods for determining F2-IsoPs in CSF Fluid

V-CSF (pg/mL) L-CSF (pg/mL)

Original GC-MS Method (16–18,20–23,34)

Modified GC-MS Method (30,31,33)

St.

Control

∆

St.

III

46

72

⫹

III

23–26

31–50

⫹

III VI iso-VI

AD

Control 41 38 15 or 25

AD

∆

49 102 66 or 68

nd ⫹ ⫹

Note: Data are the mean values from published reference (noted in parenthesis). Abbreviations used: G ⫽ GC-MS method, either O (original) or M (modified); St. ⫽ deuterated internal standard used, either III (iPF2a-III, also known as 8-isoPGF2α), VI (iPF2α-VI), or iso-VI (8-,12-iso-iPF2α-VI); V ⫽ ventricular and L ⫽ lumbar CSF (cerebrospinal fluid); AD ⫽ Alzheimer’s disease. ∆ stands for change in AD patients compared to age-matched controls; ⫹ indicates a statistically significant increase; and nd indicates no significant difference between AD and control values.

8.5 F2-ISOPS IN VENTRICULAR CEREBROSPINAL FLUID Cerebrospinal fluid (CSF) obtained from the lateral ventricles at autopsy has also been assayed for F2-IsoPs. These studies represent a bridge between postmortem tissue analysis, which still permits accurate diagnosis of LOAD and comorbid processes, and the analysis of CSF from the lumbar cistern of living patients, where processes contributing to dementia can be less accurately diagnosed but analysis can be carried out earlier in the course of disease. We and others have determined the concentration of F2-IsoPs in ventricular CSF obtained postmortem and found elevations in AD patients compared with age-matched controls38,40 (Table 8.1). Although different deuterated standards were used and slightly different results achieved, both methods had similar control values and both showed a statistically significant increase in ventricular CSF F2-IsoPs in AD patients compared with controls. Our group went on to demonstrate that ventricular CSF F2-IsoP concentrations in AD patients are significantly correlated with indices of neurodegeneration.41

8.6 F2-ISOPS IN LUMBAR CEREBROSPINAL FLUID The first study of probable AD patients early in the course of dementia showed that F2-IsoPs are significantly elevated in lumbar CSF compared with agematched hospitalized patients without neurologic disease.42 This study used the original GC-MS method and quantified those F2-IsoPs that comigrate with deuterated 8-iso-PGF2α (iPF2α-III), also including iPF2α-VI as mentioned above. The average duration of dementia in these probable AD patients at the time of CSF examination was less than 2 years, while the average duration of AD is between 9 and 12 years. This same result has been observed by us in additional

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groups of probable AD patients and controls.43–45 As expected, the concentration of F2-IsoPs was lower in lumbar CSF than ventricular CSF, owing to both a rostro-caudal gradient46 and probably lower levels earlier in the disease. Importantly, a different laboratory examining probable AD patients and controls using the modified GC-MS method and deuterated 8-,12-iso-iPF2α-VI as standard obtained similar results.37 Although different internal standards were used in these studies, there was good agreement for values obtained in control individuals and AD patients (Table 8.1). Finally, these investigators extended their studies to patients with amnestic mild cognitive impairment (MCI), a condition that appears to represent, at least in some patients, prodromal AD; individuals with MCI were reported to have 8-,12-iso-iPF2α-VI lumbar CSF concentrations that were intermediate between controls and patients with AD.47 In addition to providing mechanistic information about neurodegenerative disease pathogenesis and a means to quantitatively assess response to antioxidant therapeutics, lumbar CSF F2-IsoP levels may also provide information that is useful in diagnosis of diseases in which it is elevated early. We tested the hypothesis that quantification of lumbar CSF F2-IsoPs, along with CSF Aβ42 and total tau levels, improves laboratory diagnostic accuracy for AD in patients with probable AD, dementias other than AD, and age-matched controls.45 Individuals were classified as AD or non-AD by a commercially available test measuring CSF Aβ42 and tau levels (95% sensitivity, 50% specificity), by CSF F2-IsoP and Aβ42 levels (90% sensitivity, 83% specificity), and by combined analysis using CSF F2-IsoP, Aβ42, and tau levels (84% sensitivity, 89% specificity). These results indicate that lumbar CSF F2-IsoP quantification can enhance the accuracy of the laboratory diagnosis of AD; however, this conclusion is based on a single study and these findings need to be replicated. Another potential application of lumbar CSF F2-IsoPs is objective assessment of response to therapeutics. We pursued this question in a longitudinal assessment of lumbar CSF F2-IsoPs in a group of patients with mild probable AD followed for 1 year. Figure 8.1 shows the percent change in CSF F2-IsoPs observed in these 40 AD patients stratified for dietary supplementation with no antioxidant vitamins, α-tocopherol, or α-tocopherol plus ascorbate (no patients took ascorbate alone). Patients without dietary supplementation showed ~50% increase in CSF F2-IsoPs over the 1-year period. Consonant with recently reported epidemiologic observations on the stratification of risk for AD,48 we observed a significant pharmacologic effect only in that group that supplemented their diets with α-tocopherol and ascorbate but not α-tocopherol alone.46

8.7 F2-ISOPS IN PLASMA AND URINE Although obtaining CSF from the lumbar cistern is not associated with significant risks, even in the elderly, when performed by experienced physicians, spinal taps can be stressful and are not easily obtained in most clinics. For these reasons, several investigators have pursued quantification of F2-IsoPs in plasma or urine. Like most data for peripheral biomarkers of neurodegenerative disease, the results have been conflicting. In large measure, this is likely due to the multiple

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Change over 1 year in CSF F2-lsoPs (%)

50 40 30 20 10 0 No E plus C

E plus C

FIGURE 8.1 Patients with mild AD who took antioxidant supplements for a 1-year period in between spinal taps that were assayed for F2-IsoPs. Patients who took no supplements, or only α-tocopherol (E) (n ⫽ 20) both showed approximately 40% interval increase in CSF F2-IsoPs and are grouped together in the “No E plus C” category; no patient took only vitamin C (C). Nine patients took E plus C for the entire year and they showed a significantly suppressed interval increase in CSF F2-IsoPs (Mann–Whitney test had P ⬍ 0.01).

uncontrolled physiologic processes that influence the concentration of CSF-derived molecules in blood and urine, and the relatively small contribution of brain-derived F2-IsoPs to the total pool of peripherally derived F2-IsoPs in blood.33,35,37,47,49–52 The reasons for these conflicting data are not clear, but a few points are worth considering. F2-IsoPs are generated by every cell and, therefore, peripheral production unrelated to central nervous system (CNS) disease could easily confound interpretation of blood or urine levels in AD patients. Indeed, the amount of brain-derived F2-IsoPs contributing to plasma levels is many times smaller than peripherally derived F2-IsoPs. Moreover, several factors, such as diet, degree of exercise, and body mass index, all appear to influence systemic F2-IsoP levels. Regardless of the reasons for the difference in peripheral F2-IsoPs in AD patients by different groups of investigators, quantification of peripheral F2-IsoPs cannot be used to objectively assess response in the CNS to systemically administered therapeutics because of the lack of organ specificity for F2-IsoPs. If a new drug lowers peripheral F2-IsoPs, is that because of an antioxidant effect in liver or brain? Even if the new drug brought symptomatic relief, it would be an assumption that the mechanism was by antioxidant actions in the CNS until measurements within the CNS compartment were made. Thus, while plasma and urine F2-IsoPs as peripheral biomarkers of AD are desirable, both technical and theoretical concerns necessitate continued use of CSF.

8.8 SUMMARY The clinicopathologic entity referred to as AD is a syndrome that is (1) caused by a few rare genetic causes, (2) a common outcome in adults with Down’s

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syndrome, and (3) by far most commonly seen as a late-onset disease with multiple risk factors but no causative factors yet identified. Emerging data suggests a chronic disease model for AD with latency, prodrome, and dementia stages together lasting decades. Free radical damage to lipids in brain is one pathogenic process of AD that may be quantified with F2-IsoPs to assist in diagnosis and aid in objective assessment of disease progression and response to therapeutics. While CNS F2-IsoPs are reproducibly elevated in AD patients at both dementia and prodromal stages of disease, we conclude by a variety of analytical methods that plasma and urine F2-IsoPs, including iPF2α-VI, are not reproducibly increased in AD patients. In addition, only a very small fraction of plasma F2-IsoPs derive from CSF, and so interpretation of their changes in plasma or urine is limited with respect to CNS disease or response of the CNS to systemic drug exposure.

ACKNOWLEDGMENTS This work was supported by the Nancy and Buster Alvord Endowment and grants from the NIH (AG24011, AG05136, and AG05144).

REFERENCES 1. Tsuang, DW and Bird, TD. Genetics of dementia. Med Clin North Am. 2002; 86: 591–614. 2. U.S. Congress. Losing a Million Minds: Confronting the Tragedy of Alzheimer’s Disease and Other Dementias. 1987, Washington, DC. 3. Evans, DA. Estimated prevalence of Alzheimer’s disease in the United States. Milbank Quarterly. 1990; 68:267–289. 4. Saunders, AM, Strittmatter, WJ, Schmechel, D, George-Hyslop, PH, PericakVance, MA, Joo, SH, Rosi, BL, Gusella, JF, Crapper-MacLachlan, DR, Alberts, MJ, Hulette, C, Crain, B, Goldgaber, D and Roses, AD. Association of apolipoprotein E allele ε 4 with late-onset familial and sporadic Alzheimer’s disease. Neurology. 1993; 43: 1467–1472. 5. Ernst, RL and Hay, JW. The US economic and social costs of Alzheimer’s disease revisited. Am J Public Health. 1994; 84: 1261–1264. 6. McCormick, WC, Hardy, J, Kukull, WA, Bowen, JD, Teri, L, Zitzer, S and Larson, EB. Healthcare utilization and costs in managed care patients with Alzheimer’s disease during the last few years of life. J Am Geriatr Soc. 2001; 49: 1156–1160. 7. Welch, HG, Walsh, JS and Larson, EB. The cost of institutional care in Alzheimer’s disease: nursing home and hospital use in a prospective cohort. J Am Geriatr Soc. 1992; 40: 221–224. 8. Brookmeyer, R, Gray, S and Kawas, C. Projections of Alzheimer’s disease in the United States and the public health impact of delaying disease onset. Am J Public Health. 1998; 88: 1337–1342. 9. Katzman, R. Editorial: The prevalence and malignancy of Alzheimer disease. A major killer. Arch Neurol. 1976; 33: 217–218. 10. Martin, BK, Meinert, CL and Breitner, JC. Double placebo design in a prevention trial for Alzheimer’s disease. Control Clin Trials. 2002; 23: 93–99.

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11. Petersen, R, Doody, R, Kurz, A, Mohs, R, Morris, J, Rabins, P, Ritchie, K, Rossor, M, Thal, L and Winblad, B. Current concepts in mild cognitive impairment. Arch Neurol. 2001; 58: 1985–1992. 12. Arriagada, PV, Marzloff, K and Hyma, BT. Distribution of Alzheimer-type pathologic changes in nondemented elderly individuals matches the pattern in Alzheimer’s disease. Neurology. 1992; 42: 1681–1688. 13. Berg, L, McKeel, DW, Miller, JP, Baty, J and Morris, JC. Neuropathological indexes of Alzheimer’s disease in demented and nondemented persons aged 80 years and older. Arch Neurol. 1993; 50: 349–358. 14. Crystal, HA, Dickson, DW, Sliwinski, MJ, Lipton, RB, Grober, E, Marks-Nelson, H and Antis, P. Pathological markers associated with normal aging and dementia in the elderly. Ann Neurol. 1993; 34: 566–573. 15. Davis, DG, Schmitt, FA, Wekstein, DR and Markesbery, W. Alzheimer neuropathological alterations in aged cognitively normal subjects. J Neuropathol Exp Neurol. 1999; 58: 376–388. 16. Green, MS, Kaye, JA and Ball, MJ. The Oregon brain aging study: neuropathology accompanying health aging in the oldest old. Neurology. 2000; 54: 105–113. 17. Haroutunian, V, Purohit, DP, Perl, DP, Marin, D, Khan, K, Lantz, M, Davis, KL and Mohs, RC. Neurofibrillary tangles in nondemented elderly subjects and mild Alzheimer disease. Arch Neurol. 1999; 56: 713–718. 18. Hulette, CM, Welsh-Bohmer, KA, Murray, MG, Saunders, AM, Mash, DC and McIntyre, LM. Neuropathological and neuropsychological changes in “normal” aging: evidence for preclinical Alzheimer disease in cognitively normal individuals. J Neuropathol Exp Neurol. 1998; 57: 1168–1174. 19. Morris, JC and Price, AL. Pathologic correlates of nondemented aging, mild cognitive impairment, and early-stage Alzheimer’s disease. J Mol Neurosci. 2001; 17: 101–118. 20. Price, JL and Morris, JC. Tangles and plaques in nondemented aging and “preclinical” Alzheimer’s disease. Ann Neurol. 1999; 45: 358–368. 21. Price, JL, Davis, PB, Morris, JC and White, DL. The distribution of tangles, plaques and related immunohistochemical markers in healthy aging and Alzheimer’s disease. Neurobiol Aging. 1991; 12: 295–312. 22. Riley, KP, Snowdon, DA and Markesbery, WR. Alzheimer’s neurofibrillary pathology and the spectrum of cognitive function: findings from the nun study. Ann Neurol. 2002; 51: 567–577. 23. Schmitt, FA, Davis, DG, Wekstein, DR, Smith, CD, Ashford, JW and Markesbery, WR. “Preclinical” AD revisited. Neuropathology of cognitively normal older adults. Neurology. 2000; 55: 370–376. 24. Xuereb, JH, Brayne, C, Dufouil, C, Gertz, H, Wischik, C, Harrington, C, Mukaetova-Ladinska, E, McGee, MA, O’Sullivan, A, O’Connor, D, Paykel, ES and Huppert, FA. Neuropathological findings in the very old results from the first 101 brains of a population-based longitudinal study of dementing disorders. Ann NY Acad Sci. 2000; 903: 490–496. 25. Hardy, J and Selkoe, DJ. The amyloid hypothesis of Alzheimer’s disease: progress and problems on the road to therapeutics. Science. 2002; 297: 353–356. 26. Hansen, L, Salmon, D, Galasko, D, Masliah, E, Katzman, R, DeTeresa, R, Thal, L, Pay, M, Hofstetter, R, Klauber, M and et al. The Lewy body variant of Alzheimer’s disease: a clinical and pathologic entity. Neurology. 1990; 40: 1–8.

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27. White, L, Petrovitch, H, Hardman, J, Nelson, J, Davis, D, Ross, G, Masaki, K, Launer, L and Markesbery, W. Cerebrovascular pathology and dementia in autopsied Honolulu-Asia Aging Study participants. Ann NY Acad Sci. 2002; 977: 9–23. 28. Petrovitch, H, White, LR, Izmirilian, G, Ross, GW, Havlik, RJ, Markesbery, W, Nelson, J, Davis, DG, Hardman, J, Foley, DJ and Launer, LJ. Midlife blood pressure and neuritic plaques, neurofibrillary tangles, and brain weight at death: the HAAS. Honolulu-Asia Aging Study. Neurobiol Aging. 2002; 21: 57–62. 29. Markesbery, WR. Oxidative stress hypothesis in Alzheimer’s disease. Free Radic Biol Med. 1997; 23: 134–147. 30. Montine, TJ, Neely, MD, Quinn, JF, Beal, MF, Markesbery, WR, Roberts, LJ, 2nd and Morrow, JD. Lipid peroxidation in aging brain and Alzheimer’s disease. Free Radic Biol Med. 2002; 33: 620–626. 31. Porter, NA, Caldwell, SE and Mills, KA. Mechanisms of free radical oxidation of unsaturated lipids. Lipids. 1995; 30: 277–290. 32. Morrow, J, Hill, K, Burk, R, Nammour, T, Badr, K and Roberts, L. A series of prostaglandin-like compounds produced in vivo in humans by a non-cyclooxygenase, free radical catalyzed mechanism. Proc Natl Acad Sci USA. 1990; 87: 9383–9387. 33. Feillet-Coudray, C, Tourtauchaux, R, Niculescu, M, Rock, E, Tauveron, I, Alexandre-Gouabau, MC, Rayssiguier, Y, Jalenques, I and Mazur, A. Plasma levels of 8-epiPGF2α, an in vivo marker of oxidative stress, are not affected by aging or Alzheimer’s disease. Free Rad Biol Med. 1999; 27: 463–469. 34. Pratico, D, Barry, OP, Lawson, JA, Adiyaman, M, Hwang, SW, Khanapure, SP, Iuliano, L, Rokach, J and Fitzgerald, GA. IPF2α-I - an index of lipid peroxidation in humans. Proc Natl Acad Sci. 1998; 95: 3449–3454. 35. Bohnstedt, K, Karlberg, B, Wahlund, L, Jonhagen, M, Basun, H and Schmidt, S. Determination of isoprostanes in urine samples from Alzheimer patients using porous graphitic carbon liquid chromatography-tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci. 2003; 796: 11–19. 36. Morrow, JD and Roberts II, LJ. Mass spectrometry of prostanoids: F2-isoprostanes produced by non-cyclooxygenase free radical catalyzed mechanism. Meth Enzymol. 1994; 233: 163–174. 37. Pratico, D, Clack, CM, Lee, VMY, Trojanowski, JQ, Rokach, J and FitzGerald, G. Increased 8,12-iso-iPF2α-IV in Alzheimer’s disease: correlation of a noninvasive index of lipid peroxidation with disease severity. Ann Neurol. 2000; 48: 809–812. 38. Pratico, D, Lee, VM, Trojanowski, JQ, Rokach, J and Fitzgerald, GA. Increased F2-isoprostanes in Alzheimer’s disease: evidence for enhanced lipid peroxidation in vivo. FASEB J. 1998; 12: 1777–1784. 39. Reich, EE, Markesbery, WR, Roberts, LJ, II, Swift, LL, Morrow, JD and Montine, T. Brain regional quantification of F-ring and D/E-ring isoprostanes and neuroprostanes in Alzheimer’s disease. Am J Pathol. 2001; 158: 293–297. 40. Montine, TJ, Markesbery, WR, Morrow, JD and Roberts, LJ. Cerebrospinal fluid F2-isoprostanes are increased in Alzheimer’s disease. Ann Neurol. 1998; 44: 410–413. 41. Montine, TJ, Markesbery, WR, Zackert, W, Sanchez, SC, Roberts, LJ and Morrow, JD. The magnitude of brain lipid peroxidation correlates with the extent of degeneration but not with density of neuritic plaques or neurofibrillary tangles,

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42.

43.

44.

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46.

47. 48.

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5!.

52.

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and Alzheimer’s 9 Aspirin Disease Protection Sven E. Nilsson and Stig Berg Jönköping University Jönköping, Sweden

CONTENTS 9.1 9.2 9.3 9.4

Effects of Salicylic Compounds in Plants ...............................................159 Aspirin and the Modern Successors ........................................................160 Side Effects and Risks of Aspirin............................................................162 Aspirin and Non-aspirin NSAIDS in Modern Pharmacotherapy............163 9.4.1 Cardiovascular Diseases ..............................................................163 9.4.2 Diabetes........................................................................................163 9.4.3 Malignancy ..................................................................................164 9.5 Cognitive Decline and Alzheimer’s Disease............................................164 9.5.1 Circulation ..................................................................................164 9.5.2 Oxidative Stress ...........................................................................165 9.5.3 Inflammation................................................................................165 9.5.4 Brain Insulin ................................................................................166 9.5.5 Amyloid Deposits ........................................................................166 9.5.6 Glutamate Excitoxicity ................................................................167 9.6 Clinical Trials...........................................................................................167 9.7 The Octo-Twin Study...............................................................................168 9.8 Results......................................................................................................169 9.9 Conclusion ...............................................................................................174 References .........................................................................................................174

9.1 EFFECTS OF SALICYLIC COMPOUNDS IN PLANTS Salicylic compounds occur widely in plants. Salicylic alcohol and its glucosides characteristically occur in such species of salix as willow, spirea, and filipendula.1 However, salicylic acid is the dominating form in botany, and is frequently found in medical herbs. Free salicylic acid was first demonstrated in hyacinth, tulip, and violet, and later on in orange, plum, grape, and other cultivated plants.2 In a systematic investigation, Raskin et al.3 found salicylic acid in 20 out of 27 cultivated 159

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plants. It is instructive to study the effects of salicylic acid and other salicylic compounds in the plants where they are produced:2 ●

●

●

●

Improved resistance. When microorganisms such as viruses, fungi, and bacteria attack plants, the systemic acquired resistance (SAR) is strengthened by salicylic acid. Exogenous supply of salicylic acid also seems to affect SAR. By the injection of a 0.2% solution of salicylic acid, White4 demonstrated that he could protect tobacco plants against the infection of the tobacco mosaic virus. Maintained territory. Salicylic acid is secreted from the root cells of some plants (e.g., corn) to protect the immediate environment against invaders. The effect is achieved by a change of pH and depolarization of the membrane potential of newly formed root cells; both effects are injurious for competitors. Heat production. Some plants will flower at a low external temperature. If there is an increased production of salicylic acid, the local temperature can increase as much as 20ºC,5 which facilitates flowering and production of volatile aroma compounds that attract pollinating insects. The production of salicylic acid increases a few days before blooming. Flowering. Salicylic acid, when introduced into the plant’s milieu, stimulates formation of buds and flowers. This effect, however, is nonspecific and can also be achieved by other types of chemicals.

The concentration of salicylic acid in plants is found to be low (0.03 to 37 µg/g fresh weight),3 a level that is pharmacologically insignificant but physiologically interesting. More salicylic acid is excreted in the urine of vegetarians than of nonvegetarians,6 which may be a reflection of how important vegetables and fruits are as a source of salicylate in humans. It should be noted that a regimen of Aspirin as low as 40 to 50 mg/day,7,8 in congruence with what bioavailable salicylates in vegetarian diets can provide,9 has been found to be equal to and even more effective than higher doses to prevent vascular lesions and colon tumors. It is possible that the effect of salicylic compounds on the metabolic processes of plants, including stabilization and repair of genes involved in the regulation of metabolism, has correspondence in human biology. It should be noted that there are also other herbal compounds with similar effects. Gingko biloba has an Aspirin-like effect on thrombocytes, and an additive effect should therefore be expected if Aspirin is used at the same time.10

9.2 ASPIRIN AND THE MODERN SUCCESSORS Hippocrates (460–377 BC) believed that extracts of willow alleviated pain and fever, and he recommended that women chew its bark in order to reduce labor pains. In other ancient cultures, for example in China11 and pre-Columbian America,12 willow was used to treat fever and rheumatism. In Europe, the signature doctrine, in the form developed by Paracelsus (1493–1541), was

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generally applied during the early phase of New Time. Since the branches of willow were flexible it was considered that they might be useful to make rigid joints movable. Also since the trees grew in a moist environment, they were also thought to protect against marsh fever, which was gradually associated with the malaria midge. Thus, in 1763, the English clergyman Edward Stone wrote that he had successfully treated more than 50 patients against ague with willow extracts.13 He ends his letter to the British Royal Society with the statement “I have no other motives for publishing this valuable specific, than that it may have a fair and full trial in all its variety of circumstances and functions, and that the world may reap the benefits accruing from it.” During the 19th century, the experience of folk medicine and the progress of chemistry worked together in a rapid pharmacological evolution. In 1859, Hermann Kolbe demonstrated a method to synthesize salicylic acid, which reduced the interest in the naturally existing form of the substance. The primary applications for the new compound were in preserving meat, milk, and beer, and as amedical antiseptic.14 In 1897, Felix Hoffman, a chemist in Bayer Pharmaceutical Industry, synthesized acetylsalicylic acid using a method applicable for industrial production. This finding started a triumphal progress for the new substance, which got the name of Aspirin: “A” from acetyl and “aspirin” from the plant name of Spirea. Soon it was widely used against pain, aching, fever, rheumatic diseases, gout, and so on. Fifty years later, a new generation of Aspirin-like compounds was introduced into medical practice with the main ambition of specifically increasing the painrelieving and anti-inflammatory components. The first of these non-steroidal antiinflammatory drugs (NSAID) was phenylbutazone, made in 1952. In the 1960s, ibuprofen, naproxen, ketoprofen and indomethacin, among others, were introduced. Ten years later diclofenac joined the list, and in the 1990s, a new generation of compounds with a specific effect on Cyclo oxygenase (COX-2) was included in the list. In 1971 John Vane,15 who was awarded the Nobel Prize in 1982, showed that the NSAIDs inhibited the biosynthesis of prostaglandins (PGs) and proposed this as their main mechanism of action. Both Aspirin and the non-Aspirin NSAIDs inhibit the formation of endoperoxides (PGG and PGH) from arachidonic acid through interaction with the COX. After the characterization of the COX-1 enzyme in 1976, a second COX gene (COX-2) was discovered in 1991. The constitutive isoform, COX-1, has several normalphysiological functions, while the inducible isoform, COX-2, is provoked by pro-inflammatory stimuli in migratory cells and inflamed tissues. The range of activities of NSAIDs against COX-1 compared with COX-2 explains the variations in therapeutic results as well as the side effects of NSAIDs at their anti-inflammatory doses. Drugs introduced later with high inhibitory potency on COX-2 and less effect on COX-1 were intended to have a retained anti-inflammatory activity with fewer side effects. The limitation, however, turned out to be complicated, accompanied by an increased risk of vascular events.16 Little is known about the distribution of NSAIDs and prostaglandin rebuilding synthetases in organs and tissues. Histochemical tissue investigations, however,

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have given some information about the occurrence of COX,and it is of interest that studies in Alzheimer’s disease reveal intense staining for COX-1 in certain regions of hippocampus.17 Even if there may be other effects of NSAIDs, the COX inhibition seems to be the main consequence and induces a reduced level of prostaglandins, including thromboxane and prostacyclin, which are of special interest for the present discussion. Prostacyclin, which is produced in the vesselwall endothelial cells, has a vasodilating and anti-coagulation effect, while its opponent, thromboxane, produced in the thrombocytes, has a vasoconstricting and coagulation-inducing capacity. Aspirin has a very short half-life time (1/2 h) in the circulation and is rapidly hydrolyzed to salicylic acid and acetate. It has been demonstrated in vitro that a standard dose of Aspirin exerts its action on platelet COX by irreversibly acetylating the enzyme, and thereby inactivating it for the rest of the platelet’s lifetime. This is paralleled by a long-lasting inhibition of thromboxane synthesis in vivo; [from 2 to 3 days with very low levels, which then slowly returns to pre-dose levels in about 10 days]. The non-Aspirin NSAIDs do not acetylate COX but are reversible inhibitors of the enzyme. Further, the inhibition of the COX activity causes reduced formation of prostacyclin and other prostaglandins; for example, PGE, which is an important mediator in pro-inflammatory signaling with a muscle-relaxing and, like prostacyclin, vasodilating effect. A reduced effect of PGE brings about an impaired blood-flow in kidneys and gastric mucosa. After the broad introduction of Aspirin as an antiplatelet drug, cases with reduced vascular effect have been observed. The Aspirin resistance seems to occur with a frequency of 5 to 10% and results in a two- to threefold increase in vascular events.18

9.3 SIDE EFFECTS AND RISKS OF ASPIRIN Perspiration and tinnitus were early observed as side effects of treatment with salicylic compounds. In 1877, gastrointestinal bleeding was reported, which appeared to result from peptic ulcers secondary to an increased acid production, which also caused heartburn and dyspeptic discomfort. The gastric effect has been referred to as a reduced gastric mucosal COX-activity with a secondary impaired production of mucosa-protecting prostaglandins.19 Further, Aspirin was earlier shown to influence renal function, causing proteinuria and slight increase in blood pressure. The risk, however, seems to be marginal, and a recent report showed that there was no decline in kidney function during an observation period of 14 years.20 Hyperuricemia and gout tend to increase renal risk,21 which may be associated with the observation of increased frequency of gout in treatment with Aspirin.22 In 1902 acute angioedema/urticaria was first reported,23 and it was also realized that Aspirin could provoke bronchial obstruction. Later, other symptoms similar to allergic reactions turned out as side effects. Nasal polyposis is especially common with a prevalence of about 4%.24,25 Even if clinical asthma is seen in only 2% , provocation indicates an increased bronchial irritability in more than 20% of persons taking the drug.26 These symptoms can also be seen as side effects to berries like lingonberry and to fruits like banana, which contain salicylic acid or the

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closely related benzoic acid. Both asthma and angioedema/ urticaria may be due to inhibition of COX-1 and are typically associated with overproduction of leukotrienes27 by the alternative lipoxygenase pathway for degradation of arachidonic acid.

9.4 ASPIRIN AND NON-ASPIRIN NSAIDS IN MODERN PHARMACOTHERAPY It was shown earlier that salicylate therapy was effective for rheumatic fever and other rheumatic diseases.28 Until about 1970 the highest tolerable dose of Aspirin, usually 3 to 6 g per day, was routinely used for rheumatic diseases. [This is still the most frequently used dose in many countries] even though there are new therapeutic approaches. In modern pharmacotherapy new indications have been added. Aspirin therapy has been used in cardiovascular diseases, diabetes, malignancy, and Alzheimer’s disease, and positive results have also been reported for some other diagnoses such as gall bladder disease,29 Parkinson’s disease,30 amyotrophic lateral sclerosis,31 peridontitis,32,33 and macular degeneration.34 Controversial reports35–37 exist for ocular cataract.

9.4.1 CARDIOVASCULAR DISEASES The balance between various counter-acting prostaglandins such as thromboxane and prostacyclin is of basic importance for the pathogenesis of vascular diseases. In 1942 dicumarole was introduced in the therapy of thrombosis. Since salicylic acid is a degradation product of dicumarole, it was also studied for a possible antithrombotic effect and was shown to prolong the prothrombin time.38 During this time, Lawrence Craven, an otorhinolaryngologist in private practice, observed an increased tendency of bleeding after tonsillectomy in patients taking Aspirin. This observation gave him the idea to use Aspirin as a prophylactic against myocardial infarction. In 1950, he reported 600 friends and patients treated with Aspirin,39 and 6 years later he reported as many as 8000 without infarction or major stroke.40 Ironically, in spite of Aspirin medication, Lawrence Craven himself, before the study was fulfilled, died from a cardiac infarction.41 During the last 15 years, Aspirin has become a well-established routine therapy after episodes of myocardial infarction, and in meta-analyses a risk reduction of 20 to 30% has been registered. Similar protective effects have been achieved as secondary prophylaxes after stroke and transient ischemic attacks (TIA).42

9.4.2 DIABETES As far back as 1875, a reduction in diabetic glucosuria in association with salicylate intake was observed.43 The interest was renewed in 1957 when Reid described normalized blood sugar and interrupted insulin therapy in a young diabetic man who took 6 g of Aspirin a day as therapy for a newly contracted

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rheumatic fever.44 Later, various clinical trials, recapitulated by Baron,45 have been moderately successful. It has also been proven that Aspirin antagonizes glycation of collagen structures,46 which may reduce the risk of diabetic microangiopathy. Another related effect of Aspirin is decreased serum cholesterol. However, the lipid-lowering effect is still more obvious for para-aminosalicylic acid (PAS), a therapeutic for tuberculosis with a lipid-effect similar to that of clofibrate.

9.4.3 MALIGNANCY There is an increasing interest in the role of prostaglandins in carcinogenesis. A possible therapeutic effect of Aspirin has been obtained in various gastrointestinal tumors as well as in prostate47 and lung cancer.48 The tumor tissue usually contains both COX-1 and COX-2, although in some types of malignancy only COX-2 has been observed. This provides the prerequisites for an increased production of prostaglandins, which are important for the neovascularization necessary for tumor growth. Aspirin also counteracts various tumor-stimulating factors that enhance COX-2 production, for example, epidermal growth factor (EGF), tumor necrosis factor, hypoxia, and ultraviolet light. Microsatellite instability (MSI) occurs in tumors, and a possible gene stabilizing effect49 is another aspect of Aspirin in the pathogenesis of malignancies. Thus, nonusage of Aspirin or non-Aspirin NSAIDs has been shown to double the frequency of MSI, which is similar to the effect of smoking.50 A preventative effect of Aspirin and some other NSAIDs has been found in colon cancer, rectal cancer, and colon polyposis.51,52 Curiously, a lower dose of Aspirin (81 mg per day) seemed to be more effective than a higher dose (325 mg per day) to counteract such tumors.53 A similar protective effect was seen in breast cancer.54 Both colon and breast cancer are associated with increased serum levels of insulin and insulin growth factor-1(IGF1), which can be counteracted by a decrease in the insulin resistance from the anti-inflammatory effect of Aspirin. Further, it has been proposed that the Aspirin effect may depend on modification of the IGF1 genotype.52

9.5 COGNITIVE DECLINE AND ALZHEIMER’S DISEASE Various mechanisms may be applicable to explain the effect of Aspirin on brain function, cognitive decline, and the development of Alzheimer’s disease.

9.5.1 CIRCULATION Atherosclerosis is associated with an increased risk of both Alzheimer’s disease and vascular dementia, and vascular lesions are often found in the brains of Alzheimer patients.55 As mentioned at the outset, Aspirin has been shown to reduce the risk for all types of vascular events by influencing the synthesis of thromboxane, produced in the thrombocytes, and of prostacyclin, produced in

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vascular epithelium cells.56 While the lowering of the vasoconstricting thromboxane lasts for several days, the vasodilating prostacyklin remains depressed only for hours. An optimal effect on serum concentrations of thromboxane was achieved with 80 mg of Aspirin taken during an interval of 24 up to 3 days.57 The most reliable method to determine thromboxane synthesis in vivo is by measuring urine excretion of 2,3-dinor thromboxane B2 (dinor TXB2). An investigation of this metabolite in patients with probable Alzheimer’s disease compared with nondemented controls indicated an increased thromboxane production.58 An increased thromboxane level has also been demonstrated for the poststroke cases, who developed dementia.59 Naproxen, owing to a long half-life time, seems to inhibit several branches of the arachidonic acid cascade over a prolonged period of time.60,61 On the contrary, paracetamol, another frequently used analgesic, has been shown to be ineffective in this respect.

9.5.2 OXIDATIVE STRESS It is important to keep in mind that the COX enzymes catalyze highly oxidative reactions. Thus, through activation of two oxygen molecules, the endoperoxides PGG and PGH are formed. PGG also contains a hydroxyperoxy group. During these reactions, parts of the enzyme molecules are inactivated by oxidation. Both PGG and PGH are highly reactive and can be enzymatically transformed into prostanoids, and also disintegrate nonenzymatically. It seems likely that the activated oxygen radicals as well as PGG and PGH could contribute to oxidative stress in the cell, a phenomenon that might be blocked by Aspirin. A method to study the level of oxidative stress is to follow urinary isoprostane (8-epi-PGF2), which has been found to be lowered by Aspirin; [however this effect is less than that observed by vitamin E62]. In a study of the influence on various markers of oxidative stress, Ristimae et al.63 reported Aspirin to inhibit linoleic acid peroxidation , which has been considered to be another good indicator of the total antioxidant capacity. Alzheimer’s disease may be associated with the accumulation of reactive oxygen species leading to lipid peroxidation and neuronal degeneration in the brain.64 The protective effect of Aspirin in the ischemic brain seems mainly to depend on salicylic acid.65 However, there is also a report of Acetyl Salicyclic Acid-induced impairment of the antioxidant system.66

9.5.3 INFLAMMATION Alzheimer’s disease seems to be provoked mainly by inflammatory processes in cognitive brain centers, the microglia accumulating around the senile plaques being of central importance.67 Probably there is a vicious circle where the microglia, activated by deposits of amyloid, induces a further amyloid production by synthesis of inflammatory factors. NSAIDs may depress the inflammatory process. This explains a reduced risk for Alzheimer’s disease after prolonged treatment with drugs prescribed for chronic rheumatic diseases. The effect is associated with the inhibition of COX-1 and COX-2 and has an important impact on most inflammatory processes. McGeer,68 however, found COX-2 inhibitors to

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have no effect in the treatment of Alzheimer’s disease, which indicates that the appropriate target for inhibition should be COX-1. Further, the neurotoxic effect of the dopamine-selective neurotoxin MPTP was totally prevented by systemic administration of Aspirin and salicylate in mice, while paracetamol, diclofenac, ibuprofen, and indomethacin turned out to be ineffective.69–71

9.5.4 BRAIN INSULIN In 1855, Claude Bernard showed that he could provoke glucosuria by pricking the bottom of the fourth ventricle of dogs, and for some decades, the observation localized the primary cause of diabetes to the brain and its interplay with liver function. Thirty-five years later, following the discovery by von Mering and Minkowski of insulin production in the pancreatic β cells, the brain theory became obsolete. Today brain insulin once more has a major role in the discussion of energy and glucose homeostasis.72 In the interplay with multiple other factors such as leptin and serotonin, it regulates satiation feelings and controls the constancy of body weight. There is an abundance of insulin receptors in the hippocampus area, indicating that insulin is a potential neuromodulator involved in cognitive processes related to feeding such as specific memories and nonconscious calculations of food intake necessary to preserve body weight. It is interesting to note some similarities between β cells and brain neurons, particularly in response to hormonal stimuli and glucose, by depolarization and exocytosis in a process that resembles neurotransmitter release from synaptic vessels. Since there is a changing congruence between brain and blood insulin, locally stored insulin may be mobilized in association with neural activity.73 A disturbed blood insulin to insulin ratio in cerebrospinal fluid or in the brain has been reported in the case of metabolic dysfunction. In Alzheimer’s disease cerebrospinal insulin tends to decrease whereas blood insulin often increases.74 High blood insulin levels are not only indicators of increased risk of diabetes but also signals of a future tendency of cognitive decline.75,76 A common therapeutic effect of Aspirin in diabetes and Alzheimer’s disease may therefore depend on reduced insulin resistance. Owing to its inhibiting effect on inflammation-provoking cytokines, Aspirin improves the transmission of intracellular signals from the insulin receptors, and by higher receptor effect it counteracts various consequences of hyperglycemia.

9.5.5 AMYLOID DEPOSITS The prevailing theory of the pathogenesis of Alzheimer’s disease contends that the primary event is intraneural and extraneural deposits of the highly amyloidogenic Abeta-42-peptide (Aβ ), which forms the senile plaques characterizing the disorder. Histological evidence of amyloid infiltration can be seen in the temporal lobe up to 40 to 50 years before the onset of dementia.77 Biochemical and genetic evidence indicate an altered balance of biogenesis/clearance of Aβ, which is derived, by two sequential cleavages, from the amyloid precursor protein (APP).

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The proteases involved are β - and γ -secretase. Secreted Aβ becomes either degraded by metalloproteases such as the insulin-degrading enzyme (IDE) or metabolized through receptor uptake mediated by apolipoprotein E. Environmental risk factors such as viruses and metals may contribute to the process. Thomas et al.78 found that Aspirin has an amyloid-degradating effect in the brain just as in the tissue of the pancreatic islets . Various NSAIDs, including Aspirin, were shown to ameliorate the Aβ aggregation induced by aluminum.79 Ibuprofen, indomethacin, and sulindac sulfide have been reported to directly affect amyloid pathology in the brain by reducing the γ -secretase activity.80 IDE breaks down insulin as well as cerebral Aβ deposits.81,82 However, the insulin effect has priority, and with high cerebral insulin levels there is not enough IDE left for amyloid breakdown. The relative insufficiency of IDE with regard to Aβ may be most pronounced in insulin-treated diabetics, where insulin is introduced into the circulation without the regulative effects in the pancreas and liver. Consequently, the increased risk of cognitive decline and Alzheimer’s disease in diabetes is mainly limited to the group receiving insulin. Also, hyperglycemia and other mechanisms such as obesity causing higher insulin levels in nondiabetic subjects may interfere with the utilization of IDE with impaired Aβ degradation as a secondary result. Amylin (islet amyloid polypeptide, IAPP) is produced in the pancreatic β cells parallel with insulin and acts partly as a separate satisfaction factor. In hyperglycemia the production not only of insulin but also of amylin is enhanced. An increased amylin level is associated with a tendency for amyloid deposits in pancreatic β cells, inducing suppressed insulin production and thereby leading to an increased risk of diabetes. Aspirin reduces these amyloid depositions78 in brain as well as in pancreatic β cells. Recently, Janson et al.83 reported the joint occurrence of amyloid deposits in brain and pancreatic islets from the autopsy specimens of subjects with Alzheimer’s disease and diabetes, respectively.

9.5.6 GLUTAMATE EXCITOXICITY Glutamate excitoxicity has been implicated in Alzheimer’s disease as well as in Parkinson’s disease. Both Aspirin and ibuprofen attenuate this effect.84 The increase in the glutamate concentration seen secondary to brain ischemia can be distinctly reduced by Aspirin, and also in the low-dose prophylactic regime.85

9.6 CLINICAL TRIALS There are several problems to be addressed in studies of medication and dementia. The association of dementia with low analgesic use may be due to a lower pain perception with reduced brain function. One has also to consider whether patients with dementia have the same access to drug therapy as nondemented, and the fact cannot be denied that it is harder for demented persons to establish medical contacts and to describe their sensations. There may also be practical difficulties for the demented to buy nonprescribed drugs. Data on paracetamol which

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like Aspirin can be bought over the counter with similar indications and frequency, show both the objection of a possibly lower sensitivity for pain in the demented and the matter of availability. If reduced pain sensitivity is of importance for the low association between Alzheimer’s disease and Aspirin usage the same should be found for paracetamol. Individuals with dementia have an increased risk of death during the followup period. To overcome this potential bias, investigations should be limited to those who survive the total observation period. An optimized method would then be to compare those who survive with those who remain cognitively intact. A mirror picture for this subgroup, compared with the Alzheimer’s subgroup, strengthens a true association between the cognitive function and Aspirin, unbiased from mortality. However, the most important bias in studies of Aspirin influence on the Alzheimer’s risk is the indication for Aspirin use. Prescribed low-dose Aspirin is usually intended to prevent circulatory events, including episodes of stroke and TIA, which per se indicate an increased risk for cognitive decline. Also other cardiovascular diagnoses such as coronary disease and congestive heart failure may be associated with impaired brain function.86 Therefore, studies on low-dose Aspirin limited to pharmacy statistics are hard to estimate, and it is of some advantage to limit studies to nonprescribed Aspirin even if such treatment may be influenced by a growing layman’s awareness of the prophylactic capacity of the drug. Numerous reports have described a protective effect of Aspirin and other NSAIDs on the risk of developing dementia in patients suffering from inflammatory diseases such as arthritis.87 Since Aspirin has been widely used in vascular prophylactics, it was tempting to examine its effect in the treatment of vascular dementia. In a Cochrane report, however, it was concluded that there have been no randomized controlled trials and no evidence that Aspirin is protective in this context.88 In contrast, for Alzheimer’s disease, there have been indications of a positive effect for NSAIDs and potentially for Aspirin, in most studies (see Table 9.1). In the Swedish OCTO-Twin study99 that will be described below, the frequency of dementia was reduced by about 50% among those taking Aspirin, both at base-line and over an observation period of 6 years. No corresponding effect was registered for paracetamol and dextropropoxyphene. The results of some of the other studies are hard to evaluate as they include only prescribed lowdose Aspirin, which usually refers to secondary prophylaxis after vascular events such as stroke, TIA, and myocardial infarction. Asimilar objection can be raised to some degree against a recent meta-analysis100 showing a pooled relative risk for Aspirin of 0.87 (0.70 to 1.07) and for non-Aspirin NSAIDs of 0.72 (0.50 to 0.94).

9.7 THE OCTO-TWIN STUDY The analyses were based on data from 702 individuals 80 years of age and older, investigated at four occasions over a 6-year period. Information about diseases and health problems was drawn from medical records, self-reports, and registrations of

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TABLE 9.1 Effects of Aspirin and NSAIDs in Various Studies of the Risk for Alzheimer’s Disease Aspirin Authors 89

Breitner et al. Henderson et al.90 Stewart et al.91 Beard et al.92 Anthony et al.93 Broe et al.94 In´t Veld et al.95 Lindsay et al.96 Zandi et al.97 Landi et al.98 Nilsson et al.99

NSAIDs

Year

Country

OR/RR

CI 95%

OR/RR

CI 95%

1994 1997 1997 1998 2000 2000 2001 2002 2002 2003 2003

USA Australia USA USA USA Australia The Netherlands Canada USA Italy Sweden

0.56 1.79 0.85 0.90 0.44 0.57 0.76 0.85 0.82 0.50 0.54

0.16–1.81 0.72–4.45 0.53–1.37 0.54–1.50 0.29–0.67 — 0.40–1.19 0.55–1.34 0.54–1.23 0.23–1.10 0.33–0.90

0.50 1.66 0.46 0.79 0.47 0.35 0.20 0.62 0.67 0.33 0.54

0.10–2.23 0.64–4.32 0.24–0.86 0.45–1.38 0.24–0.90 — 0.05–0.83 0.37–1.04 0.40–1.06 0.11–0.98 0.26–1.13

medication use. When nonprescribed Aspirin and paracetamol (acetaminophen) were not primarily registered, questions were specifically addressed about possible use. In Sweden, Aspirin and paracetamol can be bought over the counter without a doctor’s prescription (while a low-dose formulation of Aspirin, usually at 75 mg, given for cardiovascular prophylaxis, does need such a prescription!). Information about medication in individuals unable to provide information was obtained through proxies or caregivers. A multibased diagnosis was determined for each individual on a lifetime basis using all three sources of information.101 Except for dementia all diagnoses were classified according to ICD-10.102 A consensus diagnosis of dementia was determined for each individual on a lifetime basis using various tests, interviews, and medical records, and based on DSM-III-R criteria.103 Alzheimer’s disease was diagnosed according to NINCDS/ADRDA104 and vascular dementia according to NINDS/AIREN.105 Intact cognitive level was based on several cognitive tests, including the Mini Mental State Examination (MMSE),106 in the range 26 to 30 for repeated examinations.

9.8 RESULTS Use of various analgesic regimes over the entire study period is shown in Table 9.2. Wave 1 represents the use at baseline (1991–1994) and Waves 2, 3, and 4 the subsequent 2-year follow-ups. There is a remarkable increase in the use of low-dose Aspirin, in Sweden officially recommended as a secondary and later as a primary prophylactic, for coronary and cerebrovascular diseases. Thus, in Wave 4 about 40% were taking Aspirin, usually 75 mg/day. In 131 cases treated with NSAIDs on some occasion, 42 had taken naproxen, 24 diclofenac, and 18 ibuprofen.

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TABLE 9.2 Prevalence of Various Analgesic Regimes over the Study Period Wave 1 (N ⫽ 702) 1991–94

Aspirin.total Aspirin.occasional Aspirin.low-dose Aspirin.prescrib.500 NSAID Naproxen Paracetamol Dextropropoxyphene

Wave 2 (N ⫽ 623) 1994–96

Wave 3 (N ⫽ 438) 1996–98

Wave 4 (N ⫽ 315) 1998–2001

n

%

n

%

n

%

n

%

175 77 90 10 54 25 143 69

24.9 11.0 12.8 1.4 7.7 3.6 20.4 9.8

182 67 111 6 56 19 165 73

29.0 10.7 17.7 1.0 8.9 3.0 26.3 11.6

139 36 106 2 35 11 147 69

31.8 8.2 24.3 0.5 8.0 2.5 33.6 15.8

110 19 91 1 22 6 116 40

51.9 9.0 42.9 0.5 10.4 2.8 54.7 18.9

Abbreviations: Aspirin.total ⫽ Aspirin, all types of regimes; Aspirin.occasional ⫽ Aspirin occasional use; Aspirin.low-dose ⫽ low-dose regimes with a weekly dose lower than 3500 mg; Aspirin. prescrib.500 ⫽ Aspirin 500 mg prescribed once or more than once in a day; NSAID ⫽ all non-aspirin NSAIDs.

The cross-sectional analysis (see Table 9.3), using baseline information, demonstrates an association of Aspirin and non-Aspirin NSAIDs with Alzheimer’s disease, but not for paracetamol and dextropropoxyphene. Thus, there were fewer subjects with Alzheimer’s disease among those identified as Aspirin users, and nonsignificant fewer also for those who had used non-Aspirin NSAIDs. No such risk reduction was seen for vascular dementia. The longitudinal observations (see Table 9.4) show the association of analgesic usage at any time as well as at multiple occasions during the longitudinal observation period. The ORs indicate a protective effect of Aspirin, but nonsignificant for non-Aspirin NSAIDs, with the risk to develop Alzheimer’s disease, and for Aspirin users to retain an intact cognitive function (see Table 9.5). Paracetamol and dextropropoxyphene, used to relieve pain with roughly the same indications as Aspirin, showed no effect. To evaluate whether the association of cognition with various regimens of analgesics, including prophylactic low-dose Aspirin, was influenced by cerebrovascular and cardiovascular diseases, ORs were compared also for cases without stroke and TIA, and alternatively without stroke, TIA, myocardial infarction, angina pectoris, and congestive heart failure. After considering these types of biases, we found even low-dose Aspirin, typically 75 mg/day, to have a possibly protective effect against cognitive decline and development of Alzheimer’s disease at least among the oldest patients. Table 9.5 shows calculations for subjects with intact cognitive function over the time of observation. There is no difference in the Aspirin effect for cases with or without ApoE4.

N

175 77 90 54 25 143 69

Abbreviations: See Table 9.2.

Aspirin.total Aspirin.occasional Aspirin low-dose NSAIDs Naproxen Paracetamol Dextropropoxyphene

5 2 3 2 1 10 9

N.alz

0.31 0.32 0.40 0.48 0.53 0.98 2.17

OR 0.011 ns ns ns ns ns ns

Significance

Alzheimer’s Disease

N 139 66 68 37 17 108 49 4 2 2 1 0 7 6

N.alz ns ns ns ns ns ns

1.01 2.26

Significance

0.55 0.45 0.37 0.48

OR

Alzheimer’s Disease NonStroke or TIA

N 176 77 92 54 25 143 69

15 5 12 7 2 15 14

N.dem

0.73 0.48 1.12 1.10 0.63 0.87 2.05

OR

All Dementia

ns ns ns ns ns ns 0.025

Significance

TABLE 9.3 Association (OR) between Various Analgesics and Alzheimer’s Disease, with and without Cases with a Diagnosis of Stroke, TIA, and Dementia before Inclusion

Aspirin and Alzheimer’s Disease Protection 171

N.alz

30 12 21 16 9 43 14

N

230 116 156 147 82 242 79

Abbreviations: See Table 9.2.

Aspirin.total Aspirin.occasional Aspirin⬍3500 Aspirin.total.24 NSAID Paracetamol Dextropropoxyphene

0.54 0.47 0.65 0.48 0.54 1.09 1.03

OR

Total Sample

0.016 0.023 ns 0.013 ns ns ns

Sign.

N 166 84 111 102 57 170 57 20 7 15 9 5 29 8

N.alz 0.60 0.42 0.82 0.43 0.49 1.43 0.90

OR

NonStroke or TIA

ns 0.042 ns 0.030 ns ns ns

Sign.

95 50 59 58 41 86 33

N

10 5 6 4 4 16 5

N.alz

0.43 0.53 0.52 0.30 0.53 1.78 1.00

OR

NonCardiovasc. Disease

0.054 ns ns 0.029 ns ns ns

Sign.

TABLE 9.4 Association (OR) between Alzheimer’s Disease and Various Analgesics over the Follow-Up Period, Corrected for Vascular Diagnoses

172 Oxidative Stress and Age-Related Neurodegeneration

144 94 81 91 51 155 52

99 66 61 69 31 97 39

N.i1

1.52 1.51 2.08 1.13 0.94 0.88 1.88

OR ns 0.013 0.012 ns ns ns ns

Significance 105 65 70 60 41 129 42

N 77 50 52 45 27 82 32

N.i1 1.88 2.11 1.78 1.81 1.02 0.83 1.86

OR 0.025 0.024 ns ns ns ns ns

Significance

NonStroke or TIA

(*a) Cases with stroke, TIA, myocardial infarction, angina pectoris, congestive heart failure excluded. Abbreviations: See Table 9.2.

Aspirin.total Aspirin.total.24 Aspirin.occasional Aspirin low-dose NSAID Paracetamol Dextropropoxyphene

N

Total Sample

63 37 46 32 31 89 30

N

49 32 38 25 19 55 22

N.i1

2.47 4.28 3.23 2.10 0.78 0.68 1.53

OR

0.012 0.003 0.005 ns ns ns ns

Significance

NonCardiovascular Diseasea

TABLE 9.5 Association (OR) between Intact Cognitive Function (i1) and Various Analgesics over the Follow-Up Period, Corrected for Vascular Diagnoses

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9.9 CONCLUSION The OCTO-Twin study, like several other studies, proved an inhibiting effect of Aspirin on the brain processes leading to cognitive decline and finally to Alzheimer’s disease. Aspirin seems to be comparable with non-aspirin NSAIDs, and it is obvious that the drug has a manifold effect in humans just as various salicylate compounds have in plants. The nonexpensive and extremely well-tested Aspirin has a considerable prophylactic potential for various diseases and ailments of the elderly such as aching, rheumatic disorders, cardiovascular diseases, myocardial infarction, stroke, diabetes, colon cancer, and dementia. The list might be further extended. All the diagnoses above are so serious that prophylactic measures are indicated. If a single drug can achieve this, it is most desirable both for convenience and for reduction of the risk of interactions. Lower dosage, which usually gives an optimized effect, may minimize adverse reactions such as dyspepsia, bleeding tendency, bronchial obstruction and so on. Owing to the early start of subclinical disturbances, prophylactic treatment for diseases among the elderly should start in midlife as already discussed.107 At a time when controlled drug studies of the therapy of dementia 108 question the socio-economic worth of newly introduced pharmaceutics such as donezepil, it is important to conduct such studies with corresponding controlled studies of Aspirin. It seems most plausible that this drug will be shown to have a superior cost–benefit effect and substantially reduce the cost of social and medical care in the elderly. However, to get an optimized prophylactic effect against morbidity during old age, the usage should start early, perhaps at about 50 years of age, which implies a study period through several decades. While we are waiting for such a decisive investigation, which will perhaps never be performed, it seems to be a good idea to try Aspirin to protect the cognitive function and reduce co-morbidity risks. In congruence with plants, humans may also benefit from low concentrations of Aspirin. One of the authors (SEN) reveals: Once a year, I buy one hundred Aspirin tablets at 500 mg for a cost of roughly $15. Every Sunday night I split one tablet in three parts, which I take on Monday, Wednesday, and Friday. Now and then, there is a little bleeding after shaving but I have no experience with other side effects. Sometimes I think of my late colleague, Dr. Craven, and his fate, and also of his 6000 patients and friends and what happened to them.

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78. Thomas T, Nadackal GT, Thomas K. Aspirin and diabetes: Inhibition of amylin aggregation by nonsteroidal anti-inflammatory drugs. Exp Clin Endocrinol Diabetes, 2003; 111:8–11. 79. Thomas T, Nadackal GT, Thomas K. Aspirin and non-steroidal anti-inflammatory drugs inhibit amyloid-β aggregation. Neuropharmacoles Neurotoxicol, 2001; 12:3263–3266. 80. Weggen S, Eriksen JL, Das P, Sagi SA, Wang R, Pietrzik CU, Findlay KA, Smith TE, Murphy MP, Bulter T, Kang DE, Marquez-Sterling N, Golde TE, Koo EH. A subset of NSAIDs lower amyloidogenic Abeta42 independently of cyclooxygenase activity. Nature, 2001; 414:212–216. 81. Prince JA, Feuk L, Gu HF, Johansson B, Gatz M, Blennow K, Brookes AJ. Genetic variation in a haplotype block spanning IDE influences Alzheimer disease. Hum Mut, 2003; 22:363–371. 82. Edland SD. Insulin-degrading enzyme, apolipoprotein E, and Alzheimer’s disease. J Mol Neurosci, 2004; 23:213–217. 83. Janson J, Laedtke T, Parisi JE, O’Brien P, Petersen RC, Butler PC. Increased risk of type 2 diabetes in Alzheimer disease.Diabetes, 2004; 53:474–481. 84. Casper D, Yaparpalvi U, Rempel N, Werner P. Ibuprofen protects dopaminergiv neurons against glutamat toxicity in vitro. Neurosci Lett, 2000; 289:201–204. 85. De Cristobal J, Moro MA, Davalos A, Castillo J, Leza JC, Camarero J, Colado MI, Lorenzo P, Lizasoain I. Neuroprotective effect of aspirin by inhibition of glutamate release after permanent focal cerebral ischaemia in rats. Neurochemistry, 2001; 79:456–459. 86. Acanfora D, Trojano L, Iannuzzi GL, Furgi G, Picone C, Rengo C, Abete P, Rengo F, CHF Italian Study. The brain in congestive heart failure. Arch Gerontol Geriatr, 1996; 23:247–256. 87. McGeer PL, Schulzer M, McGeer EG. Arthritis and anti-inflammatory agents as possible protective factors for Alzheimer’s disease: A review of 17 epidemiologic studies. Neurology, 1996; 47:425–432. 88. Williams PS, Rands G, Orrel M, Spector A. Aspirin for vascular dementia. Cochrane Database Syst Rev, 2000; CD001296. 89. Breitner JC, Gau BA, Welsh KA, Plassman BL, McDonald WM, Helms MJ, Anthony JC. Inverse association of anti-inflammatory treatments and Alzheimer’s disease: initial results of a co-twin control study. Neurology, 1994; 44:227–232. 90. Henderson AS, Jorm AF, Christensen H, Jacomb PA, Korten AE. Aspirin, antiinflammatory drugs and risk of dementia. Int J Geriat Psychiatry, 1997; 12:926–930. 91. Stewart WF, Kawas C, Corrada M, Metter EJ. Risk of Alzheimer’s disease and duration of NSAID. Neurology, 1997; 48:46–47. 92. Beard CM, Waring SC, O´Brien PC, Kurland LT, Kokmen E. Nonsteroidal antiinflammatory drug use and Alzheimer’s disease: a case-control study in Rochester, Minnesota, 1989 through 1984. Mayo Clin Proc, 1998; 73:951–955. 93. Anthony JC, Breitner JC, Zandi PP, Meyer MR, Jurasova I, Norton MC, Stone SV. Reduced prevalence of AD in users of NSAIDs and H2 receptor antagonists; The Cache County study. Neurology, 2000; 54:2066–2071. 94. Broe GA, Grayson DA, Creasey HM, Waite LM, Casey BJ, Bebbett HP, Brooks WS, Halliday GM. Anti-inflammatory drugs protect against Alzheimer disease at low doses. Arch Neurol, 2000; 57:1586–1591.

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Disposition of 10 The Lipid-Derived Carbonyls in Alzheimer’s Disease Matthew J. Picklo, Sr. University of North Dakota Grand Forks, North Dakota

CONTENTS 10.1 10.2

Introduction ............................................................................................181 Formation of Lipid-Derived Carbonyls..................................................182 10.2.1 α, β -unsaturated Aldehydes ....................................................182 10.2.2 γ -keto Aldehyde Prostanoids ...................................................183 10.3 Lipid-Derived Carbonyls and AD ..........................................................184 10.4 Mechanisms of Detoxification ...............................................................185 10.4.1 Phase I Metabolism..................................................................186 10.4.1.1 Reductive Enzymes...................................................186 10.4.1.2 Oxidative Enzymes ...................................................186 10.4.2 Phase II Metabolism ................................................................187 10.5 Potential Therapeutic Avenues ...............................................................189 Acknowledgments .............................................................................................190 References .........................................................................................................190

10.1 INTRODUCTION Alzheimer’s disease (AD) is a multifaceted neurodegenerative disease that, in most instances, does not have a clear cause. Numerous studies have examined the cellular, biochemical, and molecular changes that occur as a result of AD pathology. Oxidative stress and the excessive formation of chemically reactive species can be one of the pathways that link the biochemical changes and cellular dysfunction observed in AD.1,2 The elevated formation of chemically reactive species including lipid-derived carbonyls, peroxynitrite, and advanced glycation endproducts is well documented in AD.3–12 A wealth of data has been collected concerning the biochemical, neurotoxic, and cell signaling aspects of lipid-derived

181

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carbonyls. These studies have been reviewed elsewhere.8,9,12–14 In this chapter, the formation and metabolism of lipid-derived carbonyls will be discussed.

10.2 FORMATION OF LIPID-DERIVED CARBONYLS The central nervous system (CNS) is enriched in polyunsaturated fatty acids such as arachidonic acid and docosahexaenoic acid (DHA). Nonenzymatic lipid peroxidation is a chain reaction process in which a single oxidation event can lead to the damage of many molecules of polyunsaturated fatty acids. This process results in the generation of multiple chemically reactive carbonyls, including α, β-unsaturated aldehydes and γ-keto aldehydes. In addition, as detailed below, enzymatic oxidation of lipids also leads to the formation of γ -ketoaldehydes.

10.2.1 α, β -UNSATURATED ALDEHYDES Trans-4-hydroxy-2-nonenal (HNE; see Figure 10.1) has gained the most attention with respect to AD pathogenesis. HNE is a product of the nonenzymatic oxidation of arachidonic acid and linoleic acids.15 The exact chemical mechanism of HNE production is still not clarified although multiple pathways have been postulated.15 Trans-4-hydroperoxy-2-nonenal (HPNE) appears to be a precursor for HNE.16 The C4 carbon of HNE is stereogenic. (S)-HNE is preferentially detoxified by glutathione-S-transferases (GST) and inactivates glyceraldehyde-3-phosphate dehydrogenase more rapidly than (R)-HNE.17,18 Data from our laboratory demonstrate that (R)-HNE is selectively metabolized by respiring rat brain mitochondria.19 Studies by Schneider and coworkers20,21 have examined the stereochemistry of HNE formation, and have shown that (S)-HNE arises from (S)-hydroxy and (S)-hydroperoxy derivatives of arachidonate.20 Incubation of microsomal lipids in a test tube leads to a racemic mixture of HNE.22 However, in a biological system, others factor may influence the presence of one enantiomer over another. For example, it is not known whether hydroperoxy fatty acids are reduced by glutathione (GSH) peroxidases in a stereoselective manner. HNE, like other α, β -unsaturated aldehydes, possess an electrophilic C3 carbon (see Figure 10.1). The aldehyde moiety is an electron-withdrawing group that OH

O O

Trans-4-hydroxy-2-nonenal (HNE)

O Trans-4-oxo-2-nonenal (4-ONE) O

O 2-Propenal (Acrolein)

COOH O

OH

One of 64 isoketal regioisomers

FIGURE 10.1

Highly reactive lipid-derived carbonyls.

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enhances the strength of the C3 electrophilic center. The C4 hydroxyl group further increases its electrophilic nature. The C3 carbon of HNE forms Michael adducts with primary amines (e.g., ε-amino group of lysine), secondary amines (histidine), and thiols (cysteine). Michael adducts can condense resulting in a cyclic hemiacetals that are in equilibrium with the open-chain aldehydes.23 The C1 aldehyde is critical to the toxicity and reactivity of HNE. Oxidation of this aldehyde to a carboxyl group prevents cytotoxicity and protein adduction.24 Similarly, reduction of the aldehyde to a primary alcohol, yielding trans-1,4-dihydroxy-2-nonene, prevents protein adduction.24 HNE is a bifunctional adduction agent that forms an imine via the reaction of the aldehyde and a primary amine. The imine adduct can cyclize, resulting in a 2pentylpyrrole.23,25,26 [13C]-NMR studies using 13C-labeled HNE demonstrated that about 20% of HNE adducts are imines.23 HNE induces protein–protein crosslinks potentially through a pyrrole–pyrrole reaction or a 2-pentyl-2-hydroxy-1,2dihydropyrrol-3-one iminium cross-link.23,27 Besides HNE, other α, β -unsaturated aldehydes are generated from lipid peroxidation. 4-Hydroxyalkenal derivatives are detected that are still attached to the phospholipid backbone.28 In addition to HPNE, trans-4-oxo-2-nonenal (4-ONE) is formed, which is reactive with nucleophiles similar to HNE, and also forms adducts with arginine.29,30 Owing to the γ -keto group, 4-ONE has a higher rate of reaction with nucleophiles than HNE, like the isoketals (see below), and easily cross-links proteins.29 2-Propenal (acrolein) is derived from arachidonate and DHA.31,32 Acrolein is several times more reactive than HNE with model proteins and in biological systems.33,34 Like HNE, it is able to form adducts with primary amines, secondary amines, and thiols; however, unlike HNE, two molecule of acrolein react with the ε -amino group of lysine to form N-ε (3-formyl-3,4,-dehydropiperidino)lysine.31 Trans-2-hexenal, trans-2-nonenal, and malondialdehyde are also products of lipid peroxidation.34,35 However, these products exhibit little or no toxicity in neuronal systems.36,37

10.2.2 γ -KETO ALDEHYDE PROSTANOIDS An exciting development in lipid oxidation biochemistry is the finding that nonenzymatic and enzymatic oxidation of arachidonate by cyclooxygenase (COX) can give rise to highly reactive prostanoid-like γ -keto aldehydes termed “isoketals” or “isolevuglandins” (see Figure 10.1).38–40 Isoketals are by-products of H2-isoprostanes (lipid peroxidation mechanism) and PGH2 (COX-mediated oxidation), both of which have a bicyclic endoperoxide structure.40 This endoperoxide rearranges to a series of 64 isoketal isomers. The formation of isoketals from PGH2 is enhanced in lipophilic environments and may actually comprise a major product of COX activity. Because of their structure, isoketals are highly reactive toward amines (100-fold greater than HNE), leading to the formation of pyrroles, lactams, hydroxylactams, and pyrrole cross-links. In addition to protein, isoketals forms adducts with the phosphatidylethanolamine headgroups of phospholipids.41 Similarly, nonenzymatic oxidation of DHA yields γ -ketoaldehydes termed “neuroketals” owing to the enrichment of DHA in the brain.42,43

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The formation of the isoketals through COX-dependent activity forges a relationship between protein adduction, carbonyl formation, and inflammation. Although nonenzymatic oxidation of arachidonate leads to a 0.1% yield of isoketals, between 20 and 70% of enzymatically formed PGH2 may yield isoketals.40 Furthermore, isoketals are also formed as products of myeloperoxidase activity.44

10.3 LIPID-DERIVED CARBONYLS AND AD The protein adducts of HNE, acrolein, and isoketals are immunogenic and this property has aided in assessing which cells are targets of lipid-derived carbonyl toxicity in AD. As detailed below, these studies demonstrate different cellular and subcellular localizations that depend upon the lipid-carbonyl adduct in question and the patient genotype. Several laboratories have demonstrated that the presence of HNE–protein adducts is elevated in diseased areas of brain in patients with AD.6,7,45,46 Studies by Montine and coworkers45,47 show that the cellular localization of specific forms of HNE–protein adducts is dependent upon the APOE4 genotype, a genetic risk factor for AD. HNE–pyrrole adducts, almost exclusively localized to neurofibrillary tangles, were found in all ten patients homozygous for APOE4, but only in one out of nine patients homozygous for APOE3.6,7 On the other hand, reducible hemiacetal protein adducts were found in both genotypes with diffuse cytoplasmic staining.45 In this case, astrocytic immunoreactivity was present in six out of seven patients homozygous for APOE3, but was not found in patients homozygous for APOE4.45 However, studies by Sayre and coworkers46 found no association with the APOE genotype. The immunoreactivity of amyloid plaques for lipid–carbonyl adducts is controversial. Studies by Ando and coworkers45,48 found HNE–protein adduct immunoreactivity in plaques as opposed to those by Montine and coworkers. Immunolocalization of protein-bound carbonyls also failed to label amyloid plaques.4 Immunolocalization of other lipid–carbonyl–protein adducts has been performed. A single-chain antibody recognizing isoketal-amine adducts demonstrate immunoreactivity in almost all hippocampal neurons in sections from patients with AD, whereas there was no labeling in hippocampal sections from control individuals.40 Acrolein-protein adducts have been localized to neurofibrillary tangles, but not to amyloid plaques.49 The effect of APOE genotype was not addressed in these studies. Levels of unreacted lipid-derived carbonyls have also been measured in brain tissue and in CSF. Concentrations of unreacted HNE are doubled in diseased regions of brain and CSF of patients with AD.50,51 One study demonstrated higher levels of HNE in female patients with AD than in male patients with AD.52 Levels of unreacted acrolein are elevated over threefold in the amygdala and hippocampus of patients with AD vs controls.53 In vivo data describing the turnover of arachidonate and DHA resulting from lipid peroxidation in control and pathological situations, however, are lacking. Furthermore, given the chemical reactivity

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or subsequent metabolism (see below) of carbonyls, it is likely that the measurement of the unreacted parent carbonyl represents only a fraction of the amount of lipid-derived carbonyl load at a given time.

10.4 MECHANISMS OF DETOXIFICATION The brain possesses multiple metabolic pathways that potentially act to detoxify carbonyl species (see Figure 10.2). Phase I processes that oxidize or reduce carbonyls are present in neurons and glia. Lipid-derived carbonyls containing electrophilic centers such as the C3 carbon of HNE and acrolein are metabolized by phase II metabolism through GSTs and GSH. A wealth of literature exists regarding the metabolism of lipid-derived carbonyls, particularly HNE, in peripheral organs including liver and heart.54–60 In contrast, less is known about carbonyl metabolism in the brain and how it may be altered in disease. To date, analyses for metabolites of HNE, acrolein, or other lipid-derived carbonyls in the postmortem brain have not been reported. Furthermore, enzymes that detoxify lipid-derived carbonyls are expressed in the CNS with species-, region-, cell-, and organelle-specific expression. For example,

HNE ALDHs

OH O AKRs

GSTs OH

OH

OH

HNE Acid O– ALDHs OH O GSH

GSH GSHNE

∗ AKRs


-glutamyl transpeptidase (
GT) Aminopeptidase N

OH

O– #

OH OH GSH

OH

O CyS

HNE Diol

O– #

OH

OH

O

O

O CyS

∗

OH CyS

FIGURE 10.2 Known and potential metabolites of HNE in the CNS. HNE undergoes phase I and phase II metabolism. Oxidation is catalyzed by aldehyde dehydrogenases (ALDHs) and reduction by aldo-keto reductases (AKRs). Glutathione conjugation can occur by the activity of multiple glutathione-S-transferases (GSTs). GSHNE undergoes subsequent phase I oxidation/reduction or may be subsequently cleaved to cysteinyl adducts. Note that the glutathione and cysteinyl adducts of HNE (*) exist mostly as a hemiacetal whereas the potential adducts containing the carboxylate group (#) can lactonize.

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aldo-keto reductase (AKR) 7A2 is expressed mainly in glial cells in the human cerebral cortex, but is equally expressed in neurons and glia in the rat cerebral cortex.61 Therefore, interpretation and analysis of carbonyl metabolic routes in the brain must be cognizant of these differences.

10.4.1 PHASE I METABOLISM 10.4.1.1 Reductive Enzymes Aldo-keto reductases reduce aldehydes and ketones to primary and secondary alcohols, respectively, in an NADPH-dependent manner. AKR family members associated with the detoxification of lipid-derived carbonyls in the human brain include AKR1A1 (aldehyde reductase), AKR1B1 (aldose reductase), and AKR7A2 (succinic semialdehyde reductase). These enzymes have differing activities toward HNE, acrolein, 4-oxo-nonenal, or other carbonyls.62,63 Immunolocalization studies show that these reductases are present in human brain. However, AKR1A1 expression is not found in frontal cortex, temporal cortex, or hippocampus, but is expressed in cerebellar Purikinje cells.64 AKR1B1 expression is found in most neurons but not in glia.64 AKR7A2 has a varied expression. In human cerebral cortex and hippocampus, it is localized primarily to astrocytes and microglia although approximately 1% of neurons are immunopositive.61 Expression of these AKRs is not altered in AD except for AKR7A2.61,64 Expression of AKR7A2 is elevated in AD as a likely result of glial activation. Senile plaques are heavily immunoreactive to AKR7A2 antisera.61 In cases of dementia, Lewy bodies in the neurons of the substantia nigra are immunopositive for AKR7A2 but not for AKR1B1.61 In part, this may be due to the subcellular localization of AKR7A2 to the Golgi apparatus; however, AKR1B1 is a free cytosolic protein.65 Carbonyl reductase is a member of the short-chain dehydrogenase/reductase family and utilizes 4-oxo-nonenal, but not HNE, as a substrate.66 Carbonyl reductase is localized to neurons and glia in the brain and one study observed an increase in carbonyl reductase content in diseased regions of the brain in individuals with AD.67,68 10.4.1.2 Oxidative Enzymes Oxidation of an aldehyde to its corresponding carboxylic acid protects against imine formation. The oxidation of the C1 aldehyde of HNE, to form 4-hydroxy-trans-2nonenoic acid (HNEAcid), prevents HNE-protein adduct formation by decreasing the electrophilic nature of the C3 carbon and preventing imine formation.24 Multiple enzymes in the brain catalyze aldehyde oxidation. In the rat brain, oxidation of HNE is catalyzed predominantly by the mitochondrial aldehyde dehydrogenases (ALDHs), ALDH2 and ALDH5A.24,69,70 These two enzymes are NAD+-dependent and are localized to the mitochondrial matrix. Although responsible for the oxidation of acetaldehyde in the liver, ALDH2 is more known for its metabolism of aldehydes derived from dopamine, norepinephrine, and serotonin

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in the brain. ALDH5A has a narrower substrate specificity. ALDH5A is also known as succinic semialdehyde dehydrogenase (SSADH) and plays a critical role in γ -aminobutyric acid (GABA) metabolism.71–73 Although HNE is a substrate for these enzymes, extended incubation of ALDHs with HNE eventually leads to modification of the active-site cysteine of the enzyme, leading to inactivation. Acrolein is not a substrate for ALDHs and is a potent irreversible inhibitor of ALDH2 and ALDH5A.74,75 ALDH2 is expressed in the human brain in a cell- and region-specific manner. In the cerebral cortex and hippocampus, ALDH2 expression is restricted to glia, whereas the dopaminergic neurons of the substantia nigra are immunopositive.64 In rodents, ALDH2 appears to be expressed in glia and neurons,76,77 and ALDH5A in neurons. However, immunolocalization experiments have not been performed on human brain tissue.78,79 ALDH3A is a cytosolic enzyme that is able to utilize HNE and, in contrast to ALDH2 and ALDH5A, utilizes NADP+ as a cofactor.80 NADP +-dependent HNE oxidation is present in the human brain, although immunolocalization of this enzyme in the human brain has not been reported.81 In AD, ALDH2 immunoreactivity is elevated as a result of glial activation with ALDH2 immunoreactivity within the astrocytes and microglia of senile plaques.64 One study suggests that deficiency of ALDH2 activity, such as is found in Oriental populations, is a risk factor for AD.82 These data would indicate that astrocytic aldehyde detoxification is a factor in the development of AD. Knockout of ALDH2 activity in PC12 cells does increase vulnerability to HNE.83 One report found that ALDH5A activity is not altered in patients with AD.84 ALDH5A deficiency occurs in humans, and these individuals have severe neurodevelopmental problems owing in part to excessive (1000-fold greater) levels of γ -hydroxybutyrate.71,72 The extent to which the metabolism of lipid-derived carbonyls is affected in humans with ALDH2 or ALDH5A deficiencies is not known. Owing to their localization to the mitochondrial compartment, the activities of ALDH5A and ALDH2 are limited by the availability of NAD+. The ALDH pathway comprises 40 to 50% of the mitochondrial detoxification of HNE,85 which is indirectly decreased by the presence of Kreb’s cycle substrates and blockade of complex I activity.85 Under these conditions, the NADH/NAD+ ratio is greatly elevated. A 15% inhibition of complex I-linked respiration by rotenone blocks HNEAcid formation by 30%.85 These findings are highly relevant to Parkinson’s disease; complex I activity of the substantia nigra is inhibited 40% in patients with Parkinson’s disease vs control individuals.86 Complex I-linked respiration is sensitive to inhibition by oxidative and nitrative damages.87–89 We hypothesize that in environments of elevated oxidative stress, brain mitochondria are unable to detoxify HNE or other aldehydes efficiently, owing in part to indirect inhibition of ALDH activity, which may lead to further depletion of GSH.

10.4.2 PHASE II METABOLISM Phase II metabolism through the conjugation of GSH to electrophilic carbons is another major detoxification pathway for lipid–derived carbonyl species. GSH

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conjugation is catalyzed by GSTs, of which eight main separate classes are found in humans (alpha, mu, pi, kappa, sigma, theta, omega, and zeta).90,91 Class alpha (GST A), mu (GST M), and pi (GST P) show the most activity toward lipidderived carbonyls but with different substrate specificities.92 GST P1-1 utilizes acrolein more readily than GST A1-1 or GST M1-1. However, the activity of members within a class is dependent upon the carbonyl substrate. GST A4-4 is over 40 times more active toward HNE than GST A1-1.93 GST M1-1 also have high catalytic activity toward HNE.90 The formation of GSH–HNE adduct (GSHNE) is well characterized.56,58,90,92 GSH adducts may be further metabolized to cysteinyl and mercapturyl products and undergo phase I metabolism.56,58,94 The aldehyde group of GSHNE is significantly metabolized to a primary alcohol in the rat heart by aldose reductase.58 GSTs are localized to both neurons and astrocytes in the human CNS.95 GSTMu is localized to neurons and astrocytes and elevated astrocytic GST-Mu expression is noted in diseased regions of brain in AD patients, potentially as a result of astrocyte activation.95 GSTA4-4 is localized predominantly to astrocytes vs neurons in the human cerebral cortex.96 In rat and mouse brain, GSTA4-4 expression is inducible by stimulation of the antioxidant response element pathway and is in part localized to the mitochondrial matrix.97–101 Studies from our laboratory demonstrate that GSH conjugation is a minor route of HNE detoxification in brain mitochondria compared with the oxidation pathway.85 The extent to which the GSH pathway contributes to neuronal detoxification of HNE in control or pathological conditions is not known. Studies by Sidell et al.95 examined the formation of hydrophilic HNE metabolites in human brain following supplementation of human brain homogenate with HNE. These studies suggested that metabolites were formed through the GSH pathway, but did not characterize the metabolites further. The formation of these putative GSH pathway metabolites was elevated slightly in the frontal cortex of patients with AD vs controls. Importantly, this work found that that the capacity to form putative GSH pathway products is not blocked and points to a discrepancy of detoxification capacity and the presence of elevated HNE levels and adducts in AD. Although the ability to conjugate HNE to GSH may not be altered in AD, decreases in GST activities using 1,choro-2,4,-dinitrobenzene (CDNB) or 7chloro-4-nitrobenzo-2-oxa-1,3diazole (NBD-Cl), artificial substrates for multiple GSTs, were found in diseased regions of brain from patients with AD vs controls.102,103 Studies by Lovell and coworkers102 found a decrease in GST M1-1 protein content by immunoblot analysis. Studies by Sultana and Butterfield103 demonstrate that although GST A protein content is more than doubled in the hippocampus of patients with AD, the total GST activity using CDNB as a substrate was almost 40% lower in AD patients vs controls. These authors found that immunoprecipitated GST A from the hippocampi of patients with AD was modified by HNE. These data suggest that lipid-derived carbonyls essentially block their own detoxification. However, it is not known to what extent this decrease in GST activity is rate-limiting regarding the metabolism of electrophilic species in the brain. These data suggest that the detoxification of carbonyls by alternative

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pathways such as oxidation by mitochondrial ALDHs play an important role in the metabolism of lipid-derived carbonyls.

10.5 POTENTIAL THERAPEUTIC AVENUES Protection against the toxicity of lipid-derived carbonyls in neurodegenerative diseases might occur at three levels: by inhibiting carbonyl formation, using carbonyl scavengers, or increasing the expression of the detoxification pathways enzymes. Given that excessive oxidative damage may be a pathological effector of AD pathology, two large clinical studies were implemented to determine whether consumption of the antioxidants α-tocopherol (vitamin E) and ascorbate (vitamin C) may be beneficial for the treatment or prevention of AD.104,105 Supplementation with vitamin E (2000 IU/day for 2 years) delayed but did not stop the progression of disease symptoms in patients with moderate AD.105 The Rotterdam Study demonstrated that high intake of vitamins C and E reduced the risk of developing AD.104 The influence of vitamin E and vitamin C supplementation in decreasing indices oxidative damage in living patients with mild AD was examined by Quinn and coworkers.106 Supplementation with vitamin E and vitamin C decreased the elevation in F2-isoprostanes, products of oxidative damage to arachidonate, in the CSF over a 1-year period. This study did not address therapeutic responses; however, it suggests that the therapeutic benefit noted in the previous larger trials may be through inhibition of lipid peroxidation. Increasing the expression of carbonyl detoxification enzymes is another potential method of protection against carbonyl toxicity. The promoters for multiple carbonyl-metabolizing enzymes such as NAD(P)H:quinone oxidoreductase (NQO1), GST A4-4, those of the GSH synthetic pathway, AKR1B1, and ALDH3A contain antioxidant response elements (AREs). Binding of the Nrf-2 transcription factor to the ARE increases the transcription of multiple cytoprotective proteins as a response to oxidative stress.107,108 Activation of the ARE pathway can occur in astrocytes and neurons, is stimulated by dietary isothiocyanate sulforaphane,100,109 and can protect neurons against mitochondrial toxins.110 Furthermore, astrocytic ARE activation is critical to protection of neurons.100 In part this may be a result of astrocytes supplying GSH precursors to neurons. The use of ARE stimulation in AD, however, may be of limited benefit. As noted before, GST A content is already elevated in the hippocampus of patients with AD.103 In addition, elevated immunoreactivity for NQO1 is found in the hippocampus of patients with AD vs controls.111 These studies suggest that the ARE pathway is already responding to the increased oxidative stress in AD. A third potential avenue is to supply carbonyl scavengers that will chemically react with the carbonyl moiety itself (e.g., a primary amine or hydrazine) or will act as nucleophiles (e.g., thiols and primary and secondary amines) to neutralize the electrophilic C3 carbon of HNE, acrolein, or other α, β -unsaturated aldehydes.112 Obvious candidate scavengers include GSH and its congeners, GSH precursors, or other thiols that directly react with electrophilic carbons.113,114 N-acetylcysteine and its ester derivatives are protective against HNE toxicity in

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multiple in vitro systems.114 γ -Glutamylcysteine ethyl ester protects against Aβ (1-42) toxicity in vitro potentially through elevating intracellular GSH concentrations.113 The amino acid carnosine, which contains a histidine residue, can form an adduct with HNE and potentially with other related aldehydes.115–117 Aminoguanidine also has HNE-scavenging properties.118 Pyrodoxamine is a potent scavenger of the isoketals and a probable scavenger of 4-oxo-nonenal.119 Although these carbonyl scavengers show promise in vitro, extensive preclinical and clinical trials need to be performed.

ACKNOWLEDGMENTS The author thanks Dr. Alena Kubátová for her critical editorial assistance. This work was supported by NIH grants K22 ES 369 ZES1 and P20 RR17699-01.

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61. Picklo, M. J., Sr., Olson, S. J., Hayes, J. D., Markesbery, W. R., and Montine, T. J., Elevation of AKR7A2 (succinic semialdehyde reductase) in neurodegenerative disease, Brain Res., 916, 229, 2001. 62. Doorn, J. A., Srivastava, S. K., and Petersen, D. R., Aldose reductase catalyzes reduction of the lipid peroxidation product 4-oxonon-2-enal, Chem. Res. Toxicol., 16, 1418, 2003. 63. O'Connor, T., Ireland, L. S., Harrison, D. J., and Hayes, J. D., Major differences exist in the function and tissue-specific expression of human aflatoxin B1 aldehyde reductase and the principal human aldo-keto reductase AKR1 family members, Biochem. J., 343, 487, 1999. 64. Picklo, M. J., Olson, S. J., Markesbery, W. R., and Montine, T. J., Expression and activities of aldo-keto oxidoreductases in Alzheimer disease, J. Neuropathol. Exp. Neurol., 60, 686, 2001. 65. Kelly, V. P., Sherratt, P. J., Crouch, D. H., and Hayes, J. D., Novel homodimeric and heterodimeric rat gamma-hydroxybutyrate synthases that associate with the Golgi apparatus define a distinct subclass of aldo-keto reductase 7 family proteins, Biochem. J., 366, 847, 2002. 66. Doorn, J. A., Maser, E., Blum, A., Claffey, D. J., and Petersen, D. R., Human carbonyl reductase catalyzes reduction of 4-oxonon-2-enal, Biochemistry, 43, 13106, 2004. 67. Wirth, H. and Wermuth, B., Immunohistochemical localization of carbonyl reductase in human tissues, J. Histochem. Cytochem., 40, 1857, 1992. 68. Balcz, B., Kirchner, L., Cairns, N., Fountoulakis, M., and Lubec, G., Increased brain protein levels of carbonyl reductase and alcohol dehydrogenase in Down syndrome and Alzheimer’s disease, J. Neural. Transm., 61, 193, 2001. 69. Murphy, T. C., Amarnath, V., Gibson, K. M., and Picklo, M. J. S., Oxidation of 4-hydroxy-2-nonenal by succinic semialdehyde dehydrogenase (ALDH5A), J. Neurochem., 86, 298, 2003. 70. Murphy, T., Amarnath, V., and Picklo, M. J., Oxidation of 4-hydroxynonenal in rat brain slices, Chem. Biol. Interact., 143-144, 101, 2003. 71. Gibson, K. M., Hoffmann, G. F., Hodson, A. K., Bottiglieri, T., and Jakobs, C., 4-Hydroxybutyric acid and the clinical phenotype of succinic semialdehyde dehydrogenase deficiency, an inborn error of GABA metabolism, Neuropediatrics 29, 14, 1998. 72. Gibson, K. M., Sweetman, L., Nyhan, W. L., and Rating, D., Succinic semialdehyde dehydrogenase deficiency, J. Neurogenet., 1, 213, 1984. 73. Ryzlak, M. T. and Pietruszko, R., Human brain glyceraldehyde-3-phosphate dehydrogenase, succinic semialdehyde dehydrogenase and aldehyde dehydrogenase isozymes: substrate specificity and sensitivity to disulfiram, Alcohol Clin. Exp. Res., 13, 755, 1989. 74. Mitchell, D. and Petersen, D., Inhibition of rat liver aldehyde dehydrogenase by acrolein, Drug Metab. Disp., 16, 37, 1988. 75. Nguyen, E. and Picklo, M. J., Inhibition of succinic semialdehyde dehydrogenase activity by alkenal products of lipid peroxidation, Biochim. Biophys. Acta, 1637, 107, 2003. 76. Zimatkin, S. M., Rout, U. K., Koivusalo, M., Buhler, R., and Lindros, K. O., Regional distribution of low-Km mitochondrial aldehyde dehydrogenase in the rat central nervous system, Alcohol Clin. Exp. Res., 16, 1162, 1992. 77. Zimatkin, S., Histochemical study of Aldehyde Dehydrogenase in the Rats CNS, J. Neurochem., 56, 1, 1991.

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78. Chambliss, K. L., Zhang, Y. A., Rossier, E., Vollmer, B., and Gibson, K. M., Enzymatic and immunologic identification of succinic semialdehyde dehydrogenase in rat and human neural and nonneural tissues, J. Neurochem., 65, 851, 1995. 79. Chambliss, K. L., Lee, C. F., Ogier, H., Rabier, D., Jakobs, C., and Gibson, K. M., Enzymatic and immunological demonstration of normal and defective succinic semialdehyde dehydrogenase activity in fetal brain, liver and kidney, J. Inherit. Metab. Dis., 16, 523, 1993. 80. Pappa, A., Chen, C., Koutalos, Y., Townsend, A. J., and Vasiliou, V., Aldh3a1 protects human corneal epithelial cells from ultraviolet- and 4-hydroxy-2-nonenalinduced oxidative damage, Free Radical Biol. Med., 34, 1178, 2003. 81. Murphy, T. C., Poppe, C., Porter, J. E., Montine, T. J., and Picklo, M. J., Sr., 4-Hydroxy-trans-2-nonenoic acid is a gamma-hydroxybutyrate receptor ligand in the cerebral cortex and hippocampus, J. Neurochem., 89, 1462, 2004. 82. Kamino, K., Nagasaka, K., Imagawa, M., Yamamoto, H., Yoneda, H., Ueki, A., Kitamura, S., Namekata, K., Miki, T., and Ohta, S., Deficiency in mitochondrial aldehyde dehydrogenase increases the risk for late-onset Alzheimer’s disease in the Japanese population, Biochem. Biophys. Res. Commun., 273, 192, 2000. 83. Ohsawa, I., Nishimaki, K., Yasuda, C., Kamino, K., and Ohta, S., Deficiency in a mitochondrial aldehyde dehydrogenase increases vulnerability to oxidative stress in PC12 cells, J. Neurochem., 84, 1110, 2003. 84. Gluck, M. R., Thomas, R. G., Davis, K. L., and Haroutunian, V., Implications for altered glutamate and GABA metabolism in the dorsolateral prefrontal cortex of aged schizophrenic patients, Am. J. Psychiatry, 159, 1165, 2002. 85. Meyer, M. J., Mosely, D. M., Amaranth, V., and Picklo, M. J. S., Metabolism of 4-hydroxy-trans-2-nonenal by CNS mitochondria is dependent on age and NAD+ availability, Chem. Res. Toxicol., 17, 1272, 2004. 86. Schapira, A. H., Cooper, J. M., Dexter, D., Jenner, P., Clark, J. B., and Marsden, C. D., Mitochondrial complex I deficiency in Parkinson’s disease, Lancet, 1, 1269, 1989. 87. Murray, J., Taylor, S. W., Zhang, B., Ghosh, S. S., and Capaldi, R. A., Oxidative damage to mitochondrial complex I due to peroxynitrite: identification of reactive tyrosines by mass spectrometry, J. Biol. Chem., 278, 37223, 2003. 88. Riobo, N. A., Clementi, E., Melani, M., Boveris, A., Cadenas, E., Moncada, S., and Poderoso, J. J., Nitric oxide inhibits mitochondrial NADH:ubiquinone reductase activity through peroxynitrite formation, Biochem. J., 359, 139, 2001. 89. Zhang, Y., Marcillat, O., Giulivi, C., Ernster, L., and Davies, K. J., The oxidative inactivation of mitochondrial electron transport chain components and ATPase, J. Biol. Chem., 265, 16330, 1990. 90. Alin, P., Danielson, U. H., and Mannervik, B., 4-Hydroxyalk-2-enals are substrates for glutathione transferase, FEBS Lett., 179, 267, 1985. 91. Hayes, J. D. and Pulford, D. J., The glutathione S-transferase supergene family: regulation of GST and the contribution of the isoenzymes to cancer chemoprotection and drug resistance, Crit. Rev. Biochem. Mol. Biol., 30, 445, 1995. 92. Berhane, K., Widersten, M., Engstrom, A., Kozarich, J. W., and Mannervik, B., Detoxication of base propenals and other alpha, beta-unsaturated aldehyde products of radical reactions and lipid peroxidation by human glutathione transferases, Proc. Natl. Acad. Sci. USA, 91, 1480, 1994. 93. Hubatsch, I., Ridderstrom, M., and Mannervik, B., Human glutathione transferase A4-4: an alpha class enzyme with high catalytic efficiency in the conjugation of

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95.

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Sulfoxide 11 Methionine Reductase System: Possible Roles in Protection against Neurodegenerative Diseases Jackob Moskovitz University of Kansas Lawrence, Kansas

Ashley I. Bush

Harvard Medical School Boston, Massachusetts

CONTENTS Abstract..............................................................................................................199 11.1 Introduction..............................................................................................200 11.2 Protein Aggregation .................................................................................201 11.3 Methionine Oxidation and α-Synuclein ..................................................202 11.4 Soluble Aβ Levels are Closely Linked to Alzheimer’s Disease Pathogenesis ...............................................................................204 11.5 Methionine Oxidation and Amyloid-β ....................................................205 11.6 Methionine Oxidation and Prion .............................................................206 11.7 Conclusion ...............................................................................................207 References .........................................................................................................207

ABSTRACT Damaged and aggregated protein accumulations, mediated by reactive oxygen species (ROS), are associated with the aging process and the development of neurodegenerative diseases. Protein-methionine oxidation is involved in altering the 199

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native structure of certain brain proteins that were found to become toxic to cells owing to their aggregation. Amyloid-β (Aβ ) in Alzheimer’s disease (AD), α-synuclein in Parkinson’s disease (PD), and prion in Creutzfeldt-Jakob disease (CJD) are among the proteins that are targeted for methionine oxidation in vitro. Recently, more evidence is accumulating, suggesting that the methionine oxidation modification in these proteins may be involved in the early events leading to their toxicity. Unlike most oxidative damage to proteins, the methionine oxidation modification can be readily reversed by the methionine sulfoxide reductase (Msr) system. The Msr system is important for cell survival in various organisms when they are exposed to conditions of oxidative stress. Therefore, the capacity of Msr cells to protect neuronal cells from the damaging effects of protein-methionine oxidation accumulation is of special interest. In this chapter, the involvement of methionine oxidation and reversal in neurodegenerative diseases will be discussed.

11.1 INTRODUCTION Accumulative posttranslational modification of proteins, mediated by the action of reactive oxygen species (ROS), is thought to be one of the major causes of aging and age-related diseases. Thus, mechanisms have evolved to prevent or reverse these protein modifications. While most protein damage by ROS is irreversible, methionine oxidation of proteins can be reversed by the methionine sulfoxide reductase (Msr) system (consisting of MsrA [which reduces S-MetO (methionine sulfoxide)] and MsrB [which reduces R-MetO], thioredoxin reductase, thioredoxin, and NADPH).1,2 The action of the Msr system may prevent irreversible protein damage (e.g., protein carbonylation), contribute to cellular antioxidant resistance and, as a result, extend an organism’s life span. Evidence for the possible role of the Msr system in antioxidant defense is demonstrated by the adverse effects of MsrA ablation on several organisms.3–6 Furthermore, overexpression of MsrA in human T cells, plants, and flies protects them from oxidative stress toxicity and leads to an almost doubling of the life span of flies.6–8 A major biological role of the Msr system is suggested by the fact that the MsrA null mouse (MsrA⫺/⫺) is more sensitive to oxidative stress, accumulates higher levels of carbonylated protein, and has a shorter life span (by ~40%) than wild-type mice.5 In addition, selenium and MsrA are positive expression regulators of the selenoprotein form of MsrB and thioredoxin reductase.5,9 Weaned pups of first mouse generation (F1) fed with selenium-deficient (SD) diet exhibited higher protein-MetO and carbonyl levels.9 The protein-MetO and carbonyl levels observed in tissues of young mice fed with SD diet resembles the levels seen in middle-aged mice fed with seleniumadequate diet.9 Therefore, it is suggested that selenium deficiency decreases antioxidant capacity in the cell, which in turn, increases the levels of faulty proteins to the levels observed with older age. Previously, we have shown that exposing mice to 100% oxygen caused a higher elevation of protein-carbonyl adducts in various tissues of MsrA⫺/⫺ in comparison with control mice.5 These results correlated with the loss of MsrA activity in Alzheimer’s disease (AD) brains, while higher levels of protein-carbonyl were observed in AD brains in comparison with

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normal brains.10 Two major proteins that are involved with the toxicity associated with AD and Parkinson’s disease (PD) are amyloid-β (Aβ ) and α-synuclein, respectively. Both proteins have been shown to lose their fibrillation rate owing to MetO,11,12 and in the case of MetO–α-synuclein, to refibrillate in the presence of metal ions and ROS in vitro.13 Moreover, it seems that oxidation of Met35 in Aβ contributes significantly to its toxicity in vitro14–16 and in vivo, as demonstrated by the fact that MetO–Aβ adducts were found postmortem in senile plaques.17 Taken together, the current hypothesis is that the occurrence of neurodegenerative diseases is linked to accumulative damages to proteins, which cause them to unfold and aggregate, thereby interfering with normal neuronal functions and survival under oxidative stress conditions. In this chapter, the possible mechanisms involving MetO in proteins that may enhance the appearance of signs associated with neurodegenerative diseases will be discussed. Special emphasis will be given to the possible involvement of MetO in the toxicity of Aβ in AD. The involvement of MetO in the aggregation of α-synuclein and prion in PD and Creutzfeldt-Jakob disease (CJD), respectively, will also be addressed.

11.2 PROTEIN AGGREGATION Protein aggregation is a basic phenomenon that is commonly observed in proteins that are exposed to various denaturing conditions such as heat, oxidation, pH changes, etc., in vitro. However, the in vivo condition requirements that promote the aggregation of cellular proteins are yet to be defined. Several proteins have been shown to aggregate in vivo as a result of oxidation. Among them are crystallins, Aβ, prion protein, and α-synuclein. The common hypothesis is that protein aggregation is a result of the conformational changes triggered by cellular exposure to ROS. Some of the targeted proteins for oxidation-related aggregation contain MetO residues either in vitro or in vivo. Although other amino acid residues have been shown to be oxidized in the aggregated proteins, except for oxidized cysteine, MetO is the only amino acid that can be repaired by an enzymatic system in vivo. Moreover, methionine oxidation can locally change the physical property of a specific protein region from being hydrophobic to hydrophilic. This change may lead to enhanced structural alteration of a protein upon exposure to oxidation. Therefore, an open question still remains whether methionine oxidation in aggregated proteins is the initial modification causing the aggregation or whether it is a consequence of the aggregation itself. If methionine oxidation plays a pivotal role in the initiation of protein aggregation, it will have a major significance in our understanding of biological processes and pathways that lead to the accumulation of aggregated toxic proteins in cells. The level of neurodegeneration positively correlates with the accumulation of aggregated protein in neuronal cells. Several of these proteins are implicated in the development or progression of neurodegenerative diseases, such as α-synuclein in PD. Therefore, the fact that methionine oxidation is a naturally occurring event that can affect a vast range of proteins broadens its impact on processes that are common in causing protein

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aggregation. The hypothesis based on the role of methionine oxidation in protein aggregation in vivo is described as follows: 1. A native protein is exposed to a high level of ROS that oxidize mainly surface-exposed methionine residues. 2. The linked Msr–thioredoxin systems can reduce the MetO residues to methionine. 3. Failure of the Msr or thioredoxin system leads to protein unfolding and exposure of amino acid residues, which are otherwise hidden in its hydrophobic core. 4. The resulting methionine-oxidized proteins can be divided into two groups: a major group of proteins that require further posttranslational modifications to other amino acids (e.g., carbonylation) that are mediated by metal ions to foster aggregation (e.g., α-synuclein, prion protein, and Aβ ); and a minor group of proteins that are more resistant to unfolding but also susceptible to aggregation owing to extensive methionine oxidation (e.g., crystallins). 5. Accumulation of the aggregating proteins can inhibit the proteasome that is responsible for degrading the faulty proteins prior to their aggregation. Moreover, some of the aggregating proteins can become resistant to degradation. In summary, there is an elevation in the accumulation of aggregating proteins over time that is prompted by enhanced methionine residue oxidation. The massive protein aggregation and inhibition of degradation causes disruption of cellular functions; for example, the cell cycle is impaired owing to insufficient proteasome function. Impairment could be due to the compromised ability of the proteasome to degrade ubiquitinated and oxidized proteins. A secondary effect of the massive protein aggregation is that structural proteins may malfunction owing to their cross-linking to the aggregated proteins. As a result of all the above events, cell death is enhanced. A schematic summary of the events in protein aggregation involving methionine oxidation is presented in Figure 11.1. A strong correlation between methionine oxidation and protein aggregation in vivo may suggest the development of new strategies to repair cellular protein-MetO. One possibility is to enrich the cellular activities of the Msr and thioredoxin system by enhancement of their expression levels. The current knowledge on age-dependent processes causing cellular protein aggregation is still incomplete and poorly understood. However, determining the early steps leading to MetO-mediated protein aggregation in general will greatly contribute to our knowledge about processes that are strongly associated with the appearance of neurodegenerative diseases.

11.3 METHIONINE OXIDATION AND α -SYNUCLEIN The protein α-synuclein is highly expressed in brain and is affected by methionine oxidation in vitro.11,13 Once critical methionines in α-synuclein are oxidized,

Methionine Sulfoxide Reductase System

203 NH2

M

NH2 M COOH

Msr & Trx systems MO

M

ROS

s ion tal n) me actio S & n re RO ento (F

MO

M

Proteasome

NH2

NH2

COOH

(D) COOH NH2 “Small peptides” COOH NH2 + “Free amino-acids”

S

O

R

MO NH2

NH2

M

Tr x

NH2

sr &

MO

sy st em

s

(B1) “Unfolded and oxidized protein”

(A) “Native protein”

M S RO

MO CAR

(C) “Oxidized and aggregated protein”

(B2) “Partially-unfolded and oxidized protein” (E) “Cell death due to accumulation of aggregated proteins”

FIGURE 11.1 Methionine oxidation and its involvement in protein aggregation. A cellular native protein (A) can be exposed to high level of oxidant via ROS production, which in turn oxidizes mainly surface-exposed methionines (M) to form methionine sulfoxides (MO). As a consequence, the methionine-oxidized protein can be either largely unfolded (B1) or partly unfolded (B2). In either case, the methionine sulfoxide reductase (Msr) or the thioredoxin (Trx) systems can reduce the protein-methionine sulfoxides and keep the native protein in its intact form (A). If the protein is part of the B1 category-then its exposure to further oxidation by ROS in the presence of metal ions (Fenton reaction) can result in enhanced modification to its other amino acids, creating protein-carbonyl groups (CAR). Following this oxidation the modified protein may aggregate (C). If the protein is a part of the B2 category (in which the methionine-oxidized protein is more resistant to unfolding)-further oxidation by extensive ROS production (marked with the small arrow) will also lead to the formation of protein aggregates (C). It seems that most proteins will be included in category B1 (emphasized by the thicker arrow directed from the native protein (A) towards the unfolded and oxidized protein (B1). The B1-group protein can be degraded by the proteasome to small peptides and free amino acids (D). However, massive accumulation of oxidized and aggregated proteins may inhibit the proteasome functions. Taking it all together-cell death is enhanced(E).

there is a reduction in its fibrillation capability. According to recent studies performed in vitro, insufficient reduction of these MetO residues by the Msr system in the presence of metal ions can cause further, irreversible, posttranslational modification of α -synuclein, promoting its refibrillation and aggregation.13 It has been proposed that the intact form of α-synuclein may act as an antioxidant in vivo.18 One possibility for its action is the cyclic oxidation (by ROS) and reduction (by the Msr system) of its methionine residues. Supporting evidence for α-synuclein’s antioxidant capability is the fact that overexpression of α-synuclein in mice can protect them from neurological damage triggered by toxic insults.18 However, to date, no direct information is yet available regarding the fate of α-synuclein upon irreversible MetO damage in vivo. A suggestion regarding the role of methionine oxidation in α-synuclein toxicity and aggregation came

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recently from clinical studies: PD is sporadic in most cases, while the familial cases of PD are associated with specific genes’ mutations. The latter includes α-synuclein, parkin, DJ-1, and UCH-L1 (an ubiquitin C-terminal hydrolase). In the case of α-synuclein, the mutated α-synuclein leads to an aberrant accumulation of this protein and its toxic effects, triggering early onset of PD. Although specific mutations of these genes greatly contribute to the development of PD, a recent study has shown that moderate elevation of the wild-type α-synuclein protein in Iowan kindred (an α-synuclein locus triplication with an autosomaldominant form of this disease) causes PD.19 This unexpected finding draws more attention to the possibility that posttranslational modifications to α-synuclein may contribute to the development of PD, especially when its cellular levels are elevated. It is speculated that a failure in reversing MetO modification in α-synuclein may play a pivotal role in its toxicity, caused by its aggregation or mal-folding in vivo. Moreover, it has been shown that α-synuclein accumulates higher levels of carbonyl groups and that its degradation seems to be compromised with age.20 Therefore, it is postulated that a combination of poor protein repair and degradation mechanisms with old age could lead to the aggregation of α-synuclein, thereby enhancing its deleterious effects manifested in PD. If indeed, reversal of methionine oxidation in α-synuclein can prevent its further irreversible damage, it may be a new target for therapeutic intervention aiming at maintaining α-synuclein’s methionines in their reduced form.

11.4 SOLUBLE Aβ LEVELS ARE CLOSELY LINKED TO ALZHEIMER’S DISEASE PATHOGENESIS The pathological hallmarks of AD are marked accumulation of extracellular Aβ protein and intraneuronal neurofibrillary tangles (NFTs) in the neocortex21,22 with accompanying neocortical atrophy. Aβ is produced23–25 by constitutive cleavage of its transmembrane parent molecule, the amyloid protein precursor (APP),26,27 as a mixture of polypeptides manifesting C-terminal heterogeneity — the products of the combined action of proteases, β -28 and γ -secretases.29 The length of the Aβ species is considered to be one important factor since Aβ 1–42 is enriched in amyloid deposits21,25,30,31 and is produced in larger amounts in some, but not all, forms of familial AD (FAD).32,33 The pathogenicity of A β 1–42 has been traditionally associated with its more fibrillogenic properties.34,35 Although excess Aβ 1–42 production and deposition has been implicated as the sole culprit involved in the evolution of amyloid, there are several reasons to suggest that other neurochemical events must contribute to amyloid formation in AD. If elevated cortical Aβ concentrations were solely responsible for the initiation of amyloid, then it would be difficult to explain why the amyloid deposits are focal (related to synapses and the cerebrovascular wall) and not uniform in their distribution. Also, amyloid deposition is an age-dependent phenomenon. Since amyloid deposition does not occur in childhood, even when accelerated in FAD, it is likely that age-related stochastic neurochemical changes play a role in amyloidogenesis.

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In the normal brain, Aβ is found in a membrane-associated insoluble fraction, and soluble Aβ is undetectable.36 Recent findings have suggested that soluble forms of Aβ, elevated in AD, may be most relevant for pathogenesis,3–5 but their origin is uncertain. Soluble Aβ levels correlate with other pernicious features of the neuropathology such as NFTs, and there is even an inverse correlation of soluble brain Aβ with longevity.36 Several groups have described modified (sodium dodecyl sulfate [SDS]-resistant oligomeric) soluble forms of Aβ from AD brain tissue with enhanced toxicity.37–40 However, the nature of the modification of Aβ that converts this normal peptide into an oligomer and toxin is not clear. While self-aggregation has been a prominent theory of Aβ oligomer formation,41 evidence to support intrinsic aggregation in vivo as the cause of AD pathology is not compelling. What is missing is an explanation of how Aβ, as an intrinsically membrane-associated peptide, escapes that compartment and forms increased, soluble and aggregated forms in AD.

11.5 METHIONINE OXIDATION AND AMYLOID-β The brain in AD is under severe oxidative attack.42 Many of the oxidative products of protein, nucleic acid, and lipid attack have been described as elevated in the AD brain and in APP Tg mice. Oxidative stress-handling enzymes (SOD1, heme oxygenase-1, catalase, glutathione peroxidase, and glutathione reductase) are elevated in AD brain.43–45 Aβ itself may contribute to the ROS burden in AD since its toxicity in cell culture has been shown to be largely mediated by H2O259,60 attack and rescued by antioxidants.46–49 It was found that Aβ is unable to generate H2O2 and is therefore not toxic in cell culture unless it is bound to Cu2⫹ or Fe3⫹.46,50,51 Antioxidants may therefore be of therapeutic interest in AD. Curcumin (a potent radical scavenger) has been reported to inhibit amyloid deposition in APP Tg mice.52 Vitamin E (a lipid peroxidation scavenger) has been reported to have clinical benefit in delay nursing home admission in AD,53 and the combination of vitamins E and C has been reported to reduce the risk of AD. The potential benefits of more specific or potent antioxidants await further investigation. Importantly, oxidative stress markers in the brain are markedly elevated before amyloid deposition begins in AD, Down syndrome, and in APP Tg mice.54,55 This is compatible with the observation that vitamin E supplementation inhibits amyloid deposition in young but not aged APP Tg mice.56 The possibility that elevated ROS production is upstream in the pathogenic events that lead to Aβ deposition is supported by recent data that APP Tg mice have accelerated brain amyloid deposition when carrying the heterozygote allele for MnSOD (i.e., MnSOD⫹/ ⫺), an important catalytic superoxide scavenger in mitochondria.57 MnSOD⫹/ ⫺ mice have increased ROS burden, and several reports have shown that antioxidants can ameliorate the adverse effects of the MnSOD⫹/ ⫺ allele in mouse models of brain disease.58 Oxidized forms of Aβ represent nearly 50% of the Aβ in diffuse form and almost 100% of dense core plaque deposits.61 Aβ in plaque has been reported to

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have several side-chain modifications (e.g., isoaspartyl62 and pyroglutamate,62 which can be partially mimicked by Cu2⫹-mediated redox attack.63 Methionine oxidation denatures proteins and converts the hydrophobic properties of methionine into hydrophilic properties causing structural alterations. The sole methionine at residue 35 of Aβ is particularly vulnerable to oxidation (forming MetO or a sulfone). This modification is abundant in the species of Aβ purified from postmortem AD brain, where it has been shown to be complexed with Zn2⫹ and Cu2⫹ in aggregates.17 This is remarkable, because MetO-Aβ is highly soluble, and suggests that reaction with Cu2⫹ and Zn2⫹ has fostered the aggregation. Oxidative stress increases Aβ release into cell culture media64 and accumulation within neuroblastoma cells.65 Gain of hydrophilicity is probably the reason why MetO-Aβ cannot remain embedded in the lipid of the membrane, perhaps explaining why MetO (which still produces H2O2) is toxic.66 One intriguing possibility in preventing this oxidation may be mediated by the Msr system that may enable retaining the Aβ in its normal structure, thereby preventing Aβ deposition. Oxidation of Met35 (MetO) of Aβ alters Aβ oligomerization, inhibits its fibrillation,12 and causes it to dissociate from the membrane and become redox-active (in vitro).67 In the normal brain, Aβ is found in a membrane-associated insoluble fraction, and soluble Aβ is undetectable. Gain of hydrophilicity caused by Met-Aβ oxidation may explain its release from the membrane and MetO toxicity. MetO modification in proteins is a reversible process that is mediated by the Msr system.2,68 MsrA levels and activity are significantly decreased in AD, making MetO-Aβ more likely to be generated in brains of patients with AD.10,17 On the basis of these observations, we propose the hypothesis that methionine oxidation inappropriately releases Aβ from neuronal membrane and leads to enhanced amyloid deposition in vivo. Therefore, oxidative stress and deficient mechanisms for MetO repair (by the Msr system) may be critical upstream pathogenic events in AD.

11.6 METHIONINE OXIDATION AND PRION The normal prion protein (PrP[c]) is transformed to its pathogenic isomer (PrP (Sc)) probably through conformational changes involving posttranslation modifications.69 Like amyloid-β peptide, PrP(c) can bind copper70,71 and has a particular Met129 that is surface-exposed. A polymorphism (M129V) at amino acid 129 of the prion protein results in either a methionine residue (Met) or a valine residue (Val). This polymorphism may affect the susceptibility of an individual to develop sporadic or acquired CJD.72 A higher frequency of 129 Met-homozygotesis, in certain populations, has been found to be a risk factor for developing CJD.72–74 One possible explanation for the importance of methionine in the progression of the disease is that oxidation of Met129 in the presence of copper can produce free radicals that may further oxidize PrP(c) and lead to its enhanced aggregation and toxicity to neuronal cells. So far, only one report has shown that prion’s methionine residues are susceptible to oxidation in the presence of copper in vitro.75 Additionally, oxidation of prion’s histidine residues has been enhanced

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in the presence of copper in vitro, as well.76 However, the consecutive steps leading to prion oxidation in the presence of copper and whether prion’s Met129 and its other methionine residues may play a major role in this cascade of events have still to be determined. It has been suggested that prion may function as an antioxidant77 or serve as a signal for cellular elevation of antioxidants in vivo.78 Therefore, it will be of great interest to find out whether its methionines play a role in its antioxidant capabilities and, if so, how the Msr system is involved in this process. Recent studies have shown that in mice lacking MsrA gene (MsrA⫺/⫺), the expression of the prion protein has been elevated under normoxic and especially under hyperoxic conditions in comparison to their control wildtype mice.79 One possible explanation for this phenomenon could be that the oversensitivity of the MsrA⫺/⫺ mice to oxidative stress is a trigger for the activation of compensatory pathways involving the overexpression of certain antioxidant genes, including PrPc.

11.7 CONCLUSION Methionine oxidation seems to be involved in protein toxicity associated with neurodegenerative diseases. Therefore, determining the conditions and possible treatments promoting the elimination of toxic protein-MetO will greatly contribute to the development of new therapeutic interventions for these diseases.

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Antioxidant 12 Nutritional Enrichment and Improved Cognitive Function in Canines Elizabeth Head and Carl W. Cotman University of California Irvine, California

CONTENTS 12.1 12.2

Introduction ............................................................................................213 The Canine Model of Human Aging ....................................................215 12.2.1 Age-dependent Cognitive Decline in Canines .........................215 12.2.2 Neuropathology in Aged Canines.............................................215 12.3 Treatment With an Antioxidant-Enriched Diet Improves Cognition in Aged Canines ....................................................................216 12.4 Summary ................................................................................................217 Acknowledgments .............................................................................................218 References .........................................................................................................218

12.1 INTRODUCTION An important goal of aging research is to identify interventions that will result in the preservation of cognitive function throughout the life span of an individual. This goal is becoming more critical as our population ages, particularly because there are a rising number of individuals affected by Alzheimer’s disease (AD), one of the most common causes of dementia in the elderly. Age is the biggest risk factor for the development of AD. This suggests that neurobiological changes associated with aging may leave the brain vulnerable to AD-associated neurodegeneration. The brain consumes 20% of the body’s oxygen, which results in the normal production of free radicals that can damage lipids, proteins, and nucleotides.1 Neurons are nondividing and are vulnerable to cumulative oxidative damage.2 One of the primary sources of free radicals within neurons is from mitochondria, which become less efficient with age, leading to increased oxidative 213

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damage.1,3,4 These observations have led to the hypothesis that a diet rich in antioxidants (e.g., vitamins E and C), the use of antioxidant supplements, and dietary enrichment with mitochondrial cofactors (e.g., lipoic acid, carnitine) may be protective against AD and promote successful aging.3,5,6 In human observational studies, a positive effect of antioxidant supplementation on cognition7–13 in the elderly has been reported. Further, antioxidant supplement use also reduces the risk of developing AD and slows cognitive decline associated with AD.10,14–17 Supplementation with the use of single antioxidants such as vitamins E or C alone may have more limited effectiveness than a combination of antioxidants on cognition or risk of development of AD.7,13,16 Moreover, intake of antioxidants through food (e.g., dietary phytochemicals18) may be superior to supplements (i.e., pills) in human studies (i.e., tablets).10 However, not all study outcomes have been positive; several studies report a lack of significant beneficial effects of antioxidant supplements on cognition or the development of AD.19–21 Differential outcomes in observational studies may reflect sample biases. For example, women more frequently take supplements than men, and more highly educated individuals more often take supplements. There is also the possibility that antioxidants obtained through daily diet may exceed those obtained through supplements and are not included in calculations of antioxidant intake. Outcomes of different epidemiological studies may also depend on critical factors such as the type of population being studied, and the validity and clinical relevance of the outcome parameters. Studies in animals under more tightly controlled conditions provide a means to test the hypothesis that antioxidants improve cognition and reduce oxidative damage to the brain. In rodents, antioxidants in the form of supplements or contained within fruits and vegetables, can improve learning, memory, and motor and neuronal functions, and reduce oxidative damage (e.g. 22–25). Vitamin E and blueberry extracts rich in polyphenolics can significantly improve behavior and brain function26 in rats. More recently, treatment with curcumin, a spice that has antioxidant properties, reduced oxidative damage and neuropathology in a transgenic mouse model of AD, Tg2576 mice.27 The majority of the antioxidants explored in these previous studies were not directed at the mitochondria specifically, where free radicals are generated. Thus, several researchers have been exploring antioxidant strategies that are specifically directed toward improving mitochondrial function.28–30 Mitochondrial cofactors, particularly α-lipoic acid, carnitine, and coenzyme Q, have all been shown to be effective in targeting mitochondria and reducing ROS production both in tissue culture and in animal models.5,28–33 It is hypothesized that with age, many of the enzymes in the mitochondrial respiratory chain have reduced affinity for cofactors (i.e., an increased Km) and according to this concept, cofactor loading improves the efficiency of the electron-transport system (ETS) and reduces ROS production.6 Mitochondrial cofactors alone can markedly improve cognitive function, reduce oxidative damage, and improve mitochondrial function in rodents.6,29,31,32 However, rodents do not naturally develop some forms of neuropathology observed in human brain aging, suggesting the need for extension

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to transgenic mouse models34,35 or higher mammalian model systems.36,37 In most rodent experiments, dose levels of individual antioxidant components are increased approximately tenfold to account for differences in metabolic rate. This leads to difficulties in translating the outcomes of animal studies for future human clinical trials.

12.2 THE CANINE MODEL OF HUMAN AGING Despite successful modeling of numerous specific aspects of aging, no single animal model has fully replicated all aspects of human aging. Thus, it is necessary to continue to develop other models, to take advantage of unique aspects of each model, and to combine information from convergent studies. In contrast to rodents, canines (dogs) can be provided with similar if not lower doses of antioxidants than those used in human clinical trials, thus allowing a more direct translation of intervention studies to human aging. Our group has been studying a canine model of brain aging that naturally develops neuropathology and cognitive dysfunction similar to that observed in humans.37,38

12.2.1 AGE-DEPENDENT COGNITIVE DECLINE

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CANINES

Aged canines show declines in both simple and complex learning tasks including size concept learning,39,40 oddity discrimination learning,41,42 and size discrimination learning.43,44 Tasks sensitive to prefrontal cortex function, including reversal learning and visuospatial working memory, are also age-sensitive.43–45 A measure of spatial attention, originally developed in nonhuman primates, is also age-sensitive.46,47 In addition to learning deficits with age, aged canines have difficulty with object recognition memory tasks.48 Spatial memory appears to decline at an earlier age than object recognition memory.49,50,51 Spatial memory deficits may not only reflect deficits in memory, but also an impaired ability to modify an existing strategy.52 Possibly and more importantly, old canines can be categorized into three groups: (1) successful agers, (2) learning impaired canines, whose scores fell 2 standard deviations above the mean of the young animals and (3) severely impaired canines, who fail to learn the task.37 This clustering of aged canines on the basis of cognitive ability is consistent with aging in rats and nonhuman primates.53–57 Cognitive dysfunction is also not an inevitable consequence of aging in humans.58 Recent research has focused on the distinction between those that retain function and those that show a decline such as mild cognitive impairment (MCI).59,60 Our work with the aged-canine model suggests that aged canines show both mild and relatively severe cognitive impairments that may be consistent with MCI in humans.

12.2.2 NEUROPATHOLOGY

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AGED CANINES

In parallel with cognitive decline, aged canines also progressively accumulate human-type neuropathology. AD is characterized by the presence of senile

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plaques and neurofibrillary tangles.61 Although canines do not naturally develop neurofibrillary tangles, they appear to model early AD-like senile plaque formation observed in human brains.62–64 Senile plaques contain the β -amyloid (Aβ ) peptide, which has an identical amino acid sequence in canines and humans.65 Specific brain regions show differential vulnerabilities to Aβ deposition, and the canine parallels the aged human brain in this respect.66–73 Aβ deposition occurs at an earlier age in the prefrontal cortex and at a later age in temporal and occipital cortices. It is critical to note that initial Aβ deposits occur in a 3- to 4-year window of time between the ages of 6 and 9 years, which suggests that longitudinal studies for evaluating interventions to slow or halt human-type Aβ in canines are feasible. In contrast to nonhuman primates, the canine primarily accumulates the longer, more toxic species of Aβ1–42 similar to humans, whereas in monkeys, the primary species is the less toxic and soluble Aβ1–40.74 Age and cognitive status can predict Aβ pathology in discrete brain structures. For example, canines with reversal learning deficits show significantly higher amounts of Aβ protein in the prefrontal cortex, an area critical for proficient reversal learning.75 On the other hand, canines deficient on a size discrimination learning task, thought to be sensitive to temporal lobe function, show large amounts of Aβ deposition in the entorhinal cortex.43 Thus, cognitive decline in aged canines is associated with the accumulation of Aβ as it is in humans.43,76–83 As with humans and other animals, the canine brain accumulates oxidative damage with age. Lipid peroxidation and protein oxidation rises with age along with a corresponding decrease in endogenous antioxidant activity.84–87 If oxidative damage leads to neuronal dysfunction and subsequent cognitive decline, then we hypothesize that providing aged canines with antioxidants may reverse or slow pathological aging.

12.3 TREATMENT WITH AN ANTIOXIDANT-ENRICHED DIET IMPROVES COGNITION IN AGED CANINES To determine if reducing oxidative damage in the brain will lead to improvements in cognitive function, we have completed a longitudinal study of aging in canines. We hypothesized that an antioxidant diet would improve cognition in aged dogs and also maintain cognitive function. A group of 48 aged and 17 young beagle dogs were used in this study. All dogs were given a standardized set of baseline cognitive tests to evaluate learning (discrimination learning), executive function (reversal learning), and spatial memory (nonmatching to sample). Subsequently, animals were placed into either an antioxidant-enriched diet treatment group or served as controls counterbalanced for sex and baseline cognitive ability. The antioxidant diet was enriched with DL-α-tocopherol acetate (20 mg/kg), L-carnitine (6 mg/kg), DL-α-lipoic acid (2.7 mg/kg), and ascorbic acid in the form of Stay-C (2 mg/kg). In addition, 1% inclusions of each of the following (1 to 1 exchange for corn) — spinach flakes, tomato pomace, grape pomace, carrot granules, and citrus pulp — were added, which was approximately equivalent to going from 3 to 5–6 servings of fruits and vegetables/day. The control diet was formulated to meet the

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nutrient profile recommended for adult canines by the Association of American Feed Control Officials.88 Animals were tested for visuospatial attention46 within 2 weeks of the start of the study. Aged but not young animals improved significantly with the antioxidant-enriched diet,46 and animals on the enriched diet learned significantly faster than the animals on the control diet. After 6 months on treatment with the antioxidant diet, aged canines receiving the antioxidant treatment showed significantly improved learning on an oddity discrimination task relative to untreated aged controls, particularly when the problem became more difficult.41 Subsequently, after 1 year on treatment, animals were tested on a size discrimination learning and reversal task.39,43,44 This task was intended to be more difficult than the baseline discrimination task to prevent floor effects. After 2 years on the treatment, canines were given an intensity discrimination learning and reversal task, which we have previously found to be equivalent in difficulty to the size task. Over time, untreated animals showed progressive increases in error scores on the discrimination and reversal learning tasks. This is one of the few studies in the aging literature on a higher mammalian model showing decline in cognition over time.89 The antioxidant diet improved the performance of old canines in both the size discrimination and reversal tasks.90 One year later, intensity discrimination learning also improved significantly.89 This suggests that at least one prefrontaldependent task (reversal learning) can be improved with the appropriate intervention. Further, whereas control diet-fed animals showed a progressive decline in learning ability over time, error scores from antioxidant-treated animals were consistently lower. These results suggest that the antioxidant-enriched diet reduced the rate of cognitive decline in aged dogs. Performance on a spatial memory task was also evaluated each year of the study beginning with baseline testing. During the year 1 retest, the treatment groups did not differ. In year 2, however, the control animals’ performance showed deterioration, while performance of the animals on the enriched diet gradually improved.37 The improved performance overall is not surprising, given that animals are repeatedly tested on the same task over 3 years. The impairment seen in the controls, however, is likely due to age-related degenerative changes that were prevented when the canines were treated with an antioxidant-enriched diet. Thus, these results are consistent with the suggestion that antioxidants may be involved with neuroprotection and possibly promote neuroplasticity.

12.4 SUMMARY Progressive oxidative damage occurs in the brain with age in humans and in animals. This has led to the hypothesis that antioxidant supplementation may be beneficial for brain aging. Studies in canines may complement existing observational studies in humans and laboratory studies using rodents. Canines naturally develop impairments in learning and memory along with the progressive accumulation of human-type Aβ and oxidative damage. Our results using the canine

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model strongly support the hypothesis that a diet enriched in both cellular antioxidants and mitochondrial cofactors can improve age-associated cognitive decline, can maintain cognitive function, and may, over time, reverse memory decline. In combination, studies in humans and animals suggest that dietary antioxidants and mitochondrial cofactors may promote healthy brain aging.

ACKNOWLEDGMENTS Funding provided by NIH/NIA AG12694 and U. S. Department of the Army, Contract No. DAMD17-98-1-8622. Hills Pet Nutrition, Topeka, KS, supported some animals for the cognitive testing portion of the study.

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Tea and Resveratrol 13 Green as Protective Agents against Neurotoxins Stéphane Bastianetto, Han Ying-Shan and Quirion Rémi Douglas Hospital Research Center Montreal, Canada

CONTENTS Abstract..............................................................................................................225 13.1 Introduction ............................................................................................226 13.2 In Vitro Neuroprotective Effects.............................................................227 13.2.1 Resveratrol ...............................................................................227 13.2.2 Epigallocatechin Gallate ..........................................................228 13.3 In Vivo Neuroprotective Effects .............................................................228 13.3.1 Resveratrol ...............................................................................228 13.3.2 Epigallocatechin Gallate ..........................................................230 13.4 Conclusion..............................................................................................231 Acknowledgments .............................................................................................232 References .........................................................................................................232

ABSTRACT Recent findings from epidemiological studies support the view that polyphenols play a key role in the purported beneficial effects of natural extracts, food, and beverages in age-related neurological disorders such as stroke, Parkinson’s, and Alzheimer’s diseases. Among the numerous dietary constituents present in the plant kingdom, resveratrol and epigallocatechin gallate (EGCG) — two polyphenols that are abundant in red wine and green tea, respectively — have been the subjects of intensive investigation in various in vitro and animal models of toxicity. It is well established that antioxidant actions contribute to their neuroprotective activities, but these actions do not fully explain their overall benefits. Hence, more recent studies suggest that these polyphenols modulate various signal transduction pathways and the expression of genes that are associated with cell 225

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death/survival. In this chapter, we first review the neuroprotective properties of EGCG and resveratrol in cell culture and animal models. We then discuss the potential mechanisms underlying their global neuroprotective effects.

13.1 INTRODUCTION Epidemiological studies indicate that a moderate consumption of red wine and green tea may lower the incidence of dementia1–6 or Parkinson’s disease.7–9 Most of the in vitro and animal studies have aimed to study at the possible contribution of the green tea polyphenol known as epigallocatechin gallate (EGCG) and the red wine constituent resveratrol to the purported beneficial effects of these substances in humans. EGCG is the most abundant green tea flavanol whereas resveratrol (trans-3,4⬘,5-trihydroxystilbene) is a naturally occurring stilbene that is particularly found in grape skin (Figure 13.1).10 Recent in vitro and a limited number of in vivo studies have demonstrated that resveratrol and EGCG exert neuroprotective effects against various models of toxicity induced by oxidative stress,11–17 β -amyloid peptides (Aβ ),16,18,19 and hypoxia/ischemia,20–30 suggesting that they contribute to the purported beneficial effects of red wine and green tea.16,31–33 Interestingly, recent studies raised the hypothesis that the inhibition of free radicals is not the sole mechanism underlying neuroprotective effects of these molecules, and may involve their purported modulatory effects on various intracellular enzymes (e.g., protein kinase C [PKC], extracellular signal-regulated kinases [ERK1], mitogen-activated protein kinase [MAPK], heme oxygenase 1 etc.) as well as on the expression of various genes and transcription factors, including nuclear factor-kappaB (NF-κB).19,25,29,31,34–39 These data are of particular interest in the clinical context, given that these intracellular effectors may play a role in neurodegenerative processes occurring, for example, in AD and stroke. O O O

O

O O

O O O

O O O

O O Resveratrol

Epigallocatechin gallate

FIGURE 13.1 Chemical structures of resveratrol and epigallocatechin gallate.

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We review here the most recent in vitro and animal studies supporting the neuroprotective effects of these polyphenols. We also discuss the potential mechanisms of their neuroprotective actions, and the possible relevance of these studies to the treatment of neurodegenerative diseases.

13.2

IN VITRO NEUROPROTECTIVE EFFECTS

13.2.1 RESVERATROL Data from cultured neuronal cells studies have shown that resveratrol possesses protective effects against toxicities induced by oxidative stress, Aβ, and glutamate. Using rat primary hippocampal cell cultures, we have found that resveratrol (1–25 µM) was able to protect and even reverse hippocampal cell death induced by the nitric oxide (NO) donor sodium nitroprusside (SNP). These protective effects were accompanied by the inhibition of reactive oxygen species (ROS) accumulation (but not PKC activation) stimulated by SNP, suggesting that resveratrol’s neuroprotective action results mainly from its antioxidant properties.12 Using the electron paramagnetic resonance spin-trapping technique, Karlsson et al.11 demonstrated that the free radical-scavenging properties of resveratrol were responsible for its neuroprotective effects against toxicity induced by the prooxidant tert-butyl hydroperoxide in rat mesencephalic dopaminergic neurons. Han et al.19 found that resveratrol (20 µM) protected hippocampal neurons before, during, or after exposure to Aβ (Aβ25–35, Aβ1–40, and Aβ1–42). Pretreatment with a PKC inhibitor (GF 109203X) — but not inhibitors of other kinases — blocked the effect of resveratrol, suggesting that PKC is involved in the protective effects in this model of toxicity. Similarly, data from human neuroblastoma cells reported that resveratrol (15 µM) protected against cell death produced by Aβ25–35, this effect being accompanied by its ability to enhance the antioxidant protein glutathione.16 Using the same types of neuronlike cells, Nicolini et al.38 showed that the neuroprotective effect of trans-resveratrol against paclitaxel-induced apoptosis is not due to its antioxidant action but to its ability to reverse the phosphorylation of stress-activated protein kinase/cJun N-terminal kinase (SAPK/JNK) as well as the inhibition of the activation of caspase 7 and the degradation of poly(ADP-ribose)polymerase. Moosmann and Behl40 reported that resveratrol protected mouse neuronal HT22 cells against glutamate toxicity and SK-N-MC cells against H2O2 toxicity at a concentration (20 µM) that induced estrogen-responsive element (ERE)-directed gene transcription. Finally, De Ruvo et al41 showed that resveratrol displayed antiapoptotic effects in primary cultures of cerebellar granule neurons exposed to glutamate. Interestingly, these authors showed that both trans- and cis-resveratrol (5–200 µM) did not exert any protective effects against apoptosis induced by low K+ concentrations. According to this study, the lack of effect may be explained by the inability of resveratrol to reach intracellular sites generating ROS and to block caspase 3 activity. Interestingly, resveratrol (5–100 µM) was also shown to be able to significantly induce heme oxygenase 1 in primary cortical neuronal

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cultures.37 These authors raised the hypothesis that the increase in heme oxygenase activity induced by resveratrol is a molecular pathway underlying its neuroprotective actions.37

13.2.2 EPIGALLOCATECHIN GALLATE Numerous in vitro studies have focused on the most abundant green tea ingredient, EGCG (which constitutes up to 25% of the total extract).42 For example, EGCG (5–10 µM) was shown to protect neuronal cells against excitoxicity through the inhibition of AMPA-induced intracellular Ca2+ increase and the reduction of the production of malondialdehyde (MDA, a molecule that reflects polyunsaturated fatty acid peroxidation), suggesting an involvement of its antioxidant and free radical-scavenging properties.14,17 EGCG also attenuated neuroblastoma SH-SY5Y apoptotic cell death induced by 3-hydroxykynurenine (3-HK) and blocked increase in ROS production and caspase activities produced by 3HK.43 Using the same type of cells, Levites et al.36 reported that EGCG (1 µM) exerted neuroprotective action against cell death induced by 6-OHDA, possibly by inhibiting the induction of proapoptotic genes such as Bax, Bad, gadd45, and the fas ligand, whereas the expression of antiapoptotic effectors was unaltered. These authors also found that EGCG was able to restore reduced PKC and ERK1/2 activities induced by 6-OHDA, while not affecting on its own the levels of this kinase.36 Interestingly, EGCG (10 µM) was also shown to protect hippocampal neuronal cells against the toxic effects of Aβ25–35.18 These effects were associated with a decrease in the levels of MDA and caspase activity. However, EGCG failed to modulate proapoptotic (i.e., p53 and Bax), antiapoptotic (i.e., Bcl-XL), and proinflammatory proteins (i.e., cyclooxygenase, COX), suggesting that its effects were attributable to its ROS-scavenging properties in this model of toxicity.18 Our previous data from the same types of cultured neuronal cells indicated that treatment with EGCG (10 µM) exerted neuroprotective effects against Aβ peptides (Aβ25–35 and Aβ1–42), possibly through the inhibition of the formation of Aβ oligomers. Using the dichlorofluorescein (DCF) assay, we also found that there was no correlation between the antioxidant activities of green tea flavanols, such as epicatechin and EGCG, and their neuroprotective capacities, indicating that ROS inhibition is not directly involved in the neuroprotective effects of EGCG, at least in our model.44 Finally, Youdim’s group showed that the neuroprotective effects of EGCG against Aβ peptides (Aβ25–35 and Aβ1–42) involved PKC pathway45 (see Table 13.1).

13.3 IN VIVO NEUROPROTECTIVE EFFECTS 13.3.1 RESVERATROL Animal models also reported potent neuroprotective effects of peripheral administration of resveratrol in rodent models of ischemia, excitotoxicity, and Alzheimer’s disease (AD), suggesting that this compound is able to cross the

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TABLE 13.1 Summary of In vitro and In vivo Neuroprotective Effects of Resveratrol Models of Toxicity In vitro models Glutamate, H2O2 Tert-butyl hydroperoxide Glutamate Sodium nitroprusside Paclitaxel

Aβ25–35 Aβ peptides Animal models Kainic acid Focal ischemia Ischemia Streptozotocin

Biological Activities Induction of ERE-directed gene transcription Inhibition of free radicals Attenuation of intracellular reactive oxygen species accumulation Reversal of the sustained phosphorylation of JNK/SAPK, inhibition of the activation of caspase 7 and degradation of poly(ADP-ribose)polymerase Enhancement of glutathione Activation of PKC Reduction in MDA brain levels Decrease in levels of MDA and reduced glutathione Possible stimulation of the expression of PPARα Rise in brain glutathione and decrease in brain MDA levels

References (40) (11) (41) (12) (38)

(16) (19) (22) (24) (25) (15)

ERE ⫽ estrogen-responsive element; JNK ⫽ c-Jun N-terminal kinase; SAPK ⫽ stress-activated protein kinase; PKC ⫽ protein kinase C; MDA ⫽ malondialdehyde; PPARα ⫽ α isotype of peroxisome proliferator-activated receptors.

blood-brain barrier. Hence, chronic administration of resveratrol (8 mg/kg) partially but significantly protected rat olfactory cortex and hippocampus against kainic acid-induced toxicity.20 Moreover, pretreatment with trans-resveratrol (20 mg/kg, i.p. for 21 days) prevented motor impairments and decreased the volume of infarct in rat subjected to focal ischemia.24 These protective effects of trans-resveratrol were accompanied by a decrease in the levels of MDA and reduced glutathione, suggesting a role for its antioxidant activities.24 Similar protective effects were observed in a gerbil model of transient, global, cerebral ischemia.23 Chronic treatment with resveratrol (30 mg/kg, i.p.) during and after the occlusion of common carotid arteries decreased hippocampal neuronal cell death, as observed 4 days later.23 These authors also found that resveratrol (30 mg/kg, i.p.) could be detected in the brain, reaching a peak at 4h, providing direct evidence for its capacity to cross the blood-brain barrier, at least in rodents.23 Resveratrol was also able to reduce both the incidence of convulsions and MDA brain levels produced by kainic acid in mice.22 These effects were seen upon chronic administration of kainic acid (20 and 40 mg/kg, i.p., 5 min prior to

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and 30 and 90 min after kainite), but no effect was observed after only a single administration of 40 mg/kg i.p., 5 min prior to kainate).22 Moreover, resveratrol (20 mg/kg/day, 3 days) reduced infarct volume by 36% after middle cerebral artery occlusion in wild-type mice.25 It was also demonstrated that resveratrol requires the expression of the α isotype of peroxisome proliferator-activated receptors (PPARα, a nuclear hormone receptor family of ligand-dependent transcription factor) to exert its neuroprotective action, as it failed to protect against cerebral ischemia in PPARα gene knockout mice.25 A co- and post- treatment with the resveratrol derivative named oxyresveratrol (trans-2,3⬘,4,5⬘-tetrahydroxystilbene, 10–20 mg/kg, i.p.) reduced by about half brain infarct volume in MCAO rats.29 Histological analysis revealed that oxyresveratrol diminished cytochrome c release and decreased caspase-3 activation in treated rats.29 Finally, Sharma and Gupta15 reported that trans-resveratrol treatment (10 and 20 mg/kg, i.p., 21 days) significantly prevented against cognitive impairments induced by streptozotocin in a model of sporadic dementia in rats. This effect was associated with a rise in brain glutathione and decreased brain levels of MDA.15

13.3.2 EPIGALLOCATECHIN GALLATE EGCG shared the ability to prevent mice striatal dopamine depletion and substantia nigra dopaminergic neuronal losses induced by MPTP with green tea extracts.13 These effects may be explained by its free radicals and iron-chelating properties as well as its purported ability to stimulate the activity of the two antioxidant enzymes, superoxide dismutase and catalase.13 Moreover, a peripheral administration of EGCG was shown to protect against neuronal toxicity induced by ischemia,17, 26–28, 30 supporting the hypothesis that EGCG (or its metabolites) can cross the blood-brain barrier.46 Hence, gerbils treated with EGCG (25–50 mg/kg, i.p.) just after transient global ischemia displayed reduction in hippocampal CA1 neuronal damage.26 According to Lee et al., the ability of EGCG to reduce xanthine oxidase activity could be the primary mechanism of action, explaining its neuroprotective effects as well as its antioxidant activities and inhibition of the expression of inducible nitric oxidase synthase (iNOS).26 Similar protective effects — as evidenced by a reduction in the infarct volume, brain edema, and hippocampal neuronal losses — were observed in gerbils or rats treated before or after injury with EGCG (50 mg/kg, i.p.).17,27,30 These effects were associated with a reduction in brain MDA and putrescine levels, as well as reduction in the level of the oxidized/total glutathione ratio.17,27,30 Taken together, these results suggest that EGCG’s antioxidant action and, to a lesser extent, its modulatory effect on polyamine metabolism, contribute to its neuroprotective properties.17,27,30 Finally, Nagai et al.28 reported the inhibitory effect of a pretreatment with EGCG (50 mg/kg, i.p.) on rat hippocampal NO concentrations following ischemia, without any effects on cerebral blood flow. According to these authors, the reduction in NO levels in EGCG-treated rats is due to its reducing effects on NO⫺3 /NO⫺2 conversion from NO and not its inhibitory action on NOS28 (see Table 13.2).

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TABLE 13.2 Summary of In vitro and In vivo Neuroprotective Effects of EGCG Models of Toxicity In vitro models Excitoxicity 3-Hydroxy kynurenine 6-OHDA

Aβ25–35 Aβ peptides Aβ peptides Animal models MPTP

Ischemia Ischemia Ischemia

Biological Activities Reduction in intracellular [Ca2+] and MDA levels Attenuation of the increase in the ROS production and caspase activities Inhibition of the induction of proapototic genes bax, bad, gadd45, and fas ligand. Reversal of decline in ERK1/2 Decrease in levels of MDA and caspase activity. Inhibition of the formation of Aβ oligomers Modulation of PKC activity Blockade of free radicals and iron Elevation of the activity of superoxide dismutase and catalase in mice Reduction in xanthine oxidase and iNOS activities Reduction in brain MDA and putrescine levels, attenuation of oxidized/total glutathione ratio level Inhibition of rise in hippocampal NO concentrations

References (14,17) (43) (36)

(18) (44) (45) (13)

(26) (17,27,30) (28)

MDA ⫽ malondialdehyde; ROS ⫽ reactive oxygen species; PKC ⫽ protein kinase C; iNOS ⫽ inducible nitric oxidase synthase.

13.4 CONCLUSION Taken together, these data strongly suggest that the neuroprotective effects of resveratrol are mainly due to direct (free radical-scavenging activities) and indirect (activation of endogenous antioxidant enzymes) effects. Recent studies, in particular, those on PC12 cells, also reveal resveratrol’s antioxidant activities are unlikely to be the sole explanation of its neuroprotective properties as resveratrol is also able to modulate various signaling pathways. As for resveratrol, recent studies suggested that the antioxidant activities of EGCG significantly contribute to its neuroprotective effects, in addition to various intracellular pathways associated to cell death/survival. Considering the role of oxidative stress and intracellular effectors in neurodegenerative processes, it is likely that resveratrol and EGCG contribute to the purported beneficial effects of food and beverages (e.g., red wine, green tea) in slowing down age-related neuropathological processes. However, additional in vivo studies are necessary to establish the influence of these polyphenols on the expression of various genes and proteins with regard to their neuroprotective effects. Proteomics and genomics approaches could prove most useful in that regard. It will also be of particular interest to investigate the biological and behavioral effects of resveratrol, EGCG, and other natural products in transgenic mouse models of AD,

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in order to obtain further insight into their possible beneficial impacts in this disease. This may also help to design more effective neuroprotective agents to prevent and treat neurodegenerative diseases.

ACKNOWLEDGMENTS This work was supported by research grants from the Canadian Institutes of Health Research (CIHR) to R. Quirion.

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16. Savaskan, E. et al. Red wine ingredient resveratrol protects from beta-amyloid neurotoxicity. Gerontolology., 49, 380, 2003. 17. Lee, H., Bae, J.H. and Lee, S.R. Protective effect of green tea polyphenol EGCG against neuronal damage and brain edema after unilateral cerebral ischemia in gerbils. J. Neurosci. Res., 77, 892, 2004. 18. Choi, Y.T. et al. The green tea polyphenol (-)-epigallocatechin gallate attenuates beta-amyloid-induced neurotoxicity in cultured hippocampal neurons. Life Sci., 70, 603, 2001. 19. Han, Y.S. et al. Neuroprotective effects of resveratrol against beta-amyloidinduced neurotoxicity in rat hippocampal neurons: involvement of protein kinase C. Br. J. Pharmacol., 141, 997, 2004. 20. Virgili, M. and Contestabile, A. Partial neuroprotection of in vivo excitotoxic brain damage by chronic administration of the red wine antioxidant agent, transresveratrol in rats. Neurosci Lett., 281, 23, 2000. 21. Huang, S.S. et al. Resveratrol reduction of infarct size in Long-Evans rats subjected to focal cerebral ischemia. Life Sci., 69, 1057, 2001. 22. Gupta, Y.K., Chaudhary, G. and Srivastava, A.K. Protective effect of resveratrol against pentylenetetrazole-induced seizures and its modulation by an adenosinergic system. Pharmacology, 65, 170, 2002. 23. Wang, Q. et al. Resveratrol protects against global cerebral ischemic injury in gerbils. Brain Res, 958, 439, 2002. 24. Sinha, K., Chaudhary, G. and Gupta, Y.K. Protective effect of resveratrol against oxidative stress in middle cerebral artery occlusion model of stroke in rats. Life Sci., 71, 655, 2002. 25. Inoue, H. et al. Brain protection by resveratrol and fenofibrate against stroke requires peroxisome proliferator-activated receptor alpha in mice. Neurosci. Lett., 352, 203, 2003. 26. Lee, S., Suh, S. and Kim, S. Protective effects of the green tea polyphenol (-)-epigallocatechin gallate against hippocampal neuronal damage after transient global ischemia in gerbils. Neurosci. Lett., 287, 191–194, 2000. 27. Lee, S.Y. et al. Effects of delayed administration of (-)-epigallocatechin gallate, a green tea polyphenol on the changes in polyamine levels and neuronal damage after transient forebrain ischemia in gerbils. Brain Res. Bull., 61, 399, 2003. 28. Nagai, K. et al. (-)-Epigallocatechin gallate protects against NO stress-induced neuronal damage after ischemia by acting as an anti-oxidant. Brain Res., 956, 319, 2002. 29. Andrabi, S.A. et al. Oxyresveratrol (trans-2,3⬘,4,5⬘-tetrahydroxystilbene) is neuroprotective and inhibits the apoptotic cell death in transient cerebral ischemia. Brain Res., 1017, 98, 2004. 30. Choi, Y.B. et al. Protective effect of epigallocatechin gallate on brain damage after transient middle cerebral artery occlusion in rats. Brain Res., 1019, 47, 2004. 31. Bastianetto, S. and Quirion, R. Resveratrol and red wine constituents: evaluation of their neuroprotective properties. Pharm. News, 8, 33, 2001. 32. Sun, A.Y., Simonyi, A. and Sun, G.Y. The “French Paradox” and beyond: neuroprotective effects of polyphenols. Free Radical Biol. Med., 32, 314, 2002. 33. Weinreb, O. et al. Neurological mechanisms of green tea polyphenols in Alzheimer’s and Parkinson’s diseases. J. Nutr. Biochem., 15, 506, 2004. 34. Tredici, G. et al. Resveratrol, map kinases and neuronal cells: might wine be a neuroprotectant? Drugs Under Exp. Clin. Res., 25, 99, 1999.

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35. Miloso, M. et al. Resveratrol-induced activation of the mitogen-activated protein kinases, ERK1 and ERK2, in human neuroblastoma SH-SY5Y cells. Neurosci. Lett., 264, 141, 1999. 36. Levites, Y. et al. Involvement of protein kinase C activation and cell survival/ cell cycle genes in green tea polyphenol (-)-epigallocatechin 3-gallate neuroprotective action. J. Biol. Chem., 277, 30574, 2002. 37. Zhuang, H. et al. Potential mechanism by which resveratrol, a red wine constituent, protects neurons. Ann. N. Y. Acad. Sci., 993, 276, 2003. 38. Nicolini, G. et al. Effect of trans-resveratrol on signal transduction pathways involved in paclitaxel-induced apoptosis in human neuroblastoma SH-SY5Y cells. Neurochem. Int., 42, 419, 2003. 39. Mandel, S. et al. Cell signaling pathways in the neuroprotective actions of the green tea polyphenol (-)-epigallocatechin-3-gallate: implications for neurodegenerative diseases. J. Neurochem., 88, 1555, 2004. 40. Moosmann, B. and Behl, C. The antioxidant neuroprotective effects of estrogens and phenolic compounds are independent from their estrogenic properties. Proc. Natl. Acad. Sci. U.S.A., 96, 8867, 1999. 41. De Ruvo, C. Amodio, R. Algeri, S. Martelli, N. Intilangelo, A. D’Ancona, GM. Esposito, E. Nutritional antioxidants as antidegenerative agents. Int. J. Dev. Neurosci., 18, 359, 2000. 42. Wang, L.F., Kim, D.M. and Lee, C.Y. Effects of heat processing and storage on flavanols and sensory qualities of green tea beverage. J. Agric. Food Chem., 48, 4227, 2000. 43. Jeong, J.H. et al. Epigallocatechin 3-gallate attenuates neuronal damage induced by 3-hydroxykynurenine. Toxicology, 195, 53–60, 2004. 44. Bastianetto, S. and Quirion, R. unpublished data, 2004. 45. Levites, Y. et al. Neuroprotection and neurorescue against Abeta toxicity and PKC-dependent release of nonamyloidogenic soluble precursor protein by green tea polyphenol (-)-epigallocatechin-3-gallate. FASEB J., 17, 952, 2003. 46. Suganuma, M. et al. Wide distribution of [3H](-)-epigallocatechin gallate, a cancer preventive tea polyphenol, in mouse tissue. Carcinogenesis, 19, 1771, 1998.

Stress and 14 Oxidative Aβ PP Processing Tabaton Massimo University of Genoa Genoa, Italy

Tamagno Elena University of Turin Turin, Italy

CONTENTS 14.1 Introduction..............................................................................................235 14.2 Neurodegeneration and AD .....................................................................237 14.3 Oxidative Stress and AD..........................................................................238 14.4 Oxidative Stress and Aβ Overproduction: A Toxic Loop........................238 14.5 Hypothetical Sequence of the Pathogenetic Steps of AD .......................239 14.6 Conclusions..............................................................................................241 References .........................................................................................................243

14.1 INTRODUCTION Alzheimer’s disease (AD) is a neurodegenerative disorder that currently affects nearly 2% of the population in industrialized countries. The risk of AD increases dramatically in individuals above the age of 70, and it is predicted that the incidence of AD will augment three fold within the next 50 years. AD can be classified into two forms: sporadic AD, which accounts for the vast majority of cases, and a rare familial form of AD (FAD), in which gene mutations have been identified. Cortical regions involved in learning and memory processes are reduced in size in AD patients as a result of degeneration of synapses and death of neurons.1 The pathological hallmarks of AD include intraneuronal neurofibrillary tangles (NFT) composed of hyperphosphorylated tau protein, and deposition of β -amyloid plaques (Aβ ) in the extracellular space. The pathological diagnosis of AD is dependent on the detection of neurofibrillary pathology in the form of NFT or neuritic plaques.2,3 Central to the disease is an altered proteolytic processing of the amyloid precursor protein (APP), resulting in overproduction and aggregation of neurotoxic forms of Aβ. APP is an integral membrane protein with a single, membrane-spanning domain; a large, extracellular N-terminus; and a shorter, cytoplasmic C-terminus. Aβ 235

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is generated from the sequential cleavage of APP: a C-terminal membrane-bound fragment of 99 (C99, β CTFs) is produced by the β -secretase cleavage of APP, which is subsequently cleaved within the transmembrane domain4 (Figure 14.1 Panel A). BACE-1 was identified as the β -secretase that cleaves APP within the ectodomain.5–9 BACE-1 is expressed in all tissues, with the highest level of expression in the brain. BACE-1 is an intracellular type I transmembrane protein detected in the trans-Golgi network and endosomes.7 The role of BACE-1 in the generation of Aβ peptides was confirmed by the absence of Aβ peptides in mice with homozygous deletion of BACE-110 (Figure 14.1 Panel B). The importance of Aβ in the pathogenesis of AD is suggested by several clues. Aggregation of Aβ is specifically toxic to cultured neurons in vitro, and it initiates a series of downstream events, including the hyperphosphorylation of tau, which results in neuronal dysfunction and death.11 In addition, all genes that are now known to increase susceptibility to AD, namely the gene for the Aβ PP, (A)

sAPP

APP

A

Bace-1

40/42


Secretase

APP intracellular domain Nucleus Modulation of gene expression (B) Lumen Pro-peptide

G

Aspartyl protease motif

G

G

G

Aspartyl protease motif

Cytoplasm

Dileucine motif

FIGURE 14.1 (A) Amyloidogenic process of the amyloid precursor protein (APP). The amyloidogenic pathway involves BACE-1 (β -site APP cleaving enzyme) activity, which releases βAPPs, and, after cleavage of the C-terminal fragment by γ secretase, Aβ peptides. Aβ 42 peptide is prone to aggregation and formation of amyloid plaques, accumulation of which is the primary factor driving AD pathogenesis. (B) BACE-1 activity. Sites for potential N-linked glysosylation are indicated (G). Aspartyl protease signature sequences are boxed and their relative locations in the polypeptide are shown. The extreme C-terminus of the molecule is also enlarged to demonstrate the dileucine motif that has been implicated in the endosomal targeting of the protein.

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located on chromosome 21; the gene for apolipoprotein E (apoE), on chromosome 19; and the genes for presenilin 1 (PS1) and presenilin 2 (PS2), on chromosomes 14 and 1, respectively, modulate various events of Aβ production, aggregation, and clearance.12 Mutations in the cited genes, as well as the presence of the allele ε 4 of apoE, lead to accumulation of the longer form of Aβ, ending at residue 42.13 The causes of altered APP processing and Aβ accumulation in sporadic cases of AD are not known, but are likely to encompass a series of age-related events, such as oxidative stress, impaired energy metabolism, and perturbed calcium homeostasis. This issue, i.e, the mechanisms of Aβ accumulation in late-onset, sporadic AD, is a crucial need to identify potential molecular targets for the development of therapies for the large majority of AD cases.

14.2 NEURODEGENERATION AND AD Apoptosis controls cell populations in normal development, inflammation, and cancer. Excessive apoptosis is viewed to play a role in a number of neurological disorders in addition to AD, including stroke and Parkinson’s disease (PD).14 Evidence for frank cellular apoptosis in AD brain is controversial, but there is growing recognition that apoptotic mechanisms may contribute to neurodegeneration in the absence of overt apoptosis.15 The first clue arguing in favor of apoptosis in the pathogenesis of AD came from experiments in vitro, that showed the induction of programmed cell death in neurons exposed to fibrillar Aβ. Recent evidence suggests that it is not the fibrillar form of Aβ that has toxic properties, but rather small aggregates of Aβ, including protofibrils and oligomers (also referred to as Aβ -derived diffusible ligands or ADDLs).16,17 An important body of data suggests that the neurotoxic properties of Aβ are mediated by oxygen free radicals. In in vitro systems, cells exposed to Aβ generate increased levels of hydrogen peroxide18 that in the presence of transition metal give rise to destructive hydroxyl radicals.19,20 Induction of oxygen free radicals may be initiated by the binding of Aβ to cell surface receptors such as the receptor for advanced glycation end products (RAGE),21 although involvement of other receptors or receptor-independent mechanisms may also be implicated.22 In addition, impaired Ca2⫹ homeostasis that appears to follow oxidation of Ca2⫹ membrane pumps23 has been demonstrated after exposure of cells to Aβ.24 Among various adverse effects on neurons,25 increased intracellular Ca2⫹ leads to activation of calmodulin-dependent nitric oxide synthase and increased intracellular nitrous oxide (NO),26 which in turn reacts with superoxide anions to form peroxynitrates. These nitrates can exist in activated transitional forms (ONOO−) with reactive potentials comparable with those of hydroxyl radicals. The demonstration of widespread nitration of proteins in brains affected with AD27,28 suggests the involvement of ONOO− radicals. Although the contribution from each of the potential sources of free radicals to the overall degree of oxidative damage is not certain, exposure of cells to Aβ causes oxidation of key cellular components, which culminates in profound metabolic changes and death of neurons.

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14.3 OXIDATIVE STRESS AND AD The free radical hypothesis of aging29 claims that the age-related accumulation of reactive oxygen species (ROS) results in damage of the major cell components: nucleus, mitochondrial DNA, membranes, and cytoplasmic proteins. Some authors suggest that the imbalance between the generation of free radicals and antioxidants may be the primary cause of neurodegenerative diseases. The fact that age is the most important risk factor of sporadic AD provides considerable support for the free radical hypothesis. There is increasing evidence that the very earliest neuronal and pathological change in AD brain is oxidative damage,30–33 indicating that oxidative stress represents a very early contributor to the disease. A series of findings support this hypothesis. One line of evidence is the presence of oxidative markers that colocalize with the neuropathological lesions of AD. The identification of antioxidant enzymes, key indicators of oxidative stress, was the first documented evidence of oxidative neurotoxicity in the AD lesions.34,35 Abnormal expression of these markers of oxidation was observed associated with senile plaques, NFTs, and vulnerable neurons. These included heat shock proteins,36,37 lysosomal enzymes,38 increased protein carbonyls,39 and lipid peroxides.40 Involvement of oxygen free radicals in AD is also supported by the identification of increased products of lipid peroxidation,41 mitochondrial42 and nuclear DNA lesions,43 and neuronal membrane damage in AD brains.44

14.4 OXIDATIVE STRESS AND Aβ OVERPRODUCTION: A TOXIC LOOP Oxidative stress and Aβ are linked to each other since Aβ aggregation induces oxidative stress in vivo and in vitro45 and oxidants increase the production of Aβ.46,47 While there are observations that Aβ may initiate oxidative stress at micromolar concentrations and may reduce the antioxidant defense system, there is also some evidence that, in turn, oxidative stress increases the expression of APP and thus induces increased formation of Aβ peptides.45,46 During the process of aggregation Aβ generates hydrogen peroxide, a process that requires oxygen and that is greatly potentiated by Fe2⫹ and Cu⫹.48,49 Lipid peroxidation induced by Aβ impairs the function of ion-motive ATPases, glucose and glutamate transporters, and also GTP-binding proteins as the result of covalent modification of the proteins by the aldehyde 4-hydroxynonenal (HNE).50 On the other hand, multiple lines of evidence demonstrate that oxidative stress is an early event in AD, occurring prior to cytopathology.51 Indeed, a systematic examination of the spatiotemporal relationship between the presence of oxidative modification and the hallmark of AD lesions at early stages of the disease suggests that many markers of oxidative damage are present in susceptible neurons even when they lack neurofibrillary pathology.52 The availability of a transgenic mouse (Tg2576) that overproduces human Aβ peptides from birth and develops Aβ plaques at 6–8 months of age represents a

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unique in vivo model to address questions on the role of oxidative stress in triggering Aβ deposition. Apelt and collaborators53 demonstrated that the age-related occurrence of various parameters of oxidative stress in transgenic mice was correlated with the development pattern of Aβ production as well as activities and expression of the β secretase (BACE-1), the limiting enzyme for Aβ production.

14.5 HYPOTHETICAL SEQUENCE OF THE PATHOGENETIC STEPS OF AD We propose a hypothetical sequence of events linking oxidative stress, stressactivated protein kinases, BACE-1 induction, and apoptotic cell death through over production of Aβ (Figure 14.2). We have recently studied the effects of oxidative stress on BACE-1 expression and activity in NT2 neurons. These neurons are derived from a human teratocarcinoma cell line, Ntera 2/c1. Ds, and after treatment with retinoic acid, they are irreversibly committed to a neuronal phenotype. NT2 neurons represent a widely accepted experimental model to study the regulation of APP metabolism and the pathogenesis of AD54,55 and are highly susceptible to pro-oxidant-induced lipid peroxidation.56 We have found that exposure of NT2 neurons to two classic pro-oxidant stimuli such as ascorbate /FeSO4

Oxidative stress

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BACE-1 Overexpression

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FIGURE 14.2 A hypopthetical sequence of the pathogenetic steps of AD linking oxidative stress, stress-activated protein kinases, BACE-1 induction, and apoptotic cell death through overproduction of Aβ.

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500 µM/5 µM or H2O2/FeSO4 10 µM/100 µM as well as HNE 1–5 µM significantly increased BACE-1 expression, protein levels, and activity without affecting the levels of full-length precursor protein. Notably, these events are followed by a significant production of intracellular Aβx-42 as well as by morphological signs of apoptotic cell death (Figure 14.3). Our findings are confirmed by other authors who show that H2O2 oxidative stress caused a marked increase in BACE1 expression.57 The direct relationship between oxidative stress and BACE-1 induction is confirmed by the preventive effects exerted by antioxidant compounds such as α-tocopherol, the most important chain-breaking antioxidant known to block the generation of aldehydic end products of lipid peroxidation.58 The same results have been observed by exposure of neurons to dehydroepiandrosterone (DHEA), which was able to rescue expression, protein levels and activity of BACE-1 induced by oxidative stress through its antioxidant properties.59

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FIGURE 14.3 Exposure of NT2 neurons to two classic pro-oxidant stimuli such as ascorbate/FeSO4 500 µM/5 µM or H2O2/FeSO4 10 µM/100 µM as well as 4-hydroxynonenal 1–5 µM significantly increased BACE-1 expression (A), protein levels (B), and activity (C), without affecting the levels of full length precursor protein (D). Notably these events are followed by a significant production of intracellular Aβx-42 (E) as well as by morphological signs of apoptotic cell death (F). 쐒 : Significantly different from control cells (p⬍0.05).

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The cellular signaling pathways that mediate this event are unknown. Recently, Christensen and collaborators60 showed that transcription factor Sp1 plays an important role in regulating BACE-1 gene expression, providing the first clue on the molecular mechanisms by which human BACE-1 gene expression is regulated. Mitogen activated protein kinase (MAPK) family members, including extracellular signal regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and P38MAPK, have been proposed to be important signaling components linking extracellular stimuli to cellular responses. JNK and P38 MAPK (also known as stress-activated protein kinases, SAPKs) are highly activated in response to a variety of stress signals, including proinflammatory cytokines, ultraviolet radiation, and oxidative stress.61 Activation of SAPK is often associated with induction of apoptosis. We showed that SAPK signaling pathways modulate the expression of BACE-1 induced by oxidative stress. Indeed, oxidative stress-dependent activation of JNK and P38MAPK signaling pathways is needed for the over expression of BACE-1, since specific pharmacological inhibition of these two pathways results in the abrogation of up-regulation of BACE-1 protein levels as well as in a significant decrease in Aβx-42 production and apoptotic cell death (Figure 14.4). Notably, activation of SAPK family members has been described in AD brain tissue, and they correlate with oxidative stress. JNK is not only activated but also redistributed,62 from nuclei to the cytoplasm, concomitantly with the progression of the neurofibrillary pathology. Moreover, JNK is strongly activated in mutant APP transgenic mice with extensive oxidative damage, but not in mutant APP transgenic mice with little oxidative damage.63 The activation of P38MAPK signaling in AD is also well documented. P38MAPK are increased significantly after ischemic injury in APP over expressing mice,64 indicating a role in oxidative, stress-induced cell damage. The essentially identical staining pattern for phospho-JNK and phospho-P38MAPK in AD cases suggests that JNK and P38MAPK are activated by the same signal.65

14.6 CONCLUSIONS Alterations in gene expression and enzyme activity induced by oxidative stress are mediated through the interplay of multiple signaling pathways. In neuronal cells, potentially deleterious stimuli such as free radicals provoke an intracellular stress response that either leads to apoptosis or to defensive-protective adaptations. SAPK and their downstream effectors are the major molecules involved in the bipartite response, which can lead to either neurodegeneration or neuroprotection, depending on the cellular and environmental conditions as well as cooperation with other signaling pathways.66 In this scenario, BACE-1 over expression mediated by oxidative stress may shift the signaling equilibrium toward cell damage and apoptosis. According to this view, neuronal apoptotic cell death can depend on a significant increase of BACE-1 activity, and Aβ may be the active player of neuronal damage induced by oxidative stress through activation of SAPK cascade.

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FIGURE 14.4 Pretreatment of NT2 neurons to pharmacological inhibitor of JNK isoforms (SP600125) or of P38MAPK (SB203580) protects the HNE-mediated induction of BACE-1 protein levels (A) as well as the related Aβ x-42 intracellular over production (B) and apoptotic cell death (C). 쐒 : Significantly different from control cells (p⬍0.05).

The relationship between oxidative stress, SAPK pathways, BACE-1, and Aβ outlined in this study supports the hypothesis that the overproduction of Aβ, dependent on the over expression of BACE-1 induced by oxidative stress, contributes to the pathogenesis of the common, sporadic, late-onset form of AD, in which the primary cause of Aβ accumulation is unknown. Because oxidative stress can induce amyloidogenic processing of APP, resulting in accumulation of potentially neurotoxic

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forms of Aβ, this mechanism could contribute to increased Aβ accumulation in the late-onset form of AD. Consistently, Holsinger et al.67 found a significant increase of BACE-1 protein levels and activity in brains of patients affected by sporadic forms of AD. Fukumoto et al.68 delineated the anatomical pattern of BACE-1 dysfunction, observing that BACE-1 activity remains elevated in the temporal cortex throughout the course of the illness. The same results were obtained very recently by Yang et al.69 by using a very sensitive ELISA assay.

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18. Behl, C. et al. Amyloid beta peptide induces necrosis rather than apoptosis. Brain Res., 645, 253, 1994. 19. Behl, C. et al. Hydrogen peroxide mediates amyloid protein toxicity. Cell, 77, 817, 1994. 20. Imlay, J.A., Chin, S.M. and Linn, S. Toxic DNA damage by hydrogen peroxide through the Fenton reaction in vivo and in vitro. Science, 240, 1302,1988. 21. Yan, S.D. et al. RAGE and amyloid-peptide neurotoxicity in Alzheimer’s disease. Nature, 382, 685, 1996. 22. Verdier, Y. and Penke, B. Binding sites of amyloid beta-peptide in cell plasma membrane and implications for Alzheimer’s disease. Curr. Protein Pept. Sci., 5, 19, 2004. 23. Mark, R.J. et al. Amyloid-peptide impairs ion-motive ATPase activities: evidence for a role in the loss of neuronal Ca2⫹ homeostasis and cell death. J. Neurosci., 15, 6239, 1995. 24. Mattson, M.P. et al. Amyloid peptides destabilize calcium homeostasis and render human cortical neurons vulnerable to excitoxicity. J. Neurosci., 12, 376, 1992. 25. Mattson, M.P. et al. Cellular and molecular mechanisms underlying perturbed energy metabolism and neuronal degeneration in Alzheimer’s and Parkinson’s diseases. Ann. N Y Acad. Sci., 893, 154, 1999. 26. Morel, F., Doussiere, J. and Vignais, P.V. The superoxide-generating oxidase of phagocytic cells. Eur. J. Biochem., 201, 523, 1991. 27. Smith, M.A. et al. Widespread peroxynitrite-mediated damage in Alzheimer’s disease. J. Neurosci., 17, 2653, 1997. 28. Good, P.F. et al. Evidence for neuronal oxidative damage in Alzheimer’s disease. Am. J. Pathol., 149, 21, 1996. 29. Harman, D. Free radical theory of aging. Mutat. Res., 275, 257,1992. 30. Nunomura, A. et al. Neuronal oxidative stress precedes amyloid-beta deposition in Down syndrome. J. Neuropathol. Exp. Neurol., 59, 1011, 2000. 31. Nunomura, A. et al. Oxidative damage is the earliest event in Alzheimer’s disease. J. Neuropathol. Exp. Neurol., 60, 759, 2001. 32. Perry, G. and Smith, M.A. Is oxidative stress damage central to the pathogenesis of Alzheimer’s disease? Acta Neurol. Belg., 98, 175, 1998. 33. Perry, G. et al. Comparative biology and pathology of oxidative stress in Alzheimer’s and other neurodegenerative diseases: beyond damage and response. Comp. Biochem. Physiol. Part C Pharmacol. Toxicol., 133, 507, 2002. 34. Pappolla, M.A. et al. Immunohistochemical evidence of oxidative stress in Alzheimer’s disease. Am. J. Pathol., 140, 62, 1992. 35. Furuta, A. et al. Localization of superoxide dismutases in Alzheimer’s disease and Down dyndrome neocortex and hippocampus. Am. J. Pathol., 146, 357, 1995. 36. Calabrese, V. et al. Redox regulation of heat shock protein expression in aging and neurodegenerative disorders associated with oxidative stress: a nutritional approach. Amino Acids, 25, 437, 2003. 37. Kitamura, Y. and Nomura, Y. Stress proteins and glial functions: possible therapeutic targets for neurodegenerative disorders. Pharmacol. Ther., 97, 35, 2003. 38. Omar, R.A. et al. Acid phosphatase in senile plaque and CSF of Alzheimer’s disease patients. Arch. Pathol. Lab. Med., 117, 166, 1993. 39. Smith, M.A. et al. Carbonyl-related posttranslational modification of neurofilament protein in the neurofibrillary pathology of Alzheimer’s disease. J. Neurochem., 64, 2660, 1995.

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40. Montine, K.S. et al. 4-Hydroxy-2-nonenal pyrrole adducts in human neurodegenerative disease. J. Neuropathol. Exp.Neurol., 56, 866, 1997. 41. Subbarao, K.V., Richardson, J.S. and Ang, L.C. Autopsy samples of Alzheimer’s cortex show increased lipid peroxidation in vitro. J. Neurochem., 55, 342, 1990. 42. Mecocci, P. MacGarvey, U. and Beal, M.F. Oxidative damage to mitochondrial DNA is increased in Alzheimer’s disease. Ann. Neurol., 36, 747, 1994. 43. Dickson, D.W. Apoptotic mechanisms in Alzheimer neurofibrillary degeneration: cause or effect? J. Clin. Invest.,114, 23, 2004. 44. De Keyser, J., Ebinger, J. and Vanguelin G. D1-receptor abnormality in frontal cortex points to a functional alteration of cortical cell membranes in Alzheimer’s disease. Arch. Neurol., 46, 761, 1990. 45. Harkany, T. et al. Mechanisms of beta-amyloid toxicity: perspectives of pharmacoteraphy. Rev. Neurosci., 11, 329, 2000. 46. Paola, D. et al. Oxidative stress induces increase in intracellular amyloid β -protein production and selective activation of β I and β II PKCs in NT2 cells. Biochem. Biophys. Res. Commun., 268, 642, 2000. 47. Misonou, H., Morishima-Kawashima, M. and Ihara, Y. Oxidative stress induces intracellular accumulation of amyloid β -protein (Aβ) in human neuroblastoma cells. Biochemistry, 39, 6951, 2000. 48. Cotman, C.W. and Berchtold, N.C. Exercise: a behavioral intervention to enhance brain health and plasticity. Trends Neurosci., 25, 295, 2002. 49. Morgan, D. et al. A beta vaccination prevents memory loss in an animal model of Alzheimer’s disease. Nature, 408, 982, 2000. 50. Mattson, M.P. Cellular actions of beta-amyloid precursor protein and its soluble and fibrillogenic derivatives. Physiol. Rev., 77, 1081, 1997. 51. Zhu, X. et al. Oxidative stress signaling in Alzheimer’s disease. Brain Res., 1000, 32, 2004. 52. Nunomura, A. RNA oxidation is a prominent feature of vulnerable neurons in Alzheimer’s disease. J. Neurosci., 19, 1959, 1999. 53. Apelt, J. et al. Aging-related increase in oxidative stress correlates with developmental pattern of beta-secretase activity and beta-amyloid plaque formation in transgenic Tg2576 mice with Alzheimer-like pathology. Int. J. Devl. Neurosci., 22, 475, 2004. 54. Uetzuki, T. et al. Activation of neuronal caspase-3 by intracellular accumulation of wild-type Alzheimer amyloid precursor protein. J. Neurosci., 19, 6955, 1999. 55. Yang, S.D., Schmidt, A.M. and Stern, D. Deficiency of complement defense protein CD59 may contribute to neurodegeneration in Alzheimer’s disease. J. Neurosci., 20, 7505, 2000. 56. Tamagno, E. et al. NT2 neurons, a classical model for Alzheimer’s disease, are highly susceptible to oxidative stress. Neuroreport, 11, 1865, 2000. 57. Kao, S.C. et al. BACE1 suppression by RNA interference in primary cortical neurons. J. Biol. Chem., 279, 1942, 2004. 58. Tamagno, E. et al. Oxidative stress increases expression and activity of BACE in NT2 neurons. Neurobiol. Dis., 10, 279, 2002. 59. Tamagno, E. et al. Dehydroepiandrosterone reduces expression and activity of BACE in NT2 neurons exposed to oxidative stress. Neurobiol. Dis., 14, 291, 2003. 60. Christensen, M.A. et al. Trancriptional regulation of BACE-1, the β -amyloid precursor protein β -secretase, by Sp1. Mol. Cell. Biol., 24, 865, 2004.

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61. Wei, W., Wang, X. and Kusiak, W. Signaling events in amyloid β -peptide-induced neuronal death and insulin-like growth factor I protection. J. Biol. Chem., 277, 17649, 2002. 62. Zhu, X. et al. Activation and redistribution of c-Jun N-terminal kinase/stress activated protein kinase in degenerating neurons in Alzheimer’s disease. J. Neurochem., 76, 435, 2001. 63. Smith, M.A. et al. Amyloid-β deposition in Alzheimer’s transgenic mice associated with oxidative stress. J. Neurochem., 70, 2212, 1998. 64. Koistinaho, M. et al. Beta-amyloid precursor protein transgenic mice that harbor diffuse A beta deposits but do not form plaques show increased ischemic vulnerability: role of inflammation. Proc. Natl. Acad. Sci. USA, 99, 1610, 2002. 65. Zhu, X. et al. Differential activation of neuronal ERK, JNK/SAPK and p38 in Alzheimer: the “two hit” hypothesis. Mech. Ageing Dev., 123, 39, 2001. 66. Mielke, K. and Herdegen, T. JNK and p38 stress kinases-degenerative effectors of signal transduction-cascades in the nervous system. Prog. Neurobiol., 61, 45, 2000. 67. Holsinger, R.M. et al. Increased expression of the amyloid precursor β -secretase in Alzheimer’s disease. Ann. Neurol., 51, 783, 2002. 68. Fukumoto, H. et al. β -secretase protein and activity are increased in neocortex in Alzheimer disease. Arch. Neurol., 59, 1381, 2002. 69. Yang, L.B. et al. Elevated β -secretase expression and enzymatic activity detected in sporadic Alzheimer. Nat. Med., 9, 3, 2003.

Phenolics and 15 Apple Alzheimer’s Disease Ho Jin Heo

Jeollanamdo Innovation Agency Suncheon, South Korea

Dae-Ok Kim

Kyung Hee University Yongin, South Korea

Chang Yong Lee Cornell University Geneva, New York

CONTENTS 15.1 Alzheimer’s Disease and Amyloid β Protein ..........................................247 15.2 Production of Oxidative Stress in Alzheimer’s Disease ..........................248 15.2.1 Aβ and Oxidative Stress ..............................................................248 15.2.2 Naturally Occurring Oxidative Stress..........................................249 15.2.3 High Fragility of Brain on Oxidative Stress................................250 15.3 Phytochemicals ........................................................................................250 15.3.1 Health Benefits ............................................................................250 15.3.2 Action of Antioxidants in Alzheimer’s Disease ..........................251 15.4 Apple Phenolics .......................................................................................252 15.4.1 Composition and Antioxidant Capacity in Apple........................252 15.4.2 The Action of Quercetin ..............................................................253 15.5 Conclusion ...............................................................................................257 References .........................................................................................................258

15.1 ALZHEIMER’S DISEASE AND AMYLOID β PROTEIN Many scientists who are working on Alzheimer’s disease (AD) have focused mainly on determining the mechanisms underlying the biological toxicity associated with amyloid β (Aβ ) proteins. Aβ is a normal physiological product of amyloid precursor protein (APP) processing1,2 as well as a soluble component of the plasma and cerebrospinal fluid.3 The aggregation of soluble Aβ peptide into fibrillar β -sheet conformation is generally considered to be a critical event in the pathology of AD.4 Aβ peptides may show their toxic actions even before fibril formation.5 247

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Several reports have indicated that synaptic, physiological, and behavioral abnormalities precede Aβ plaque deposition in AD transgenic mice, supporting the possibility that Aβ plaques may not be the critical pathogenic matter. However, potential roles for pre-amyloid protofibrils and intraneuronally accumulated Aβ may prove to be important for the pathogenic process.6,7 There are two major C-terminal variants of Aβ. Aβ1–40 is the major species secreted from cultured cells and is found in cerebrospinal fluid, whereas Aβ1–42 is the major component of amyloid deposits in brain with AD.8 Increases in Aβ1–42, which is more susceptible to aggregation and formation of fibrils, have also been detected in transgenic mice and cells expressing familial AD (FAD) mutations of both APP and presenilins.9 These results suggest a link between this variant of Aβ and AD pathogenesis in that the polymerization of Aβ into protease-resistant fibrils is a significant step in the pathogenesis of AD.9,10 The neurotoxicity exerted by aggregated Aβ can be mediated by several mechanisms, such as the generation of reactive oxygen species (ROS), dysregulation of calcium homeostasis, inflammatory response, and activation of some signaling pathways.11–14

15.2 PRODUCTION OF OXIDATIVE STRESS IN ALZHEIMER’S DISEASE 15.2.1 Aβ AND OXIDATIVE STRESS The AD brain is subjected to increased oxidative stress resulting from free-radical damage.15 The sites in the AD brain where neurodegeneration occurs and oxidative stress exists are reported to be associated with increased Aβ deposits.16 Although the mechanism of Aβ -associated free-radical formation is not fully understood, Aβ is believed to contact or insert into the neuronal and glial membrane bilayer and then generate oxygen-dependent free radicals that cause lipid peroxidation and protein oxidation.17 It has been shown that Aβ causes H2O2 accumulation in cultured hippocampal neurons18 and in neuroblastoma cultures.19 Electron paramagnetic resonance analysis of gerbil synaptosomes using a 12-nitroxyl stearate spin probe demonstrated that Aβ induced lipid peroxidation.20 Increased oxidative modifications of proteins such as advanced glycation end-products have been found to increase DNA oxidation, and increased peroxidation of membrane lipids has been found in the brains of patients with AD upon autopsy.21 Furthermore, Koppaka and Axelsen22 demonstrated that phospholipid membranes with oxidative damage promoted β -sheet formation by Aβ, suggesting the possible role of lipid peroxidation in the pathogenesis of AD. In addition, it has been shown recently that oligomeric Aβ, not monomeric or fibrillar Aβ, promoted the release of lipid, cholesterol, phospholipids, and mono-sialoganglioside from cultured neurons and astrocytes in a dose- and time-dependent manner. These findings indicate that oligomeric Aβ promotes lipid release from the neuronal membrane, which may lead to the disruption of neuronal lipid homeostasis and the loss of neuronal function23. Loss of membrane integrity resulting from Aβ -induced free-radical damage may lead

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to cellular dysfunction such as inhibition of ion-motive ATPase, imbalance of calcium homeostasis, inhibition of Na⫹-dependent glutamate uptake of glial cells, disturbance of signaling pathways, activation of nuclear transcription factors, and apoptotic pathways. Aβ -associated free-radical generation might be strongly influenced by the aggregation state of the peptides.24 Neuronal death may be the ultimate consequence of these cellular dysfunctions.17

15.2.2 NATURALLY OCCURRING OXIDATIVE STRESS It is known that 95% of oxygen that humans inhale is transferred to the mitochondria, and 2 to 5% of this amount is fluxed into the generation of free radicals. It has been estimated that during a normal lifetime a 70-kg man will produce 3 tons of superoxide radical anion (O2.⫺) alone.25 Free radicals are generated in the brain during the normal intake of oxygen, infection, and normal oxidative metabolism. During normal aerobic respiration, the mitochondria of one rat nerve cell will process about 1012 oxygen molecules and reduce them to water. During . this process, various ROS, including superoxide radical anion (O2 ⫺), H2O2, and . 25,26 hydroxyl (OH ), are produced. In addition, partially reduced oxygen, which represents about 2% of consumed oxygen, leaks out from the mitochondria and . generates about 20 billion molecules of O2 ⫺ and H2O2 per cell per day.26 During bacterial or viral infection, phagocytic cells produce high levels of nitric oxide . (NO), O2 ⫺, and H2O2 in order to kill infective agents; however, these radicals can also damage normal cells.26 During degradation of fatty acids and other molecules by peroxisomes, H2O2 is generated as a by-product. During oxidative metabolism of absorbed toxins, free radicals are also provoked. Some brain enzymes, such as monoamine oxidase, tyrosine hydroxylase, and L-amino acid oxidase, make H2O2 as a normal by-product of their activities.27 Moreover, auto-oxidation of ascorbate and catecholamines produces H2O2.28 Oxidative stress can also be produced by Ca2⫹-mediated activation of glutamate receptors. The Ca2⫹-dependent activation of phospholipase A2 by N-methyl-Daspartate (NMDA) releases arachidonic acid, which then releases O2 during the biosynthesis of eicosanoid.29 Another radical, NO, is formed by nitric oxide . synthase stimulated by Ca2⫹. NO can react with O2 ⫺ to form peroxynitrite anions . that can form OH having the highest activity among ROS. NMDA receptor stim. . ulation produces marked elevations in O2 ⫺ and OH levels.30 Some enzymes, such as xanthine oxidase and flavoprotein oxidase (e.g., aldehyde oxidase), also develop superoxide radical anions during metabolism of their respective substrates. Oxidation of hydroquinone and thiol as well as synthesis of uric acid from purines yields superoxide radical anions. Certain external agents elevate oxidative stress. For example, cigarette smoking increases the level of NO by about 1000 ppm by each cigarette and decreases antioxidant levels.31,32 Free iron and copper increase the levels of free radicals.33 Some plants consumed as food contain large amounts of phenolic compounds, such as chlorogenic and caffeic acids, which can be oxidized to form radicals.34,35

250

15.2.3 HIGH FRAGILITY

Oxidative Stress and Age-Related Neurodegeneration OF

BRAIN

ON

OXIDATIVE STRESS

The human brain performs the huge arrangement of cognitive and behavioral functions through an intricate network of approximately 100 billion neurons and supporting glial cells (i.e., astrocytes and microglia). Although it comprises only 2% of the adult body weight, it requires and utilizes a large amount (20%) of energy. In fact, it has been reported that the human brain receives almost 15% of the cardiac output and accounts for up to 30% of the resting metabolic rate.36 This high energy requirement may well be necessary to maintain efficient functioning of its complex and diverse active chemical processes. It is the changes in the functional integrity and optimum efficiency of these processes that contribute toward impairments in brain function, neuronal signaling, and subsequent behavioral changes. The brain is considered abnormally sensitive to oxidative damage,37 and early studies demonstrating the ease of peroxidation of brain membranes38 support this notion. The brain is enriched with more easily peroxidizable fatty acids (arachidonic acid and docosahexaenoic acid), but is not particularly enriched with antioxidant defenses.37 The brain has about 90% lower activity of catalase, which detoxifies toxic H2O2 into H2O and O2 compared to liver.39 Additionally, human brain has higher levels of iron (Fe) in certain regions and in general has high levels of ascorbate. Thus, if constructive disruption of tissue occurs, the Fe–ascorbate mixture is expected to be an abnormally potent pro-oxidant for brain membranes.40 In conclusion, a number of studies suggest that the brain generates high levels of ROS and reactive nitrogen species (RNS) every day. In addition, the brain has the highest levels of polyunsaturated fatty acids (PUFA), which are easily oxidizable by free radicals. Paradoxically, the brain is least prepared to handle this excessive load of reactive free radicals, which results in oxidative stress. It has low levels of both antioxidant enzyme systems and dietary antioxidants. These inherent biological features make the brain very vulnerable to oxidative and nitrosylative stress.30,41 Despite this, the risk of idiopathic AD becomes significant only after the age of 65 or more. This is due to the fact that neurons exhibit a high degree of plasticity in maintaining normal brain functions. Only when a significant number of neurons are lost, clinical symptoms of neurological diseases, including AD, appear, which supports the view that plasticity of the neurons plays a valuable role in maintaining normal brain function.

15.3 PHYTOCHEMICALS 15.3.1 HEALTH BENEFITS Antioxidants stabilize free radicals formed in the ordinary process of oxygen metabolism, so they can prevent the oxidation of various macromolecules such as DNA, proteins, lipids, and carbohydrates. Free radicals are very reactive substances that can damage cellular function. Scientific research indicates that some naturally occurring, nonnutritive materials, commonly referred to as

Apple Phenolics and Alzheimer’s Disease

251

phytochemicals or nutraceuticals, exhibit antioxidant activity. Phytochemicals are generally found in fresh fruits, vegetables, and their processing products. They may reduce the risk of some chronic diseases, such as cancer and cardiovascular disease. Phenolics are one of the major phytochemical groups that have impor-tant biochemical properties, including antioxidant activity. For instance, some phenolics scavenge free radicals, e.g., superoxide radical anions, singlet oxygen, and lipid peroxyl radicals. Polyphenols inhibit lipoxygenase, cyclo-oxygenase, and lipid peroxidation,42 and also have various beneficial effects on immune and inflammatory cell functions.43 Certain phenolics also display antihemolytic activities, inhibit oxidation of low-density lipoprotein (LDL) by macrophages, and prevent cytotoxicity of oxidized LDL on lymphoid cell lines.44 In addition, some flavonoids, one of the major phenolic groups, have been reported to affect capillary permeability, cellular secretory processes, cell membrane receptors, and carriers. Mutagenic, antiviral, antibacterial, and antifungal properties of flavonoids have also been demonstrated. Flavonoids, which are present in regularly consumed foods such as tea, onions, and red wine, may reduce the risk of coronary heart disease.45 There is also a large body of evidence that supplementing the diet with micronutrients has positive health benefits. Many of these micronutrients mayoriginate from plants. Moreover, plant macro-components, such as fiber, may reduce cancers of some tissues, including the colon. In 1982, the National Research Council advised that increasing consumption of carotene-rich fruits and cruciferous vegetables, including cabbage and broccoli, might reduce cancer risk. This recommendation has been confirmed by epidemiological studies that people with a low fruit and vegetable intake experienced twice the risk of cancer.46 Some phenolics have been shown to exhibit synergistic interactions with other nutrients. Therefore, phytochemicals, including phenolics, will play an important role in optimal nutrition for supplying bio-functional active compounds to our diet in this new era as the food and nutritional sciences develop. As consumers become more aware of the anticancer potential of some plant foods, the demand for natural and processed foods that have these health-promoting benefits will grow. Several stone fruits and berries are also reported to contain significant amounts of phytochemicals. In particular, apples contain many beneficial polyphenols (e.g., flavonol glycosides, phenolic acids, catechins, dihydrochalcones, and procyanidins), and most of these compounds are known to have antioxidant activity and anticarcinogenic activity in vitro.47,48 In less significant amounts, glutathione and ascorbic acid also occur as antioxidants in apples.

15.3.2 ACTION

OF

ANTIOXIDANTS

IN

ALZHEIMER’S DISEASE

Many studies show that oxidative damage to neurons plays an important role in AD pathogenesis. Therapeutic strategies to reduce oxidative stress and increase antioxidant protection might slow down and prevent the onset of the disease. Preclinical studies suggest that some antioxidants may have therapeutic potential for AD. Ginkgo biloba extracts protect neurons from H2O2-induced oxidative

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Oxidative Stress and Age-Related Neurodegeneration

stress.49 A particular extract of Ginkgo biloba, EGb761, was examined to assess the efficacy and safety in patients with AD and multi-infarct dementia.50 AD patients who were treated with EGb761 (120 mg/d) for 52 weeks showed improvements on the AD Assessment Scale-Cognitive subscale and on the Geriatric Evaluation of Relative’s Rating Instrument. Melatonin reduces neuronal damage induced by ROS in experimental models of AD. In addition to its antioxidant effect, melatonin also has antiamyloidogenic activities.51 Research with cultured cells or animal models revealed that Aβ -induced neurotoxicity and cognitive impairments can be attenuated by either vitamin E or idebenone (coenzyme Q10 analog), which is an antioxidant and free-radical scavenger.52,53 The efficacy and safety of idebenone was examined in 450 patients with mild to moderate AD. The beneficial effects of idebenone in the patient were maintained until 2 years after treatment.54 In the double-blind, controlled, clinical, 2-year study of patients with moderately advanced AD, the primary outcome of the disease progression was delayed with treatments of selegiline (10 mg/day, monoamine oxidase B inhibitor) or vitamin E (2000 IU/day, antioxidant), or both.55 Although there were no significant effects on cognitive ability, these results suggest that the use of selegiline or vitamin E might play some useful roles in delaying clinical deterioration related to AD. Several in vitro studies showed that some kinds of anticholinesterase, such as tacrine, huperzine A, and dehydroevodiamine, can attenuate Aβ -induced oxidative damage, thereby enhancing their therapeutic efficacy for AD.56,57 Consequently, it is believed that antioxidant or free-radical scavengers might have some beneficial activities for the prevention and treatment of AD.

15.4 APPLE PHENOLICS 15.4.1 COMPOSITION AND ANTIOXIDANT CAPACITY

IN

APPLE

Apples are one of the major fruits consumed by Americans. Among fresh fruits consumed in the United States, bananas (12.7 kg/person/year) ranked first, and apples (8.8 kg/person/year) second, more than fresh oranges, grapes, or grapefruits. When fresh and processed products are combined, the per capita consumption of apples (21.3 kg) exceeds that of bananas. Therefore, apple phenolics as antioxidant sources in the American diet may provide major protection against free-radical damage in the human body.58 Lee et al.47 studied the antioxidant activity in various apple cultivars and their antioxidant contribution to the American diet. Table 15.1 shows the composition and concentrations of the major phenolics of six apple cultivars grown in New York State in 2001.47 Different apple cultivars showed different concentrations of phenolics. Among the apple cultivars, Rhode Island Greening showed the highest content in all phenolic phytochemicals analyzed. Average concentrations (mg/100 g of fresh weight) of the major phenolics were as follows: quercetin glycosides, 13.20; procyanidin B2, 9.35; chlorogenic acid, 9.02; epicatechin, 8.65; and phloretin glycosides, 5.59. Chlorogenic acid and phloretin glycosides presented lower phenolic contents

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253

TABLE 15.1 Composition and Quantification of Major Antioxidants of Six Apple Cultivars Fresh Apples with Skins (mg/100 g) Antioxidant

Vitamin C Chlorogenic acid Epicatechin Phloretin glycosides Glucoside Xyloglucoside Procyanidin B2 Quercetin glycosides Arabinoside Xyloside Glucoside Galactoside Rhamnoside Total

Golden Delicious

Cortland

Monroe

Rhode Island Greening

Empire

NY674

av

16.60 8.48 7.12

12.17 5.36 8.32

9.00 10.08 10.72

14.22 14.28 19.16

13.22 11.52 2.28

11.62 4.40 4.32

12.80 9.02 8.65 5.59

1.80 1.92 6.28

1.44 3.20 11.32

2.40 4.92 8.32

2.08 5.88 21.68

2.80 1.72 3.44

1.84 3.56 5.04

2.16 1.68 2.40 4.20 3.84 56.48

2.40 1.08 1.56 3.36 2.28 52.49

4.44 2.28 2.40 4.80 3.12 62.48

2.88 1.92 1.20 4.32 4.08 91.70

2.76 2.16 2.40 4.20 3.84 50.34

1.56 1.20 0.36 1.92 2.40 38.22

9.35 13.20

58.61

Note: av ⫽ average concentration. Source: Modified from Lee KW et al. J Agric Food Chem, 2003; 51:6516–6520. With permission from the American Chemical Society.

compared to quercetin glycosides and procyanidin B2. Several phenolics in apples were present as glycosides. In particular, a wide variety of quercetin glycosides were present in the apple cultivars. Galactoside was the most abundant form among the glycosides identified in most of the tested cultivars except NY674, in which rhamnoside was most abundant. Moreover, xyloglucoside was a plentiful form of phloretin glycoside. Antioxidant capacities of the major apple phenolics are shown in Table 15.2.47 The relative total antioxidant capacity of phenolics evaluated by using the 2,2⬘-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical chromogens compared with vitamin C was as follows: quercetin (3.06) ⬎ epicatechin (2.67) ⬎ procyanidin B2 (2.36) ⬎ phloretin (1.63) ⬎ vitamin C (1.00) ⬎ chlorogenic acid (0.97). The data show that quercetin has the most powerful antioxidant capacity (lowest EC50 value) among the major phenolics in apples.

15.4.2 THE ACTION

OF

QUERCETIN

There is increasing evidence that flavonoids can be assimilated into the human body in amounts that are, in principle, sufficient to exert antioxidant or other biological

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Oxidative Stress and Age-Related Neurodegeneration

TABLE 15.2 Contributions of Major Antioxidants to the Total Antioxidant Activity of Apples Phytochemical

Quercetin glycosides Epicatechin Procyanidin B2 Vitamin C Phloretin glycosides Chlorogenic acid Total

av (mg/100 g of fresh weight)

EC50

Relative VCEAC Value

13.20 8.65 9.35 12.80 5.59 9.02 58.61

0.56 0.64 0.72 1.71 1.05 1.76

3.06 2.67 2.36 1.00 1.63 0.97

Total Antioxidant Activity (mg of VCEAC/100 g) 40.39 23.10 22.07 12.80 9.11 8.75 116.22

Relative Contribution (%)

34.7 19.9 19.0 11.0 7.8 7.6 100.0

Note: av ⫽ average concentration; EC50 ⫽ median effective dose; and Relative VCEAC value ⫽ vitamin C equivalent antioxidant capacity (VCEAC) of each compound/antioxidant capacity of vitamin C. Source: Modified from Lee KW et al. J Agric Food Chem, 2003; 51:6516–6520. With permission from the American Chemical Society.

activities in vitro.59–61 Chlorogenic acid is absorbed with no structural change in the small intestine,62 whereas procyanidin dimmers as B2 & B5 are digested as epicatechin.63 In general, derivatives of flavonoids and isoflavones were found to have lower biological activities in glycosides compared with their parent aglycons in vivo. However, gastrointestinal hydrolase discharged the sugar moiety from flavonoid glycosides, and their aglycons are released to be absorbed in the gut.59 Intestinal conjugation seemed to be an important process for the absorption because only conjugated forms were detected in the mesenteric vein blood.60 Furthermore, when quercetin glycosides and genistin were fed to rats or humans, quercetin and genistein, their only respective aglycon forms, were detected in the urine.60,61 The central nervous system (CNS) is especially vulnerable to free-radical attack. The two major oxidative-stress inducers among ROS are O2.⫺ and H2O2. Although H2O2 is a reactive nonradical molecule, it can easily pass through biological cell membranes, while O2.⫺ can only move through an anion channel.64 Recent study demonstrated that quercetin had a protective effect on pheochromocytoma (PC12) cell against H2O2-induced neurotoxicity.65 The protective efficacy of quercetin and vitamin C was demonstrated by exposing PC12 cells to H2O2. The effect of quercetin and vitamin C on H2O2-induced toxicity was tested in PC12 cells by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay (Table 15.3). This test is used as a marker for cell viability, because intact mitochondrial enzymes can only reduce MTT. One of the most sensitive primary targets of oxidative stress may be mitochondria in neuronal

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255

TABLE 15.3 Effects of the Apple Phenolics on the Viability of PC12 Cells with Exposure of H2O2a % of Control µ M) Unit (µ 400 µM H2O2 Quercetin 10 30 60 100 Vitamin C 10 30 60 100

MTT Reductionb

LDH Release into Mediumc

Trypan Blue Exclusiond

45

180

54

55 109 187 208

168 164 145 126

81 108 117 132

48 87 131 190

177 172 169 148

57 71 93 99

a

The cell viability was evaluated using three different methods (MTT reduction, LDH release, and trypan blue exclusion assays). PC12 cells were preincubated for 10 min with various concentrations of apple extracts (100 µg extract ⫽ 500 mg fresh apple), and then the cells were treated with H2O2 (400 µM) for 2 h. The control group received treatment with neither H2O2 nor apple extracts. Results are shown as mean ⫾SD (n ⫽ 3). Significant difference (P ⬍ 0.05) was observed on the H2O2-induced cell death. b

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay is used as a marker for cell viability, because intact mitochondrial enzymes can only reduce MTT.

c

Lactate dehydrogenase (LDH) assay is a means of measuring either the number of cells via total cytoplasmic LDH or membrane integrity. Damage of the plasma membrane was evaluated by measuring the amount of the intracellular enzyme LDH released into the medium.

d Trypan blue exclusion assay was based on the capability of viable cells to exclude the dye. Since viable PC12 cells maintained membrane integrity, the cells did not allow trypan blue dye to pass through the cell membrane.

Source: Modified from Heo HJ et al. J Agric Food Chem, 2004; 52:7514–7517. With permission from the American Chemical Society.

cells.66 Since mitochondrial DNA does not encode for any repair enzymes and, unlike nuclear DNA, is not shielded by protective histones, mitochondrial defects were suggested in the pathogenesis of AD.67 Hydrogen peroxide caused a significant decrease in cell viability (45%), but preincubation with these nutrients dose dependently protected PC12 cells from H2O2-induced toxicity. Neuronal cell protection effects were observed over 10 µM quercetin, and quercetin also showed higher cell viability effect over 30 µM than the control. In contrast, the protective

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Oxidative Stress and Age-Related Neurodegeneration

effects of vitamin C were lower than those of quercetin (10 to ~60 µM), but both showed almost the same protective effects at the highest concentration (100 µM). Another study also showed that quercetin has higher antioxidative activity than vitamin C.68 Therefore, these studies strongly suggest that H2O2-induced apoptosis in neuronal cells will be suppressed by pretreatment with quercetin, and partially protected due to the mitochondrial protection mechanisms. It has been reported that superoxide dismutase and hemoxygenase-1, biomarkers of oxidative stress, were promoted by Aβ /H2O2-induced oxidative stress in aged transgenic mice and PC12 cells.69 This Aβ protein decreases the MTT dye reduction by an increased instability of the plasma membrane,70 and it has been identified as a possible source of oxidative stress in the AD brain because it can acquire a free-radical state contributing to its toxic effects.69 Quercetin, as one of the major phenolics in apples, also showed its neuronal cell membrane protection.65 H2O2induced oxidative stress increased plasma membrane damage but quercetin and vitamin C protected the PC12 cells. The study also showed that quercetin has more protective effect in PC12 cells than vitamin C (Table 15.3). These results indicate that quercetin and vitamin C have excellent antioxidant activities that could protect the neuronal cell membrane against H2O2-induced neurotoxicity, although the protective effect of vitamin C was not as strong as that of quercetin. Lipid peroxidation is increased in neurodegenerative diseases such as AD. PUFA levels in neuronal cell membrane, especially arachidonic acid and docosahexaenoic acid, which are more vulnerable to attack by ROS, are found to be decreased in pathogenesis of AD. Lipid peroxidation can lead to changes in the membrane integrity and fluidity.71 In terms of chemical structure, it has been reported that the 3-OH in the chroman ring of flavonoids is important for antioxidant activity,67,68 and that this activity can be boosted by electron-donating substituents (e.g., OH or OCH3 groups) at the 5 and 7 positions. Moreover, the flavonoids with a catechol structure in ring B are known to be very beneficial inhibitors of lipid peroxidation, and the hydroxyl groups on both 3⬘ and 4⬘ are very important in cell protection (Figure 15.1).72 Consistent with this notion, quercetin was found to prevent lipid peroxidation via the prevention of oxidative stress.72 These studies suggest that the strong protective effect of quercetin on oxidative stress-induced membrane damage may be mainly due to the inhibition of lipid peroxidation. In another study, quercetin protected the mouse hippocampal cell line HT-22 from glutamate-induced oxidative toxicity by two distinct mechanisms that prevent intracellular glutathione (GSH) loss and block ROS production.73 The loss in GSH up to 85% of the control level causes a five- to tenfold increase in levels of ROS.73 A greater GSH loss stimulates mitochondria to produce a 100-fold increase in ROS, resulting in programed cell death.74 Quercetin increases levels of GSH in HT-22 cells in the presence of glutamate.73 The structural determinants as in quercetin are required to protect neuronal cells from oxidative stress: the presence of the hydroxyl group on the C-3 position, an unsaturated C ring, and hydrophobicity (Figure 15.1). The hydroxyl group on the C-3 position dramatically increases cell viability. The unsaturation of the C ring in a flavonoid skeleton is essential for protection from oxidative glutamate toxicity. The unsaturation of the C ring, which

Apple Phenolics and Alzheimer’s Disease

257 OH 3′ 2′

HO

8 7

9

A

C

10

6

1′

O

B 6′

2

OH 4′ 5′

3

5

4

OH

O

OH

FIGURE 15.1 Quercetin (C15H10O7) structure.

may facilitate electron delocalization across the molecule for the stabilization of the free radical, is also an important factor for antioxidants in the cell-free systems.75 The decrease in hydrophobicity by glycosylation (e.g., rutin) or ionization (e.g., cyanidin) inactivates the protective activities on oxidative glutamate toxicity. In addition, a hydrophobic antioxidant may easily pass into the cytoplasm where ROS are generated and accumulate in oxidative glutamate toxicity.73 Quercetin has such specific structure that has the preventive effect on GSH to protect against oxidative stress-induced neurotoxicity as mentioned above. Heo and Lee65 showed that the protective effect of quercetin against oxidative stress in PC12 cells was more apparent than that of vitamin C. This may be due to the structural properties and variable physiological benefits of quercetin. Futhermore, blood–brain barrier (BBB), formed by the endothelium of brain microvessels, is a regulatory interface, and selectively limits drug delivery to the CNS. Hence, drug permeability into the brain will be controlled by the BBS’s physicochemical characteristics such as hydrophobicity or lipophilicity.76 It has been reported that quercetin can flux into brain regions.77 Therefore, it is possible that quercetin with its beneficial antioxidant and biological functions is able to penetrate the BBB and offer protection against H2O2induced neurotoxicity.

15.5 CONCLUSION ROS play an important role in the progression of neurodegenerative disorders such as AD. Natural foods possessing antioxidative components (vitamins and phenolics) have received a great deal of attention because they are considered to be safe and nonmedicinal; some of these are known to function as chemopreventive agents against oxidative damage. Hence, natural, food-derived antioxidants would be more useful. Apples have shown a strong antineurodegenerative activity in vitro. Since apples are one of the richest sources of flavonoids among fruits that have many important bioactive activities, daily consumption of an apple may provide many beneficial effects that may reduce the risk of various chronic diseases, including neurodegenerative diseases such as AD.

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Oxidative Stress and Age-Related Neurodegeneration

Although there have been many scientific reports on the biological activity of selected phytochemicals in foods in recent years, the major questions still remain: which chemical constituents are responsible for physiological benefits, and what kinds of guidelines are needed for consumers in selecting and consuming the beneficial plant foods? The nutritional value and health-related biological activity of various fruits and vegetables depend not only on the concentration of certain nutrients and bioactive phytochemicals, but also on the amount of such foods consumed in the diet. No matter how high the concentration of a certain bioactive compound in a food, if the level of consumption of that food is low, the contribution of the bioactive compound in the diet is insignificant. For example, spinach and Brussels sprouts are relatively high in major vitamins and minerals, but their contribution to our diet is low because the amounts we consume are very small. On the other hand, tomatoes are relatively low in concentration of vitamins and minerals but make a major contribution to the American diet because of large per capita consumption. In the same way, apple phenolics may contribute significantly to our health. Eating one apple (~150 g) each day may provide ~0.23 g of phenolics, and 0.15 g of flavonoids, and a significant amount of healthy antioxidant activity.78 In a recent study, it was estimated that the average person living in the West consumes about 0.5 to 1 g of phenolics a day, with the major portion coming from apples, oranges, bananas, potatoes, and tomatoes.48,79 Since we do not yet know the daily requirement of antioxidants, we have to be cautious in recommending the high doses of antioxidants with supplements. The evidence shown here is that apple, as a natural food, contains important flavonoids such as quercetin, which exhibits significantly high antioxidant and neuroprotective activities. Flavonoids are mainly located in apple skins; therefore, consumption of apples with skins is highly desirable in order to maximize apple’s physiological benefits.

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23. Michikawa M, Gong JS, Fan QW, Sawamura N, Yanagisawa K. A novel action of Alzheimer’s amyloid β-protein (Aβ ): oligomeric Aβ promotes lipid release. J Neurosci, 2001; 21:7226–7235. 24. Monji A, Utsumi H, Ueda T, Imoto T, Yoshida I, Hashioka S, Tashiro K, Tashiro N. The relationship between the aggregational state of the amyloid-β peptides and free radical generation by the peptides. J Neurochem, 2001; 77:1425–1432. 25. Chance B, Sies H, Boveris A. Hydroperoxide metabolism in mammalian organs. Physiol Rev, 1979; 59:527–605. 26. Ames BN, Shigenaga MK, Hagen TM. Oxidants, antioxidants, and the degenerative diseases of aging. Proc Natl Acad Sci USA, 1993; 90:7915–7922. 27. Coyle JT, Puttfarcken P. Oxidative stress, glutamate, and neurodegenerative disorders. Science, 1993; 262:689–695. 28. Graham DG. Oxidative pathways for catecholamines in the genesis of neuromelanin and cytotoxic quinones. Mol Pharmacol, 1978; 14:633–643. 29. Chan PH, Fishman RA. Transient formation of superoxide radicals in polyunsaturated fatty acid-induced brain swelling. J Neurochem, 1980; 35:1004–1007. 30. Lafon-Cazal M, Pietri S, Culcasi M, Bockaert J. NMDA-dependent superoxide production and neurotoxicity. Nature, 1993; 364:535–537. 31. Reznick AZ, Cross CE, Hu ML, Suzuki YJ, Khwaja S, Safadi A, Motchnik PA, Packer L, Halliwell B. Modification of plasma proteins by cigarette smoke as measured by protein carbonyl formation. Biochem J, 1992; 286:607–611. 32. Duthie GG, Arthur JR, James WP. Effects of smoking and vitamin E on blood antioxidant status. Am J Clin Nutr, 1991; 53:1061S–1063S. 33. Winterbourn CC. Toxicity of iron and hydrogen peroxide: the Fenton reaction. Toxicol Lett, 1995; 82–83:969–974. 34. Ames BN, Profet M, Gold LS. Dietary pesticides (99.99% all natural). Proc Natl Acad Sci USA, 1990; 87:7777–7781. 35. Gold LS, Slone TH, Stern BR, Manley NB, Ames BN. Rodent carcinogens: setting priorities. Science, 1992; 258:261–265. 36. Sokoloff L, Fitzgerald GG, Kaufman EE. Determinants of the availability of nutrients to the brain. In: Wurtman JR, Wurtman JJ, eds. Nutrition and the Brain. New York: Raven Press, 1977:87–139. 37. Floyd RA, Carney JM. Free radical damage to protein and DNA: mechanisms involved and relevant observations on brain undergoing oxidative stress. Ann Neurol, 1992; 32:S22–S27. 38. Zaleska MM, Floyd RA. Regional lipid peroxidation in ray brain in vitro: possible role of endogenous iron. Neurochem Res, 1985; 10:397–410. 39. Marklund SL, Westman NG, Lundgren E, Roos G. Copper- and zinc-containing superoxide dismutase, manganese-containing superoxide dismutase, catalase, and glutathione peroxidase in normal and neoplastic human cell lines and normal human tissues. Cancer Res, 1982; 42:1955–1961. 40. Zeleska MM. Nagy K, Floyd RA. Iron-induced peroxidation and inhibition of dopamine synthesis in striatum synaptosomes. Neurochemistry, 1989; 14:597–605. 41. Mutisya EM, Bowling AC, Beal MF. Cortical cytochrome oxidase activity is reduced in Alzheimer’s disease. J Neurochem, 1994; 63:2179–2184. 42. Laughton MJ, Evans PE, Moroney MA, Hoult JRS, Halliwell B. Inhibition of mammalian 5-lipoxygenase and cyclo-oxygenase by flavonoids and phenolic dietary additives. Biochem Pharmacol, 1991; 42:1673–1681.

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43. Decharneux T, Dubois F, Beauloye C, De Coninck SW, Wattiaux R. Effect of various flavonoids on lysosomes subjected to an oxidative or an osmotic stress. Biochem Pharmacol, 1992; 44:1243–1248. 44. Negre-Salvayre A, Salvayre R. Quercetin prevents the cytotoxicity of oxidized LDL on lymphoid cell lines. Free Radic Biol Med, 1992; 12:101–106. 45. Hertog MGL, Feskens E, Hollman PCH, Katan MB, Kromhout D. Dietary antioxidant flavonoids and risk of coronary heart disease: the Zutphen elderly study. Lancet, 1993; 342:1007–1011. 46. Block G, Patterson B, Subar A. Fruit, vegetables, and cancer prevention: a review of the epidemiological evidence. Nutr Cancer, 1992; 18:1–29. 47. Lee KW, Kim YJ, Kim DO, Lee HJ, Lee CY. Major phenolics in apple and their contribution to the total antioxidant capacity. J Agric Food Chem, 2003; 51:6516–6520. 48. Liu RH. Health benefits of fruit and vegetables are from additive and synergistic combinations of phytochemicals. Am J Clin Nutr, 2003; 78:517S–520S. 49. Oyama Y, Chikahisa L, Ueha T, Kanemaru K, Noda K. Ginkgo biloba extract protects brain neurons against oxidative stress induced by hydrogen peroxide. Brain Res, 1996; 712:349–352. 50. Le Bars PL, Katz MM, Berman N, Itil TM, Freedman AM, Schatzberg AF. A placebo-controlled, double-blind, randomized trial of an extract of Ginkgo biloba for dementia. North American EGb Study Group. J Am Med Assoc, 1997; 278:1327–1332. 51. Pappolla MA, Chyan YJ, Poeggeler B, Frangione B, Wilson G, Ghiso J, Reiter RJ. An assessment of the antioxidant and the antiamyloidogenic properties of melatonin: implications for Alzheimer’s disease. J Neural Transm, 2000; 107:203–231. 52. Yamada K, Tanaka T, Han D, Senzaki K, Kameyama T, Nabeshima T. Protective effects of idebenone and α-tocopherol on β -amyloid-(1–42)-induced learning and memory deficits in rats: implication of oxidative stress in β -amyloid-induced neurotoxicity in vivo. Eur J Neurosci, 1999; 11:83–90. 53. Huang HM, Ou HC, Hsieh SJ. Antioxidants prevent amyloid peptide-induced apoptosis and alteration of calcium homeostasis in cultured cortical neurons. Life Sci, 2000; 66:1879–1892. 54. Gutzmann H, Hadler D. Sustained efficacy and safety of idebenone in the treatment of Alzheimer’s disease: update on a 2-year double-blind multicentre study. J Neural Transm Suppl, 1998; 54:301–310. 55. Sano M, Ernesto C, Thomas RG, Klauber MR, Melville R, Schafer K, Grundman M, Woodbury P, Growdon J, Cotman CW, Pfeiffer E, Schneider LS, Thal LJ. A controlled trial of selegiline, α-tocopherol, or both as treatment for Alzheimer’s disease. The Alzheimer’s Disease Cooperative Study. N Engl J Med, 1997; 336:1216–1222. 56. Xiao XQ, Wang R, Tang XC. Huperzine A and tacrine attenuate β -amyloid peptide-induced oxidative injury. J Neurosci Res, 2000; 61:564–569. 57. Park CH, Lee YJ, Lee SH, Choi SH, Kim HS, Jeong SJ, Kim SS, Suh YH. Dehydroevodiamine HCl prevents impairment of learning and memory and neuronal loss in rat models of cognitive disturbance. J Neurochem, 2000, 74:244–253. 58. Putnam JJ, Allshouse JE. Food Consumption, Prices, and Expenditures (1970–1993). Washington, DC: Department of Agriculture, 1994. 59. Day AJ, Gee JM, DuPont MS, Johnson IT, Williamson G. Absorption of quercetin3-glucoside and quercetin-4⬘-glucoside in the rat small intestine: the role of lactase

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of Nicotine in 16 Effects Models of Alzheimer’s Disease Baolu Zhao

Institute of Biophysics Chinese Academy of Science Beijing, China

CONTENTS Abstract..............................................................................................................265 16.1 Antioxidant Actions of Nicotine and the Scavenging Effect of Nicotine on Oxygen Free Radicals..........................................................266 16.2 Nicotine Inhibits β -Amyloidosis .............................................................267 16.3 Nicotine Breaks Down Preformed Alzheimer’s Amyloid β Fibrils ........268 16.4 Nicotine Protects Hippocampal Neuronal Cells against Aβ -Induced Apoptosis .............................................................................269 16.5 Chronic Nicotine Treatment Reduces β -Amyloidosis in the Brain of a Mouse Model with Alzheimer’s Disease................................271 16.6 Conclusion ...............................................................................................272 References .........................................................................................................272

ABSTRACT Recent studies indicate that neuronal loss in Alzheimer’s disease (AD) is accompanied by the deposition of amyloid β protein (Aβ ) in senile plaques. Previous findings also suggest that generation of free radicals and increase of intracellular free Ca2⫹ can be induced by Aβ. It has been suggested that nicotine, a major component of cigarette smoke, protects neurons from Aβ neurotoxicity through mechanisms probably mediated by nicotinic receptors or antiamyloidosis. This chapter reviews the effect of nicotine in models of AD. Evidence suggests that nicotine possesses antioxidant properties and can scavenge hydroxyl radicals, superoxide free radicals, and even free radicals in gas-phase cigarette smoke. Nicotine can dose-dependently inhibit fibrillar Aβ (fAβ ) formation from fresh Aβ (1–40) and fAβ (1–42). Moreover, nicotine can disrupt preformed fAβ. Experimental results show that nicotine can protect cultured hippocampal neurons against Aβ -induced apoptosis 265

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partly via its antioxidant properties and partly via nicotinic receptors. In vivo experiments show that chronic nicotine treatment reduces β -amyloidosis in the brain of a transgenic mouse model of Alzheimer’s disease (APPsw). Evidence suggests that nicotine may be beneficial in retarding the neurodegenerative processes in AD. Alzheimer’s disease (AD) is one of the most common forms of dementia, and one of the neuropathological hallmarks of AD is the neuronal degeneration associated with senile plaques.1 Such plaques are composed of compacted amyloid β -peptide (Aβ ), which is a 40–43-amino-acid peptide.2,3 Aβ is formed by the proteolytic processing of a transmembrane glycoprotein called β -amyloid precursor protein ( β -APP), which can be secreted4 or cleaved, releasing Aβ by the action of β - and γ -secretases.5 The deposition of soluble Aβ produces the aggregation of the peptide-forming amyloid fibrils, which have been reported to be neurotoxic in vitro6–8 and in vivo.3,9,10 It is true that fibrillar materials have toxic effects on a range of cells.11–13 The intrinsic toxicity of high levels of fibrils themselves could result from the generation of oxygen free radicals by Aβ fibrils (in the absence of any cellular elements)14 or their destabilization of membranes resulting in changed intracellular calcium homeostasis and eventual cell death.11 Numerous epidemiological studies have reported a highly significant negative association between cigarette smoking and neurodegenerative disorders, especially in Parkinson’s disease (PD)15,16 and AD.17–19 Nicotine is a predominant component of cigarette smoke and is currently being used in pilot clinical studies for the treatment of AD.20 α 4β 2 nicotinic receptor activation plays an important role in neuroprotection against Aβ cytotoxicity.21–23 Nicotine could exert potent neuroprotective effects by inhibiting arachidonic acid-induced apoptotic cascades (caspases-3 activation and cytochrome c release) of spinal cord neurons. The α 7 nicotinic acetylcholine receptor subtype mediates nicotine protection against N-methyl-D-aspartate (NMDA) excitotoxicity in primary hippocampal cultures through a Ca2⫹-dependent mechanism.24 We have also studied the scavenging effect of nicotine on oxygen free radicals and found that it could effectively scavenge superoxide, hydroxyl radicals, and even the free radicals in the gas phase of cigarette smoke.25 The key pathological change in the brain linked to the emergence and progressive development of dementia is the gradual degeneration of nerve cells and the related loss of specific synaptic connections. Only a highly specific subset of nerve cells shows vulnerability to degeneration, especially in hippocampal and cortical neurons. A number of studies on the effect of nicotine in models of AD have appeared in the literature, focusing on its antioxidant effects, inhibition of fAβ formation, and disruption of fAβ, protective effects against neuron cellular apoptosis, and protective effects of nicotine in in vivo models. The effect of nicotine in the models of AD is reviewed and discussed in this chapter.

16.1 ANTIOXIDANT ACTIONS OF NICOTINE AND THE SCAVENGING EFFECT OF NICOTINE ON OXYGEN FREE RADICALS An inverse relationship appears to exist between cigarette smoking and the risk of PD and AD. Since both diseases are characterized by enhanced oxidative stress,

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Linert et al.26 investigated the antioxidant potential of nicotine, a primary component of cigarette smoke. Initial chromatographic studies suggest that nicotine can affect the formation of the neurotoxin 6-hydroxydopamine resulting from the addition of dopamine to Fenton’s reagent (i.e., Fe2⫹ and H2O2). Thus, under certain circumstances, nicotine can strongly affect the course of the Fenton reaction. In in vivo studies, adult male rats being treated with nicotine showed greater memory retention than controls in a water maze task. However, neurochemical analysis of neocortex, hippocampus, and neostriatum from these same animals revealed that nicotine treatment had no effect on the formation of reactive oxygen species (ROS) or on lipid peroxidation for any brain region studied. In an in vitro study, addition of various concentrations of nicotine to rat neocortical homogenates had no effect on lipid peroxidation compared with saline controls.26 We investigated the scavenging effect of nicotine on free radicals by electron spin resonance (ESR) techniques. It was found that nicotine could scavenge the hydroxyl radicals, superoxide free radicals, and free radicals in gas-phase cigarette smoke. The hydroxyl radical was generated from the Fenton reaction and trapped by 5,5-dimethyl-1-pyroline-N-oxide (DMPO). It was found that nicotine had a much stronger scavenging effect on the hydroxyl radical than vitamin C. The IC50 (the concentration of drug when scavenging rate is 50%) of nicotine (25 µmol/L) was approximately 20 times higher than that of vitamin C (500 µmol/L). The superoxide radical was generated from irradiation of riboflavin/DETAPAC system and trapped by DMPO. The scavenging rate is defined as above. The results show that the scavenging effect of nicotine on superoxide radical was also much stronger than that of vitamin C. The IC50 of nicotine (50 µmol/L) was approximately six times higher than that of vitamin C (300 µmol/L). It was also found that the scavenging effect of nicotine on hydroxyl free radical is about two times higher than that on superoxide free radicals. The free radicals generated from gas-phase of cigarette smoking (GPCS) were trapped by N-tert-butyl-2-phenyl-nitrone (PBN). The scavenging rate of nicotine on free radicals generated from gas-phase cigarette smoke was strong, and the scavenging efficiently increased with the concentration of nicotine. The scavenging effect of vitamin C on free radicals generated from GPCS was not significant.25

16.2 NICOTINE INHIBITS β -AMYLOIDOSIS Aβ peptide is a nontoxic, soluble, 39–43-amino-acid peptide that is generated from the enzymatic cleavage of a transmembrane amyloid precursor protein (APP).27,28 The excessive production and deposition of Aβ is the key event leading to the neurodegenerative changes in AD.29 The intracerebral accumulation of the amyloid peptide as senile plaques or vascular amyloid plays a key role in the pathogenesis of AD. It is important to find a suitable inhibitor that either slows down or inhibits Aβ formation (β -amyloidosis). During β -amyloidosis, structural changes associated with the conversion of monomeric Aβ peptide building blocks into the aggregated fibrillar β -sheet structures occur (α-helix3β -sheet or random, extended chain3β -sheet). Salomon et al.30 established that nicotine inhibits β -amyloidosis of Aβ (1–42), which may result from nicotine binding to

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the α-helical structure. These conclusions were based on solution nuclear magnetic resonance (NMR) spectroscopic studies with the nonnative 28-residue Aβ (1–28). This information suggests that when administered therapeutically to AD patients, nicotine may not only affect cholinergic activation, but could also conceivably alter amyloid deposition.30 NMR studies by Zeng et al.31 were augmented with the naturally occurring Aβ (1–42) under conditions in which the peptide folds into a predominantly α-helical or random, extended chain structure. The major result is that nicotine shows only modest binding to these conformations, indicating that the nicotine inhibition of β -amyloidosis probably results from binding to a small, soluble, β -sheet aggregate that is NMR invisible.31 Naiki et al.32 studied the effects of apolipoprotein E (apoE) and antioxidants (nordihydroguaiaretic acid and rifampicin) on the formation of Aβ aggregation and found that antioxidants have effects similar to those of nicotine. But apoE extended t1/2 of Aβs in a dose-dependent manner, and the antioxidants did not. On the other hand, the final amount of fAβ formed was dose-dependently decreased by both apoE and antioxidants. It was also found that although apoE extended the time to proceed to equilibrium in a dose-dependent manner, the antioxidants did not. These results indicate that apoE and antioxidants inhibit formation in vitro by different mechanisms and suggest the existence of multiple pharmacological targets for the prevention of fAβ formation.32 Ono et al.33 also reported that when Aβ (1–40) was incubated with increasing concentrations of nicotine, fAβ (1–40) formation decreased dose dependently. A significant inhibition effect of nicotine was observed at concentrations above 10 mmol/L. Similar results were obtained with Aβ (1–42). N-methylpyrrolidine similarly inhibited fAβ (1–40) and fAβ (1–42) formation from fresh Aβ (1–40) and Aβ (1–42) in a dose-dependent manner. Pyrrolidine had these inhibit effects on fAβ (1–40) and fAβ (1–42).33

16.3 NICOTINE BREAKS DOWN PREFORMED ALZHEIMER’S AMYLOID β FIBRILS After fAβ is formed, it is important to find a material or a technique to disrupt it. Using fluorescence spectroscopy, electron microscopy, and polarized light microscopy, Ono et al. found that incubation with 10 and 100 mmol/L nicotine could concentration-dependently decrease the fluorescence of fAβ (1–40) and fAβ (1–42) owing to the disruption of the Aβ fibrils. fAβ assumed a nonbranched, helical filament structure of ~220 nm. After incubation of 25 µmol/L of fresh fAβ (1–40) with 1.0 mol/L nicotine for 24 h, many short, sheared fibrils were observed. At 72 h, the number of fibrils was markedly reduced, and small, amorphous aggregates were occasionally observed. Similarly, 100 mmol/L nicotine also disrupted the preformed fAβ (1–40) and fAβ (1–42). N-methylpyrrolidine had similar disruption of fAβ (1–40) and fAβ (1–42) to nicotine, but pyrrolidine had no disruption effect on fAβ (1–40) and fAβ (1–42).34 Nicotine dose-dependently inhibited fAβ (1–40) and fAβ (1–42) formation from fresh fAβ (1–40) and fAβ (1–42), respectively, as well as the extension reaction of both fAβs. Moreover, nicotine disrupted preformed fAβ (1–40) and fAβ (1–42). These effects of nicotine were observed at

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concentrations above 10 mmol/L and were similar to those of N-methylpyrroline. The antiamyloidogenic effect of nicotine may be exerted not only by the inhibition of fAβ formation, but also by the disruption of preformed fAβ. Additionally, this effect may be attributed to N-methylpyrroline moieties of nicotine. Fluorescence spectroscopic analysis with thioflavin T and electron microscopic studies showed that nordihydroguaiaretic acid disrupts fAβ (1–40) and fAβ (1–42) within a few hours at pH 7.5 at 37°C. The activity of nordihydroguaiaretic acid in breaking down fAβ (1–40) and fAβ (1–42) in comparison with other molecules, reported to inhibit fAβ formation from Aβ, was in the order: nordihydroguaiaretic acid ⬎⬎ rifampicin ⫽ tetracycline ⬎ poly (vinylsulfonic acid, sodium salt) ⫽ 1,3-propanedisulfonic acid, disodium salt ⬎ β -sheet breaker peptide (iAβ 5). Moreover, in cell culture experiments, fAβ disrupted by nordihydroguaiaretic acid was significantly less toxic than intact fAβ.33

16.4 NICOTINE PROTECTS HIPPOCAMPAL NEURONAL CELLS AGAINST Aβ -INDUCED APOPTOSIS The function of nicotine obtained from a chemical system needs to be tested in a biological system. The cell is the basic model of life for testing the biological function of nicotine. We have studied the protective effect of nicotine on hippocampal neuronal cell apoptosis induced by Aβ.35 Mitochondrial function was measured by the 3-[4,5-dimethylthiazol-2]-2,5 diphenyl tetrazolium bromide (Determination of cell survival (MTT) conversion assay, which may also serve as a general indicator of cell viability. Protective effects of different concentrations of nicotine on Aβ -induced alterations of mitochondrial function of cultured hippocampal neurons was found. Treatment with 50 µg/mL aged Aβ (25⫺35) or Aβ (1⫺40) for 24 h markedly decreased MTT conversion in cultured hippocampal neuronal cells. In addition, treatment with 10 µM of nicotine significantly attenuated aged Aβ (25–35)- or Aβ (1–40)-induced decrease in MTT conversion. There were no effects on cell viability for nicotine alone at 1 or 10 µM. DNA fragmentation was used as a marker of cell apoptosis in most cases.36 In order to evaluate the effects of nicotine on DNA fragmentation in aged Aβ (25–35)- or Aβ (1–40)-induced apoptosis, an enzyme-linked immunosorbent assay (ELISA) kit was used to analyze DNA degradation quantitatively. Histoneassociated DNA fragmentation (mono- and oligonucleosomes) was detected using antihistone monoclonal antibody. When the cells were incubated with aged Aβ (25–35) or Aβ (1–40) for 48 h, the Enrichment Factor (EF) values of DNA fragmentation were significantly increased concentration-dependently (10 and 50 µg/mL, respectively). When nicotine was added to the culture with aged Aβ (25–35) or Aβ (1–40), DNA fragmentation was significantly decreased, especially when the concentration of nicotine was 10 µM. There was no effect on EF for nicotine alone at 1 or 10 µM. Aged Aβ (25–35)-induced decrease in viability of hippocampal cells may be caused by induction of apoptosis. To clarify the interaction of nicotine with the intracellular downstream signaling cascade of Aβ (25–35), the activity of caspase-3,

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which is an enzyme involved in the executive phase of apoptosis, was measured in hippocampal cultures exposed to aged Aβ (25–35) and nicotine. Treatment with 50 µg/mL Aβ (25–35) or Aβ (1–40) for 24 h markedly increased caspase-3 activity. However, treatment with 10 µM nicotine decreased the aged Aβ (25–35)- or Aβ(1–40)-induced elevation of caspase-3 activity to control levels. Aβ (1–40) had no effect on caspase-3 activity in the presence or absence of nicotine. However, mecamylamine significantly antagonized nicotine-induced protection against Aβ induced caspase-3 activation. Mecamylamine alone had no effect on Aβ -induced caspase-3 activation. Some researchers have suggested the involvement of oxidative stress in the pathogenesis of hippocampal neuronal cell death in AD.37 By using ROS fluorescent dye 2⬘,7⬘-dichlorofluorescin (DCF), it was found that exposure of cultured hippocampal cells to aged Aβ (25–35) (50 µg/mL) for 24 h resulted in a highly significant (100%) increase in DCF fluorescence in the cells. The increase in DCF fluorescence was essentially eliminated in culture cells co-treated with nicotine (10 µM). Aged Aβ (25–35)-induced, concentration-dependent ROS increase and low concentration of aged Aβ (25–35)- (10 µg/mL) induced ROS increase was almost completely inhibited by 10 µg/mL nicotine. Aβ (1–40) induced accumulation of ROS. Aβ (1–40) had no effect on the accumulation of ROS in the presence or absence of nicotine. However, mecamylamine significantly antagonized nicotine-induced protection against Aβ -induced accumulation of ROS. Mecamylamine alone had no effect on Aβ -induced accumulation of ROS. It was found that 10 µM nicotine had no effect on the generation of ROS in the cells. Since the mechanism of toxicity of aged Aβ (25–35) is mediated in part by elevations of [Ca2⫹]i,38,39 we determined whether nicotine affected aged Aβ (25–35)-induced increase in [Ca2⫹]i. Exposure of hippocampal neuronal cells to aged Aβ (25–35) for 24 h resulted in an about fourfold elevation of [Ca2⫹]i. Nicotine alone had little effect on [Ca2⫹]i; however, the [Ca2⫹]i in hippocampal neuronal cells co-treated with nicotine (10 µM) was significantly less than that in hippocampal neuronal cells treated with aged Aβ (25–35) (50 µg/mL) alone. Thus, nicotine attenuated aged Aβ (25–35)- or Aβ (1–40)-induced elevation of free [Ca2⫹]i. In a similar experiment using soybean isoflavan genistein, a natural antioxidant, we found that Aβ (25–35)-induced apoptosis, indicated by decreased cell viability and neuronal DNA condensation and fragmentation, is associated with an increase in the level of intracellular free Ca2⫹, the accumulation of ROS, and the activation of caspase-3. All these phenotypes induced by Aβ (25–35) are reverted by genistein. Our results further show that at nanomolar level (100 nm), genistein protects neurons from Aβ (25–35)-induced damages largely via the estrogen receptor (ER)-mediated pathway, and at micromolar level (40 µM), the neuroprotective effect of genistein is mainly mediated by its antioxidative properties.40 The study by Levites et al.41 shows that (-)-epigallocatechin-3-gallate(EGCG) enhances (approximately sixfold) the release of the nonamyloidogenic soluble form of the amyloid precursor protein (sAPPα) into the conditioned media of human SHSY5Y neuroblastoma and rat pheochromocytoma (PC12) cells. sAPPα release was blocked by the hydroxamic acid-based metalloprotease inhibitor Ro31.9790, which

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indicated mediation via α-secretase activity. Inhibition of protein kinase C (PKC) with the inhibitor GF109203X, or by down-regulation of PKC, blocked EGCGinduced sAPPα secretion, suggesting the involvement of PKC. Indeed, EGCG induced the phosphorylation of PKC, thus identifying a novel PKC-dependent mechanism of EGCG action by activation of the nonamyloidogenic pathway. EGCG is not only able to protect, but it can rescue PC12 cells against the Aβ toxicity in a dose-dependent manner. In addition, administration of EGCG (2 mg/kg) to mice for 7 or 14 days significantly decreased membrane-bound holoprotein APP levels, with a concomitant increase in sAPPα levels in the hippocampus. Consistently, EGCG markedly increased PKCα and PKCε in the membrane and the cytosolic fractions of mice hippocampus. Thus, EGCG has protective effects against Aβ -induced neurotoxicity and regulates secretory processing of nonamyloidogenic APP via the PKC pathway.41 The above results show that the protection of nicotine is partly via antioxidant properties and partly via nicotinic receptors.

16.5 CHRONIC NICOTINE TREATMENT REDUCES β -AMYLOIDOSIS IN THE BRAIN OF A MOUSE MODEL WITH ALZHEIMER’S DISEASE There are presently initiatives to develop treatment strategies in AD that effectively lower the Aβ load in brain. Nordberg et al.42 treated transgenic mice carrying the Swedish mutation of human amyloid precursor protein (APPsw), which develop brain Aβ deposits, with nicotine in drinking fluid (200 µg/mL) from 9 to 14.5 months of age (Five and half months). A significant reduction in Aβ (1–42) positive plaques by more than 80% (p ⬍ 0.03) was observed in the brains of nicotine-treated compared to sucrose-treated transgenic mice. In addition, there was a selective reduction in extractable Aβ in nicotine-treated mice; cortical-insoluble Aβ (1–40) and Aβ (1–42) peptide levels were lower by 48 and 60%, respectively (p ⬍ 0.005), while there was no significant change in soluble Aβ (1–40) or Aβ (1–42) levels. The expression of glial fibrillary acidic protein was not affected by nicotine treatment. These results indicate that nicotine may effectively reduce Aβ aggregation in brain and that nicotinic drug treatment may be a novel protective therapy in AD.42 Ten-days treatment with nicotine reduced insoluble Aβ (1–40) and Aβ (1–42) peptides by 80% in the cortex of 9-month-old APPsw mice, which is more than that observed in 14.5-month-old mice following nicotine treatment for 5.5 months. A reduction in Aβ associated with cerebral vessels was observed in addition to that deposited as paranchymal plaques after 5.5-months treatment. The diminution in Aβ peptides observed was not accompanied by changes in brain α-, β -, or γ - secretaselike activities, nerve growth factor (NGF) or brain-derived neurotrophic factor (BDNF) protein expression measured in brain homogenates. A significant increase in sAPP was observed after nicotine treatment of SH-SY5Y neuroblastoma cells that could be blocked by the nicotine antagonist mecamylamine. Attenuation of

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elevated[125I]-α−bungarotoxin binding(α 7) in APPsw mice was observed after 5.5months nicotine treatment. Both these observations suggest that the reduction in insoluble Aβ by nicotine might be in part mediated via the α 7 nicotine receptor. Further studies are required to identify potential mechanisms of the nicotine’s amyloid-reducing effect.43 Nicotinic cholinergic receptor stimulation induces neuroprotection against glutamate cytotoxicity by its inhibitory action on NO formation. However, there are different reports about the effect of nicotine on the generation of NO. It was reported that nicotine evoked NO release in the rat hippocampal slice,44 and that NOS expression in the ventromedial hypothalamic nucleus increased by nicotine treatment. However, it was found that nicotine administration was effective in limiting the enhancement on NOS expression following food restriction in another report. The mismatch results were reported about the effects of nicotine on NO production and NOS expression determined by biochemical and histological methods.45 The contradictory results indicate that there is no conclusion and its mechanism is not clear. We are studying the production of NO free radicals in transgenic mice and the effect of nicotine. The preliminary results showed that nicotine may be involved in the protection of the brain against AD by regulation of NO and ROS.46

16.6 CONCLUSION In conclusion, the protective effect of nicotine on AD in model systems is positive. In the in vitro systems, nicotine shows antioxidant, antiamyloidogenic, and disruption effects on fAβ. In the cell system, nicotine protects neurons against fAβ -induced apoptosis. In the in vivo system, nicotine shows reduction of β -amyloidosis in the brain of a mouse model of Alzheimer’s disease (APPsw). Further study is essential for understanding the protective mechanisms of nicotine in neurodegenerative processes in AD and development of an effective technique for human treatment.

REFERENCES 1. Vickers JC, Dickson TC, Adlard PA, Saunders HL, King CE, Mccormack G. The cause of neuronal degeneration in Alzheimer’s disease. Prog Neurobiol, 2000; 60: 139–165. 2. Soto C, Braens MC, Alvarez J, Inestrosa NC. Structural determinants of the Alzheimer’s amyloid-β -peptide. J Neurochem, 1994; 63: 1191–1198. 3. Soto C, Sigurdsson EM, Morelli L, Kumar RA, Castana O EM, Frangione B. β -sheet breaker peptides inhibit fibrillogenesis in a rat brain model of amyloidosis: implications for Alzheimer’s therapy. Nat Med, 1998; 4: 822–826. 4. Saitoh T, Sundsmo M, Roch JM, Kimura N, Cole G, Schubert D, Oltersdorf T, Schenk DB. Secreted form of amyloid-β -protein precursor is involved in the growth regulation of fibroblasts. Cell, 1989; 58: 615–622. 5. Hass C, De Strooper B. The presenilins in Alzheimer’s disease-proteolysis holds the key. Science, 1999; 286: 916–919.

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6. Yankner BA. Mechanisms of neuronal degeneration in Alzheimer’s disease. Neuron, 1996; 16: 921–932. 7. Alvarez A, Alarcoa R, Opaza C, Campos EO, Munoz FJ, Calderon FH, Dajas F, Gentry MK, Doctor BP, De Mello FG, Inestrosa NC. Stable complexes involving acetylcholinesterase and amyloid-β peptide change the biochemical properties of the enzyme and increase the neurotoxicity of Alzheimer’s fibrils. J Neurosci, 1998; 18: 3213–3223. 8. Munoz FJ, Inestrosa NC. Neurotoxicity of acetylcholinesterase-amyloid-β -peptide aggregates is dependent on the type Aβ -peptide and the AChE concentration present in the complexes. FEBS Lett, 1999; 450: 205–209. 9. Inestrosa NC, Reyes AE. Acetylcholinesterase induces amyloid formation and increases neurotoxicity of Alzheimer’s fibrils. Neurobiol Aging, 1998; 19(Suppl. 4): S44. 10. Inestrosa NC, Larrondo LF. Oxidative stress in Alzheimer’s disease, in Health, Antioxidants and Ageing, Leighton F, Urquiaga I. Eds, Vice Rectory of Catholic University of Chile, Santiago, Chile, 2000. 11. Mark R, Blanc E, Mattson M. Amyloid beta-peptide and oxidative cellular injury in Alzheimer’s disease. Mol Neurobiol, 1996; 12: 915–924. 12. Pollard H, Arispe N, Rojas E. Ion channel hypothesis for Alzheimer amyloid peptide neurotoxicity. Cell Mol Neurobiol, 1995; 15: 513–526. 13. Pike C, Weinstein S, Velaquez P, Cotman C. All-D-enantiomers of beta-amyloid exhibit similar biological properties to all-L-beta-amyloids. J Biol Chem, 1997; 272: 7431–7436. 14. Hensley K, Carney J, Mattson M, Aksenova M, Harris M, Wu J, Floyd R, Butterfield D. A model for beta-amyloid aggregation and neurotoxicity based on free radical generation by the peptide: Relevance to Alzheimer disease. Proc Natl Acad Sci, 1994; 91: 3270–3274. 15. Morens DM, Grandinetti A, Reed D, White LR, Ross GW. Cigarette smoking and protection from Parkinson’s disease: false association or etiologic clue? Neurology, 1995; 45: 1041–1051. 16. Ramon SO, Estefanya MA. Studies on the interaction between 1,2,3,4-tetrahydroβ -carboline and cigarette smoke: a potential mechanism of neuroprotection for Parkinson’s disease. Brain Res, 1998; 802: 155–162. 17. Barrantes GE, Murphy CT, Westwick J, Wonnacott S. Nicotine increases intracellular calcium in rat hippocampal neurons via voltage-gated calcium channels. Neurosci Lett, 1995; 196: 101–104. 18. Hillier V, Salib E. A case-control study of smoking and Alzheimer’s disease. Int J Geriatr Psychiatr, 1997; 12: 295–300. 19. Ulrich J, Johannson-Locher G. Does smoking protect from Alzheimer’s disease? Alzheimer-type changes in 301 unselected brains from patients with known smoking history. Acta Neuropathol, 1997; 945: 450–454. 20. Emilien G, Beyreuther K, Masters CL, Maloteaux JM. Prospects for pharmacological intervention in Alzheimer disease. Arch Neurol, 2000; 57:454–459. 21. Kihara T, Shimohama S, Sawada H, Kimura J, Kume T, Kochiyama H. Nicotinic receptor stimulation protects neurons against β -amyloid toxicity. Ann Neurol, 1997; 42: 159–163. 22. Garrido R, Malecki A, Hennig B, Toborek M. Nicotine attenuates arachidonic acid-induced neurotoxicity in cultured spinal cord neurons. Brain Res, 2000; 861: 59–68.

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23. Garrido R, Mattson MP, Hennig B, Toborek M. Nicotine protects against arachidonic-acid-induced caspase activation, cytochrome c release and apoptosis of cultured spinal cord neurons. J Neurochem, 2001; 76: 1395–1403. 24. Dajas-Bailador FA, Lima PA, Wonnacott S. The alpha7 nicotinic acetylcholine receptor subtype mediates nicotine protection against NMDA excitotoxicity in primary hippocampal cultures through a Ca2⫹ dependent mechanism. Neuropharmacology, 2000; 39: 2799–2807. 25. Liu Q, Tao Y, Zhao B-L. ESR study on scavenging effect of nicotine on free radicals. Appl Mag Reson, 2003; 24: 105–112. 26. Linert W, Bridge MH, Huber M, Bjugstad KB, Grossman S, Arendash GW. In vitro and in vivo studies investigating possible antioxidant actions of nicotine: relevance to Parkinson’s and Alzheimer’s diseases. Biochim Biophys Acta, 1999; 1454: 143–152. 27. Robakis NK, Ramakrishna N, Wolfe G, Wisniewski HM. Molecular cloning and characterization of a CDNA encoding the cerebrovascular and the neuritic plaque amyloid peptide. Proc Natl Acad Sci USA, 1987; 84: 4190–4194. 28. Selkoe DJ. The cell biology of β -amyloid precursor protein and presenilin in Alzheimer’s disease. Trends Cell Biol, 1998; 8: 447–453. 29. Seloke DJ. Cell biology of the amyloid beta-protein precursor and the mechanism of Alzheimer’s disease. Annu Rev Cell Biol, 1994; 10: 373–403. 30. Salomon AR, Marcinowski KJ, Friendland RP, Zagorski MG. Nicotine inhibits amyloid formation by b-peptides. Biochemistry, 1996; 35: 13568–13578. 31. Zeng H, Zhang Y, Peng L-J, Shao H, Menon NK, Yang J, Salomon AR, Freidland RP, Zagorski MG. Nicotine and amyloid formation. Biol Psychiatr, 2001; 49: 248–257. 32. Naiki H, Hasegawa K, Yamaguchi I, Nakamura H, Gejyo F, Nakakuki K. Apolipoprotein E and antioxidants have different mechanisims of inhibiting Alzheimer’s amyloid fibril formation in vitro. Biochemistry, 1998; 37: 17882–17898. 33. Ono K, Hasegawa K, Yoshiike Y, Takashima A, Yamada M, Naiki H. Nordihydroguaiaretic acid potently breaks down preformed Alzheimer’s amyloid fibrils in vitro. J Neurochem, 2002; 81: 434–440. 34. Ono K, Hasegawa K, Yamada M, Naiki H. Nicotine breaks down preformed Alzheimer’s β -amyloid fibrils in vitro. Biol Psychiatr, 2002; 52: 880–886. 35. Liu Q, Zhao B-L. Nicotine attenuates β -amyloid peptide induced neurotoxicity, free radical and calcium accumulation in hippocampal neuronal cultures. Brit J Pharmocol, 2004; 141: 746–754. 36. Earnshaw WC. Nuclear changes in apoptosis. Curr Opin Cell Biol, 1995; 7: 337–343. 37. Miranda S, Opazo C, Larrondo LF, Munoz FJ, Ruiz F, Leighton F, Inestrosa NC. The role of oxidative stress in the toxicity induced by amyloid beta-peptide in Alzheimer’s disease. Prog Neurobiol, 2000; 62: 633–648. 38. Brorson JR, Bindokas VP, Iwama T, Marcuccilli CJ, Chisholmand JC, Miller RJ. The Ca2⫹ influx induced by beta-amyloid peptide 25–35 in cultured hippocampal neurons results from network excitation. J Neurobiol, 1995; 26: 325–338. 39. Mattson MP, Cheng B, Davis DK, Bryant K, Lieberburg I, Rydel RE. β -amyloid peptides destabilize calcium homeostasis and render human cortical nervous vulnerable to excitotoxicity. J Neurosci, 1992; 12:379–389. 40. Zeng H, Chen Q, Zhao B-L. Genistein ameliorated β -amyloid peptide-induced hippocampal neuronal apoptosis. Free Radic Biol Med, 2004; 36: 180–188. 41. Levites Y, Amit T, Mandel S, Youdim MBH. Neuroprotection and neurorescue against Aβ toxicity and PKC-dependent release of non-amyloidogenic soluble precursor protein by green tea polyphenol (−)- epigallocatechin-3-gallate. FASEB J, 2003; 17:952–954, Published online - www.fasebj.org.

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42. Nordberg A, m-Lindahl EH, Lee M, Johnson M, Mousavi M, Perry RHE, Bednar I, Court J. Chronic nicotine treatment reduces β -amyloidosis in the brain of a mouse model of Alzheimer’s disease (APPsw). J Neurochem, 2002; 81: 655–658. 43. Hellstrom-Lindahl E, Court J, Keverne J, Svedberg M, Lee M, Marutle A, Thomas A, Perry E, Bednar I, Nordberg A. Nicotine reduces Aβ in the brain and cerebral vessels of APPsw mice. Eur J Neurosci, 2004; 19: 2703–2710. 44. Kaiser S, Wonnacott S. in Neuronal Nicotinic Receptors: Pharmacology and Therapeutic Opportunities. Arneric SP, Brioni JD. Eds, Wiley-Liss, NY, 1999; 141–159. 45. Shimohama S, Greenwald DL, Shafron DH, Akaike A, Maeda T, Kaneko S. Nicotinic receptor-mediated protection against β -amyloid neurotoxicity. Brain Res, 1998; 779: 359–363. 46. Zhao B-L. Nitric oxide in neurodegenerative diseases. Front Biosci, 2005; 10: 454–461.

Essentiality of Iron 17 The Chelation in Neuroprotection: A Potential Role of Green Tea Catechins Silvia A. Mandel, Tamar Amit, Hailin Zheng, Orly Weinreb, and Moussa B.H. Youdim Technion Israel Institute of Technology Haifa, Israel

CONTENTS Abstract..............................................................................................................277 17.1 Introduction..............................................................................................278 17.2 Neuroprotective Activity of Tea Catechins via Chelation of Divalent Metals ........................................................................................280 17.2.1 Neuroprotective Effects In Vivo................................................280 17.2.2 Neuroprotective Effects In Vitro...............................................281 17.2.3 Tea Catechins as Brain-Permeable, Nontoxic Iron Chelators to “Iron Out” Iron from the Brain............................282 17.2.3.1 Effect of Tea Catechins on Iron and Hypoxia-Regulated Genes .......................................287 17.2.4 Effects of Tea Catechins on Cell Signaling Pathways .............289 17.3 Conclusions..............................................................................................291 References .........................................................................................................291

ABSTRACT Neurodegeneration in Parkinson’s, Alzheimer’s, or other neurodegenerative diseases appears to be multifactorial, in that a complex set of toxic reactions such as 277

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oxidative stress, inflammation, glutamatergic neurotoxicity, reduced expression of trophic factors and endogenous antioxidants, dysfunction of the ubiquitin– proteasome system with a concomitant accumulation of protein aggregates, and increase of nitric oxide, lead to the demise of neurons. A cardinal chemical pathology observed in these disorders is the accumulation of iron at sites where the neurons die. The buildup of an iron gradient in conjunction with reactive oxygen species (ROS) (superoxide, hydroxyl radical, and nitric oxide) is thought to constitute a major trigger of neuronal toxicity and demise in these diseases. Therefore, promising future treatment of neurodegenerative diseases and aging depends on the availability of effective brain-permeable, iron-chelatable/radical scavenger, neuroprotective drugs that will prevent the progression of neurodegeneration. Tea flavonoids (catechins) have been reported to possess potent divalent metal-chelating, antioxidant, and anti-inflammatory properties; to penetrate the brain barrier and to protect neuronal death in a wide array of cellular and animal models of neurological diseases. Additional mechanisms include activation of signaling pathways responsible for cell survival, growth, and differentiation. These activities make tea catechins promising substances for the treatment of brain deterioration during aging and neurodegeneration. This chapter aims to shed light on the multifunctional properties of green tea catechins, especially (-)-epigallocatechin-3-gallate (EGCG), with special emphasis on their transitional metal (iron and copper)-chelating property and inhibition of oxidative stress.

17.1 INTRODUCTION Oxidative damage to neuronal biomolecules, accumulation of iron in specific brain areas, and inflammatory processes with proliferation of reactive microglia, are considered major pathological aspects of the aging process and neurodegenerative disorders such as Parkinson’s and Alzheimer’s diseases (PD and AD, respectively).1 Transitional metal alterations (e.g., iron, copper, and zinc) have been described in the brains of people suffering from PD and other neurodegenerative diseases, which maybe caused, to a large degree, by endogenous dysregulation of iron uptake, transport, distribution, and storage.2,3 The redox-active metals are promoters of membrane-associated oxidative stress (OS), including lipid peroxidation and oxidative modifications of membranes and associated proteins such as receptors. In line with this, high intake of iron has been associated with increased risk of PD and AD,4 while persistent iron deprivation, has been shown to protect cortex and hippocampal cells from kainate-induced damage.5 It is not surprising that antioxidants were the first drugs to be studied in an attempt to retard the progress of PD. Recently, coenzyme Q10, an intrinsic component of the mitochondrial respiratory chain acting as a bioenergizer and an antioxidant, was studied as a putative neuroprotective agent in PD. This double-blind, placebo-controlled pilot study demonstrated that high doses of coenzyme Q10 (1200 mg/d) were associated with a reduced rate of deterioration in motor function from baseline over the 16-month course of the trial.6 Indeed, the metal-protein

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attenuating compound iodochlorhydroxyquin (clioquinol) has been shown to slow down the clinical progression of AD dementia in a small-scale clinical study.7 Thus, promising future treatment of neurodegenerative diseases and aging depends on availability of effective brain-permeable, iron-chelatable/radical scavenger, neuroprotective drugs that will prevent the progression of neurodegeneration. In this context, we have recently developed novel nontoxic, lipophilic, brain-permeable iron chelators, VK-28, HLA20, and M30, which are neuroprotective in animal and cell culture models of neurological diseases.8,9 Several plant flavonoids have been reported to possess potent iron-chelating, radical scavenging, and anti-inflammatory properties. They also prevent neuronal death in a wide array of cellular and animal models of neurodegeneration. Indeed, increasing evidence from epidemiological studies, animal research, clinical trials, and research in nutritional biochemistry suggests that some dietary supplements may be used beneficially in aging,10 PD, AD,11 as well as coronary heart disease,12 cancer,13 osteoporosis,14 and diabetes.15 The beneficial effects of the plantand fruit-derived compounds are ascribed to their high content of polyphenolic flavonoids. These compounds have been shown to possess antioxidant/radical scavenging activity and increase the capacity of endogenous antioxidant defenses, thus modulating the cellular redox state. Fresh tea (Camellia sinensis) leaves contain a high amount of catechins, a group of flavonoids or flavanols, known to constitute 30 to 45% of the solid green tea extract.16,17 The favorable properties ascribed to tea consumption are believed to derive from its bioactive components: catechins and their derivatives, demonstrated to act directly as radical scavengers and exert indirect antioxidant effects through activation of transcription factors and antioxidant enzymes (see reviews18,19). The most abundant polyphenolic compound, (-)-epigallocatechin-3-gallate (EGCG), is thought to contribute to the beneficial effects attributed to green tea, such as its anticancer, cardiovascular function improvement, and antioxidant anti-inflammatory properties. EGCG accounts for more than 10% of the extract dry weight (30 to 130 mg per cup of tea) followed by (-)-epigallocatechin (EGC) ⬎ epicatechin (EC) ⱖ (-)epicatechin-3-gallate (ECG).16 In addition to their radical scavenging action, green tea catechins possess well-established metal-chelating properties. Structurally important features defining their chelating potential are the 3⬘,4⬘-dihydroxyl group in the B ring20 as well as the gallate group,21,22 which may neutralize ferric iron to form redox-inactive iron, thereby protecting cells against oxidative damage.23 There is evidence that polyphenol metabolites and their parent compounds have access to the brain.24,25 It has also been shown that both the methylated and glucuronidated derivatives of EC are detected in rat brain following oral administration.26 Research from our laboratory suggests that the antioxidant iron-chelating activity of major green tea polyphenol EGCG plays a major role in preventing neurodegeneration in a variety of cellular and animal models of neurodegenerative diseases.27,28 Furthermore, collective studies indicate that beyond these properties, flavonoids appear to regulate various signaling pathways involved in cellular survival, growth, and differentiation. Thus, some flavonoids have been

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shown to modulate activities of protein kinases, such as mitogen activated protein kinase (MAPK) and protein kinase C (PKC). They have also been shown to regulate the activity of the signal transducers, to serve as ligands for transcription factors, and to alter protease activities.29 This chapter is mainly an overview of the multipharmacological neuroprotective activities of green tea with respect to their relevance for iron-induced brain neurodegeneration.

17.2 NEUROPROTECTIVE ACTIVITY OF TEA CATECHINS VIA CHELATION OF DIVALENT METALS 17.2.1 NEUROPROTECTIVE EFFECTS IN VIVO There is a growing recognition that polyphenolic catechins exert a protective role in neurodegeneration. An experimental study conducted in an N-methyl-4phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of PD has shown that both green tea extract and EGCG effectively prevent mice striatal dopamine (DA) depletion and substantia nigra (SN) dopaminergic neuron loss.30 The protection exerted by green tea polyphenols in vivo may involve direct scavenging of reactive oxygen species (ROS) and regulation of antioxidant protective enzymes. EGCG was found to elevate the activity of two major oxygen-radical species metabolizing enzymes, superoxide dismutase (SOD) and catalase in mice striatum.30 This is supported by a previous finding where 1-month administration of a catechin-containing antioxidant preparation increased SOD activity in the mitochondria fraction of striatum and midbrain, and decreased thiobarbituratereactive substance formation in the cortex and cerebellum of aged rats.31 The structural resemblance of EGCG to cathechol may explain a recently reported inhibitory effect of green tea polyphenols on the DA presynaptic transporters. This inhibition lead to 1-methyl-4-phenylpyridinium (MPP⫹) uptake blockade (because of competition for the vesicular transporter), thereby protecting DAcontaining neurons against MPP⫹-induced injury.32 In addition, EGCG greatly inhibited catechol-O-methyltransferase (COMT) activity in rat liver cytosol at a low IC50 concentration (0.2 µM).33 This action may be of particular significance for PD patients, given that DA and related catecholamines are physiological substrates of COMT. Thus, its inhibition will result in increased DA in the synapse. Green tea polyphenols have also been shown beneficial in animal models of cerebral ischemia: intraperitoneal (IP) injection of EGCG reduced hippocampal neuronal damage and brain edema caused by global34 or unilateral35 cerebral ischemia in gerbils. Insights into the possible mechanism of neuroprotection by EGCG in the infarct area of ischemic rats revealed that it acts by reducing inducible nitric oxidase synthase (iNOS) expression, neutrophil infiltration, and peroxynitrite formation36 and by increasing endothelial and neuronal NOS and preserving mitochondrial complex activity and integrity.37 In this context, the decrease in the activity of the transcription factor signal transducer and activator

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of transcription-1α (STAT-1α) by EGCG in ischemic rat cardiac myocytes may well account for the reduced mRNA levels of iNOS, a target of STAT-1.38 Other investigators have recently shown that EGCG reduced brain inflammation and neuronal damage in experimental autoimmune encephalomyelitis (EAE) when given at initiation or after the onset of EAE.39

17.2.2 NEUROPROTECTIVE EFFECTS IN VITRO An extensive number of studies regarding neuroprotection by green tea flavonoids in cellular models of neurodegenerative diseases are starting to accumulate. Hence, the flavonoid EC was shown to attenuate the toxicity induced by oxidized, low-density lipoprotein in mouse-derived striatal neurons40 or fibroblasts,41 and to confer protection to a primary culture of mesencephalic neurons challenged with 6-hydroxydopamine (6-OHDA).42 Recently, catechin was shown to reduce injury produced by hydrogen peroxide, 4-hydroxynonenal, rotenone, and 6-OHDA in primary rat mesencephalic cultures, as shown by increases in cellular viability and [3H]DA uptake.43 Similarly, EGCG was reported to protect human neuroblastoma cells from damage induced by 6-OHDA and MPP⫹.44 EGCG also protects primary hippocampal neurons45 and rescues rat pheochromocytoma (PC12) cells from amyloid-beta peptide (Aβ )-induced toxicity, presumably through the scavenging of ROS.46 More recently, EGCG was reported to exert a neurorescue activity in long-term, serum-deprived PC12 cells and to promote neurite outgrowth, as manifested by the expression of a surrogate marker of cell differentiation, growth-associated protein (GAP)-43.47 This could have important implications with regard to aging, PD, and AD, suggesting a potential therapeutic use of EGCG in regenerating injured neuronal cells. Studies from our and other laboratories have shown that EGCG displays a concentration-dependent window of neuroprotective action. It protects at low micromolar concentrations, whereas it becomes pro-oxidant and pro-apoptotic at concentrations above 10 to 20 µM.44,48 This bell-shaped pattern is typical of antioxidative drugs, such as vitamin C,49 R-apomorphine,50 and DA,51 which are neuroprotective at low (1 to 10 µM) concentrations, while showing pro-oxidant/pro-toxic activity at higher (10 to 50 µM) concentrations. This latter effect is apparently responsible for the well-characterized, anticarcinogenic/proliferative action of green tea polyphenols, leading to inhibition of cell cycle progression effecters, promotion of ROS and nitrogen species (thereby collapsing the mitochondrial membrane potential), induction of p53 and apoptogenic factors, and inactivation of protein kinases that contribute to survival-associated signal transduction (for extensive reviews see19,52,53). A more recent study has shown that EGCG is an inhibitor of dihydrofolate reductase (DHFR) activity in vitro and in lymphoma cell lines at concentrations found in the serum and tissues of green tea drinkers (0.1 to 1.0 µmol/L).54 Employing a customized cDNA microarray, it was revealed that a protective concentration of EGCG decreased the expression of pro-apoptotic genes bax, bad, caspases-1 and 6, cyclin-dependent kinase inhibitor p21, cell-cycle

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inhibitor gadd45, fas-ligand, and tumor necrosis factor-related apoptosis-inducing ligand TRAIL in SH-SY5Y neuronal cells.44 The same group has recently reported that EGCG reduced the expression of several apoptogenic factors when given after long-term serum deprivation of PC12 cells.47 These findings are supported by an in vivo study showing that oral consumption of EGCG (2 mg/kg) for 2 weeks alone caused a complete disappearance of Bax immunoreactivity, specifically in the dopaminergic neurons of the SNpc27 and counteracted the robust increase of Bax protein when administered before MPTP intoxication in the same area. The decline in Bax and Bad expression caused by EGCG may favor the increase in the ratio of Bcl-2/Bcl-xL to Bax/Bad proteins, thereby contributing to mitochondrial stability and regulation of MPTP.55 Protection of mitochondrial integrity is of major importance, especially in the case of postmitotic cells such as neurons and heart muscle cells, which are commonly not renewed. Thus, it is not surprising that one of the major neuroprotective strategies in PD, AD, and other neurodegenerative diseases, where increased OS, perturbed cellular energy, and ion homeostasis have been implicated, includes pharmacological agents directed to specific mitochondrial targets. In this respect, Gingko biloba extract EGb 761 or its individual components were shown to protect mitochondria integrity by protecting against uncoupling of oxidative phosphorylation, thereby increasing ATP levels and increasing the expression of the mitochondrial DNA-encoded cytochrome oxidase III subunit (for review see [56]). Flavonoids may also affect mitochondrial integrity by increasing reduced glutathione (GSH) levels and preventing the influx of calcium, as reported previously.57,58

17.2.3 TEA CATECHINS AS BRAIN-PERMEABLE, NONTOXIC IRON CHELATORS TO “IRON OUT” IRON FROM THE BRAIN Various metals have been implicated in the pathophysiology of certain neuropsychiatric diseases — copper and iron in Wilson’s disease; aluminum, zinc, and iron in AD; iron in PD, Friedreich’s ataxia, and Hallervorden-Spatz syndrome, to mention a few59–61 (Table 17.1). Studies on human and animal brains have shown that the distribution of brain iron is uneven when compared with other metals in the brain. Thus, iron is present in substantia nigra (SN), globus pallidus, and dentate gyrus at a concentration equal to or greater than that found in the liver. These three brain regions are known to be associated with neurodegenerative diseases.62 Specifically, redox-active iron has been observed in the rim of Lewy body, the morphological hallmark of PD, which is composed of lipids, aggregated α-synuclein (concentrating in its peripheral halo), and ubiquitinated, hyperphosphorylated, neurofilament proteins.63 α-Synuclein has been shown to form toxic aggregates in the presence of iron and is considered to contribute to the formation of Lewy body via OS.64–66 Furthermore, iron promotes both deposition of Aβ and induction of OS, which is associated with the plaques. Indeed, it has been demonstrated that amyloid deposits are enriched with zinc, iron, and copper.61

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TABLE 17.1 Increased Localized Brain Ferritin and Iron in Neurodegenerative Diseases Aceruloplasnemia Alzheimer’s disease Amyotrophic lateral sclerosis Acquired immune deficiency syndrome Freidriech ataxia Hallervorden syndrome Huntington’s chorea Juvenile Parkinson’s disease Multiple sclerosis Parkinson’s disease Prion diseases Source: From Gotz ME et al., Ann NY Acad Sci, 2004; 1012:193–208.

Recently, iron has taken center stage in AD as a consequence of the studies by Rogers and coworkers67 who described the existence of an iron responsive element (IRE-type II) in the 5⬘-untranslated region (UTR) of amyloid precursor protein (APP) mRNA. APP is post-transcriptionally regulated by iron regulatory proteins (IRPs), which are labile iron pool-sensitive cytosolic RNA proteins binding specifically to the IRE located in the 5⬘ or 3⬘ UTRs of iron metabolism-associated mRNAs. Changes in iron status (iron overload or depletion) lead to compensating changes in the IRP/IRE system of translational control of iron homeostasis. For example, the APP 5⬘-UTR conferred translation was selectively down-regulated upon intracellular iron chelation, in a similar manner as the ironstorage protein ferritin, which also possesses an IRE in its 5⬘UTR mRNA.67 The involvement of metals in protein deposition in neurological disorders has encouraged the development of iron chelators as a major new therapeutic strategy. In fact, intramuscular administration of the prototype iron chelator desferrioxamine (DFO) slowed down the clinical progression of AD dementia.68 Some success has also been achieved with another metal-complexing agent, clioquinol.7 However, clioquinol is highly toxic,69 and DFO has poor penetration across the blood–brain barrier (BBB).70 A novel and promising therapeutic approach for treating AD, PD, and amyotrophic lateral sclerosis (ALS) with nontoxic, brainpermeable metal chelators could be the use of the natural occurring polyphenols, such as EGCG and curcumin, which, by being of natural origin, may not exert toxic side effects inherent to synthetic drugs. Both compounds have well-characterized antioxidant, metal (iron and copper) chelating, and anti-inflammatory properties,11,71 and have been demonstrated to exert neuroprotective activity against a variety of neurotoxic insults, as well as to regulate APP processing and Aβ burden in vivo and in cell culture.11,72 Our recent studies have shown that prolonged administration of EGCG to mice induced a reduction in holoAPP

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levels in the hippocampus.46 Indeed, this effect may be related to the ironchelating properties of EGCG, leading to a decrease in the free-iron pool. This in turn, would result in the suppression of APP mRNA translation by targeting the IRE-II sequences in the APP 5⬘-UTR,67 as was recently shown for DFO and the bifunctional amyloid-binding/metal-chelating drug XH173 (Figure 17.1). Comparative analysis of the Fe2⫹-chelating potency of EGCG, the prototype DFO, and our newly developed nontoxic, lipophilic, brain-permeable iron chelators, VK-28, HLA20, and M30 has revealed similar binding potency29 (Figure 17.2).

Disruption of BBB

Ferritin

Neurotoxic events Genetic-enviroment

Released iron

Fe 2+

Serum Fe 2+

EGCG Curcumin VK28 M30 HLA20 DFO XH1

Fe 3+

Fe 3+ + A


EGCG curcumin

Labile Fe pool H2O2, OH ?

Degradation of IRP2

Prolyl hydroxylase key iron and oxygen sensor

Oxidative reactions

Degradation of HIF

IRE II APP mRNA 5′

3′ AAAn

Angiogenesis Cell proliferation/survival Glucose/iron metabolism

A


Neurodegeneration

FIGURE 17.1 Iron-induced neurodegeneration in AD via transcriptional activation of APP mRNA. Neurodegeneration can result from abnormal serum iron transport to the neurons because of a disruption in the blood-brain barrier (BBB) or release of its storage protein ferritin, thereby increasing the free-labile iron pool (ionic iron). Labile iron can increase the production of APP by down-regulating the activities of iron regulatory proteins (IRP1 and IRP2, inactivation and proteasomal degradation, respectively), thereby promoting the translation of APP mRNA from its 5⬘-UTR-typeII). Ionic iron may also cause aggregation of Aβ to form toxic aggregates, which, in turn, can initiate OH• generation, causing OS. Increased iron and OS may activate the prolyl hydroxylase enzymes, which are key iron and oxygen sensors, leading to proteasomal-mediated degradation of the transcription factor hypoxia-inducible factor 1α, a master regulator orchestrating the coordinated induction of a wide array of survival genes. It has also been suggested that IRP2 can be a substrate for prolyl hydroxylase. Neuroprotective agents that can be used to prevent iron-induced neurodegeneration include: M30 and HLA20 (bifunctional iron chelator-MAO inhibitors), XH1 (amyloid-binding/metal-chelating), DFO, VK-28 (iron chelator), EGCG and curcumin (iron chelators-antioxidant). HIF. For a more detailed explanation read the text.

The Essentiality of Iron Chelation in Neuroprotection

285

0.7 0.6

∆A at 562 nm

0.5 0.4 0.3 0.2 0.1 0

C

DFO EGCG HLA20 VK28

M30

Novel iron chelators

FIGURE 17.2 Comparison of the Fe2⫹-chelating potency of EGCG to other iron chelators. The metal-binding capacity of EGCG was compared to that of DFO and the novel iron chelators VK28, HLA-20, and M30, by assessing their ability to compete with ferrozine for the ferrous ions, resulting in a decrease in the absorbance at 562 nm. Ferrozine can quantitatively react with Fe2⫹ to form Fe2⫹-ferrozine complex with a strong absorbance at 562 nm. In the presence of other chelating agents, the complex formation is disrupted, resulting in the absorbance at 562 nm being decreased. Drug measuring 0.1 mM was mixed with 0.1 mM ferrozine in 5% ammonium acetate (pH 7) followed by the addition of 0.02 mM FeSO4. After 2 h incubation, the absorbance (at 562 nm) of resulting solutions was read. Considering that the purpose of this assay was to evaluate the ability of drugs to compete with the iron indicator ferrozine, drugs and ferrozine were used at equal concentrations. Chelating effect of drug on Fe2⫹ was calculated as follows: Chelating effect (%) ⫽ [1−(absorbance of sample at 562 nm)/(absorbance of control at 562 nm)] ⫻ 100. The order of chelating potency for complexing Fe2⫹ in solution is Desferal (DFO) ⬎ M30 ⱖ VK28 ⱖ HLA20 ⬎ EGCG.

Moreover, both EGCG and the iron chelator M30 were shown by us to induce a significant down-regulation of membrane-associated holoAPP level in the mouse hippocampus (Figure 17.3) and in vitro, in neuroblastoma SH-SY5Y and Chinese hamster ovary (CHO) cells expressing the APP “Swedish” mutation (data not shown). This may have a direct influence on Aβ levels and plaque formation, as shown in preliminary studies for XH1.73 Using a nucleation-dependent polymerization model, it has been shown that wine and green tea polyphenols are able to inhibit formation, extension, and destabilization of Aβ fibrils.74 Another potential beneficial effect of EGCG in AD may be related to our previous studies demonstrating the ability of EGCG to promote the nonamyloidogenic pathway via a PKC-dependent activation of α-secretase, thereby increasing the production of the nontoxic sAPPα.46 As sAPPα and Aβ are formed by two mutually exclusive mechanisms, stimulation of the secretory processing of sAPPα might prevent the formation of the amyloidogenic Aβ. Thus, EGCG may influence

286

Oxidative Stress and Age-Related Neurodegeneration (B)

holoAPP

holoAPP

β-actin

β-actin 120

120

100

100

80 ∗∗

60 40

% of control

% of control

(A)

80

∗∗

60 40 20

20

0

0 Control

EGCG (2 mg/kg)

Control

M30 (5 mg/kg)

FIGURE 17.3 Effect of EGCG and M30 on APP processing in mice hippocampus. Representative Western blots of levels of holoAPP in the membrane compartment, obtained from the hippocampus of mice treated with (A) EGCG (2 mg/kg), or with (B) M30 (5 mg/kg) for 14 days, detected with 22C11 antibody (directed to the APP Nterminus) or with a C-terminal APP antibody, respectively. Densitometric analysis is expressed as percent of the control, untreated animals after normalizing to the levels of β -actin. Data are expressed as the mean ⫾SEM (n⫽6 mice in each group). p ⬍ 0.03 vs. control. Adapted from Levites Y et al., Faseb J, 2003; 17:952–954.

Aβ levels, either via translational inhibition of APP or by regulating APP processing. Finally, iron chelation by EGCG may ablate Fe3⫹-induced aggregation of hyperphosphorylated tau (PHFτ), the major constituent of neurofibrillary tangles in AD brains75 (a descriptive explanation is depicted in Figure 17.4). In spite of the absence of clinical trials regarding tea polyphenols and PD, epidemiological studies have shown reduced risk of PD associated with consumption of 2 cups/day or more of tea76 and a much lower prevalence of PD in the Chinese population than in white people.77,78 Experimental studies have shown that iron chelation prevents cytochrome c-induced α-synuclein aggregation and toxicity in vitro79 and attenuates dopaminergic neurotoxicity in response to neurotoxins MPTP and 6-OHDA in vivo.80,81 In line with the iron-chelating feature of EGCG, this polyphenol was shown to prevent α-synuclein accumulation in the SNpc of mice intoxicated with MPTP, when given orally for a period of 2 weeks.27 This may be associated with our previous studies where green tea extract or EGCG prevented DA-containing neuron degeneration and decrease in tyrosine hydroxylase activity.30 Additional studies examining the effect of iron chelation, by either transgenic expression of the iron-binding protein, ferritin, or oral administration of the metal chelator clioquinol, have shown significant attenuation of MPTP-induced neurotoxicity.82 These findings are substantiated by a recent study demonstrating that the brain-permeable iron chelator, VK-28 given intracerebroventricular (i.c.v.)

The Essentiality of Iron Chelation in Neuroprotection

287 HO OH B

HO

O OH A

C O

EGCG

OH

OH O

?

PKC ( , )

Labile Fe2+

D OH OH

 -Secretase

IRP

APP

PHF solubilization

A
plaques destabilization

sAPP

A


Neuroprotection Neurorescue

FIGURE 17.4 Proposed schematic model for EGCG neuroprotective/neurorescue effect via regulation of APP processing and Aβ formation. ↑ Indicates increased levels/activity, ↓ decreased levels/activity. For full explanation see the text.

or intraperitoneally (i.p.) to rats, induces neuroprotection against 6-OHDA.8 Moreover, nutritional iron deficiency protects rats against kainate and 6-OHDA.5 Figure 17.5 summarizes the mechanism of neurotoxin-induced iron uptake, release and interaction with α-synuclein resulting in OS-initiated neurodegeneration and its prevention by iron-chelating/antioxidant agents. 17.2.3.1 Effect of Tea Catechins on Iron and Hypoxia-Regulated Genes Hypoxia and iron chelation have similar effects on genes regulated by the transcription factor hypoxia-inducible factor-1 α (HIF-1α), a master regulator orchestrating the coordinated induction of an array of hypoxia-sensitive genes.83 The target genes of HIF are especially related to angiogenesis, cell proliferation/survival and glucose/iron metabolism.84 A recent study found that ECG strongly activates HIF-1 and induces the expression of HIF-1 target genes including GLUT-1, VEGF, and CDKN1A, in T47D human breast carcinoma cells, probably through the chelation of iron.85 Iron removal by metal chelators has the effect of mimicking hypoxia, whereas many of the effects of iron overload may be the result of signaling associated with OS. The mechanism of HIF-1α activation by iron chelation is not well understood. Fe(II)/2-oxoglutarate-dependent dioxygenases have been identified that hydroxylate critical proline and asparagine residues in HIF and, under conditions of high oxygen levels and iron overload, target HIF for degradation.86 Thus,

288

Oxidative Stress and Age-Related Neurodegeneration Ferritin 6-OHDA MPTP

Released iron Degradiation of IRP2 by ubiquitination Fe BBB

2+

EGCG DFO VK28 R-APO

Fe 3+

Fe 3+ ↑DMT1 Fe

Serum iron

2+

Labile Fe pool (IRPs) + −Synuclein→−Synuclein-Fe2+aggregate

M30, HLA20

Dopamine

R-CHO + NH3 + NADP+

NADPH + H+

2GSH

GSSG

Fe 2+

H2O2

Other oxidative processes

Fenton chemistry

OH· + OH− EGCG, R-APO Melatonin, Vit E

H2O + 1/2 O2 Membrane lipid peroxidation DNA damage Protien oxidation and misfolding

Oxidative stress Vitamin E Decreased GSH GSH/GSSG Glutathione peroxidase

Cell death

FIGURE 17.5 Possible mechanism of neurotoxin-induced iron uptake, release and interaction with α-synuclein resulting in OS initiated neurodegeneration and its prevention by iron-chelating/antioxidants. The mechanism by which 6-OHDA and MPTP induce the increase of iron in substantia nigra pars compacta and within the melanin-containing neurons is not known. These neurotoxins may (a) activate the divalent metal transporter 1 (DMT1), which is responsible for iron transport into the brain across the cell membrane; (b) may alter the BBB, thereby allowing iron access to the brain; (c) may induce release of iron from ferritin, which enters the labile (redox-active) pool of iron. It is the labile pool of iron, which can initiate the Fenton chemistry in response to the presence of hydrogen peroxide, thus generating the highly reactive hydroxyl radical (OH⭈). The resultant effect is the depletion of cell reduced glutathione (GHS), the rate limiting cofactor of glutathione peroxidase, the main enzymatic pathway in the brain, to eliminate hydrogen peroxide. The labile pool of iron can also cause aggregation of α-synuclein to the neurotoxic form, which can generate OH·. The net effect is oxidative-stress-dependent damage to neuron antioxidant mechanism, membrane lipid peroxidation, demise of cell and mitochondrial membrane, protein misfolding, and ultimately cell death. Neuroprotective agents that can be used to prevent iron-induced neurodegeneration include M30 and HLA20 (bifunctional iron chelator-MAO inhibitors); DFO, VK28, R-APO (R-apomorphine) and EGCG (iron chelators); R-APO, EGCG, melatonin and vitamin E (radical scavengers). Sharp arrows indicate positive inputs, whereas blunt arrows are for inhibitory inputs (Adapted from Youdim and Buccafusco, Trends Pharmacol Sci, 2005; 26: 27–35.)

these prolyl hydroxylase enzymes act as key iron and oxygen sensors. This may explain the decrease in cell survival genes described in neurodegenerative diseases, such as phosphofructokinase and the angiogenic factor VEGF, both regulated by the HIF proteins.87 Interestingly, the free iron-induced, proteasomal-mediated degradation of IRP2 also involves the activation of a prolyl hydroxylase and is inhibited by

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289

iron chelators.88,89 Thus, it is possible that IRP2 is a substrate for this enzyme, in a similar way as HIF, signaling it for protein degradation. This suggests a convergence of both iron and OS on a common pathway, triggering the neurotoxic degenerative cascade (for a detailed explanation see Figure 17.1). Our recent high-throughput gene expression study in the SNpc of parkinsonian brains employing Affymetrix chip technology90 has revealed a significant increase in the key iron and oxygen sensor EGLN1 gene coding for an isoform of 2-oxoglutarate-dependent dioxygenase hydroxylase (PDH2). Excessive production of PDH2 in the SNpc may lead to a decrease in IRP2 and subsequent decrease in transferrin receptor (TfR) mRNA, and an increase in ferritin levels, both subjected to positive and negative transcriptional regulation by IRP2, respectively.91,92 Recent studies in knockout mice for IRP2 have revealed accumulation of iron in the striatum with substantial bradykinesia and tremor.93

17.2.4 EFFECTS

OF

TEA CATECHINS

ON

CELL SIGNALING PATHWAYS

A wealth of accumulated experimental data indicates that catechin polyphenols regulate a variety of signaling pathways involved in cellular survival, growth, and differentiation, such as PKC, MAPK, and phosphatidylinositide 3⬘-OH kinase (PI3-K)-AKT.29 The induction of PKC activity in neurons is thought to be a prerequisite for neuroprotection against several exogenous insults. PKC expression has been previously coupled with the preservation of cell survival and the formation and consolidation of different types of memory.94–96 Indeed, PKCε activation after ischemic preconditioning or pharmacologic preconditioning (with PKCε, N-methyl-D-aspartate (NMDA), or adenosine A1 receptor (A1AR) agonists) was shown to be essential for neuroprotection against oxygen/glucose deprivation in organotypic slice cultures.97 In accordance, activation of PKC by estrogen or the grape flavonoid resveratrol in rat cortical or hippocampal neurons, respectively, protects against Aβ toxicity.98,99 Also, we have recently shown that the anti-Parkinson/monoamine oxidase-B (MAO-B) inhibitor drug, rasagiline (Teva Pharmaceutical Industries Ltd),100 prevented PC12 cell death induced by serum deprivation via PKC signaling cascade.101 Similarly, we have reported that phosphorylative activation of PKC by EGCG is responsible for the protective effects against 6-OHDA- and Aβ -induced cell death in SH-SY5Y and PC12 cells, respectively,44,46 and for the neurorescue effect against long-term growth factor withdrawal in PC12 cells.47 This is supported by the observation that EGCG could not overcome cell death under PKC pathway blockade, which was determined both morphologically and by monitoring various apoptotic markers, suggesting that this cascade is essential for the neuronal protection and rescue effects of EGCG. Consistent with these findings, recent animal studies have shown that the consumption of EGCG (2 mg/kg) for 2 weeks leads to a highly significant up-regulation of PKCα isoform in mice striatum27 and to a significant increase in PKC isoenzymes α and ε in the membrane and cytosolic fractions of mice hippocampus.46 The implication of PKCα in neuronal survival by EGCG is further demonstrated in vitro by the rapid translocation of PKCα to the membrane compartment

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in PC12 cells, in response to EGCG. PKCα is a well-established neuron cell survival factor that also participates in cell growth and differentiation.102,103 In support of these findings, a recent report shows that the treatment of human cells with EGCG induces a specific translocation of PKCα to the membrane.104 More direct evidence implicating PKC in EGCG mechanistic action has come from a recent study employing solid-state NMR, showing that EGCG interacts with the head group region of the phospholipids within lipid bilayers from liposomes.105 The interaction pattern of EGCG in terms of rotational motion within the lipid bilayers was similar to that described for 12-O-tetradecanoylphorbol-13acetate (TPA),106 a phorbol ester. Phorbol esthers are prototype activators of PKC, suggesting that direct interaction of green tea catechins with cell membranes may be sufficient for the rapid activation of PKC by EGCG as reported by us previously.44 The impact of EGCG on membrane fluidity may give rise to activation of other membrane-associated signaling pathways (e.g., G proteins), which can also contribute to its protective action. This clearly needs to be examined. In addition to PKC, other cell signaling pathways have also been implicated in the action of green tea catechins, such as the MAPKs and (PI3K)/AKT signaling cascades. These cascades have been shown to play central functions in neuronal protection against a variety of extracellular insults and to be essential for neuronal differentiation and survival.107–109 OS seems to be a major stimulus for the MAPK cascade, which might lead to cell survival/cell death (for review see [110]). Among MAPKs, the extracellular signal-regulated kinases (ERK1/2) are mainly activated by mitogen and growth factors,111 while p38 and c-jun-N-terminal kinase (JNK) respond to stress stimuli.112 However, there have been reports of situations in which activation of ERK1/2 is thought to mediate neuronal injury, such as in focal ischemia,113 in glutamate and oxidized low-density, lipoproteininduced toxicity,40,114 and in cytotoxicity and activation of caspase-3 in the extraneuronal hepatoma HepG2115 and HeLa116 cell lines. Increasing evidence shows that catechins can protect against neuronal cell death caused by exogenous OS-inducing agents through modulation of ERK activity.40,44 In this regard, a number of flavonoids and phenolic antioxidants, at their respective low, protective concentrations, were demonstrated to activate the expression of some stress-response genes, such as phase II drug metabolizing enzymes, glutathione-S-transferase, and heme oxygenase 1,116most likely via activation of the MAPK pathway.117 More recently, PI3K/AKT, protein kinase A (PKA), and calcium, have been implicated in the protective action of catechin flavonoids. These studies have been conducted mainly in extraneuronal tissue such as skin and heart. For example, topical application of EGCG induces proliferation of human normal epidermal keratinocytes through stimulation of ERK1/2 and AKT kinase (AKT).118 Other investigators reported a rapid activation of endothelial NOS after EGCG treatment by a process that involves PI3K, PKA, and AKT in endothelial cells119 and a decrease in iNOS expression via inactivation of STAT-1α in epithelial and colon cell lines.120 Consistent with this, Townsend et al.38 have recently reported that in cardiac myocytes EGCG protects against ischemia/reperfusion-induced apoptosis through a mechanism involving reduction of STAT-1 phosphorylation

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(inactivation) and of its downstream pro-apoptotic target gene, Fas. The discrepancy or divergence in the different signal pathway activation by EGCG may reflect differences in cell tissue (e.g., neuronal vs. peripheral), or the fact that the downstream pathways are under the control of the different kinases, thereby providing cell function diversity.

17.3 CONCLUSIONS Diets rich in antioxidants appear to be a promising approach to fortify the physiologic antioxidant defense system and help protect against the aging process and chronic diseases. The multifactorial nature of neurodegenerative diseases makes the use of compounds with polypharmacological activities or cocktail of drugs a promising therapeutic approach for the treatment of these disorders. Indeed, a wealth of new data indicates that green tea catechins are being recognized as multifunctional compounds for neuroprotection: they act as antioxidantradical scavengers, modulators of prosurvival genes and of the PKC signaling pathway, and as metal chelators. Considering the pathological role iron plays in a number of neurological conditions, the use of EGCG as a natural, nontoxic, lipophilic, brain-permeable, neuroprotective drug, could offer potential therapeutic benefits to “iron out” iron from those brain areas where it preferentially accumulates.121 Its worldwide consumption and lack of toxicity in man provide a great impetus to determine its disease-modifying activity in the neurodegenerative diseases.

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40. Schroeter H, Spencer JP, Rice-Evans C, and Williams RJ. Flavonoids protect neurons from oxidized low-density-lipoprotein-induced apoptosis involving c-Jun N-terminal kinase (JNK), c-Jun and caspase-3. Biochem J, 2001; 358: 547–557. 41. Spencer JP, Schroeter H, Kuhnle G, Srai SK, Tyrrell RM, Hahn U, and Rice-Evans C. Epicatechin and its in vivo metabolite, 3⬘-O-methyl epicatechin, protect human fibroblasts from oxidative-stress-induced cell death involving caspase-3 activation. Biochem J, 2001; 354: 493–500. 42. Nobre Junior HV, Cunha GM, Maia FD, Oliveira RA, Moraes MO, and Rao VS. Catechin attenuates 6-hydroxydopamine (6-OHDA)-induced cell death in primary cultures of mesencephalic cells. Comp Biochem Physiol C: Toxicol Pharmacol, 2003; 136: 175–180. 43. Mercer LD, Kelly BL, Horne MK, and Beart PM. Dietary polyphenols protect dopamine neurons from oxidative insults and apoptosis: investigations in primary rat mesencephalic cultures. Biochem Pharmacol, 2005; 69: 339–345. 44. Levites Y, Amit T, Youdim MBH, and Mandel S. Involvement of protein kinase C activation and cell survival/cell cycle genes in green tea polyphenol (-)-epigallocatechin-3-gallate neuroprotective action. J Biol Chem, 2002; 277: 30574–30580. 45. Choi YT, Jung CH, Lee SR, Bae JH, Baek WK, Suh MH, Park J, Park CW, and Suh SI. The green tea polyphenol (-)-epigallocatechin gallate attenuates beta-amyloid-induced neurotoxicity in cultured hippocampal neurons. Life Sci, 2001; 70: 603–614. 46. Levites Y, Amit T, Mandel S, and Youdim MBH. Neuroprotection and neurorescue against amyloid beta toxicity and PKC-dependent release of non-amyloidogenic soluble precursor protein by green tea polyphenol (-)-epigallocatechin-3-gallate. FASEB J, 2003; 17: 952–954. 47. Reznichenko L, Amit T, Youdim MB, and Mandel S. Green tea polyphenol (-)-epigallocatechin-3-gallate induces neurorescue of long-term serum-deprived PC12 cells and promotes neurite outgrowth. J Neurochem, 2005 101111/j14714159200503085x 2005. 48. Weinreb O, Mandel S, and Youdim MBH. cDNA gene expression profile homology of antioxidants and their anti-apoptotic and pro-apoptotic activities in human neuroblastoma cells. FASEB J, 2003; 17: 935–937. 49. Halliwell B. Vitamin C: antioxidant or pro-oxidant in vivo?. Free Radi Res, 1996; 25: 439–454. 50. Gassen M, Pinchasi B, and Youdim MB. Apomorphine is a potent radical scavenger and protects cultured pheochromocytoma cells from 6-OHDA and H2O2induced cell death. Adv Pharmacol (NY), 1998; 42: 320–324. 51. Gassen M, Gross A, and Youdim MB. Apomorphine enantiomers protect cultured pheochromocytoma (PC12) cells from oxidative stress induced by H2O2 and 6-hydroxydopamine. Mov Disord, 1998; 13: 242–248. 52. Galati G and O’Brien PJ. Potential toxicity of flavonoids and other dietary phenolics: significance for their chemopreventive and anticancer properties. Free Radi Biol Med, 2004; 37: 287–303. 53. Wiseman S, Mulder T, and Rietveld A. Tea flavonoids: bioavailability in vivo and effects on cell signaling pathways in vitro. Antioxid Redox Signal, 2001; 3: 1009–1021. 54. Navarro-Peran E, Cabezas-Herrera J, Garcia-Canovas F, Durrant MC, Thorneley RN, and Rodriguez-Lopez JN. The antifolate activity of tea catechins. Cancer Res, 2005; 65: 2059–2064.

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73. Dedeoglu A, Cormier K, Payton S, Tseitlin KA, Kremsky JN, Lai L, Li X, Moir RD, Tanzi RE, Bush AI, Kowall NW, Rogers JT, and Huang X. Preliminary studies of a novel bifunctional metal chelator targeting Alzheimer’s amyloidogenesis. Exp Gerontol, 2004; 39: 1641–1649. 74. Ono K, Yoshiike Y, Takashima A, Hasegawa K, Naiki H, and Yamada M. Potent anti-amyloidogenic and fibril-destabilizing effects of polyphenols in vitro: implications for the prevention and therapeutics of Alzheimer’s disease. J Neurochem, 2003; 87: 172–181. 75. Yamamoto A, Shin RW, Hasegawa K, Naiki H, Sato H, Yoshimasu F, and Kitamoto T. Iron (III) induces aggregation of hyperphosphorylated tau and its reduction to iron (II) reverses the aggregation: implications in the formation of neurofibrillary tangles of Alzheimer’s disease. J Neurochem, 2002; 82: 1137–1147. 76. Checkoway H, Powers K, Smith-Weller T, Franklin GM, Longstreth Jr., WT, and Swanson PD. Parkinson’s disease risks associated with cigarette smoking, alcohol consumption, and caffeine intake. Am J Epidemiol, 2002; 155: 732–738. 77. Li SC, Schoenberg BS, Wang CC, Cheng XM, Rui DY, Bolis CL, and Schoenberg DG. A prevalence survey of Parkinson’s disease and other movement disorders in the People’s Republic of China. Arch Neurol, 1985; 42: 655–657. 78. Zhang ZX and Roman GC. Worldwide occurrence of Parkinson’s disease: an updated review. Neuroepidemiology, 1993; 12: 195–208. 79. Hashimoto M, Takeda A, Hsu LJ, Takenouchi T, and Masliah E. Role of cytochrome c as a stimulator of alpha-synuclein aggregation in Lewy body disease. J Biol Chem, 1999; 274: 28849–28852. 80. Ben-Shachar D, Eshel G, Finberg JP, and Youdim MB. The iron chelator desferrioxamine (Desferal) retards 6-hydroxydopamine-induced degeneration of nigrostriatal dopamine neurons. J Neurochem, 1991; 56: 1441–1444. 81. Lan J and Jiang DH. Desferrioxamine and vitamin E protect against iron and MPTP-induced neurodegeneration in mice. J Neural Transm (Budapest), 1997; 104: 469–481. 82. Kaur D, Yantiri F, Rajagopalan S, Kumar J, Mo JQ, Boonplueang R, Viswanath V, Jacobs R, Yang L, Beal MF, DiMonte D, Volitaskis I, Ellerby L, Cherny RA, Bush AI, and Andersen JK. Genetic or pharmacological iron chelation prevents MPTPinduced neurotoxicity in vivo: a novel therapy for Parkinson’s disease. Neuron, 2003; 37: 899–909. 83. Sharp FR and Bernaudin M. HIF1 and oxygen sensing in the brain. Nat Rev Neurosci, 2004; 5: 437–448. 84. Lee JW, Bae SH, Jeong JW, Kim SH, and Kim KW. Hypoxia-inducible factor (HIF1)alpha: its protein stability and biological functions. Exp Mol Med, 2004; 36: 1–12. 85. Zhou YD, Kim YP, Li XC, Baerson SR, Agarwal AK, Hodges TW, Ferreira D, and Nagle DG. Hypoxia-inducible factor-1 activation by (-)-epicatechin gallate: potential adverse effects of cancer chemoprevention with high-dose green tea extracts. J Nat Prod, 2004; 67: 2063–2069. 86. Jaakkola P, Mole DR, Tian YM, Wilson MI, Gielbert J, Gaskell SJ, Kriegsheim A, Hebestreit HF, Mukherji M, Schofield CJ, Maxwell PH, Pugh CW, and Ratcliffe PJ. Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science, 2001; 292: 468–472. 87. Minchenko O, Opentanova I, and Caro J. Hypoxic regulation of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene family (PFKFB-1–4) expression in vivo. FEBS Lett, 2003; 554: 264–270.

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Ginkgo biloba Extract 18 EGb 761 Extends Life Span and Attenuates H2O2 Levels in a Caenorhabditis elegans Model of Alzheimer’s Disease Julie V. Smith

William Carey College Hattiesburg, Massachusetts

Yuan Luo

University of Maryland Baltimore, Maryland

CONTENTS 18.1 Introduction..............................................................................................302 18.2 Reactive Oxygen Species, Aging, and Alzheimer’s Disease...................303 18.2.1 Supplemental Antioxidant Therapies...........................................304 18.2.2 Vitamin E .....................................................................................304 18.2.3 Vitamin C.....................................................................................305 18.3 Ginkgo biloba Extract..............................................................................305 18.3.1 Standardized Ginkgo biloba Extract - EGb 761..........................305 18.3.1.1 Composition of the Extract EGb 761...........................305 18.3.1.2 Identification of Principal Active Compounds or Classes of Compounds Gives Mechanistic Clues to the Action of EGb 761 ..............306 18.3.1.3 Clinical use of the Extract EGb 761 ............................309 18.3.2 The Natural Antioxidant Properties of Ginkgo biloba ................310 301

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18.3.3 Neuroprotective Effects of EGb 761 ...........................................311 18.3.3.1 Antiapoptotic and Antioxidative Properties of EGb 761 ..................................................311 18.3.3.2 Major Biochemical/Pharmacological Activities of EGb 761...................................................312 18.3.3.3 Transcriptional Effects of EGb 761..............................312 18.4 Caenorhabditis elegans, a Valid Model Organism for Pharmacological Evaluation of EGb 761 Protection...............................313 18.4.1 The Alzheimer’s Disease-Associated Transgenic Caenorhabditis elegans Mutant...................................................313 18.4.2 Analysis of Reactive Oxygen Species Levels in Caenorhabditis elegans ...............................................................314 18.4.3 Alzheimer’s Disease-Associated Mutant Models Exhibit Higher Reactive Oxygen Species Levels .......................314 18.4.4 Alzheimer’s Disease-Associated Caenorhabditis elegans Mutants Exhibit Acceleration of Age-Related Reactive Oxygen Species Production.....................316 18.5 EGb 761 Components Show Protective Effects in Alzheimer’s Models ................................................................................317 18.5.1 Kaempferol and Quercetin Attenuate Reactive Oxygen Species Levels In Vitro and In Vivo ...............................317 18.5.2 Advancement in Recent Methodology for Reactive Oxygen Species Detection ............................................317 18.6 Ginkgo biloba Extract EGb 761 Imparts Stress Resistance and Longevity ........................................................................318 18.6.1 Oxidative Stress Resistance in Caenorhabditis elegans Fed with EGb 761...........................................................318 18.6.2 Enhanced Life Span in Caenorhabditis elegans Treated with EGb 761.....................................................318 18.7 Summary .................................................................................................319 References ........................................................................................................320

18.1 INTRODUCTION With an aging population and an estimated 20 million patients suffering from Alzheimer’s disease (AD) worldwide,1 preventing or slowing the progression of this disease is a major goal of the current biomedical research community. Research indicates that cellular insult seen in AD cases might be a direct result of the free radicals generated by the amyloid beta peptide (Aβ ).2,3 It is likely, therefore, that these insults contribute heavily to the neurotoxicity and pathology found in AD.4–6 Unfortunately, this excess in free radical production and the resulting oxidative damage may occur well in advance of clinical symptoms, damaging key cellular components and facilitating cell death by necrosis or apoptosis.7 It appears that the normal age-related increases in reactive oxygen species (ROS) are accelerated in

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AD-associated model organisms,8 which further lends support to the hypothesis that Aβ and ROS are involved in AD pathology. To combat this strong association, the medical and research communities along with leading pharmaceutical companies are giving considerable attention to the efficacy of antioxidant treatments. In addition to pharmaceutical intervention, the use of antioxidant therapies to attenuate free radical production, increase clearance of oxygen radicals, and minimize resulting damages is becoming a common component of the therapeutic repertoire used to treat neurodegenerative diseases such as AD.4,9,10

18.2 REACTIVE OXYGEN SPECIES, AGING, AND ALZHEIMER’S DISEASE Commonly referred to as free radicals, ROS are constantly being formed in the body as a by-product of normal aerobic metabolism.11 Free radicals refer to any molecule or atom containing at least one unpaired electron in one of its orbital shells. These molecules are thus very reactive, constantly searching for other molecules to oxidize in order to fill their orbital shell with an additional electron. Even though they can be important physiologically as necessary intermediates of oxygen metabolism, unchecked production of free radicals can cause damage to cellular functions and membranes, thus contributing to both normal aging and a variety of pathological processes. Among the family of free radicals, the three main toxic radicals are superoxide (O2⫺), hydrogen peroxide (H2O2), and the hydoxyl radical (OH.). Normal metabolism of glucose, and ATP production in the electron transport chain, results in the physiological production of O⫺2 . The enzyme superoxide dismutase (SOD) may neutralize O2⫺,12 resulting in the formation of H2O2, a more stable, less reactive radical. A process called the Fenton reaction converts H2O2 into the extremely reactive and damaging OH. Although endogenous antioxidant enzymes SOD and glutathione peroxidase normally act to reduce ROS levels by neutralizing these radicals, any imbalance in the system can lead to oxidative stress damage in an organism. Since strong research evidence now links AD, Aβ deposition,13,14 and oxidative stress,4,15 the scientific and medical communities are devoting increased research attention to antioxidant research. In fact, cumulative oxidative stress and the role played by free radicals in the etiology of AD and impairment of brain function have become major topical issues among the general public, who now wish to extend not only the length, but also the quality of their lives as they enter their senior years. The well-established free radical theory of aging16 suggests that cumulative oxidative damages at the cellular and tissue level arise as a consequence of normal aerobic metabolism and that damage to proteins, lipids, and nucleic acids increases with age. Therefore, in addition to theories postulating the involvement of Aβ production and aggregation in the development, progression, and neurotoxicity of AD,14,17 aging is unfortunately postulated to be the greatest risk factor associated with the development of AD.1,18,19 The involvement of ROS in limiting

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the life span of an organism has been reported with various models, including Drosophila and Caenorhabditis elegans, where organisms engineered to overexpress human SOD are living significantly longer than their wild-type counterparts.20–24 Moreover, transgenic models deficient in SOD activity display oxidative sensitivity phenotypes, higher protein carbonylation levels25 and shorter life spans than the corresponding wild types.26 These data provide direct support for the free radical theory of aging.

18.2.1 SUPPLEMENTAL ANTIOXIDANT THERAPIES In the past, the use of individual defined molecules possessing a single, well-identified mechanism of action (enzymatic inhibition, membrane receptor binding, etc.)27,28 has been a popular trend in medicine. Although the use of pure chemical compounds in modern medicine has immense value, there appears to be tremendous interest in and an expanding research effort toward studying the therapeutic benefits achieved by natural substances such as plant extracts. Since the antioxidant status, or balance of both oxidant and antioxidant functioning, is an important factor for the survival of an organism and the chief indicator of the spectrum of damage incurred by oxidative damage,29 the use of naturally derived antioxidants has received a great deal of attention by researchers studying disorders of oxidative origins. Some theories even suggest that the synergistic effects of antioxidant combinations, or antioxidant cocktails, may be more successful in preventing the onset of AD than those administered in isolation.

18.2.2 VITAMIN E Attenuation of free radical production, improved ROS clearance, and a clear reduction in cellular damage has been successfully demonstrated through the use of antioxidant therapies.9,10 One example is vitamin E, a biological antioxidant capable of preventing the oxidation of polyunsaturated fatty acids and proteins, and thus playing a protective role in the development of diseases and morbidity related to oxidative processes.30,31 Vitamin E supplementation has been found to prevent oxidative damage during the aging process to brain and lymphocyte band 3 proteins,32 which maintain anion transportation, acid–base regulation, and transport of CO2.33 In rats, vitamin E supplementation has been found to reduce age-related deterioration of brain synapse functions in nerve terminals,34 further demonstrating the protective effect on free radical-mediated damage to the brain. Another study in transgenic mice of AD demonstrated that early vitamin supplementation in young but not aged mice protected them against Aβ toxicity and deposition.35 Human studies have indicated that high doses of vitamins E C may correlate with a lower risk for the development of AD.36,37 The studies additionally implicate the effectiveness of vitamin E on life span extension in C. elegans,38 a common model organism used in research of neurological disorders. As a major chain-breaking antioxidant,30 vitamin E is widely used as one of the first lines of defense against lipid peroxidation and free radical damage, and has been the focus of many studies.20,39–42 There are strong evidence that lipid

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peroxidation can be prevented by vitamin E supplementation in a set of experiments conducted in the 1950s.31 As a lipid-soluble antioxidant, vitamin E has the advantage of being able to maneuver through the fatty parts of cellular membranes. However, this simultaneously poses a disadvantage by limiting its ability to scavenge peroxyl radicals in only hydrophobic domains. Additionally, since excessive intake of lipid-soluble vitamins is not excreted, injestion of high doses of vitamin E may be associated with potentially harmful side effects.43

18.2.3 VITAMIN C In contrast, L-ascorbic acid (vitamin C), another well-recognized antioxidant,31 is a water-soluble vitamin. Vitamin C has been called the “hub of the antioxidant network” and the “link connecting the fat-soluble antioxidants to the water-soluble antioxidants” owing to its ability to recycle the fat-soluble vitamin E.31 Normally, vitamin C levels found in the brain are significantly higher than in other body organs, suggesting the presence of a regulatory active transport mechanism for vitamin C from the serum, across the blood–brain barrier (BBB), and into the brain.44 Because of their strong antioxidant capabilities, both vitamin E and vitamin C have been used in studies of aging and disorders with known oxidative stress components.9,43

18.3 GINKGO BILOBA EXTRACT 18.3.1 STANDARDIZED GINKGO

BILOBA

EXTRACT - EGb 761

With a staggering $151 million in retail sales in 1998,45 Ginkgo biloba ranks as one of the top-selling herbal medicinal products sold in the United States. Although Ginkgo biloba has been used for thousands of years to ameliorate a wide variety of ailments, modern use of Ginkgo biloba extract has become associated more with European phytomedicine than the traditional Chinese medicine from which its use originated. This change in attitude is largely due to the development of standardized extracts and preparation techniques. The development of standardized preparations is being urged by both government entities and the general public, who are now demanding increased product safety and clinical “hard” evidence on the efficacy of herbal products. Furthermore, the rigors of scientific research demand and depend on standardization of products and reagents for consistency, reproducibility, and predictable responses among users. EGb 761 is the standardized extract from Ginkgo biloba leaves. It was initially developed by German physician–pharmacist Dr. Willmar Schwabe and registered as a medication by his company (Dr. Willmar Schwabe GmbH & Company) in 1965 in Germany.46 EGb 761 was further established by the company, along with Beaufour IPSEN (Paris, France), and was introduced in the market in the early 1980s. 18.3.1.1 Composition of the Extract EGb 761 The chemical definition of EGb 761 is rigorously controlled and standardized, mandating that the product be adjusted to a potency of 24% flavonoid glycosides

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(kaempferol, quercetin, and isorhamnetin), 6% terpene lactones (ginkgolide A, ginkgolide B, ginkgolide C, ginkgolide J, and bilobalide B) (GA, GB, GC, GJ, and BB, respectively),47 5 to 10% organic acids, ⬎ 0.5% proanthocyanidins, and less than 5 parts per million of ginkgolic acid. Analysis of the individual components by HPLC has been previously documented48,49 along with quantitative structure–activity relationship analysis of the flavonoids and other phenolic compounds50 for the hierarchy of radical-scavenging abilities. 18.3.1.2 Identification of Principal Active Compounds or Classes of Compounds Gives Mechanistic Clues to the Action of EGb 761 In addition to the determination of antioxidant properties of EGb 761 extract, much work has been done in an effort to determine the mechanism of action of its individual constituents.51–53 Logically, the efficacy of extracts prepared from natural products depends on the complexity achieved through the combination of constituents, which consequentially allows the plant to modulate its own state of homeostasis within its particular environment. This is certainly the case with EGb 761. Several of the individual constituents in EGb 761 are pharmacologically active, but it is widely believed that the polyvalent action, or combined activity of the constituents, is responsible for the wide-ranging therapeutic benefits of the extract.46

18.3.1.2.1 Flavonoid Glycosides The flavonoids are one of the most widely represented classes of plant secondary metabolites existing in the plant kingdom and refer to phenylbenzopyrones, a large group of low molecular weight substances. Over 4500 chemically distinct flavonoids have been identified and can be found, for example, in wines, teas, and berries. They are also found in many other parts of plants, and are responsible for the brilliant colors in flowers and autumn leaves. Flavonoids exhibit a wide range of biochemical and pharmacological activities in mammalian systems, including antioxidant properties, enzyme modulation, and cation chelation.54 Given that they may also possess antiallergenic, anti-inflammatory, antipoliferative, antiviral, hepato-protective, antithrombotic and anticarcinogenic activities, it is not surprising that certain plants and spices containing flavonoids have been widely used for thousands of years in traditional Eastern medicine. In the Ginkgo biloba tree, the flavonols may act as protective agents against UV-B light and radiation (probably attributed to kaempferol), as well as antioxidants and free radical scavengers. They appear to be beneficial in the survival of the plant under extremely harsh conditions, such as the radiation exposure they were subjected to following the atomic bombings.55 Since flavonoids are ubiquitous in all terrestrial plants (except the Anthoceropsidae),56 they occur frequently in our diet. Western diets are estimated to contain an average of 1 to 2 gm of mixed flavonoids per day, about half of which is absorbed.46 Flavones are found in grains and herbs; flavonols and their glycosides in fruits and vegetables; flavanones in citrus juices; and isoflavones in legumes or catechins in tea. Flavonol mono-, di-, and triglycosides and the coumaric esters of quercetin glucorhamnoside are

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present in relatively high percentages in EGb 761. EGb 761 also contains proanthocyanidins. Glycosides are acetyl derivatives that yield sugars on hydrolysis. Mechanistically, flavonoids possess phenolic hydroxyl groups, which have the ability to chelate Fe2⫹ and consequently attenuate the formation of hydroxyl radicals.57 Given this fact, it is widely accepted, and supported by both in vitro and in vivo studies, that the flavonol glycosides account for the majority of the free radical scavenging activity of EGb 761. Additionally, many of the pharmacological effects of EGb 761 (free radical scavenging, antioxidant action, influences on membrane permeability, actions on enzymes, transcriptional effects, etc.) can be attributed to the properties of its flavonoid constituents (Figure 18.1).

18.3.1.2.2 Terpene Lactones Terpenes represent one of the two principal categories of nonsaponifiable lipids of plants, the other being steroids. The terpene molecules possess three lactone functions and a tertiary butyl group, which occurs rarely in the plant kingdom. Most terpenoids are cyclic, and many contain multiple ring systems. The basic structure of these rings is governed by the highly specific synthases. Terpene lactone ginkgolides are made up of multiple units of the 5-carbon hydrocarbon isoprene (2-methyl-I, 3-butadiene). They are exclusive to the Ginkgo biloba tree, and give EGb 761 a characteristically bitter taste. Interestingly, the fat-soluble vitamins A, E, and K are terpenes, as is the steroid precursor squalene. Having a novel chemical structure, the ginkgolides found in EGb 761 are made up of four units, and are thus termed diterpenes, while bilobalide has traditionally been thought of as a sequiterpene (containing one and a half units). Both the sesquiterpene and diterpene synthases in Gingko biloba trees may generate compounds that function in plant defense (e.g., resins and olefins). The ginkgolides of EGb 761 possess two to four hydroxyl functions, and they may exist in “cage-like” molecules. Their structure is described as 20 carbon atoms (C20) arranged into 6 rings of 5 carbons each, which involve a spirononane system, a tetrahydrofuran cycle, three lactone rings, and a tert-butyl group. A lactone refers to any of a class of organic compounds formed from hydroxy acids and containing the group –C(CO)OC–, where the carbon atoms are part of a ring (Figure 18.2) The identified ginkgolides, GA, GB, GC (together comprising ~3.1% of EGb 761) and GJ (⬍ 0.5% of EGb 761) have been isolated from the leaves of Gingko biloba, and are constituents of EGb 761. Ginkgolide M, however, is only found in the roots of the Gingko biloba tree, and is not a constituent of EGb 761. The ginkgolides, especially GB, are primarily known as platelet activating factor antagonists in mammalian systems,52 thus having a potential role in EGb 761 as modulators of circulatory functions with the ability to provide increased peripheral and cerebral circulatory efficiency and decreasing incidence of thromboses.58 These properties also give the ginkgolides the ability to attenuate ROS generation, and protect sensitive brain tissues from ischemic neuronal damage. In a recent study using isolated components of EGb 761, the terpenoid constituents GB, GC, and BB were found to inhibit Glycine and GABAA receptors in the central nervous system (CNS), which normally mediate inhibitory synaptic transmission.59

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Oxidative Stress and Age-Related Neurodegeneration R1 OH

HO

R1

O

H OH OCH3

O-mono-, di-, triglycoside

Kaempferol derivatives Quercetin derivatives Isorhamnetin derivatives

OH O

R1 OH R2

R2

R1

O

H O-glc

O-glc OH

Apigenin derivatives Luteolin derivatives

O-mono-, di-, triglycoside OH

O

R1 OH HO

R1

O

OH

C

H3C HO

H

O

O

C

O

O HO

O

OH OH

R4 R1 R2

Kaempferol derivatives Quercetin derivatives H or glucose

CO O

HO

H OH R2

H

O

R3 HO

O

OH O OH

R1

R2

R3

H OCH3 OCH3 OCH3 OCH3 OCH3

H OH OCH3 OH OCH3 OH

H OH OH OCH3 OCH3 OH

R4 H H H H H OCH3

Amentoflavon Bilobetin Ginkgetin Isoginkgetin Scaiadopitysin 5′-methoxybilobetin

O

FIGURE 18.1 Chemical structure of flavonoid glycosides obtained from G. biloba. The flavonoid compounds in EGb 761, primarily found in the leaves and characterized by a C6–C3–C6 carbon skeleton, are essentially flavonol-O-glycosides, with a glycosidic linkage normally located in position 3 or 7 of a phenolic aglycon (quercetin, kaempferol, or isorhamnetin), with the carbohydrate moiety usually being D-glucose, L-rhamnose or glucorhamnose.

It is possible therefore, that modulation of CNS neurotransmission may contribute to the neuroprotective effects of the extract. Bilobalide accounts for ~2.9% of total EGb 761 extract, and possesses a unique structure. The chemical structure of bilobalide shares important features with the structures of ginkgolides, i.e., three β -lactone groups and a tert-butyl group. BB is also capable of reducing ROS-induced cellular changes, therefore exhibiting antioxidant properties.60 Taken together, these multiple pharmacological data and

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O R1 HO

H

H O

H 3C

H C (CH3)3

O R2

H H

O R1 O

R3

O

R3

Ginkgolide

OH

H

H

A

OH

OH

H

B

OH

OH

OH

C

OH

H

OH

J

H

OH

OH

M

H H

H

R2

O

O O

HO

C (CH3) O

OH

O O

O

Bilobalide A

FIGURE 18.2 Chemical structure of terpene lactone constituents of EGb 761 (ginkgolides and bilobalide). These structures are believed to occur exclusively in the G. biloba tree.58

clinical trials show Ginkgo biloba extract EGb 761 to be a potent antioxidant agent and free radical scavenger with neuroprotective effects.27,58,61 18.3.1.3 Clinical use of the Extract EGb 761 The widespread use of herbal dietary supplements such as Ginkgo biloba by the general public has become common place in our society for the prevention and treatment of almost every ailment and aging malady imaginable. Owing to the vast use of Ginkgo biloba for its perceived memory-enhancing properties, the substantial number of pharmaceutical preparations containing the extract, and demands for product safety and detailed pharmacological and clinical data is growing to elucidate the mechanisms of action and efficacy of this extract. EGb 761 is currently used extensively in clinical trials,27 and is popular in the United States as a natural dietary supplement for memory enhancement. In European countries, the extract is being prescribed for the treatment of peripheral and cerebral insufficiency. EGb 761 has been shown to inhibit coagulation,52 facilitate naturally occurring age-related deterioration of cognitive functions in rats,62 and improve degenerative dementias of the Alzheimer’s and multi-infarct type.63,64 As noted by Packer et al.,65 the following characteristics are important in studying the “ideal” antioxidant: the ability to quench free radicals, metal-chelating properties, interaction with other antioxidants, and the ability to affect gene expression. Other important factors include bioavailability, absorption, and the ability to interact in both the aqueous environment and cross the lipophilic cellular

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membranes. These characteristics have all been demonstrated in the standardized extract of Ginkgo biloba.

18.3.2 THE NATURAL ANTIOXIDANT PROPERTIES

OF

GINKGO

BILOBA

The standardized extract of Ginkgo biloba, EGb 761, may show potential for improving the survival rates of persons afflicted with neurological disorders.46 This well-known antioxidant may play a part in preventing the propagation of tissue damage, and is being investigated as a potential treatment strategy in the pathology of AD. The EGb 761 compound shows the ability to cross the BBB without the side effects of vitamin E. Hence, to date, no mutagenic, carcinogenic, teratogenic, or embryogenic side effects have been demonstrated from the use of EGb 761, with the oral LD50 in mice representing 2100 times more than the recommended daily dose.57 For physiological relevance, Maitra et al.,42 evaluated the peroxyl radicalscavenging activity of EGb 761 in both aqueous and lipid environments. Results indicate the ability of EGb 761 to gain access to both the aqueous and lipid environments in which oxidative stress can occur.42 Since EGb 761 is able to effectively scavenge free radical species in both cytosolic (hydrophilic) and membrane (hydrophobic) domains of cells,31 it is plausible to suppose that the polyvalent actions of this extract may afford more effective antioxidant protection than the lipid-soluble vitamin E or water-soluble vitamin C, and supports its use in a physiologically relevant environment. Analysis of individual components shows that the flavonoid constituents of the extract have strong antioxidant properties.31,46 This finding has now been confirmed in both cellular and whole-organism models.8 Not only do the flavonoid components of kaempferol and quercetin outperform the whole extract EGb 761, but they also provide greater attenuation of free radicals than vitamins C or E.8 One interesting finding is that the flavonoid components provide greater free radical protection in models associated with AD mutations than in those associated with their wild-type counterparts. This finding is consistent with previous reports of the protection afforded by EGb 761, particularly under stress-induced conditions.66 Therefore, one particular advantage of EGb 761 over the widely used vitamin E is the multiple directionality of its effects. In contrast to the unidirectional antioxidant activities of the lipophilic vitamin E or the hydrophilic vitamin C, for example, the biphasic effects of EGb 761 are known to be regulatory and adaptive.27 The “polyvalent” activities of EGb 761 give it the advantage of homeostasis sensitivity, such as the ability to either dilate or contract blood vessels, or induce the directionality of neurochemical or neuroendocrine indicators according to the circumstances, a benefit that may not be observed in vitamins or even pharmaceutical preparations targeting a single substrate or receptor. The scientific community holds a strong consensus that although in vitro studies are vital to the discovery of potential mechanisms of action, they do not necessarily predict in vivo actions or reactions. In this chapter, we will demonstrate a strong correlation between results demonstrated in vitro with AD-associated

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neuronal cells and in an in vivo animal model of AD-associated mutant nematodes. We will present evidence confirming the effectiveness of EGb 761 as an antioxidant and radical-scavenging agent within a whole-organism system undergoing normal metabolic processing, and therefore capable of modulating responses to external and internal stimuli.

18.3.3 NEUROPROTECTIVE EFFECTS

OF

EGb 761

In-depth research investigations into the antiapoptotic properties of EGb 761 have supported the claims of the neuroprotective properties of this extract. The induction of apoptosis in a cellular system may be initiated by a multitude of factors, each contributing to cytotoxicity by different molecular pathways. While some pathways are parallel in nature, converging into a programmed cell death,67 others conceivably act independently.53 Therefore, protection of a neuronal system against apoptosis theoretically necessitates the effective inhibition of each converging pathway to attenuate cytotoxicity. Many researchers have now substantiated the neuroprotective effects of EGb 761. EGb 761 provides neuroprotection in a multifactorial way, functioning in a synergistic manner upon multiple cellular pathways as a neuronal regulator of apoptosis under conditions of cellular stress and cytotoxicity.8,53,66 EGb 761 provides substantial antiamyloidogenic neuroprotection,68 and is able to significantly regulate the cellular apoptotic machinery.53 In 2000, Dr. Francis DeFeudis proposed several mechanisms of action that are useful in explaining how EGb 761 benefits patients with AD and other age-related, neurodegenerative disorders. In animals, EGb 761 possesses antioxidant and free radical-scavenging activities, reverses age-related losses in brain α 1-adrenergic, 5-HT1A and muscarinic receptors, protects against ischemic neuronal death, preserves the function of the hippocampal mossy-fiber system, increases hippocampal high-affinity choline uptake, inhibits the down-regulation of hippocampal glucocorticoid receptors, enhances neuronal plasticity, and counteracts the cognitive deficits that follow stress or traumatic brain injury.58 18.3.3.1 Antiapoptotic and Antioxidative Properties of EGb 761 Apoptosis has been related to neurodegenerative diseases.69 The induction of apoptosis in a cellular system by subjection to multiple cytotoxic factors is effected through several molecular pathways. In our study of neuronal cell systems, we have observed that the neuroprotective properties of EGb 761 are multifactoral, and synergistically act upon multiple cellular pathways.53 Included in these observations are the ability of EGb 761 to protect the pivotal integrity of the mitochondrial membrane; attenuation of the release of cytochrome c from the mitochondria, a critical step in the formation of the apoptosome leading to the initiation of the apoptotic caspase cascade; up-regulation of the transcription of antiapoptotic Bcl-2-like protein; down-regulation of the transcription of proapoptotic caspase-12, an endoplasmic reticulum-specific stress-mediated protein; inhibition of the cleavage and activation of caspase-3, a key effector protease in the execution of apoptosis; and inhibition of nuclear DNA fragmentation, the hallmark of apoptosis.53 Thus, the

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“polyvalent activity” of EGb 761 provides neuroprotection to a cellular system at least partially through bidirectional mediation of the existing apoptotic machinery. 18.3.3.2 Major Biochemical/Pharmacological Activities of EGb 761 Further substantiation of protection by EGb 761 is derived from its many proven biochemical and pharmacological effects. At the tissue level, the radical-scavenging action of EGb 761 compound is responsible for decreasing ROS levels in tissues and subsequent inhibition of membrane lipid peroxidation.58 Also, the bilobalide component increases the respiratory control ratio of mitochondria and increases ATP levels by protecting against the uncoupling of oxidative phosphorylation.51 Circulatory benefits of EGb 761 includes the antiplatelet activating factor activity, which improves cerebral insufficiency,52 contributes to enhanced neuronal plasticity,70 and improves vascular circulation by stimulating endothelium-derived relaxing factor.46 The benefits of EGb 761 seen in the neuronal system include decreased expression of peripheral benzodiazepine receptors in the cerebral cortex58 and anti-inflammatory protection against brain damage through its terpenoids ginkgolides,71 In neuronal cell cultures, EGb 761 has been shown to attenuate apoptosis53,72,73 and inhibit Aβ aggregation.68 Finally, increased stress resistance and extension of life span has been shown in C. elegans66 and mice74,75 when treated with EGb 761. 18.3.3.3 Transcriptional Effects of EGb 761 To decode the diverse effects of complex natural substances such as EGb 761 on biological systems, researchers are increasingly selecting a new set of molecular research methods, such as genomics and proteomics microarray techniques. These techniques have the select advantage of detecting gene expression changes and protein interaction profiles of several thousand candidate genes at once.76 Fairly recent laboratory evidence indicates that modification of gene expression may underlie the therapeutic effects of EGb 761, possibly through the complex interaction of the extract’s components, or through the actions of its pharmacologically active chemical constituents.51 Changes in gene expression from gene array assays yield molecular evidence for the actions of EGb 761 in the brain, as these studies have demonstrated a differential effect of the extract in separate brain regions.77 The heterogeneity of neuroanatomical responsiveness of the different brain regions is certain to become an important topic of future research in which specific brain regions will be targeted by herbal medicine or dietary supplementation in hopes of achieving a desired molecular, physiological, or behavioral outcome.70 Substantiation of the neuroprotective effects of EGb 761 has been provided by DNA microarray analysis. These reports indicate that gene transcription specific to the initiation of adaptive transcriptional responses may be modulated by EGb 761,78 including the improvement of the antioxidant status of cells, and the improvement of cellular tolerance to oxidative stress in a wide variety of organisms (including humans).51,79 Further, EGb 761 has the potential to inhibit DNA

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damage through the modulation of DNA synthesis proteins, as well as those involved in repair and cell cycle functions. The antiapoptotic mechanisms hypothesized to play a role in the neuroprotective properties of EGb 761 mentioned earlier have also been supported by DNA microarray analysis. For example, altered transcriptional patterns have been shown in multiple apoptotic-related genes of cells treated with EGb 761. Included in these are the increased expression of the antiapoptotic Bcl-2 protein and the down-regulation of the proapoptotic caspase-12 protein.53 Microarray analysis was also used by Watanabe et al., to demonstrate the neuromodulatory effects of EGb 761 on gene transcription in mice. These studies demonstrated significant up-regulation of multiple genes linked to neuroprotective roles, including transthyretin, AMPA-2 channel, neuronal tyrosine/threonine phosphatase 1, and microtubule-associated τ. Additionally, ten genes were shown to be up-regulated threefold or more in cerebral cortex and hippocampal tissues of mice fed with a EGb 761-supplemented diet for 4 weeks.77 Similarly, cDNA macroarrays have been used by Soulie et al., to define the transcriptional effects of EGb 761 on human hNT neurons, with results implicating genes involved in antioxidant and stress responses. Seven genes were identified whose expression was strongly modified by EGb 761 treatment. From these seven genes, three groups were distinguished: genes encoding transcription factors (increase of NF-kappaB p65 subunit and zinc finger protein 91 mRNAs, and decrease of c-myc transcripts), genes involved in antioxidant defenses (increase in the CuZn SOD mRNAs, and decrease of glutathione reductase and glutathione-S-transferase pi mRNAs), and genes involved in stress responses (upregulation of HSP70 transcripts).80

18.4 CAENORHABDITIS ELEGANS, A VALID MODEL ORGANISM FOR PHARMACOLOGICAL EVALUATION OF EGB 761 PROTECTION In the pharmacological evaluation of the effects of EGb 761, the nematode C. elegans has been shown as an apt model organism. Molecular analysis has demonstrated the presence of all the fundamental components involved in neurodegeneration and aging in this nematode, including endogenous antioxidant enzymes and genetic and cellular features of apoptotic cell death. These features make C. elegans a suitable system for the evaluation of pharmacological and drug intervention in a multicellular organism. The culture and maintenance of C. elegans colonies has been previously characterized by Wood et al.81

18.4.1 THE ALZHEIMER’S DISEASE-ASSOCIATED TRANSGENIC CAENORHABDITIS ELEGANS MUTANT The transgenic AD-associated nematode strain CL2006 was created by Dr. Chris Link of the University of Colorado.82 This transgenic strain is engineered by the gonadal microinjection technique83 to constitutively express high levels of the

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42-amino acid human amyloid beta peptide (Aβ1-42). The muscle-specific unc-54 promotor/enhancer constructs contained in these nematodes drive the expression of coding regions derived from human amyloid precursor protein (APP) cDNA clones.82 Histochemistry from fixed tissues of this C. elegans strain reveals deposits that are reactive with Congo Red and thioflavin S, both of which are amyloid-specific dyes.84 Moreover, the muscle-specific deposits are immunoreactive with anti-Aβ monoclonal and polyclonal antibodies82 and exhibit classic fibrillar amyloid ultrastructure when visualized by immunoelectron microscopy.85 Taken together, these characteristics make the transgenic C. elegans strain CL2006 a useful model in the investigation of amyloid-related factors in AD research. Although in vivo expression of Aβ in the transgenic C. elegans containing the unc-54/β -(1-42) is in muscle cells, a positive correlation is shown between ROS levels and muscle degeneration (muscle cell toxicity), which presents as a progressive paralysis of normal locomotive movement.86

18.4.2 ANALYSIS OF REACTIVE OXYGEN SPECIES LEVELS CAENORHABDITIS ELEGANS

IN

For the analysis of intracellular levels of steady state ROS in whole-organism C. elegans, we developed the following technique. Age-synchronized groups of nematodes are first collected into 100 µl phosphate-buffered saline (PBS) containing 1% Tween-20 (PBST) in groups of 30 to 45 worms per tube. The animals are then subjected to equally timed periods of both homogenization with a pellet pestle motor and sonication to break up the thick outer cuticle of the animal and provide easier access of the probe to individual groups of cells. After harvesting procedures have been completed, the C. elegans samples are transferred to a 96-well microtiter plate. Intracellular ROS can be measured using the fluorescent probe 2,7-dichlorofluorescein diacetate (H2DCF-DA), a well-established probe used for measuring intracellular ROS in cultured neuronal cells.8,87 H2DCF-DA is a freely cell permeable dye which converts readily to 2⬘7⬘-dichlorofluoroscin after being deacetylated by intracellular enzymes, and then forms the fluorescent DCF owing to the interaction with intracellular H2O2. Samples are incubated in the presence of 50 µM H2DCF-DA in PBS at physiological temperatures in a microplate fluorescent reader, and read kinetically for quantification of fluorescence (Figure 18.3).

18.4.3 ALZHEIMER’S DISEASE-ASSOCIATED MUTANT MODELS EXHIBIT HIGHER REACTIVE OXYGEN SPECIES LEVELS According to the Aβ -induced oxidative stress theory of AD, internal expression of Aβ may cause the levels of ROS to increase disproportionately,5,88 which would thus increase the extent to which the Alzheimer’s brain experiences oxidative stress. To determine whether ROS levels are increased by endogenous Aβ expression, we have studied both cellular and organismal models of AD using the technique described above. In confirmation of this theory, both cellular and invertebrate

Ginkgo biloba Extract EGb 761 Extends Life Span

Synchronized eggs

Control

315

Sonication

+ EGb 761

+ 50 µM H2DCF-DA Measurement of Intracellular H2O2

Harvest Read at 37°C in Fluorescent Microplate Reader PBST

2h

• Ex: 485 nm, Em: 530 nm

FIGURE 18.3 Schematic representation of intracellular ROS assay technique. Diagram depicting simplified example of conditions, time lines, and procedure using H2DCF-DA used to assess changes in intracellular ROS levels (108).

AD-associated models in our study exhibited significantly higher ROS levels compared with the wild-type models. In a cellular model of AD,89 the Aβ -secreting mutant cells (N2a swe/∆9) produced 68% higher intracellular ROS levels than the N2a wild-type cells.8 In cells pretreated with EGb 761, ROS levels were significantly reduced by 32% in the Aβ -secreting mutant cells and by 19% in wild type cells. Admittedly, studies of the levels of ROS in biological tissues can be challenging. By using the technique outlined above, we were able to successfully measure intracellular ROS in whole organisms using C. elegans as a model organism. We were able to definitively show that endogenous ROS levels produced in the AD-associated mutant worms (CL2006) were significantly higher than the levels found in wild-type C. elegans (N2) by 60%. The degree of agreement exhibited by these in vitro and in vivo models has increased researchers’ confidence in using this assay technique as a measure of the steady-state levels of intracellular ROS. These studies have provided direct evidence of elevated ROS in transgenic neuronal cells secreting endogenous Aβ as well as in vivo evidence of elevated ROS in transgenic C. elegans expressing constitutive Aβ.90,91 This evidence strengthens the theory that AD-associated mutations cause significant alterations in the production and modulation of free radicals, making attenuation of ROS levels a significant therapeutic target. Additionally, these observations in models expressing constitutive Aβ have significantly advanced the current understanding of the relationship between free radical production and Aβ previously set forth by researchers exposing cell cultures to exogenous synthetic Aβ (1-42).38 Since cellular damage resulting from ROS

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production is believed to be one of the early events in the progression of neurodegenerative diseases such as AD,20,92–97 the demonstration of in vitro and in vivo ROS attenuation in AD-associated models by EGb 761 is a significant finding.

18.4.4 ALZHEIMER’S DISEASE-ASSOCIATED CAENORHABDITIS ELEGANS MUTANTS EXHIBIT ACCELERATION OF AGE-RELATED REACTIVE OXYGEN SPECIES PRODUCTION It has been well accepted that oxidative stress is a feature of cellular aging,98 and intracellular free radical levels are increased in aged cells.19 To test the hypothesis that ROS levels are accumulated at a faster rate in Alzheimer’s models, we used both wild-type and AD-associated models of neuronal cells and C. elegans. The levels of ROS accumulation in the age-synchronized mutants surpass that of wildtype nematodes at each time span tested (Figure 18.4). Bearing a striking resemblance to the onset and exacerbation of symptoms seen in human AD, the rate of ROS accumulation in the mutant nematodes is dramatically increased during the midlife period. These results confirm the theory that ROS production is positively correlated with age, and that organisms bearing AD mutations accumulate ROS at 450 C. elegans CL2006

ROS levels (raw AFU values)

400

y = 66.1x - 13.7

350 300 C. elegans N2

250

y = 44.3x - 19.7

200 150 100 50 0 3

6

9 12 15 Age of worms (days)

FIGURE 18.4 Age-dependent ROS production is accelerated in AD C. elegans model. Both WT (solid columns) and AD-associated mutant nematodes (filled columns) were maintained separately, as age-synchronized groups ranging from 3 to 15 days old. Experimental groups were homogenized and sonicated, then immediately analyzed for ROS production. Results are expressed in raw data form as arbitrary fluorescence units (AFU) generated by DCF dye excitation. Trend lines and slope of each line indicate rate of ROS elevation from 3 to 12 days old for both nematode strains. Results are obtained from two independent experiments and represent a total of 1180 worms.

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a more rapid rate than others. These results indicate the potential usefulness of chronic antioxidants to scavenge circulating free radicals while reducing the rate of subsequent free radical production.

18.5

EGb 761 COMPONENTS SHOW PROTECTIVE EFFECTS IN ALZHEIMER’S MODELS

18.5.1 KAEMPFEROL AND QUERCETIN ATTENUATE REACTIVE OXYGEN SPECIES LEVELS IN VITRO AND IN VIVO Attenuation of increased intracellular ROS levels by EGb 761 has now been demonstrated in cultures exposed to exogenous Aβ as well as those expressing or secreting endogenous Aβ. Logically, we next compared the protection afforded by the extract EGb 761 to that of its main flavonoid constituents, kaempferol and quercetin. Compared with Aβ -expressing controls, ROS levels were significantly attenuated in neuronal cells by 17% with EGb 761 treatment, compared with 78% attenuation by kaempferol and 65% attenuation by quercetin. The pattern shown in the in vivo ROS study of AD-associated C. elegans expressing constitutive Aβ closely resembles that of the in vitro assay of Aβ expressing neuronal cells. In the mutant nematodes, ROS reduction was approximately 21% with EGb 761 treatment compared with controls. Attenuation of intracellular ROS levels by isolated flavonoid components was significant at 69% with kaempferol treatment and 44% with quercetin when compared with untreated AD-associated nematodes.8,99 Data analysis shows that the flavonoid components of the Ginkgo biloba extract has strong antioxidant properties,46 which has been now been confirmed in both cellular and invertebrate models.8 Not only do the flavonoid components of kaempferol and quercetin outperform the whole extract EGb 761, but they also provide greater attenuation than vitamins C and E.8 Most surprisingly, the flavonoid components provide greater free radical protection in models associated with AD mutations than wild-type counterparts measured under non-stressed physiological conditions. This finding is consistent with previous reports of the protection afforded by EGb 761 particularly under stress-induced conditions.66

18.5.2 ADVANCEMENT IN RECENT METHODOLOGY OXYGEN SPECIES DETECTION

FOR

REACTIVE

Given that expression of Aβ increases ROS production levels, and AD subjects demonstrate higher incidence of oxidative stress, numerous research groups have now conducted studies characterizing the relevance of free radical damage in the development of neurodegenerative disrders. Though free radical research has grown exponentially, a general consensus remains that the actions and measurements of free radicals in biological tissues in vivo remains a major challenge.88 Direct detection of free radicals is challenging owing to the short-lived nature of these highly reactive molecules. Ongoing oxidative damage is generally analyzed

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by measurement of secondary products, including derivatives of amino acids, nucleic acids, and lipid peroxidation.86 Therefore, the direct measurement technique of whole-organism ROS levels that we have presented here8 represents a significant advancement in this field. These research findings of increased in vivo ROS production in AD-associated C. elegans mutants8 were recently confirmed by McLellan et al.100 using multiphoton imaging to show a direct association between Aβ deposits and ROS production in vivo in live, transgenic AD-mouse models. Together, these data support the hypothesis that Aβ is responsible for the elevated production of free radicals in living AD models, and provides strong indication for the use of antioxidant therapies such as EGb 761 to neutralize these molecules and ameliorate further oxidative damage.

18.6 GINKGO BILOBA EXTRACT EGb 761 IMPARTS STRESS RESISTANCE AND LONGEVITY 18.6.1 OXIDATIVE STRESS RESISTANCE FED WITH EGb 761

IN

CAENORHABDITIS ELEGANS

It has been well noted that synthesis of a characteristic set of polypeptides called heat shock proteins may be induced in cells through exposure to varied forms of stress.101 Expression of the small heat shock protein Hsp-16 has similarly been used as a measure of stress in the C. elegans model, as the damage caused by most inducers of HSP synthesis manifests in a pattern similar to that of free radical damage. The transgenic strain of C. elegans CL2070, engineered by Dr Chris Link102 to express the green fluorescent protein (GFP)-tagged inducible small heat-shock protein (hsp-16-2), has been used to measure the effects of oxidative or thermal stress in this organism. In worms subjected to heat shock or a prooxidant chemical juglone, our research team was able to show a reduction in the levels of GFP fluorescence measured by epifluorescent imaging, and a reduction in ROS levels by the H2-DCF-DA method described above when pretreated with EGb 761.103 The expression of heat-induced hsp-16-2 in CL2070 was significantly suppressed by 33% in nematodes fed with EGb 761; and the chemicalinduced expression of hsp-16-2 was significantly suppressed by 86% in EGb 761-treated worms.103 To demonstrate that attenuation of hsp-16-2 expression is beneficial to the life span of the model, survival assays were conducted following the stress-induced event, with results indicating longer survival in both the chemically and thermally stressed colonies when pretreated with EGb 761 prior to initiation of the event.103

18.6.2 ENHANCED LIFE SPAN WITH EGb 761

IN

CAENORHABDITIS ELEGANS TREATED

According to the well-accepted free radical theory of aging, there is a strong relationship between longevity of an organism, and its ability to prevent or repair

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chronic macromolecular damage. In the event that the endogenous antioxidant defenses of an organism become impaired or overwhelmed, chronic cellular damage accumulates and manifests as typical signs of aging.66 This makes oxidative stress as another major contributor to both aging and neurodegenerative processes.24 The hypothesis that EGb 761 is able to augment the natural antioxidant defenses of an organism thereby increasing stress resistance, diminishing premature aging due to oxidative stress, and increasing longevity, has also been tested in C. elegans. Studies in our laboratory found that treatment of wild-type worms with EGb 761 can extend the median life span of the organism by 8%. A flavonoid component called tamarixetin showed dramatic results with the extension of the median life span by 25%. Furthermore, EGb 761 increased the resistance of the wild type strain to acute oxidative and thermal stress by 33 and 25%, respectively.66 Likewise, EGb 761 treatment of the prematurely aging mutant worms mev-1 increased their resistance to acute oxidative and thermal stress by 33and 11%, respectively. To support the hypothesis that improved longevity should be a by-product of the augmentation of the antioxidant system with EGb 761, oxidative stress has been induced with the chemical juglone (5-hydroxy-1, 4-naphthoquinone). This quinone is known to generate the toxic superoxide anion (O2⫺) from molecular oxygen during metabolism, thus acutely raising the levels of oxidative stress. After receiving the juglone-supplemented diet for 24 h, the nematodes were subsequently returned to a control or EGb 761-supplemented diet, and measured for survival. Pretreatment with EGb 761 in this study extended both the median and maximum life span of C. elegans by 8and 25%, respectively, after the chemically induced oxidative challenge.66 From these series of tests it appears that oxidative stress is a major determinant of life span, and can be successfully counteracted by the Ginkgo biloba extract EGb 761. These studies strengthen previous hypotheses relating the degree of oxidative damage to viability as well as the antiapoptotic and antistress benefits of EGb 761, which are now well documented in both cellular and animal behavior models.53,75,103

18.7 SUMMARY In AD, deposition of the Aβ peptide has been associated with atypically elevated levels of oxidative stress and apoptosis.104 Our studies have not only shown that Aβ initiates ROS production and subsequent apoptosis in AD models, but also that the antioxidative and radical-scavenging properties of EGb 761 may provide a therapeutic strategy for the treatment of neurodegenerative diseases such as AD, as well as those associated with the aging process.8,53,61,66,68,103 On the basis of numerous pharmacological studies using well-established methods of cell and molecular biology and biochemistry in both animals, and recently in humans, the use of EGb 761 in the abrogation of oxidative stress and age-associated neurodegenerative changes is gaining strong support.58,105 Current scientific knowledge of the neuroprotective mechanisms of EGb 761 is rapidly expanding, revealing aspects of its nature that support its use in both normal

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aging and also neurodegenerative diseases such as AD. EGb 761 is antiamyloidogenic, preventing the aggregation of Aβ, which leads to the formation of typical plaques in AD brain samples.68 EGb 761 is able to modulate the apoptotic machinery in AD models,53 which normally show elevated levels of apoptosis, and neuronal degeneration in response to Aβ expression.106 In conclusion, oxidative stress, subsequent protein damage, and viability are definitive factors in aging that appear to be heightened or accelerated in AD cases. Treatment with EGb 761 shows the potential for significant abrogation of ROS levels, thereby attenuating these mechanistic responses. Finally, the ability of EGb 761 to facilitate bidirectional adaptive responses, rather than unidirectional stimulation or inhibition107 is certainly one of the most intriguing aspects of EGb 761, and is postulated to be due to the polyvalent or synergistic activities of this multiconstituent extract. Ginkgo biloba studies ranging from the cellular to the behavioral and pharmacokinetic levels have indicated the effectiveness of EGb 761 at almost every level of life.46,107

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Proteases as 19 Cysteine β -Secretases for

Aβ Production in the Major Regulated Secretory Pathway of Neurons: Implications for Therapeutic Strategies in Alzheimer’s Disease

Vivian Hook and Thomas Toneff University of California La Jolla, California

Gregory Hook and Terry Reisine American Life Science Pharmaceuticals San Diego, California

CONTENTS 19.1 19.2 19.3 19.4 19.5 19.6

Introduction ............................................................................................328 β -Amyloid is a Major Factor Involved in Development of Alzheimer’s Disease ...............................................................................329 β -Amyloid Peptide is Generated by Proteolytic Processing of the Amyloid Precursor Protein.....................................................................329 β -Amyloid Production and Secretion in the Major Regulated Secretory Pathway of Neurons ...............................................................330 Regulated Cosecretion of β -Amyloid with Neurotransmitters from Neuronal Chromaffin Cells in Primary Culture ............................330 Chromaffin Vesicles as Regulated Secretory Vesicles for Identification of β -Secretase ..................................................................332 327

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Cysteine Proteases for β -Secretase Activity in the Regulated Secretory Pathway of Neurons .............................................................333 19.8 Kinetic Studies of the Cysteine β -Secretases Demonstrate their Preference for Cleavage of the Wild-Type β -Secretase Site, Rather than the Swedish Mutant β -Secretase Site ...............................336 19.9 BACE 1 in the Minor Constitutive Secretory Pathway for Basal Secretion of β -Amyloid..............................................................337 19.10 Unifying Hypothesis for Distinct Proteases in the Regulated Compared to the Constitutive Secretory Pathways for β -Amyloid Production and Secretion: Strategies for Drug Inhibition of β -Secretase to Treat Alzheimer’s Disease............................................339 Acknowledgments .............................................................................................339 References .........................................................................................................340 19.7

19.1 INTRODUCTION Alzheimer’s disease (AD) is a progressive, long-term, debilitating mental disorder that causes loss of memory and cognitive functions. Over 4 million Americans are afflicted with AD and the disease costs the US economy billions of dollars every year. Currently, there is no effective treatment for the disease, and therefore, there is an urgent need for effective therapeutic agents to ameliorate the detrimental conditions of AD. Amyloid plaques in the brain represent the neuropathological hallmark of AD. The plaques are composed of accumulated, extracellular β -amyloid peptides (Aβ ), which are neurotoxic and participate in the pathogenic mechanisms in AD.1–4 Aβ peptides are produced by proteolytic processing of the amyloid precursor protein (APP) by proteases known as β - and γ -secretases, which cleave N- and C-termini of Aβ within APP, respectively. Clearly, secretases represent critical steps in the production of neurotoxic Aβ peptide forms. It is believed that drugs that inhibit these secretases will reduce Aβ production and provide new therapeutic agents for the treatment of AD. It is, therefore, essential that the appropriate proteases for secretase activities be identified to provide relevant drug targets for AD. Identification of the secretases that produce Aβ requires investigation of these proteases at the primary subcellular site for Aβ production in neurons. The regulated secretory pathway represents the major source of Aβ production and secretion from brain neurons that undergo electrical, activity-dependent secretion.4–11 However, most studies of β -secretases have only investigated Aβ production in the minor constitutive secretory pathway that provides basal secretion of Aβ.12–17 Different proteases and cofactors are present in regulated secretory vesicles compared with constitutive secretory vesicles of these distinct pathways.4,18–21 It is, therefore, important to characterize β -secretase activity in regulated secretory vesicles. Results from our studies of regulated secretory vesicles indicate that cysteine proteases represent the major β -secretase of the regulated secretory pathway.4,11 These findings contrast with other studies claiming that the aspartyl protease BACE 1 functions as the only β -secretase.12–17 Results of these studies demonstrate BACE 1 as β -secretase in the minor constitutive secretory pathway.

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This chapter reviews evidence for the regulated secretory pathway as the major route for Aβ production and secretion. Moreover, cysteine proteases within regulated secretory vesicles of this pathway are hypothesized to participate as prominent β -secretases for the production and secretion of Aβ. A unifying hypothesis for β secretase activities by cysteine proteases in the major regulated secretory pathway, combined with BACE 1 in the constitutive secretory pathway, is proposed for Aβ production and secretion in neurons.

19.2 β -AMYLOID IS A MAJOR FACTOR INVOLVED IN DEVELOPMENT OF ALZHEIMER’S DISEASE A consistent neuropathological sign of AD is the appearance of extracellular neuritic plaques in limbic and cortical brain regions. The plaques contain accumulations of Aβ peptides consisting of Aβ (1–40) and Aβ (1–42). β-Amyloid (1–42) accumulates to a greater extent than Aβ (1–40) in AD. β-Peptides have been shown in experimental studies to be neurotoxic.1–3 A central hypothesis in the AD field is that accumulation of neurotoxic Aβ is responsible for neuronal degeneration in brain regions responsible for memory and cognition.1–3 The essential role of Aβ in AD has been demonstrated in transgenic mice that overexpress mutant forms of APP, which results in overproduction of Aβ. These animals have plaques and neuronal lesions in brain similar to those as found in AD. Moreover, mutant forms of presenilin result in excessive production of Aβ in AD. Furthermore, a vaccine developed against Aβ (1–42) reduced the pathology resulting from the overexpression of APP in both young and old animals.22 The vaccine improved cognitive impairments in the transgenic mice.23 The effectiveness of the vaccine indicates neutralization of Aβ (1–42), since antibodies are functional extracellularly. These findings support the hypothesis that secreted extracellular Aβ plays an important role in the development of AD. Because of the prominent role of Aβ in the pathogenesis of AD, intense research effort in the scientific community and pharmaceutical industry has focused on developing strategies and drugs to block Aβ formation for the treatment of AD.

19.3

β -AMYLOID PEPTIDE IS GENERATED BY PROTEOLYTIC PROCESSING OF THE AMYLOID PRECURSOR PROTEIN

All forms of Aβ are derived from a large precursor molecule, amyloid precursor protein (APP).24–26 Proteases produce Aβ by cleaving specific amino acid sequences within APP at or near the N- and C-termini of Aβ (Figure 19.1). Proteases that convert APP into Aβ have been named “secretases,” which are subdivided according to the cleavage sites within APP that are required to produce Aβ. The secretase that cleaves the N-terminal end of Aβ is called β -secretase and the protease that cleaves the C-termini of Aβ is called γ -secretase. The subtypes of γ -secretase activities, γ -secretase-40 and γ -secretase-42, determine whether Aβ (1–40) or Aβ (1–42) is produced.

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C-rich

KPI

Cytoplasmic A
Secretases A
Peptides

A
-secretases


-secretase

VKM - DAEFRHDSGYEVHHGKLVFFAEDVGSNKGAIIGLMVGGVV - IA - T VIVITL A
(1−40) A
(1−42)

FIGURE 19.1 Aβ peptide within APP. The structure of APP is illustrated, showing the Aβ peptides of Aβ (1–40) and Aβ (1–42). Production of Aβ requires proteolytic cleavage at γ -secretase sites, and at the β -secretase site. Cleavage at the α-secretase site precludes formation of Aβ.

19.4 β -AMYLOID PRODUCTION AND SECRETION IN THE MAJOR REGULATED SECRETORY PATHWAY OF NEURONS Cellular trafficking and proteolytic processing of APP to produce Aβ have been demonstrated to occur within the regulated secretory vesicle pathway of brain neurons in vivo (Figure 19.2).5–7,9,10 APP and its Aβ -related proteolytic products are transported from neuronal cell bodies to nerve terminals in the brain.27,28 In the brain, Aβ undergoes secretion induced by electrical depolarization, which is an essential feature of receptor-mediated secretion of APP-derived, Aβ -related peptides. Moreover, the presence of APP and Aβ products within regulated secretory vesicles of neurons indicates the presence of secretases within this organelle for the proteolytic conversion of APP into Aβ peptides.4,11,29–31 Aβ peptides must be secreted from brain neurons to form amyloid plaques and to cause toxicity. Peptides are released from neurons via two mechanisms: the regulated secretory pathway, controlled by electrical activation via which most biologically active peptide neurotransmitters are secreted, and a minor constitutive secretory pathway which releases a small amount of transmitters and Aβ. Importantly Aβ is primarily released via the regulated secretory pathway of neurons.

19.5

REGULATED COSECRETION OF β -AMYLOID WITH NEUROTRANSMITTERS FROM NEURONAL CHROMAFFIN CELLS IN PRIMARY CULTURE

To directly compare the relative amounts of Aβ secreted via the regulated secretory pathway with the constitutive secretory pathway, stimulated and basal secretion of Aβ from neuronal chromaffin cells were conducted simultaneously in the same experiment. Regulated secretion from chromaffin cells in primary

Cysteine Proteases as β -Secretases for Aβ Production

Stimulator of secretion via transmitter receptors

331

Activity-dependent secretion

APP

APP-derived peptides, A
,

APP gene

Neurotransmitters

ER,Golgi

Secretases: KPI








A


C83

APPs- APPs-


p3 C99 A


Regulated secretory vesicles Constitutive secretory vesicles

FIGURE 19.2 Aβ production and secretion in the regulated secretory pathway of neurons. Proteolytic processing of APP for the production of Aβ occurs in the regulated secretory pathway of neurons. The action potential propagates along the axon, and induces electrical depolarization dependent secretion of Aβ and neurotransmitters from neurons, known as the regulated secretory pathway. Secreted Aβ results in extracellular Aβ, accumulating in amyloid plaques, that produces toxicity indicated by cell death of neurons in brain regions responsible for memory and cognition in AD. (From Hook VYH, Reisine TD, J Neurosci Res, 2003: 74:393–405. With permission.)

culture was stimulated by nicotine or KCl depolarization (Figure 19.3a). Cholinergic receptor stimulation by nicotine induced high levels of Aβ (1–40) secretion that were nearly 50-fold greater than basal secretion. KCl depolarization also stimulated Aβ (1–40) secretion 30-fold above basal secretion (Figure 19.3). The secretion of Aβ (1–42) was also stimulated by nicotine and KCl depolarization. These results indicated that more than 90% of Aβ undergoes regulated secretion. Only a minor portion of Aβ is secreted under basal conditions of the constitutive secretory pathway. These results indicate that the regulated secretory pathway is the primary cellular route for production and secretion of Aβ from chromaffin cells as a model neuronal cell type. Moreover, Aβ is cosecreted with neuropeptide and catecholamine neurotransmitters of chromaffin cells. Nicotine and KCl stimulated the cosecretion of Aβ with (Met)enkephalin and neuropeptide Y (NPY) peptide neurotransmitters (Figures 19.3b and c). The Aβ peptide is also cosecreted with the catecholamine transmitters dopamine and norepinephrine, as well as epinephrine (Figures 19.3 d–f). These results illustrate the cosecretion of Aβ with neurotransmitter molecules by the regulated secretory pathway.

332

Oxidative Stress and Age-Related Neurodegeneration (a) A
peptide

3000

ng DA/well

ng A
/well

750 500 250

2000 1000 0

0 Control

(b) Enkephalin ngNEi/well

1500

10

0

2000

ng NPY/well

KCl

Control

Nicotine

(c) NPY

0

Control

Nicotine

KCl

Nicotine

KCl

(e) Norepinephrine

500

3000

1000

Control

1000

0

KCl

ng Epi/well

ng enk/well

20

Nicotine

(d) Dopamine

Control

Nicotine

KCl

(f) Epinephrine

2000 1000 0

Control

Nicotine

KCl

FIGURE 19.3 Cosecretion of Aβ with peptide and catecholamine neurotransmitters from neuronal chromaffin cells. Regulated secretion from neuronal chromaffin cells in primary culture was stimulated with nicotine (10 µM) or by KCl (50 mM) depolarization for 15 min, and the media was collected for measurements of Aβ (a) with the neuropeptides (Met)enkephalin (b) and NPY (c). Cosecretion of Aβ with the catecholamines dopamine, norepinephrine, and epinephrine (d–f, respectively) was also demonstrated.

Cosecretion of Aβ indicates its colocalization with neurotransmitters in regulated secretory vesicles. Further evidence for Aβ colocalization with neuropeptides was illustrated by immunoelectron microscopy (Figure 19.4) which showed that Aβ was present in regulated secretory vesicles that contain enkephalin and NPY. These Aβ -containing secretory vesicles contain full-length APP.29,31 Therefore, secretases that convert APP into Aβ peptides are present in the regulated secretory vesicle organelle, a primary subcellular site for Aβ production and secretion.

19.6 CHROMAFFIN VESICLES, REGULATED SECRETORY VESICLES, FOR IDENTIFICATION OF β -SECRETASE Because regulated secretory vesicles represent a primary subcellular site for proteolytic processing of APP to generate Aβ peptides, it is important to study secretase activities in such isolated secretory vesicles. However, it is difficult to purify a homogeneous population of brain secretory vesicles in high amounts owing to the heterogeneity of neuronal cell types in brain. Therefore, to overcome this difficulty, neuronal chromaffin cells of the sympathetic nervous system can be used

Cysteine Proteases as β -Secretases for Aβ Production A
40

333

Enk

NPY

(i)

(iii)

(v)

(ii)

(iv)

(vi) 0.5 µm

FIGURE 19.4 Colocalization of Aβ with peptide neurotransmitters in regulated secretory vesicles. Aβ, enkephalin, and NPY in regulated secretory vesicles demonstrated by immunoelectron microscopy. Regulated secretory vesicles isolated from chromaffin cells contain Aβ 40 (i,ii), enkephalin (iii,iv), and NPY (v,vi), demonstrated by detection of Aβ or neuropeptides with 6 nm gold particles.

as an abundant source for purification of regulated secretory vesicles (chromaffin vesicles) that contain authentic in vivo secretases colocalized with APP and Aβ. Chromaffin vesicles contain full-length APP as substrate for β -secretase. Full-length APP within chromaffin granules exists as transmembrane protein, membrane-associated form, and as fully soluble APP within these vesicles.29,31 Chromaffin vesicles express full-length APP695 and KPI/APP751,770.29,31 These vesicles also contain presenilin,30 which has been demonstrated to possess γ -secretase activity.32,33 Since APP and its peptide products Aβ (1–40) and Aβ (1–42) are present in these regulated secretory vesicles, β -secretase activity is present in this organelle (Figure 19.5). These regulated secretory vesicles of chromaffin cells are an ideal model system to study natural Aβ production and to identify drugs to block the synthesis of this neurotoxic peptide.

19.7 CYSTEINE PROTEASES FOR β -SECRETASE ACTIVITY IN THE REGULATED SECRETORY PATHWAY OF NEURONS To characterize the β -secretase within regulated secretory vesicles, β -secretase activity in these isolated secretory vesicles (from neuronal chromaffin cells) was tested with class-specific protease inhibitors. Assay of the β -secretase utilized a highly sensitive fluorescent assay that monitored β -secretase site cleavage of

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APP A



-Secretase: Cysteine Proteases

A
40

-Secretase: Presenilin

A
42

FIGURE 19.5 APP and processing components for Aβ production in regulated secretory vesicles of neuronal chromaffin cells. Regulated secretory vesicles isolated from neuronal chromaffin cells contain the biosynthetic machinery for Aβ production. These biosynthetic components consist of full-length APP, Aβ(1–40) and Aβ(1–42), and presenilin. Moreover, APP has been demonstrated as transmembrane APP, membrane-associated APP within the vesicles, and as full-length soluble APP.29,31 The presence of Aβ peptides and its APP precursor indicates the presence of β -secretase within these regulated secretory vesicles.

the peptide–MCA substrate Z-Val-Lys-Met-↓MCA by measuring production of fluorescent MCA.11 Importantly, tests with protease inhibitors indicated that the cysteine protease inhibitor E64c completely inhibited β -secretase activity in these vesicles. Significantly, the production of Aβ from APP within these secretory vesicles was blocked by E64c (Figure 19.6), indicating the participation of cysteine protease activity to produce Aβ. Moreover, E64c blocked production of Aβ from full-length APP in these vesicles. In primary cultures of neuronal chromaffin cells, the cell-permeable E64d reduced conversion of APP to the COOH-terminal β -secretase cleavage product.11 It was of interest to note that the β -secretase activity in these secretory vesicles was not inhibited by a specific inhibitor of BACE 1.11 These results demonstrated the participation of cysteine protease activity in the production of Aβ. Purification by chromatographic analyses of in vivo β -secretase activity from chromaffin vesicles provided evidence for multiple cysteine endoproteases as β -secretase. First, direct cleavage of the β -secretase of the model substrate Z-ValLys-Met-↓MCA was demonstrated by the cysteine protease activity of peak II. Second, another cysteine protease of peak I represented an alternative β -secretase pathway involving cleavage at Lys-↓Met, followed by methionine aminopeptidase to remove N-terminal methionine. These cysteine proteases represent two distinct proteolytic pathways (Figure 19.7) for β -secretase activity:(1) direct cleavage at

Cysteine Proteases as β -Secretases for Aβ Production

335

A
(1-40) pg/µg vesicle protein

0.20

0.15

0.10

0.05

0.00

0

4 8 Incubation time (hr.)

12

FIGURE 19.6 Cysteine protease activity is responsible for Aβ production in the regulated secretory vesicles of neuronal chromaffin cells. Inhibition of Aβ (1–40) peptide production in regulated secretory vesicles by the cysteine protease inhibitor E64c. Lysed secretory vesicles from chromaffin cells were incubated with E64c in a time-course study, and the production of Aβ (1–40) from endogenous APP was measured. Peak I

Peak II

A
peptide

- Val - Lys - Met - DAEFRH - - - - - - - -

Peak I

Peaks II-A, II-B, with BACE 1

A
peptide -Val-Lys- + Met-

DAEFRH - -

A
peptide -Val-Lys-Met + DAEFRG - -

Methionine aminopeptidase A
peptide Met + DAEFRH - -

FIGURE 19.7 Distinct cysteine proteolytic pathways in the regulated secretory pathway for Aβ production and secretion. It is hypothesized that the major portion of secreted, extracellular Aβ is produced by the regulated secretory pathway by the cysteine proteases of peaks I and II. The cysteine protease of peak II displays direct cleavage of the wild-type β -secretase site. An alternative aminopeptidase-dependent pathway for β -secretase is represented by the cysteine protease activity of peak I. (From Hook VYH, Toneff T, Aaron W, Yasothorsnrikul S, Bundey R, Reisine T, J Neurochem, 2002; 81:237–256. With permission.)

the β -secretase site achieved by cysteine protease activity of peak II, and (2) an aminopeptidase-dependent pathway consisting of the peak I cysteine protease activity that cleaves at Lys-↓Met, followed by methionine aminopeptidase to

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generate the β -secretase cleavage site. Evidence for these distinct proteolytic pathways implicates the complexity of β -secretase activity that may require coordinate regulation of multiple proteases to generate Aβ.

19.8 KINETIC STUDIES OF THE CYSTEINE B-SECRETASES DEMONSTRATE THEIR PREFERENCE FOR CLEAVAGE OF THE WILD-TYPE B-SECRETASE SITE, RATHER THAN THE SWEDISH MUTANT B-SECRETASE SITE The majority of AD patients express the wild-type β -secretase site, which is represented by the peptide-MCA substrate Z-Val-Lys-Met-MCA. A few individuals from an extended family possess the Swedish mutant β -secretase site, with the mutation Asn-Leu substituting for Lys-Met at the β -secretase site.34 Studies of the aspartyl protease BACE 1 as β -secretase have utilized the Swedish mutant β -secretase site.12–17 However, because the wild-type β -secretase site is expressed in the majority of the AD population (⬎99% of AD patients), it is important to define the specificity of the cysteine proteases in regulated secretory vesicles for the wild-type compared with the Swedish mutant β -secretase site. Therefore, rates of cleavage of the wild-type β -secretase substrate, Z-ValLys-Met-↓MCA, were compared with rates of cleavage of the Swedish mutant β -secretase substrate Z-Val-Asn-Leu-↓MCA. Cysteine protease activities of peaks II and I showed high specific activities of 547 and 185 pmol AMC/min/µg protein for cleavage of the wild-type β -secretase substrate, Z-Val-Lys-Met↓MCA (Table 19.1). However, very low rates of cleavage were found for the mutant substrate Z-Val-Asn-Leu-↓MCA. In contrast, BACE 1 showed little cleavage of the wild-type β -secretase site of the Z-Val-Lys-Met-↓MCA substrate.

TABLE 19.1 Preference of Cysteine Proteases from Regulated Secretory Vesicles to Cleave the Wild-Type β -Secretase Site, Compared with the Swedish Mutant β -Secretase Site Relative β -Secretase Activity

Protease

Wild-Type Site ⬎99% AD ↓MCA Z-V-K-M-↓

Swedish Mutant Site ⬍1% AD ↓MCA Z-V-N-L-↓

pmol AMC/min/µg enzyme Cysteine protease of peak I Cysteine protease of peak II BACE 1

185 547 0

0.2 0.2 0.01

β-Secretase activity was measured with fluorescent assays using peptide-MCA substrates containing the wild-type β-secretase cleavage site or the Swedish mutant β-secretase cleavage site.

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337

TABLE 19.2 Kinetic Studies Demonstrate High Efficiencies of Cysteine Proteases I and II from Regulated Secretory Vesicles Protease Cysteine protease of peak I Cysteine protease of peak II BACE 1

Kinetic Rate Constant kcat/Km (M⫺1 s⫺1)

Relative Efficiency Compared with BACE 1

8.4 ⫻ 103 3.2 ⫻ 104 4 ⫻ 101

200⫻ 800⫻ 1⫻

Note: The kcat/Km kinetic rate constant measures high catalytic efficiencies of the cysteine proteases I and II purified from regulated secretory vesicles of neuronal chromaffin cells. In contrast, the reported kinetic rate constant for BACE 1 is low (16).

Although BACE 1 showed some cleavage of the Swedish mutant β -secretase site, it was lower than that for the cysteine proteases represented by peaks I and II. Further kinetic characterization of cysteine protease peaks II and I showed high efficiencies for cleaving the wild-type β -secretase site, as indicated by kcat /Km kinetic constants (Table 19.2). Peaks II and I show kcat/Km constants of 31,700 and 8,620 M⫺1s⫺1, respectively. In contrast, BACE 1 showed extremely low catalytic efficiency with kcat/Km of 40 M⫺1s⫺1 (based on literature reports35); these catalytic constants demonstrate that BACE is a very low-activity enzyme. Clearly, the cysteine proteases of peaks II and I demonstrate catalytic efficiencies for processing the wild-type β -secretase site, as expected for active protease enzymes. Significantly, these results demonstrate that selective proteases exist for cleaving the wild-type β -secretase site compared with the Swedish mutant β -secretase site. Because the wild-type β -secretase site is expressed in the majority of Alzheimer’s patients (⬎99% of AD population), proteases that cleave the wild-type β -secretase site are relevant to the disease. The Swedish mutant β -secretase is expressed in only one extended family.34 These results suggest that BACE 1 as β -secretase is less relevant to the majority of AD patients. Rather, the cysteine proteases within regulated secretory vesicles of neuronal chromaffin cells demonstrate the appropriate specificities for processing the wild-type β -secretase site that is expressed in the AD population.

19.9 BACE 1 IN THE MINOR CONSTITUTIVE SECRETORY PATHWAY FOR BASAL SECRETION OF β -AMYLOID In contrast to the regulated secretory pathway of neurons, the constitutive secretory pathway provides a small portion (⬍5–10%) of secreted Aβ.4,11 Support for participation of the constitutive secretory pathway in Aβ production is provided by studies of the aspartyl protease BACE 1.12–17 BACE 1 was identified as a β -secretase for basal secretion of Aβ into conditioned media from CHO, HEK-293, or other nonneuronal cells. Moreover, non-neuronal cells lack the regulated secretory pathway,

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and therefore, studies in such cells can only provide information about β -secretase and Aβ production in the constitutive secretory pathway. Only specialized neurons and endocrine cells contain the regulated secretory pathway for stimulated secretion of neurotransmitters and endocrine hormones.35 Other cell types possess only the constitutive secretory pathway. Moreover, studies of BACE 1 in HEK-293 cells that overexpress the Swedish mutant APP provided evidence that BACE 1 prefers the Swedish mutant β -secretase site. These approaches, however, could not identify β -secretases that specifically cleave the wild-type β -secretase site, since wild-type APP was not present in the screening assays. The hypothesis that cysteine proteases are β -secretases in the regulated secretory pathway, and that BACE 1 is a β-secretase in the constitutive secretory pathway (Figure 19.8) is consistent with data from studies of BACE 1 knockout mice. One study demonstrated that resting neuronal cultures from BACE 1 knockout mouse brains showed reduced basal secretion of Aβ into conditioned media,17 indicating a role for BACE 1 in the constitutive secretory pathway for Aβ production. Another study examined the absence of BACE 1 in mice that

A
, Stimulated secretion Regulated secretion of A
, > 90%

Basal secretion Constitutive secretion Extracellular of A
, < 10% Intracellular

Regulated secretory pathway: Cysteine proteases

Constitutive secretory pathway: BACE 1

TGN Golgi apparatus

FIGURE 19.8 Unifying hypothesis for cysteine proteases as β -secretase in the regulated secretory pathway, with BACE 1 as β -secretase in the minor constitutive secretory pathway. A unifying hypothesis for β -secretase in the production of neurotoxic Aβ proposes that (1) cysteine proteases provide β -secretase activity for Aβ production and secretion in the major regulated secretory pathway, and (2) the aspartyl protease BACE 1 functions as β-secretase in the minor constitutive pathway that provides a small percentage of secreted Aβ. This is based on reported results of β -secretases in these two pathways.4,11,12–17 This hypothesis links the relative roles of cysteine proteases in regulated secretory vesicles for Aβ production in the major regulated secretory pathway, with BACE 1 for Aβ production in the minor constitutive secretory pathway. (From Hook VYH, Reisine TD, J Neurosci Res, 2003: 74:393–405. With permission.)

Cysteine Proteases as β -Secretases for Aβ Production

339

overexpress the Swedish mutant form of APP.36 On the basis of the preference of BACE 1 for cleaving the Swedish mutant β -secretase site, it is logical that the absence of BACE 1 resulted in reduced processing of Swedish mutant APP. It must also be noted that assays without a reducing agent cannot detect cysteine protease activities, which require reducing conditions.13,37 Thus, assays of β -secretase from brain that lacked reducing agents could only detect BACE 1. These data demonstrate that BACE 1 participates in the minor constitutive secretory pathway for Aβ production and prefers to cleave the Swedish mutant β -secretase site of APP, which is expressed in only several members of a family with AD.34 Recent studies have demonstrated that BACE 1 is not cotransported with APP in axons of the sciatic nerves of mice.38 The lack of cotransport of putative BACE 1 with APP argues against proposals that BACE 1 functions as a β -secretase with APP, since APP processing can only be achieved with secretases that are colocalized with APP substrate. Since regulated secretory vesicles undergo axonal transport from neuronal cell bodies to nerve terminals, the dissociation of APP transport from that of BACE 1 is consistent with our hypothesis that the majority of APP is present in regulated secretory vesicles, whereas BACE 1 is located in distinct constitutive secretory vesicles. These findings support the hypothesis for cysteine proteases as important β -secretases for APP processing in the regulated secretory pathway of neurons.

19.10 UNIFYING HYPOTHESIS FOR DISTINCT PROTEASES IN THE REGULATED COMPARED TO THE CONSTITUTIVE SECRETORY PATHWAYS FOR β -AMYLOID PRODUCTION AND SECRETION: STRATEGIES FOR DRUG INHIBITION OF β -SECRETASE TO TREAT ALZHEIMER’S DISEASE Results from studies of β -secretase in regulated secretory vesicles, compared with those of the constitutive secretory pathway, lead to a unifying hypothesis for (1) cysteine proteases as β -secretases in the major regulated secretory pathway of neurons, and (2) the aspartyl protease BACE 1 as the β -secretase in the minor constitutive secretory pathway for Aβ production and secretion (Figure 19.8). This unifying hypothesis for multiple proteolytic pathways of the regulated and constitutive secretory pathways predicts that inhibition of several proteases may be required to achieve significant reduction of secreted Aβ that accumulates in AD. On the basis of inhibitor studies in this report showing reduction of Aβ within regulated secretory vesicles by cysteine protease inhibitor E64c, it is possible that future studies of related inhibitors may be useful for reducing brain Aβ. These findings demonstrate the importance of elucidating the primary protease(s) within the complexity of multiple pathways for β -secretase activity in the production of neurotoxic Aβ of AD.

ACKNOWLEDGMENTS These studies were supported by a NIH SBIR Phase II grant AG18044 awarded to American Life Science Pharmaceuticals (ALSP), Inc., San Diego, CA, with a

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subcontract to the University of California, Skaggs School of Pharmacy and Pharmaceutical Sciences. Dr. V. Hook holds equity in ALSP, Inc., and serves on the Scientific Advisory Board of ALSP, Inc. The terms of this arrangement have been reviewed and approved by the University of California, San Diego in accordance with its conflict of interest policies. Note added in proof. The peak II β-secretase has been identified as cathepsin B, as reported in Hook et al, 2005 (Biol. Chem. 386, 931–940).

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13. Sinha S, Anderson JP, Barbour R, Basi GS, Caccavello R, Davis D, Doan M, Dovey HF, Frigon N, Hong J, Jacobson-Croak K, Jewett N, Keim P, Knops J, Lieberburg I, Power M, Tan H, Tatsuno G, Tung J, Schenk D, Seubert P, Suomensaari SM, Wang S, Walker D, Zhao J, McConlogue L, John V. Purification and cloning of amyloid precursor protein β-secretase from human brain. Nature, 1999; 402:537–540. 14. Yan R, Bienkowski MJ, Shuck ME, Miao H, Tory MC, Pauley AM, Brashler JR, Stratman NC, Mathews WR, Buhl AE, Carter DB, Tomasselli AG, Parodi LA, Heinrikson RL, Gurney ME. Membrane-anchored aspartyl protease with Alzheimer’s disease β-secretase activity. Nature, 1999; 402:533–537. 15. Hussain I, Powell D, Howlett DR, Tew DG, Meek TD, Chapman C, Gloger IS, Murphy KW, Southan CD, Ryan DM, Smith TS, Simmons DL, Walsh FS, Dingwall C, Christie G. Identification of a novel aspartic protease (Asp2) as β -secretase. Mol Cell Neurosci, 1999; 14:419–427. 16. Lin X, Koelsch G, Wu S, Downs D, Dashti A, Tang J. Human aspartic protease memapsin 2 cleaves the β-secretase site of β-amyloid precursor protein. Proc Natl Acad Sci USA, 2000; 97:1456–1460. 17. Cai H, Wang Y, McCarthy D, Wen H, Borchelt DR, Price DL, Wong PC. BACE 1 is the major β -secretase for generation of Aβ by neurons. Nat Neurosci, 2001; 4:233–234. 18. Hook V, Yasothornsrikul S, Greenbaum D, Medzihradszky KF, Troutner K, Toneff T, Bundey R, Reinheckel T, Peters C, Bogyo M. Cathepsin L and Arg/Lys aminopeptidase: a distinct prohormone processing pathway for the biosynthesis of peptide neurotransmitters and hormones. Biol Chem, 2004; 385:473–480. 19. Benjannet S, Savaria D, Laslop A, Munzer JS, Chretien M, Marcinkiewicz M, Seidah NG. Alpha1-antitrypsin Portland inhibits processing of precursors mediated by proprotein convertases primarily within the constitutive secretory pathway. J Biol Chem, 1997; 272:26210–26218. 20. Gensberg K, Jan S, Matthews GM. Subtilisin-related serine proteases in the mammalian constitutive secretory pathway. Semin Cell Dev Biol, 1998; 9:11–17. 21. Thomas G. Furin at the cutting edge: from protein traffic to embryogenesis and disease. Nat Rev Mol Cell Biol, 2002; 3:753–766. 22. Vehmas AK, Borchelt DR, Price DL, McCarthy D, Wills-Karp M, Peper MJ, Rudow G, Luyinbazi J, Siew LT, Troncoso JC. β-amyloid peptide vaccination results in marked changes in serum and brain Aβ levels in APPswe/PS1∆E9 mice, as detected by SELDI-TOF-based proteinchip technology. DNA Cell Biol, 2001; 20:713–721. 23. Younkin SG. Amyloid β vaccination: reduced plaques and improved cognition. Nat. Med, 2001; 7:18–19. 24. Kang J, Lemaire HG, Unterbeck A, Salbaum JM, Masters CL, Grzeschik KH, Multhaup G, Beyreuther K, Muller-Hill B. The precursor of Alzheimer’s disease amyloid A4 protein resembles a cell surface receptor. Nature, 1987; 325:733–736. 25. Tanzi RE, Gusella JF, Watkins PC, Bruns GA, St George-Hyslop P, Van Keuren ML, Patterson D, Pagan S, Kurnit DM, Neve RL. Amyloid β protein gene: cDNA, mRNA distribution and genetic linkage near the Alzheimer’s locus. Science, 1987; 235:880–884. 26. Robakis NK, Ramakrishna N, Wolfe G, Wisniewski HM. Molecular cloning and characterization of a cDNA encoding the cerebrovascular and the neuritic plaque amyloid peptides. Proc Natl Acad Sci USA, 1987; 84:4190–4194.

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27. Buxbaum JD, Thinakaran G, Koliatsos V, O’Callahan J, Slunt HH, Price DL, Sisodia SS. Alzheimer amyloid protein precursor in the rat hippocampus: transport and processing through the perforant pathway. J Neurosci, 1998; 18:9629–9637. 28. Kamal A, Stokin GB, Yang Z, Xia CH, Goldstein LS. Axonal transport of amyloid precursor protein is mediated by direct binding to the kinesin light chain subunit of kinesin-I. Neuron 2000; 28:449–459. 29. Vassilacopoulou D, Ripellino JA, Tezapsidis N, Hook VYH, Robakis NK. Fulllength and truncated Alzheimer amyloid precursors in chromaffin granules: solubilization of granule membrane amyloid precursor is mediated by an enzymatic mechanism. J Neurochem, 1995; 64:2140–2146. 30. Efthimiopoulos S, Georgakopoulos A, Floor E, Shioi J, Cui W, Yasothornsrikul S, Hook VYH, Wisniewski T, Buee L, Robakis NK. Enrichment of presenilin 1 peptides in neuronal large dense core and somatodendritic clathrin coated vesicles. J. Neurochem, 1998; 71:2365—2372. 31. Tezapsidis N, Li HG, Rippellino JA, Efthimiopoulos S, Vassilacopoulou D, Sambamurti K, Toneff T, Yasothornsrikul S, Hook VYH, Robakis NK. Release of non transmembrane, full-length Alzheimer’s amyloid precursor protein from the lumenar surface of chromaffin granule membranes. Biochemistry, 1998; 37:1274–1282. 32. Wolfe MS, Haass C. The role of presenilins in γ-secretase activity. J Biol Chem, 2001; 276:5413–5416. 33. Kimberly WT, Xia W, Rahmati T, Wolfe MS, Selkoe D. The transmembrane aspartates in presenilin 1 and 2 are obligatory for γ-secretase activity and amyloid β -protein generation. J Biol Chem, 2000; 275:3173–3178. 34. Citron M, Oltersdorf T, Haass C, McConlogue L, Hung AY, Seubert P, VigoPelfrey C, Lieberburg I, Selkoe D. Mutation of the β -amyloid precursor protein in familial Alzheimer’s disease increases β -protein production. Nature, 1992; 360:672–674. 35. Lodish H, Berk A, Zipursky SL, Matsudaira P, Baltimore D, Darnell J. Molecular Cell Biology, 4th ed. New York: WH Freeman, 1999, pp. 691–726. 36. Luo Y, Bolon B, Kahn S, Bennet BD, Babu-Khan S, Denis P, Fan W, Kha H, Zhang J, Gong Y, Martin L, Louis JC, Yan Q, Richards WG, Citron M, Vassar R. Mice deficient in BACE 1, the Alzheimer’s β -secretase, have normal phenotype and abolished β -amyloid generation. Nat Neurosci, 2001; 4:231–232. 37. Roberds SL, Anderson J, Basi G, Bienkowski MJ, Branstetter DG, Chen KS, Freedman S, Frigon NL, Games D, Hu K, Johnson-Wood K, Kappenman KE, Kawabe TT, Kola I, Kuehn R, Lee M, Liu W, Motter R, Nichols NF, Power M, Robertson DW, Schenk D, Schoor M, Shopp GM, Shuck ME, Sinha S, Svensson KA, Tatsuno G, Tintrup H, Wijsman J, Wright S, McConlogue L. BACE knockout mice are healthy despite lacking the primary β-secretase activity in the brain: implications for Alzheimer’s disease therapeutics. Hum Mol Genet, 2001; 10:1317–1324. 38. Lazarov O, Morfini GA, Lee EB, Farah MH, Szodorai A, DeBoer SR, Koliatsos VE, Kins S, Lee VM, Wong PC, Price DL, Brady ST, Sisodia SS. Axonal transport, amyloid precursor protein, kinesin-1, and the processing apparatus: revisited. J Neurosci, 2005; 25:2386–2395.

Molecular 20 The Mechanism of the Neuroprotective Action of Antioxidants Compared with the Anti-Parkinson Drug, Rasagiline Orly Weinreb, Tamar Amit, Silvia A. Mandel, and Moussa B.H Youdim Technion Israel Institute of Technology Haifa, Israel

CONTENTS Abstract..............................................................................................................344 20.1 Introduction..............................................................................................344 20.2 Studies of Neuroprotective Properties in Models of Parkinson’s Disease .................................................................................345 20.2.1 Antioxidant Agents ......................................................................345 20.2.2 Rasagiline.....................................................................................347 20.3 Neuroprotective Studies in Models of Alzheimer’s Disease ...................348 20.3.1 Antioxidant Agents ......................................................................348 20.3.2 Rasagiline.....................................................................................348 20.4 The Mechanisms Underlying Antioxidant-Attenuated Cell Death .........349 20.4.1 Modulation of Cell Signaling Pathways......................................349 20.4.2 Effect of Antioxidants on Cell Survival/Death Gene Expression....................................................................................351 20.5 Mechanism of Action of Rasagiline ........................................................352 20.6 Conclusions and Perspectives for the Future...........................................355 Acknowledgments .............................................................................................355 References .........................................................................................................356 343

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ABSTRACT Accumulating evidence suggests that oxidative stress resulting in reactive oxygen species generation and inflammation in the brain plays a pivotal role in neurodegenerative diseases, supporting the implementation of radical scavengers, antioxidants, transition metals, chelators, and monoamine oxidase (MAO) inhibitors in the clinic. These observations are in line with the current view that antioxidative supplements may have an impact on cognitive deficits in aging. As a consequence, green tea polyphenols and the pineal hormone, melatonin, are now being considered as therapeutic agents in well-controlled epidemiological studies aimed to alter brain-aging processes, and as possible neuroprotective agents in progressive neurodegenerative disorders such as Parkinson’s and Alzheimer’s diseases. Additionally, recent clinical studies with the MAO-B inhibitor rasagiline (Azilect) in Parkinsonian subjects have shown that patients treated with rasagiline for 12 months show significantly less functional decline than those whose treatment was delayed by 6 months. This suggests that rasagiline may possess a disease-modifying activity that could be related to cell culture and animal neuroprotective studies described so far. This chapter will discuss recent literature on the putative novel neuroprotective mechanism of several antioxidant drugs, dopamine, dopamine D1-D2-receptor agonist, R-apomorphine, green tea polyphenol (⫺)-epigallocatechin-3-gallate, and melatonin, compared with the antiParkinson drug/MAO-B inhibitor, rasagiline.

20.1 INTRODUCTION Parkinson’s disease (PD) is an age-dependent neurodegenerative disease, characterized at the cellular level by a depletion of dopamine (DA) in dopaminergic neurons of substantia nigra pars compacta (SNPC). Although the etiology of the neurodegeneration is still unclear and no casual therapy is available yet, several lines of evidence suggest that oxidative stress (OS), resulting in reactive oxygen species (ROS) generation, plays a pivotal role in chronic disorders such as PD and Alzheimer’s disease (AD).1,2 The proliferation of reactive microglia surrounding neurons in AD and PD is thought to contribute to the initiation of OS, a consequence of increased generation of reactive hydroxyl radicals (OH⫺), superoxide (O⫺2 ), and nitric oxide.3–5 Thus, the oxidative mechanisms responsible for the ROS-mediated cell injury involve mainly peroxidation of lipid membranes, DNA and protein oxidation, and subsequent neuronal cell death that contributes to disease pathogenesis.6–8 Previous reports described a decrease in the activity of the enzyme catalase, which catalyzes the breakdown of hydrogen peroxide (H2O2) within the parietal-temporal cortex and basal ganglia, as determined from postmortem AD brain tissues.9 Furthermore, an increase in monoamine oxidase (MAO)-B activity within reactive microglia in AD and PD brain tissues is considered to contribute to the formation of even higher levels of H2O2 as a by-product of amine turnover.10 Failure to remove excess H2O2 as a result of reduced glutathione (GSH) and catalase activity may result in the generation of reactive free oxygen and OH⫺, produced through the interaction of

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H2O2 with chelatable (free ionic) iron (Fenton chemistry), which would initiate OS processes.11 Indeed, high concentrations of reactive iron can increase OS-induced neuronal vulnerability, and iron accumulation might increase the toxicity of environmental or endogenous toxins. Animal models of PD, such as 6-hydroxydopamine (6-OHDA) in rats and N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in mice, support this hypothesis of OS involvement in neurodegenerative diseases.12–14 A variety of therapeutic neuroprotective strategies mainly target OS, on the basis of accumulated evidence concerning the pathogenesis of neurodegenerative diseases. Most of the neuroprotective experimental studies have been effectively conducted in cell cultures or animal models, since clinical neuroprotection is much more difficult to carry out. In this chapter, we will focus on the molecular mechanism of action involved in PD and AD of several antioxidant agents: DA, DA D1-D2-receptor agonist, R-apomorphine (R-APO), green tea polyphenol (⫺)-epigallocatechin-3-gallate (EGCG), melatonin, and the anti-Parkinson drug/MAO-B inhibitor, rasagiline (Azilect) (Figure 20.1), which are potent neuroprotective compounds in both animal and in vitro models.

20.2 STUDIES OF NEUROPROTECTIVE PROPERTIES IN MODELS OF PARKINSON’S DISEASE 20.2.1 ANTIOXIDANT AGENTS Oxidative stress-dependent apoptosis may be a contributing pathway to dopaminergic neuronal cell death in PD.3 Therefore, it is not surprising that neuroprotective therapies aimed at interfering with these cytotoxic and apoptotic processes, or alternatively, at promoting neuronal growth and function, have been employed in cellular and animal models of neurodegenerative diseases.13–15 Several catechol derivatives, especially the precursor of DA, L-dihydroxyphenylalanine (L-DOPA), or mixed-type DA D1-D2-receptor agonist, R-APO, are employed for the therapy of PD.16,17 In vitro experiments have demonstrated that R-APO and its S-isomer are potent iron chelators and radical scavengers.17 At low concentration, both are able to protect rat pheochromocytoma (PC-12) cells18,19 from the toxic effects of the parkinsonism-inducing neurotoxin 6-OHDA and H2O2, but higher doses induce cell death. Previous studies have shown that high DA concentrations can be cytotoxic to PC-1220 and human neuroblastoma (NB) cells,21 raising the question whether long-term treatment with the catechol derivative, L-DOPA, which gives rise to DA, may contribute to the degeneration of the dopaminergic neurons of the substantia nigra in PD.22 However, there is no evidence from clinical studies that long-term and continuous administration of LDOPA or DA receptor agonist, R-APO, with structural similarity to DA, has a pathogenic effect in PD. Moreover, both R- and S-APO were shown in vivo to protect against dopaminergic neurotoxin, MPTP, in mice23 and to prevent OS and cell death in substantia nigra of mice.24,25 The fact that both isomers of APO display a

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HO HO CH3 N HO

CH2

CH2

NH2

R-apomorphine

Dopamine

OH OH

O

H

O OH

O O OH

OH O

O

NH

H 3C

OH N H

OH

Melatonin Green tea polyphenol (−)-epigallocatechin-3-gallate

N H Rasagiline (Azilect)

FIGURE 20.1 The chemical structures of the antioxidants, dopamine (DA), dopamine D1D2-receptor agonist, R-apomorphine (R-APO), green tea polyphenol (−)-epigallocatechin3-gallate (EGCG), and melatonin, and the anti-Parkinson drug/monoamine oxidase (MAO)-B inhibitor, rasagiline (Azilect).

neuroprotective activity suggest that other pharmacological properties, besides DA receptor agonistic mechanism, may also play a role in neuroprotection. Thus, RAPO neuroprotective action is also attributed to its radical-scavenging, iron-chelating features.19,26,27 This agent is a catechol-derived compound, easily oxidizable, and can therefore react with ROS (for review see [28]). It was suggested that the antioxidant and iron-chelating properties and possible MAO-inhibitory actions,

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together with the activation of DA receptors, may participate in the neuroprotective mechanism of R-APO.29 The extensive evidence that the pineal indoleamine hormone, melatonin (N-acetyl-5-methoxytryptamine), may exert a neuroprotective effect also through its antioxidant and free-radical scavenger activity, has led to the suggestion that this compound may have clinical potential in the treatment of PD30 and other neurodegenerative diseases. In line with this, melatonin was shown to enhance the survival of PC-12 cells following treatment with 6-OHDA,31 and to protect DA neurons from the neurotoxic effect of MPTP in mice.32 In addition, catecholrelated phenolic compounds, such as tea extract and its major component, EGCG, have been demonstrated to prevent lipid peroxidation in membrane mitochondrial fraction33 and to protect MPTP- and 6-OHDA-induced cell death both in mice and in neuronal cell cultures.33–36 Additional potential mechanisms underlying the effectiveness of these antioxidants against MPTP animal model, may involve their iron-chelating and freeradical scavenging activities.37 This inhibition was suggested to block the uptake of the metabolic product of MPTP, the neurotoxin 1-methyl-4-phenylpyridinium (MPP⫹) (because of competition for the vesicular transporter), thereby protecting DA-containing neurons against MPP +-induced injury.38 Previous neuroprotective studies also demonstrated inhibition of MPP⫹-induced neurotoxicity in human NB cells by EGCG35 and in vivo by melatonin in Sprague–Dawley rats.39 Furthermore, both EGCG40 and melatonin inhibited the activity of the enzyme catechol-O-methyltransferase (COMT) in rat liver cytosol homogenates and in golden (Syrian) hamster adrenal medulla.41 Dopamine and related catecholamines are physiological substrates of COMT. The COMT inhibitors, entacapone and tolcapone, clinically prescribed to PD-affected individuals, dose-dependently inhibit the formation of the major metabolite of levodopa, 3-O-methyldopa, thereby improving its bioavailability in the brain.42

20.2.2 RASAGILINE Rasagiline (N-propargyl-1-(R)-aminoindan), a potent, selective, irreversible inhibitor of MAO-B, is a novel anti-Parkinsonian drug, and may also have disease-modifying properties. In light of recently reported benefits in patients with early illness, rasagiline is a promising new treatment for PD.43 Recent multicenter, double-blind, mono-therapy with rasagiline by the Parkinson Study Group44 and as adjunct therapy to L-DOPA45 have shown that rasagiline confers significant symptomatic improvement, and long-term studies suggest possible alterations in disease progression at doses of 1 and 2 mg. Rasagiline has been shown to have a broad neuroprotective activity against a variety of neurotoxins in neuronal cell cultures and in animal models. This includes attenuation of cell death in partially differentiated PC-12 cells deprived of serum and nerve growth factor (NGF),46 and neuroprotection against the endogenous neurotoxin N-methyl-(R)-salsolinol (N-M-(R)-Sal),47–49 6-OHDA,50 SIN-1 (a peroxynitrite donor),51,52 and glutamate toxicity53 in PC-12 and human NB cells. In vivo studies have described that the

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protective effect of rasagiline in MPTP model in mice and monkeys prevented its conversion to MPP+54 in focal ischemia model in rats55 and in neurotrauma model of head injury in mice .56 In addition, rasagiline suppresses the cell-death cascade initiated by pro-apoptotic mitochondrial proteins, prevents decline in mitochondrial membrane potential (∆Ψm), inhibits the apoptotic processes including activation of caspase 3, nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and DNA fragmentation.52,57,58

20.3 NEUROPROTECTIVE STUDIES IN MODELS OF ALZHEIMER’S DISEASE 20.3.1 ANTIOXIDANT AGENTS Multiple lines of evidence demonstrate that OS is an early event in AD, occurring prior to cytopathology, and therefore may play a key pathogenic role in the disease. Indeed, oxidative modification may lead to metabolic impairment in AD. One of the main characteristics of AD is the deposition of the supposedly neurotoxic amyloid β -peptide (Aβ ), which is derived from a larger amyloid precursor protein (APP). Amyloid precursor protein can be processed proteolytically via alternative pathways: (1) a nonamyloidogenic secretary pathway, which involves cleavage of APP to soluble APP (sAPP) by α−secretase within the sequence of Aβ peptide, thus preventing the formation of Aβ; and (2) a formation of amyloidogenic Aβ peptides, which is regulated by the sequential action of β - and γ -secretases.59,60 Previous study supported the notion that the neuroprotective activity of melatonin associated with its antioxidant mechanism, where melatonin attenuated neurotoxicity and apoptosis in PC-12 cells by Aβ - (Aβ 23–35 or Aβ1–42), involved OS. Melatonin suppressed Aβ1–42-induced nitric oxide formation and prevented Aβ1–40-induced intracellular calcium overload.61 Furthermore, melatonin affected the secretion of soluble Aβ in PC-12 cells.62 The antioxidant/iron chelator, EGCG, is also able to regulate the proteolytic processing of APP under in vivo and in vitro conditions,63 suggesting that green tea polyphenols might be potentially promising therapeutic agents not only for PD but also for AD. EGCG promoted the nonamyloidogenic α-secretase pathway of APP in neuronal cell cultures. The increase was dose-dependent and the stimulatory effect of EGCG on sAPPα secretion was inhibited by the hydroxamic acid-based metalloprotease inhibitor Ro31-9790, indicating that this effect was mediated via α-secretase processing. In agreement with these results, long-term treatment of mice with EGCG resulted in decreases in cell-associated, fulllength-APP levels as well as increases in sAPP levels in the hippocampus.

20.3.2 RASAGILINE A significant percentage of Parkinsonian subjects may also develop AD dementia. Indeed, the anti-Parkinson drug, rasagiline, significantly stimulates the release of

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the nonamyloidogenic α-secretase form of sAPPα from both SH-SY5Y and PC-12 cells, and reduces full-length APP in rat and mice hippocampus.64,65 The increase in sAPPα was blocked by the hydroxamic acid-based metalloprotease inhibitor Ro319790, suggesting that the effect was mediated via α-secretase activity.64 Moreover, rasagiline significantly protected rat PC-12 cells against Aβ1–42. Structural activity has clearly shown that α-secretase-dependent processing of APP is associated with the propargyl moiety of rasagiline, since propargylamine itself has similar action, but not aminoindan, the metabolite of rasagiline.65,66 In AD, increased expression and altered processing of APP causing an increase in generation of Aβ may play a central role in the amyloidogenesis process.60 Thus, the observed reduction of APP protein levels after treatment with EGCG, melatonin, and rasagiline, could be of clinical value toward accelerating nonamyloidogenic APP processing, thereby reducing Aβ levels.

20.4 THE MECHANISMS UNDERLYING ANTIOXIDANTATTENUATED CELL DEATH 20.4.1 MODULATION

OF

CELL SIGNALING PATHWAYS

Emerging evidence suggests that the antioxidant activities cannot be the sole mechanism responsible for their neuroprotective action, but rather, their ability to alter signaling pathways may significantly contribute to the cell survival effect. The modulation of cellular survival and signal transduction pathways has significant biological consequences that are important in understanding the various pharmacological and toxicological responses of antioxidant drugs. A number of intracellular signaling pathways have been described to play central functions in EGCG-promoted neuronal protection, such as the mitogen-activated protein kinases (MAPK),67 protein kinase C (PKC)35 and phosphatidylinositol-3-kinase (PI-3 kinase)-Akt68,69 pathways. Given the critical role of MAPK pathways in regulating the cellular processes that are affected in neurodegenerative diseases, the importance of MAPKs as transducers of extracellular stimuli into a series of intracellular phosphorylation cascades of disease pathogenesis is being increasingly recognized. OS seems to be a major stimulus for MAPK cascades, which might lead to cell survival/cell death. Among the MAPKs, the extracellular signal-regulated kinases (ERK1/2) are mainly activated by mitogen and growth factors,70 while p38 and c-jun-N-terminal kinase (JNK) respond to stress stimuli.71,72 Previous in vitro studies demonstrated the potency of EGCG67 to induce antioxidant-response element (ARE)-mediated defensive genes and MAPKs pathways including ERK, JNK, and p38 MAPK,73 which enhanced cell survival and beneficial homeostatic response. Similarly, treatment of NB cells with APO74 and melatonin75 attenuated 6-OHDA-induced ROS generation, the phosphorylation of JNK, and subsequent apoptotic cell death. These results demonstrate some protective properties of these antioxidants against neuronal cell degeneration and their action on the inhibition of MEK/ERK/JNK signaling cascade (Figure 20.2).

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EGCG OS

P-PKC

Bax

Melatonin OS

R-APO OS

MEK

SAPK/JNK

P-ERK1/2

NF-
B

p

p

Bcl-2

Bad

Neuronal survival

FIGURE 20.2 A neuroprotective mechanism model for green tea polyphenol (−)-epigallocatechin-3-gallate (EGCG), dopamine D1-D2-receptor agonist, R-apomorphine (R-APO), and melatonin, indicating potential signal transduction pathways with respect to their proposed modulate effect in neuronal tissues.

The neuroprotective activity of EGCG also involves the intracellular signaling mediator, PKC,35 known to have an essential role in the regulation of cell survival and programed cell death.76,77 The induction of PKC activity in neurons by EGCG (1–10 µM) was shown to be a prerequisite for neuroprotection against several neurotoxins, such as Aβ,63 serum withdrawal,78 and 6-OHDA.33,35 Inhibition of PKC phosphorylation abolished completely the protection induced by EGCG and by PKC activator, phorbol 12-myristate 13-acetate (PMA). These in vivo results were supported by a recent study,63 which showed that EGCG oral administration to mice (2 mg/kg) caused significant increase of the PKC isoenzymes α and ε protein levels in the membrane and cytosolic fractions of hippocampus. These isoforms play a crucial role in cell survival and differentiation pathways79 and may be involved in APP processing elevated pathogenesis of AD.80 Although it is not known which isoenzyme of PKC plays a major role in modulating APP processing, several lines of evidence suggest the involvement of

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PKCα and PKCε in APP processing.80,81 In agreement with these findings, melatonin selectively activated PKC α, suggesting this Ca2+-dependent isoform activation participated in neurite formation in murine NB, N1E-115 cells.82 The mechanism by which PKC activation leads to neuroprotection and cell survival has not been clearly defined. Studies with extra-neuronal tissues support a role for PKCα as a kinase of the antiapoptotic Bcl-2, probably through direct or indirect phosphorylation of this cell-survival protein.83 Recent studies reported upregulation of Bcl-2 in nigral neurons and reactive microglia after kainite by melatonin.84 Additionally, EGCG promoted cell survival in human epidermal keratinocytes, taken from EGCG-treated skin of healthy subjects, and increased the ratio of Bcl-2 to pro-apoptotic Bax and phosphorylation of another pro-apoptotic Bcl-2 family member, Bad, through ERK and Akt signaling pathways.85 Nonetheless, a study with high concentrations of EGCG reported cell proliferation arrest of tumor cells and inhibition of ERK1/2 and Akt phosphorylation, which was associated with reduced phosphorylation of Bad.86 This biphasic mode of biological activity of antioxidants, such as EGCG,87 DA,18 and R-APO,88 relies on their concentration-dependent window of pharmacological action. Antioxidants exhibit pro-oxidant and pro-apoptotic activity at high concentrations, while at low doses they are neuroprotective against a wide spectrum of neurotoxic compounds.

20.4.2 EFFECT OF ANTIOXIDANTS EXPRESSION

ON

CELL SURVIVAL/DEATH GENE

Studies based on customized cDNA and quantitative real-time reverse transcriptase–polymerase chain reaction (RT-PCR) were recently conducted to verify the molecular mechanisms involved in the cell-survival and cell-death action of EGCG.87,89 In these studies, gene expression profile of the four neuroprotective/antioxidant drugs, DA, R-APO, EGCG, and melatonin, was compared at low and high concentrations.90 Dopamine, EGCG, and R-APO (1–10 µM) behaved as potent neuroprotective agents, decreasing the expression of the pro-apoptotic genes, Bax, Bad, Gadd45, and Fas ligand. However, EGCG did not affect the expression of the antiapoptotic Bcl-w, Bcl-2, and Bcl-xL, while the other three compounds increased them. EGCG’s neuroprotective effect is thought to be mediated through down-regulation of pro-apoptotic genes.35 These findings support the assumption that complementary mechanisms, in addition to radical scavenging, are involved in their neuronal survival effect. In contrast to the antiapoptotic effect observed with low concentrations (⬍ 10 µM), a pro-apoptotic pattern of gene expression is observed with high concentrations (⬎ 20 µM) of EGCG, DA, R-APO, and melatonin. It includes the expression of Bax, Gadd45, caspase family members(-3, -6, -10) and tumor necrosis factor (TNF) receptor family member Fas and Fas-ligand mRNAs. The results revealed a significant functional group homology between these drugs in genes coding for signal transducers, transcriptional repressors, and growth factors, which may account for their mechanism of action. This might be predictable, given that EGCG, DA, and

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R-APO share similar attributes, being catechol-like derivatives, iron chelators, and protecting against neurotoxicity induced by 6-OHDA or MPTP.28,91–93 Despite the similarities of the gene changes induced by these drugs,90 the extent of apoptosis-related gene induction was higher with R-APO. This effect may be related to its low IC50 value for inducing cell death (10 µM) and its greater pro-oxidant potency.88 Indeed, R-APO is a far more potent radical-scavenger compound (IC50, 0.28 µM) than DA (IC50: 6.6 µM),88 EGCG (IC50: 1 µM,94 and melatonin (IC50: 400 µM,95 both in vivo and in vitro. The low radical-scavenging potency of melatonin is consistent with its low cell toxicity effect at high concentrations. The low index of mortality of melatonin may be partially explained by the observation that a high concentration did not significantly affect the gene expression of mitochondrial Bcl-2 family members, Bcl-2 and Bax.90 Thus, the maintenance of their basal activity levels in concert with the slight decrease in Bax mRNA and Bax protein expression may be essential for cell viability. This wide range spanning the effective neuroprotective and the toxic concentrations of melatonin may be of critical importance in terms of pharmacotherapy, since it may provide a safer dosage window.96

20.5 MECHANISM OF ACTION OF RASAGILINE A major pathway implicated in neuronal cell survival is the intrinsic or mitochondrial signaling, triggered and mediated by Bcl-2 family members that may either support cell survival (Bcl-2, Bcl-xL, Bcl-w, Mcl-1, and A1/Bfl-1) or promote cell death (Bax, Bak, Bcl-Xs, Bad, Bid, Bik, Hrk, and Bok).97 The proapoptotic proteins of the Bcl-2 family members may trigger the opening of the mitochondrial megachannel, or a specific channel in the outer mitochondrial membrane, both of which promote the fall in mitochondrial membrane potential, leading to cytochrome c release.98,99 The competitive action of the pro- and antisurvival Bcl-2 family proteins regulates the activation of the proteases (caspases) that dismantle the cell.100–102 It is apparent that members of the Bcl-2 family interact with each other to form a dynamic equilibrium between homo- and heterodimers.103 Bcl-2 overexpression was shown to prevent cell death, probably by inhibiting Bax translocation and insertion into mitochondrial membrane, or via a direct interaction with the channels.104 Rasagiline was recently shown to prevent the fall in mitochondrial membrane potential (∆Ψm) and the opening of the mitochondrial voltage-dependent anion channel via the increase in Bcl-2 and Bcl-xL proteins.50,51 This is consistent with recent study58 and provides evidence that the neuroprotection effect of rasagiline is mediated by gene regulation of the Bcl-2-related protein family. Rasagiline decreased the mRNA of the pro-apoptotic members, Bax and Bad, and increased the mRNA of the cell survival members, Bcl-2, Bcl-w, and Bcl-xL. In addition, the authors demonstrated the involvement of the PKC pathway in rasagiline-induced inactivation of the BH3-only, pro-apoptotic, Bcl-2 family member, Bad58,10,105 This is consistent with PKC-dependent promotion of cell survival via phosphorylation and inactivation of Bad-mediated cell death.106 Thus, PKCα is known to

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phosphorylate Bcl-2 in a site that increases its antiapoptotic function,83 and overexpression of PKCε results in increased expression of Bcl-2.79 Furthermore, suppression of PKCα triggers apoptosis through down-regulation of Bcl-xL.107 The role of PKC activation in the neuroprotective mechanism of rasagiline, as described for EGCG,108 is supported by the findings that rasagiline can activate p-PKC levels and upregulate essential PKC isoforms involved in cell survival pathways, PKCα and PKCε, in mice hippocampus66 and in starved PC-12 cells.58 Furthermore, rasagiline down-regulated detrimental PKCγ in serum-deprived PC12 cells.58 The inhibition of PKC activity in serum-deprived PC-12 cells blocked the neuroprotective action of rasagiline and its propargyl moiety compound. By using a specific broad-spectrum PKC inhibitor, GF109203X, which exhibited high affinity for the conventional PKCs (α, β, γ ) as well as the novel isoenzyme PKCε,109–111 the authors demonstrated that GF109203X markedly reversed rasagiline-suppressive effects on the protein expression of the pro-apoptotic regulator/cell death machinery, Bad, and on the cleavage and activation of procaspase-3 and poly (ADP-ribose) polymerase (PARP), in serum withdrawalinduced programmed cell death.58 Similarly, PKC inhibitor, GF109203X, and ERK1/ERK2 inhibitor, PD98059, prevented rasagiline activation/phosphorylation of p42 and p44 MAPK, thus indicating that rasagiline directly activates the PKC–MAPK pathway.65 The importance of the PKC pathway in rasagiline-neuroprotective activity is also supported by previous data demonstrating that rasagiline65 induced the release of the neuroprotective, neurotrophic, nonamyloidogenic sAPPα by MAPK- and PKC-dependent mechanisms in vitro.64 Rasagiline has been shown to cause upregulation of antioxidative proteins, such as superoxide dismutase (SOD) and GSH.112,113 Nonetheless, it is unlikely that the neuroprotective effect of rasagiline is related to MAO-B inhibition, because PC-12 cells contain only MAO type A.114,115 Moreover, the S-isomer of rasagiline, TVP1022, which is not an inhibitor of MAO-A or –B, also protected serum-deprived PC-12 cells from cell death, suggesting that rasagiline’s mode of action is independent of MAO inhibition. These results are consistent with previous reports providing clear evidence that neuroprotection by rasagiline and its derivatives does not depend on inhibition of MAO-B,57 but rather is associated with some intrinsic pharmacological action of the propargyl moiety in these compounds, which act on the mitochondria cell survival proteins. Propargylamine58 had similar effects with the same potency. Additional neuroprotective mechanism of rasagiline involves the upregulation of the expression levels of glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF), as demonstrated in neuronal cells.58,116 Indeed, both neurotrophic factors have been shown to induce neuroprotective and neurorescue activity in dopaminergic and cholinergic neurons, and promote survival of major neuronal types affected in AD and PD.117,118 Recently, rasagiline was proved to increase the protein and mRNA levels of GDNF in NB cells through activation of nuclear factor-kappaB (NF-κ B) transcription factor.116 Additionally, rasagiline was reported to enhance gene expression of β -NGF and its downstream

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Upregulation by Rasagiline Downregulation by Rasagiline Death ligands: FasL, TNF, TRAIL

Neurotrophic factors: e.g., NGF, BDNF, GDNF

Ras Raf PI3K

Caspase-8,-10

PKC ERK1/2

Akt

p90RSK

Bad, Bax, Bim

CREB DNA damage

tBid p53 IKK

Bcl-2

GAPDH

Bcl-X L

I
B

Bcl-w

NFkB

SOD Catalase

Opening of PTP or pore formation

Oxidative stress

NFkB Cytochrome c AIF Cytochrome c Apaf-1 Caspase-9 Caspase-3 PARP

Neuronal death

FIGURE 20.3 Schematic overview demonstrating protein and gene targets involved in the neuroprotective activity of the anti-Parkinson drug, rasagiline. Full explanation is discussed in the text. Stimulation ; Inhibition ; Translocation ; Transcription .

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transcription factors, NGF-1A-binding protein (EGR1) and the early growth response protein-3 (EGR3), in response to the pro-apoptotic action of MPTP in vivo.119 These results indicate that the induction of neurotrophic factors by rasagiline might suppress apoptosis in neurodegenerative diseases. The present findings may support a possible disease-modifying activity of rasagiline, as suggested in clinical trials of early PD patients.44

20.6 CONCLUSIONS AND PERSPECTIVES FOR THE FUTURE To date, accumulated evidence on the neuroprotective properties of R-APO, melatonin, and EGCG is associated with antioxidation and iron chelation. However, the effect of these compounds has not been evaluated enough and most studies are carried out in animal models and cell culture to assess the effect of acute and chronic administration of these drugs. Melatonin and EGCG appear to affect the mortality of neuronal cells. Given the central role mitochondria play in OS-induced apoptosis, it may be speculated that the inhibition of apoptosis might implicate mitochondrial targets. This may be a consequence of the blockade of mitochondrial permeability transition pore opening, since the antioxidants such as EGCG and R-APO have an effect on the mitochondrial protein expression, the Bcl-2 family members. Similarly, the antiParkinson drug, rasagiline, prevents the decrease in mitochondrial membrane potential and the opening of mitochondrial voltage-dependent anion channel by regulating the Bcl-2 family proteins associates with PKC–MAPK pathways, as described in Figure 20.3. In addition, the PKC activation and translocation has been proven to be involved in the neuroprotective mechanism of EGCG. The toxicity of the metabolic product of MPTP, MPP +, which is also a mitochondrial complex I inhibitor,120 induced OS, iron signaling, and α-synuclein expression,121 is attenuated by EGCG, R-APO, and melatonin probably via their metal-chelating ability. Consistent with this idea, a recent study reported a novel iron-responsive element (IRE-type II) within the 5⬘-untranslated region of the Alzheimer’s APP transcript, which can be regulated by metal chelators122 such as these antioxidants. On the basis of these accumulated data, recent studies123,124 describe use of brain-permeable iron chelators as multipharmacological neuro-protective drugs to “iron out iron” from those brain areas where it preferentially accumulates in neurodegenerative diseases. This will allow a more specific therapy design in the future, especially when a cocktail is considered for formulation, which can be more effective in clinical treatment of AD and PD.

ACKNOWLEDGMENTS The support of Teva Pharmaceutical Co. (Netanya, Israel), National Parkinson Foundation (Miami, USA), Stein Foundation (Philadelphia, USA), Rappaport Family Research Institute, Technion- Israel Institute of Technology, and Golding Parkinson Research Fund (Technion, Haifa) are gratefully acknowledged.

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99. Bernardi P, Petronilli V, Di Lisa F, Forte M. A mitochondrial perspective on cell death. Trends Biochem Sci, 2001; 26:112–117. 100. Adams JM, Cory S. The Bcl-2 protein family: arbiters of cell survival. Science, 1998; 281:1322–1326. 101. Evan G, Littlewood T. A matter of life and cell death. Science, 1998; 281:1317–1322. 102. Zamzami N, Brenner C, Marzo I, Susin SA, Kroemer G. Subcellular and submitochondrial mode of action of Bcl-2-like oncoproteins. Oncogene, 1998; 16:2265–2282. 103. Oltvai ZN, Milliman CL, Korsmeyer SJ. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell, 1993; 74:609–619. 104. Cheng EH, Wei MC, Weiler S, Flavell RA, Mak TW, Lindsten T, Korsmeyer SJ. BCL-2, BCL-X(L) sequester BH3 domain-only molecules preventing BAX- and BAK-mediated mitochondrial apoptosis. Mol Cell, 2001; 8:705–711. 105. Youdim MB, Bar Am O, Yogev-Falach M, Weinreb O, Maruyama W, Naoi M, Amit T. Rasagiline: neurodegeneration, neuroprotection, and mitochondrial permeability transition. J Neurosci Res, 2005; 79:172–179. 106. Tan Y, Ruan HD, MR, Comb MJ. p90(RSK) blocks bad-mediated cell death via a protein kinase C-dependent pathway. J Biol Chem, 1999;34859–34867. 107. Hsieh YC, Jao HC, Yang RC, Hsu HK, Hsu C. Suppression of protein kinase Calpha triggers apoptosis through down-regulation of Bcl-xL in a rat hepatic epithelial cell line. Shock, 2003; 19:582–587. 108. Mandel SA, Avramovich-Tirosh Y, Reznichenko L, Zheng H, Weinreb O, Amit T, Youdim MB. Multifunctional activities of green tea catechins in neuroprotection: modulation of cell survival genes, iron-dependent oxidative stress and PKC-signaling pathway. Neurosignals, 2005; 14:46–60. 109. Bar-Am O, Amit T, M.B.H Y. Contrasting neuroprotective and neurotoxic actions of respective metabolites of anti-Parkinson drugs rasagiline and selegiline. Neurosci Lett, 2004; 355:169–172. 110. Ku WC, Cheng AJ, Wang TC. Inhibition of telomerase activity by PKC inhibitors in human nasopharyngeal cancer cells in culture. Biochem Biophys Res Commn, 1997; 241:730–736. 111. Gekeler V, Boer R, Uberall F, Ise W, Schubert C, Utz I, Hofmann J, Sanders KH, Schachtele C, Klemm K, Grunicke H. Effects of the selective bisindolylmaleimide protein kinase C inhibitor GF 109203X on p-glycoprotein-mediated multidrug resistance. Br J Cancer, 1996; 74:897–905. 112. Carrillo MC, Minami C, Kitani K, Maruyama W, Ohashi K, Yamamoto T, Naoi M, Kanai S, Youdim MBH. Enhancing effect of rasagiline on superoxide dismutase and catalase activities in the dopaminergic system in the rat. Life Sci, 2000; 67:577–585. 113. Youdim MBH. Rasagiline: an anti-Parkinson drug with neuroprotective activity. Future Drugs, 2003; 3:737–749. 114. Youdim MBH. PC12 cells as a window for the differentiation of neural crest into adrenergic nerve ending and adrenal medulla. J Neural Transm (Suppl), 1991; 34:61–67. 115. Youdim MBH, Gross A, Finberg JPM. Rasagiline [N-propargyl-1R(+)-aminoindant], a selective and potent inhibitor of mitochondrial monoamine oxidase B. Br J Pharmacol, 2001; 132:500–506.

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Primacy of 21 Temporal Oxidative Stress in the Pathological Cascade of Alzheimer Disease Akihiko Nunomura, Kazuki Tabata, and Shigeru Chiba Asahikawa Medical College Asahikawa, Japan

Mark A. Smith and George Perry Case Western Reserve University Cleveland, Ohio

CONTENTS Abstract..............................................................................................................365 21.1 Introduction..............................................................................................366 21.2 Genes in Autosomal-Dominant Familial Alzheimer Disease and Oxidative Stress...................................................................366 21.3 Risk Factors for Alzheimer Disease and Oxidative Stress ......................366 21.4 Temporal Primacy of Oxidative Stress in the Pathological Cascade of Alzheimer Disease ...........................................369 21.5 Conclusion ...............................................................................................369 References .........................................................................................................369

ABSTRACT Most of the known mutations in specific genes, and genetic, medical, environmental, and lifestyle-related risk factors for Alzheimer disease (AD) are associated with an increase in oxidative stress. In contrast, several agents, nutrients, and behavior that reduce the risk of AD are associated with a protection against oxidative stress. This evidence strongly suggests that oxidative stress is universally involved in the pathogenesis of full-scale AD, especially in the upstream of the pathological cascade. An early involvement of oxidative stress in the pathogenesis of AD is more directly demonstrated by recent studies. Indeed, oxidative stress 365

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induces intracellular amyloid-β (Aβ ) accumulation and tau phosphorylation in cell cultures, while vitamin E, a radical scavenging antioxidant, reduces Aβ and tau lesions in the transgenic animals. Oxidative damage precedes Aβ deposition in Down syndrome patients and transgenic animal models of AD. Furthermore, individuals with mild cognitive impairment (MCI), who, at least in part, represent the prodromal stage of AD, show increased oxidative damage.

21.1 INTRODUCTION Alzheimer disease (AD) is a disease with a prevalence that increases exponentially with age, with about half of the population afflicted by the age of 95.1 This strongly supports an association between old age and AD. As in other organ systems, cells in the brain encounter a cumulative burden of oxidative and metabolic stress, which may be a universal feature of the aging process as well as a major causal factor of senesence. Each of the macromolecules, including nucleic acids, proteins, and lipids, is oxidatively modified during aging. Indeed, the brain is especially vulnerable to free radical damage because of its high oxygen consumption rate, abundant lipid content, and relative paucity of antioxidant enzymes compared with other organs.2,3 Both the aging process as well as the genetic, medical, environmental, and lifestyle-related risk factors for AD are associated with oxidative stress. This chapter focuses on the early involvement of oxidative stress in the pathological cascade of AD.

21.2 GENES IN AUTOSOMAL-DOMINANT FAMILIAL ALZHEIMER DISEASE AND OXIDATIVE STRESS Recently, an increasing number of in vitro and in vivo studies have suggested that oxidative stress is involved in the pathogenesis of AD and autosomal-dominant familial AD with amyloid β protein precursor (Aβ PP), presenilin-1 (PS-1), or presenilin-2 (PS-2) gene mutations. Indeed, increased oxidative stress, elevated vulnerability to oxidative stress-induced cell death, and reduced antioxidant defenses have been demonstrated in (1) cell lines expressing mutant human Aβ PP, PS-1, or PS-2;4–7 (2) transgenic mice expressing mutant human Aβ PP and PS-1 as well as knock-in mice expressing mutant human PS-1;8–14 (3) fibroblasts and lymphoblasts from familial AD patients with Aβ PP or PS-1 gene mutation;15 and (4) cerebral cortex of autopsied brain samples from patients with Aβ PP or PS-1 gene mutations.16,17 These mutations, however, account for only a small proportion of patients with AD.

21.3 RISK FACTORS FOR ALZHEIMER DISEASE AND OXIDATIVE STRESS There are several known risk factors for AD other than advanced age, namely, genetic, medical, environmental, and lifestyle-related factors. The major genetic risk

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factor for early- to late-onset sporadic and familial AD is the possession of one or both of the apolipoprotein E4 (ApoE4) alleles. In vitro, ApoE shows allele-specific antioxidant activity, with ApoE2 being the most effective, and ApoE4 the least effective.18 Indeed, oxidative damage in ApoE genotype-dependent manner has been demonstrated in autopsy brain samples of AD patients.19–21 Medical risk factors for AD include traumatic brain injury, cerebral infarcts, diabetes mellitus, hypercholesterolemia, and hyperhomocysteinemia. Environmental and lifestyle-related risk factors include aluminum exposure, smoking, high calorie intake, and lack of exercise and intellectual activities.3,22–24 Indeed, all these AD risk factors are associated with an increase in oxidative stress.3,25–30 With this notion, it is not surprising that agents or nutrients inhibiting free radical formation reduce the incidence of AD. Indeed, agents or nutrients such as vitamins E and C, as well as estrogen, nonsteroidal anti-inflammatory drugs, statins, n⫺3 polyunsaturated fatty acids, and wine have been proven to have an antioxidant activity and reduce the incidence of AD.22,24,31–35 Furthermore, calorie restriction, exercise, and intellectual activity have been proven to promote neuronal survival through decreased oxidative stress in experimental animals.3,23 AD-causing genetic mutations in:

Risk factors for AD

Amyloid
protein precursor gene

Advanced age

Presenilin-1 gene

Apolipoprotein E4 allele

Presenilin-2 gene

Traumatic brain injury Cerebral infarcts Diabetes mellitus Hypercholesterolemia Hyperhomocysteinemia

Oxidative stress

Aluminum exposure Smoking High calorie intake Lack of exercise Lack of intellectual activity

Agents, nutrients, and behavior against risk of AD Vitamins E and C Estrogen, nonsteroidal anti-inflammatory drugs, statins N-3 polyunsaturated fatty acids, wine consumption low calorie intake, exercise, intellectual activity

FIGURE 21.1 Genetic, medical, environmental, and lifestyle-related factors for AD: relation to oxidative stress. Most of the known genetic mutations and risk factors for AD are associated with an increase in oxidative stress. In contrast, several agents and nutrients that are known to reduce the incidence of AD have antioxidant properties per se or help to prevent/reduce free radical generation/propagation. A low-calorie diet as well as physical and intellectual activities are suggested to enhance the production of antioxidant enzymes in the brain.

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TABLE 21.1 Evidence for Temporal Primacy of Oxidative Stress in the Pathological Cascade of AD Materials/Subjects Cell culture models Transgenic animal models

Postmortem brains from patients with Down syndrome Postmortem brains from patients with AD

CSF from patients with AD CSF, plasma, urine, and peripheral leukocytes from subjects with MCI

Findings Oxidative stress induces intracellular accumulation of Aβ and phosphorylation of tau 1. Increased lipid peroxidation or protein oxidation precedes Aβ plaque deposition or Aβ fibril formation in transgenic mouse or C.elegans model of AD amyloidosis 2. Vitamin E supplementation reduces Aβ levels and Aβ plaque deposition in young but not aged Aβ PP transgenic mice 3. Vitamin E supplementation suppresses the development of tau pathology in transgenic mice overexpressing human tau 4. Dietary copper stabilizes brain copper/zinc SOD activity and reduces Aβ production in Aβ PP transgenic mice 5. Aβ PP mutant mice crossed with manganese SOD heterozygous knockout mice show increased Aβ plaque deposition in brain Oxidative damages to nucleic acid and protein precede Aβ plaque deposition in a series of Down syndrome brains, a model of AD neuropathology. 1. Oxidative damages to nucleic acid and protein are more prominent in AD patients with smaller amounts of Aβ plaque deposition or shorter disease duration 2. Oxidative damage to nucleic acid is more prominent in hippocampal neurons free of NFTs compared with neurons with NFTs 3. Oxidative damage to nucleic acid is increased in a presymptomatic case with PS1 gene mutation Oxidative damage to nucleic acid is more prominent in AD patients with shorter disease duration or higher scores in mini-mental state examination 1. Lipid peroxidation in CSF, plasma, and urine is increased. 2. Plasma antioxidants (vitamins A, C, E, carotenoids, SOD, etc.) are depleted 3. Oxidative damage to DNA in peripheral leukocytes is increased

References (42,43) (12,46)

(44)

(45)

(47)

(48)

(37)

(36)

(36)

(17) (38)

(39) (41) (40)

Note: CSF, Cerebrospinal fluid; MCI, mild cognitive impairment; NFTs, neurofibrillary tangles; SOD, superoxide dismutase

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Known genetic mutations and risk factors for AD that cause or promote oxidative damage as well as agents, nutrients, and behavior that prevent or attenuate oxidative damage are summarized in Figure 21.1. This evidence strongly suggests that oxidative stress is universally involved in the pathogenesis of full-scale AD, especially in the upstream of the pathological cascade.

21.4 TEMPORAL PRIMACY OF OXIDATIVE STRESS IN THE PATHOLOGICAL CASCADE OF ALZHEIMER DISEASE The early involvement of oxidative stress in the pathogenesis of AD is demonstrated more directly by recent studies on cell culture models, transgenic animal models, postmortem brains from patients with AD and Down syndrome, and biological fluids from patients with AD and subjects with MCI (Table 21.1). We selected an in situ approach to identify markers of nucleic acid oxidation and protein oxidation in postmortem brain samples. Surprisingly, the oxidative damage is not only more prominent in AD cases with smaller amounts of Aβ deposition or shorter disease duration,36 but also precedes Aβ deposition in a series of Down syndrome brains, a model of AD neuropathology.37 Our observation corresponds with the results of increased nucleic acid oxidation in cerebrospinal fluid from AD cases, in which the shorter the disease duration, the greater the oxidative damage.38 Moreover, individuals with MCI who, at least in part, represent the prodromal stage of AD show significantly increased levels of lipid peroxidation and nucleic acid oxidation in peripheral samples39,40 as well as decreased levels of plasma antioxidants.41 These data obtained from human subjects clearly indicate an early involvement of oxidative stress in AD pathogenesis, which is supported by the experimental studies using cell culture models and transgenic animal models of AD. Indeed, oxidative stress induces intracellular Aβ accumulation and tau phosphorylation in cell cultures,42,43 and vitamin E reduces Aβ and tau lesions in transgenic animals.44,45

21.5 CONCLUSION As we have reviewed here, a growing body of evidence supports the hypothesis that oxidative stress plays a primary role of oxidative stress in the pathogenesis of AD. This notion increases the importance of further development and testing of antioxidants as a strategy for the prevention and treatment of AD (reviewed by Moreira et al. in this book). Moreover, the induction of intracellular Aβ accumulation and tau phosphorylation with oxidative stress has led us to hypothesize a compensatory role for the Aβ and tau lesions in AD against oxidative stress (reviewed by Castellani et al. in this book).

REFERENCES 1. Hy LX, Keller DM. Prevalence of AD among whites: a summary by levels of severity. Neurology, 2000; 55:198–204.

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2. Coyle JT, Puttfarcken P. Oxidative stress, glutamate, and neurodegenerative disorders. Science, 1993; 262:689–695. 3. Mattson MP, Chan SL, Duan W. Modification of brain aging and neurodegenerative disorders by genes, diet, and behavior. Physiol Rev, 2002; 82:637–672. 4. Eckert A, Steiner B, Marques C, Leutz S, Romig H, Haass C, Muller WE. Elevated vulnerability to oxidative stress-induced cell death and activation of caspase-3 by the Swedish amyloid precursor protein mutation. J Neurosci Res, 2001; 64:183–192. 5. Guo Q, Sopher BL, Furukawa K, Pham DG, Robinson N, Martin GM, Mattson MP. Alzheimer’s presenilin mutation sensitizes neural cells to apoptosis induced by trophic factor withdrawal and amyloid β -peptide: involvement of calcium and oxyradicals. J Neurosci, 1997; 17:4212–4222. 6. Hashimoto Y, Niikura T, Ito Y, Kita Y, Terashita K, Nishimoto I. Neurotoxic mechanisms by Alzheimer’s disease-linked N141I mutant presenilin 2. J Pharmacol Exp Ther, 2002; 300:736–745. 7. Marques CA, Keil U, Bonert A, Steiner B, Haass C, Muller WE, Eckert A. Neurotoxic mechanisms caused by the Alzheimer’s disease-linked Swedish amyloid precursor protein mutation: oxidative stress, caspases, and the JNK pathway. J Biol Chem, 2003; 278:28294–28302. 8. Guo Q, Sebastian L, Sopher BL, Miller MW, Ware CB, Martin GM, Mattson MP. Increased vulnerability of hippocampal neurons from presenilin-1 mutant knockin mice to amyloid β -peptide toxicity: central roles of superoxide production and caspase activation. J Neurochem, 1999; 72:1019–1029. 9. LaFontaine MA, Mattson MP, Butterfield DA. Oxidative stress in synaptosomal proteins from mutant presenilin-1 knock-in mice: implications for familial Alzheimer’s disease. Neurochem Res, 2002; 27:417–421. 10. Leutner S, Czech C, Schindowski K, Touchet N, Eckert A, Muller WE. Reduced antioxidant enzyme activity in brains of mice transgenic for human presenilin-1 with single or multiple mutations. Neurosci Lett, 2000; 292:87–90. 11. Matsuoka Y, Picciano M, La Francois J, Duff K. Fibrillar β -amyloid evokes oxidative damage in a transgenic mouse model of Alzheimer’s disease. Neuroscience, 2001; 104:609–613. 12. Praticò D, Uryu K, Leight S, Trojanoswki JQ, Lee VM. Increased lipid peroxidation precedes amyloid plaque formation in an animal model of Alzheimer amyloidosis. J Neurosci, 2001; 21:4183–4187. 13. Smith M A, Hirai K, Hsiao K, Pappolla MA, Harris PL, Siedlak SL, Tabaton M, Perry G. Amyloid-β deposition in Alzheimer transgenic mice is associated with oxidative stress. J Neurochem, 1998; 70:2212–2215. 14. Takahashi M, Dore S, Ferris CD, Tomita T, Sawa A, Wolosker H, Borchelt DR, Iwatsubo T, Kim SH, Thinakaran G, Sisodia SS, Snyder SH. Amyloid precursor proteins inhibit heme oxygenase activity and augment neurotoxicity in Alzheimer’s disease. Neuron, 2000; 28:461–473. 15. Cecchi C, Fiorillo C, Sorb S, Latorraca S, Nacmias B, Bagnoli S, Nassi P, Liguri G. Oxidative stress and reduced antioxidant defenses in peripheral cells from familial Alzheimer’s patients. Free Radic Biol Med, 2002; 33:1372–1379. 16. Bogdanovic N, Zilmer M, Zilmer K, Rehema A, Karelson E. The Swedish APP670/671 Alzheimer’s disease mutation: the first evidence for strikingly increased oxidative injury in the temporal inferior cortex. Dement Geriatr Cogn Disord, 2001; 12:364–370.

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17. Nunomura A, Chiba S, Lippa CF, Cras P, Kalaria RN, Takeda A, Honda K, Smith MA, Perry G. Neuronal RNA oxidation is a prominent feature of familial Alzheimer’s disease. Neurobiol Dis, 2004; 17:108–113. 18. Miyata M, Smith JD. Apolipoprotein E allele-specific antioxidant activity and effects on cytotoxicity by oxidative insults and β -amyloid peptides. Nat Genet, 1996; 14:55–61. 19. Montine KS, Reich E, Neely MD, Sidell KR, Olson SJ, Markesbery WR, Montine TJ. Distribution of reducible 4-hydroxynonenal adduct immunoreactivity in Alzheimer disease is associated with APOE genotype. J Neuropathol Exp Neurol, 1998; 57:415–425. 20. Ramassamy C, Averill D, Beffert U, Bastianetto S, Theroux L, Lussier-Cacan S, Cohn JS, Christen Y, Davignon J, Quirion R, Poirier J. Oxidative damage and protection by antioxidants in the frontal cortex of Alzheimer’s disease is related to the apolipoprotein E genotype. Free Radic Biol Med, 1999; 27:544–553. 21. Tamaoka A, Miyatake F, Matsuno S, Ishii K, Nagase S, Sahara N, Ono S, Mori H, Wakabayashi K, Tsuji S, Takahashi H, Shoji S. Apolipoprotein E allele-dependent antioxidant activity in brains with Alzheimer’s disease. Neurology, 2000; 54:2319–2321. 22. Haan MN, Wallace R. Can dementia be prevented? Brain aging in a populationbased context. Annu Rev Public Health, 2004; 25:1–24. 23. Mattson MP. Gene-diet interactions in brain aging and neurodegenerative disorders. Ann Intern Med, 2003; 139:441–444. 24. Mayeux R. Epidemiology of neurodegeneration. Annu Rev Neurosci, 2003; 26:81–104. 25. Bramlett HM, Dietrich WD. Pathophysiology of cerebral ischemia and brain trauma: similarities and differences. J Cereb Blood Flow Metab, 2004; 24:133–150. 26. Maritim AC, Sanders RA, Watkins JB III. Diabetes, oxidative stress, and antioxidants: a review. J Biochem Mol Toxicol, 2003; 17:24–38. 27. Moriel P, Plavnik FL, Zanella MT, Bertolami MC, Abdalla DS. Lipid peroxidation and antioxidants in hyperlipidemia and hypertension. Biol Res, 2000; 33:105–112. 28. Perna AF, Ingrosso D, De Santo NG. Homocysteine and oxidative stress. Amino Acids, 2003; 25:409–417. 29. Gupta VB, Anitha S, Hegde ML, Zecca L, Garruto RM, Ravid R, Shankar SK, Stein R, Shanmugavelu P, Jagannatha Rao KS. Aluminium in Alzheimer’s disease: are we still at a crossroad? Cell Mol Life Sci, 2005; 62:143–158. 30. Preston AM. Cigarette smoking-nutritional implications. Prog Food Nutr Sci, 1991; 15:183–217. 31. Behl C, Skutella T, Lezoualc’h F, Post A, Widmann M, Newton CJ, Holsboer F. Neuroprotection against oxidative stress by estrogens: structure-activity relationship. Mol Pharmacol, 1997; 51:535–541. 32. Hamburger SA, McCay PB. Spin trapping of ibuprofen radicals: evidence that ibuprofen is a hydroxyl radical scavenger. Free Radic Res Commun, 1990; 9:337–342. 33. Stoll LL, McCormick ML, Denning GM, Weintraub NL. Antioxidant effects of statins. Drugs Today (Barc), 2004; 40:975–990. 34. Green P, Glozman S, Weiner L, Yavin E. Enhanced free radical scavenging and decreased lipid peroxidation in the rat fetal brain after treatment with ethyl docosahexaenoate. Biochim Biophys Acta, 2001; 1532:203–212.

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35. Commenges D, Scotet V, Renaud S, Jacqmin-Gadda H, Barberger-Gateau P, Dartigues JF. Intake of flavonoids and risk of dementia. Eur J Epidemiol, 2000; 16:357–363. 36. Nunomura A, Perry G, Aliev G, Hirai K, Takeda A, Balraj EK, Jones PK, Ghanbari H, Wataya T, Shimohama S, Chiba S, Atwood CS, Petersen RB, Smith MA. Oxidative damage is the earliest event in Alzheimer disease. J Neuropathol Exp Neurol, 2001; 60:759–767. 37. Nunomura A, Perry G, Pappolla MA, Friedland RP, Hirai K, Chiba S, Smith MA. Neuronal oxidative stress precedes amyloid-β deposition in Down syndrome. J Neuropathol Exp Neurol, 2000; 59:1011–1017. 38. Abe T, Tohgi H, Isobe C, Murata T, Sato C. Remarkable increase in the concentration of 8-hydroxyguanosine in cerebrospinal fluid from patients with Alzheimer’s disease. J Neurosci Res, 2002; 70:447–450. 39. Praticò D, Clark CM, Liun F, Rokach J, Lee VY, Trojanowski JQ. Increase of brain oxidative stress in mild cognitive impairment: a possible predictor of Alzheimer disease. Arch Neurol, 2002; 59:972–976. 40. Migliore L, Fontana I, Trippi F, Colognato R, Coppede F, Tognoni G, Nucciarone B, Siciliano G. Oxidative DNA damage in peripheral leukocytes of mild cognitive impairment and AD patients. Neurobiol Aging, 2005; 26:567–573. 41. Rinaldi P, Polidori MC, Metastasio A, Mariani E, Mattioli P, Cherubini A, Catani M, Cecchetti R, Senin U, Mecocci P. Plasma antioxidants are similarly depleted in mild cognitive impairment and in Alzheimer’s disease. Neurobiol Aging, 2003; 24:915–919. 42. Misonou H, Morishima-Kawashima M, Ihara Y. Oxidative stress induces intracellular accumulation of amyloid β-protein (Aβ) in human neuroblastoma cells. Biochemistry, 2000; 39:6951–6959. 43. Gomez-Ramos A, Diaz-Nido J, Smith MA, Perry G, Avila J. Effect of the lipid peroxidation product acrolein on tau phosphorylation in neural cells. J Neurosci Res, 2003; 71:863–870. 44. Sung S, Yao Y, Uryu K, Yang H, Lee VM, Trojanowski JQ, Praticò D. Early vitamin E supplementation in young but not aged mice reduces Aβ levels and amyloid deposition in a transgenic model of Alzheimer’s disease. FASEB J, 2004; 18:323–325. 45. Nakashima H, Ishihara T, Yokota O, Terada S, Trojanowski JQ, Lee VM, Kuroda S. Effects of alpha-tocopherol on an animal model of tauopathies. Free Radic Biol Med, 2004; 37:176–186. 46. Drake J, Link CD, Butterfield DA. Oxidative stress precedes fibrillar deposition of Alzheimer’s disease amyloid β -peptide (1–42) in a transgenic Caenorhabditis elegans model. Neurobiol Aging, 2003; 24:415–420 47. Bayer TA, Schafer S, Simons A, Kemmling A, Kamer T, Tepest R, Eckert A, Schussel K, Eikenberg O, Sturchler-Pierrat C, Abramowski D, Staufenbiel M, Multhaup G. Dietary Cu stabilizes brain superoxide dismutase 1 activity and reduces amyloid Aβ production in APP23 transgenic mice. Proc Natl Acad Sci USA, 2003; 100:14187–14192. 48. Li F, Calingasan NY, Yu F, Mauck WM, Toidze M, Almeida CG, Takahashi RH, Carlson GA, Flint Beal M, Lin MT, Gouras GK. Increased plaque burden in brains of APP mutant MnSOD heterozygous knockout mice. J Neurochem, 2004; 89:1308–1312.

Neuronal 22 Age-Related and Behavioral Deficits are Improved by Polyphenol-Rich Blueberry Supplementation Francis C. Lau, Barbara Shukitt-Hale, and James A. Joseph Tufts University Boston, Massachusetts

CONTENTS 22.1 Introduction .............................................................................................373 22.2 Oxidative Stress ......................................................................................374 22.2.1 Reactive Oxygen Species and Reactive Nitrogen Species .........374 22.2.2 Oxidant–Antioxidant Balance ....................................................375 22.2.3 Age-Related Increase in Oxidative Stress ..................................375 22.2.4 Inflammation ...............................................................................376 22.3 Neuronal and Behavioral Changes in Aging ..........................................377 22.3.1 Neuronal Changes .......................................................................377 22.3.2 Behavioral Deficits .....................................................................377 22.4 Neuroprotective Effects of Blueberries ..................................................378 22.5 Conclusions .............................................................................................382 References ........................................................................................................383

22.1 INTRODUCTION Population estimates conducted by the United Nations indicate that, in developed countries, persons aged 60 years or over are projected to increase from 20% of today’s population to 32% by the year 2050. At the same time, the elderly 373

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population will not only surpass but also double the number of children (persons aged 0–14) for the first time in history.1 It is highly possible that the aged population will exhibit some of the most common correlative motor and cognitive behavioral changes that occur in aging. These alterations occur even in the absence of age-related neurodegenerative diseases but can interact to exacerbate the behavioral aberrations found in these conditions. Alzheimer’s disease (AD) and Parkinson’s disease (PD) are the most common neurodegenerative diseases found in the aged population. Although the etiology of these diseases is largely unknown,2–4 a few risk factors have been identified, and aging is the only factor found to be common to both AD and PD.5–7 Thus, population aging causes an increase in age-related deficits, which inflict a great deal of health and economic burden on the elderly because of the diminished quality of life and the increased health care costs. In order to relieve the burden imposed by the increases in life expectancy, it is of great importance to explore methods to retard or reverse the age-related neuronal deficits. Unfortunately, very little is known about the mechanisms involved in these age-associated declines in cognitive and motor behaviors. In the 1950s, Harman proposed the free radical theory of aging.8 According to Harman,8–10 the aging process is a result of the accumulation of oxidative damage to cells and tissues . It is postulated that the behavioral and neuronal deteriorations seen in the aged population are the result of an increasing inability to protect against free radicals.6,11–14 This theory is now widely accepted in the field of nutraceutical research, which investigates the effects of dietary antioxidants as an intervention to reverse the aging process.15–22 Our research shows that supplementation with fruits and vegetables provides beneficial effects, which include both forestalling and reversing the deleterious effects of aging on neuronal functioning and behavior.23–30 This protection is probably due to the polyphenolic compounds in these fruits and vegetables, which possess antioxidant and antiinflammatory properties.31 This chapter will review the concepts of oxidative stress (OS), OS-induced neuronal and behavioral changes, and the effect of blueberries (BB) on age-related neuronal and behavioral deficits.

22.2 OXIDATIVE STRESS 22.2.1 REACTIVE OXYGEN SPECIES AND REACTIVE NITROGEN SPECIES Reactive oxygen species (ROS) collectively refer to oxygen radicals (such as superoxide and hydroxyl) and nonradicals (such as hydrogen peroxide) that are readily converted into radicals.32–34 Reactive oxygen species are the by-products of normal aerobic metabolism.19,35,36 Mitochondria are the major source of ROS in mammalian cells.37–39 Approximately 90% of the total O2 consumed by the human body is used by mitochondria.40,41 The biologically relevant oxygen ⋅ radicals are the hydroxyl radical (OH ), hydrogen peroxide (H2O2), perhydroxyl radical (HO⫺2 ), and the superoxide anion (O⫺2 ).36 Endogenous superoxide is

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formed enzymatically by NAD(P)H oxidases or xanthine oxidase, and nonenzymatically as a by-product of the reduction of oxygen by semiubiquinone.36 Under normal cellular conditions, superoxide is dismutated rapidly by superoxide dismutase (SOD) to form O2, and H2O2 nonradicals.42 H2O2 can also be generated in the brain during enzymatic degradation of neurotransmitters, such as dopamine (DA).43–45 Consequently, H2O2 is converted to water by catalase or glutathione peroxidase (GSHPX).36,46 However, at high concentrations, H2O2 reacts with ferrous or cuprous ions to produce the highly oxidizing OH.32,47,48 HO attacks almost all the macromolecules in living cells.47,49–51 Animal studies have shown that aging induces a significant increase in cellular H2O2, the source of HO.52 Nitric oxide (NO) and its derivatives are referred to as reactive nitrogen species (RNS). NO, named the molecule of the year in 1992 by Science,53 is a ubiquitous intracellular messenger that diffuses through cells and tissues.54–58 A double-edged sword, NO can be either cytotoxic or cytoprotective59–62 depending on factors such as the level and duration of its expression.63,64 Nitric oxide is formed enzymatically in mammalian systems by nitric oxide synthases (NOS). There are at least three isoforms of NOS identified thus far.65–67 The neuronal and epithelial NOS (nNOS and eNOS, respectively) are constitutively expressed, whereas inducible NOS (iNOS) is produced upon stimulation by immunological factors.68–70 Endogenous NO can also be generated nonenzymatically by reaction of NO⫺2 under acidic conditions.64 Exogenous NO exposure occurs via air pollutants and cigarette smoking.71 Nitric oxide reacts with O⫺2 to form the highly toxic oxidant peroxynitrite (ONOO⫺).72 It is also the source for other RNS, such as nitrogen dioxide radical (NO⫺2 ), nitrite (NO⫺2 ), nitrate (NO⫺3 ), and dinitrogen trioxide (N2O3).64,73,74

22.2.2 OXIDANT–ANTIOXIDANT BALANCE The detrimental effects of OS are normally kept in check by antioxidant defense systems (Figure 22.1a). The antioxidant defenses consist of enzymes (such as SOD, glutathione peroxidase, and catalase) and oxidant scavengers (such as glutathione, uric acid, trace elements, and dietary antioxidants, including vitamins and polyphenolics).35 However, this equilibrium between oxidants and antioxidant defenses tends to shift toward oxidants because the defense systems are not absolutely effective.75 Indeed, about 1% of ROS/RNS evades the antioxidant defense systems daily, thus tipping the balance in favor of oxidants.76 When the antioxidant defense systems fail to repair the damages, redox imbalance occurs. This imbalance (Figure 22.1b) leads to the oxidation of macromolecules such as DNA, proteins, and lipids.77–86 Furthermore, calcium homeostasis, cellular signaling cascades, and gene expression are perturbed by the imbalance.87–92 This redox imbalance results in a progressive OS accumulation, and the end product of the increased OS is cell death.19,93–95

22.2.3 AGE-RELATED INCREASE

IN

OXIDATIVE STRESS

The normal aging process brings about qualitative changes such as the progressive degeneration of cellular functions.36,89,96 Since oxidative damage is a major

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Oxidative Stress and Age-Related Neurodegeneration Antioxidant defenses

ROS/RNS Balanced

(a)

Antioxidant defenses Imbalanced

ROS/RNS

OS ↑

Oxidation of •DNA •Proteins •Lipids Alterations in •Calcium homeostasis •Signaling cascades •Gene expression Cell death

(b)

FIGURE 22.1 Illustration of oxidant–antioxidant homeostasis. (a) a balanced state; (b) a shift of the balance toward oxidants leading to oxidative stress.

factor contributing to the decline of cellular functions, OS is likely to increase in the aged population.35,97 An increasing body of correlative evidence suggests that oxidants produced in the mitochondria cause oxidative damage in mitochondrial DNA, lipids, and proteins and that this damage accumulates with time and induces mitochondrial dysfunctions during the aging process.14,38,98–101 Oxidative damage to nuclear and mitochondrial DNA,102 protein,103 and lipids104 has been found to increase with normal aging in human brain tissues (reviewed by [105]). A study of lipid peroxidation in healthy human subjects of different ages has revealed that the plasma level of lipid oxidation products was significantly higher (25 to 45%) in aged individuals (over 50 years of age) compared with young individuals (20 to 29 years of age).14 A similar experiment has produced comparable findings,106 suggesting that lipid peroxidation is increased during normal aging. An animal study has also indicated that an age-related increase in OS, as measured by oxidative damages to DNA and proteins, might be attributed to both an increase in the generation of oxidants and an augmented susceptibility to oxidative damage.107 The observed oxidative damage during normal aging is exacerbated by neurodegenerative diseases such as AD103,104,108 and PD.109,110

22.2.4 INFLAMMATION Inflammation also produces OS.105,111–115 Localized ROS/RNS over-production has been linked to inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease, atherosclerosis, and uremia.116–118 Oxidative stress-mediated

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inflammation has also been attributed to neurodegenerative diseases such as AD and PD.60,111,119–124 Accumulating evidence suggests that inflammation in the central nervous system (CNS) plays an important role during normal aging.105,125,126 Microglial cells serve a supportive role in the CNS. Upon activation, microglia generate high levels of radicals such as O⫺2 and NO.117,127 Activated microglia also produce inflammatory molecules such as cytokines, growth factors, and complement proteins.76,117,127–130

22.3 NEURONAL AND BEHAVIORAL CHANGES IN AGING The brain is particularly susceptible to oxidative damage6,11,105 for the following reasons. The brain weighs about 2% of the body weight but uses 20% of the total oxygen consumption. Furthermore, it is enriched with polyunsaturated fatty acids that are especially vulnerable to ROS peroxidation. Additionally, the brain has low levels of catalase activity and moderate amounts of antioxidant enzymes SOD and GSHPX. Moreover, the human brain has high concentrations of iron in certain areas, which facilitates lipid peroxidation.12,47,105,131 Research indicates that not only is the CNS particularly vulnerable to OS but this vulnerability increases during aging16,132 and may also enhance CNS vulnerability to inflammation.132,133 Thus, OS and inflammation may interact synergistically to induce the observed age-related neuronal and behavioral changes.

22.3.1 NEURONAL CHANGES There is a wealth of data suggesting that OS and inflammation increase in the CNS during aging and in neurodegenerative diseases.24,35,134 Numerous studies have shown that increased ROS/RNS activate neuronal stress signaling pathways such as the mitogen-activated protein kinase (MAPK) cascade, the nuclear factor-kappa β (NF-κ B) signal transduction pathway, and the tumor suppressor p53.135–139 Moreover, ROS/RNS have been found to modulate ion channels.90 It has been shown that intracellular calcium levels [Ca]i rise during aging.98,134,140,141 Inflammatory cytokines, such as interleukin-1β (IL-1β ) and tumor necrosis factorα (TNF-α), are also generated in the CNS in response to OS and inflammation.142–146 Microarray data on age-related changes in gene expression in the human brain have revealed an increase in the expression of TNF-α,147 NF-κ B, and IL-1β.148 These neuronal changes may ultimately lead to behavioral deficits.22

22.3.2 BEHAVIORAL DEFICITS Behavioral deficits, both cognitive149 and motor,150,151 during normal aging have been shown in a number of experiments. While the mechanisms involved in both motor and cognitive deficits during aging remain to be elucidated, it is clear that both OS152 and inflammation153,154 are involved. Motor deficits include decreases in balance, muscle strength, and coordination,150 which result from age-related

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alterations in the striatal DA system155 or in the cerebellum.156,157 Cognitive deficits are manifested in tasks involving spatial learning and memory.149,158–161 Brain regions such as the hippocampus, prefrontal cortex, and dorsomedial striatum162–166 are thought to be involved in various types of memory disruption. Animal studies indicate that young subjects exposed to OS exhibit neuronal and behavioral changes similar to those seen in aged subjects.166–169 Experiments involving the reduction of the endogenous antioxidant glutathione (GSH) with buthionine sulfoximine (BSO), followed by central administration of DA mimicking conditions seen in aging, have produced deficits in both cognitive and motor performance. When BSO was given before DA administration, psychomotor170 and cognitive performance171 were selectively impaired. However, neither BSO nor DA alone had adverse effects on behavior. Thus, these data indicate that the CNS is especially susceptible to OS and that this susceptibility may increase during aging or conditions involving reduced antioxidant capacity. Similar changes in behavior have been observed with exogenously introduced inflammatory mediators (e.g., cytokines) known to elicit the activation of glial cells, perivascular/parenchymal macrophages, and increased mobilization and infiltration of peripheral inflammatory cells into the brain.153 It appears that intrahippocampal administration of lipopolysaccharide (LPS) induces the up-regulation of several inflammatory markers and results in the degeneration of hippocampal pyramidal neurons, as well as impairments in working memory.172–174 Indeed, chronic infusion of LPS into the ventricle of young rats can reproduce many of the behavioral, inflammatory, neurochemical, and neuropathological dysfunctions seen in the brains of AD patients.153,154,172–174 Recent studies have revealed that intrahippocampal kainic acid (KA) injections induced increases in cytokines IL-1β and TNF-α, as well as NF-κβ in the hippocampus.175 Moreover, there were increases in oxotremorine (OX)-6 activation accompanied by declines in cognitive performance on a Morris water maze (MWM),176 further suggesting an important role for inflammatory processes in motor and cognitive deficits in aging.

22.4 NEUROPROTECTIVE EFFECTS OF BLUEBERRIES There is a vast interest in the utilization of nutritional antioxidants in combating the deleterious effects of OS on aging and neurodegenerative diseases.22,27,177–179 Here we focus on the neuroprotective effects of BB supplementation. Blueberries are rich in anthocyanins, which are among the plant polyphenols that have potent antioxidant and anti-inflammatory activities.180,181 By using fruits and vegetables that were high in antioxidant activity as determined by the oxygen radical absorbance capacity assay (ORAC),182–184 we showed that dietary supplementation (for 8 weeks) with spinach, strawberry, or BB extracts in an AIN-93 diet was effective in reversing age-related deficits in neuronal and cognitive function in aged (19 month-old) Fischer 344 (F344) rats.24 However, only the BB-supplemented group exhibited improved performance on tests of motor function that assessed balance and coordination (e.g., rod walking and the accelerating rotarod), while none of the other supplemented groups differed from control on these tasks.

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However, examinations of the striata from the supplemented groups showed only small levels of antioxidant activity, which was insufficient to account for the observed significant beneficial effects of BB supplementation on motor and cognitive function. Findings from the current24 and a subsequent study185 suggested that there are additional benefits, in addition to those involving antioxidant or anti-inflammatory effects, of BBs on both motor and cognitive behavior. We hypothesized that these beneficial properties may involve alterations in neuronal signaling and communication. A recent study carried out in the APP/PS1 (amyloid precursor protein/presenilin1) transgenic mice confirmed the hypothesis.20 The APP/PS1 transgenic mice serve as a murine model for AD since these mutations promote the production of beta amyloid (Aβ ) and, subsequently, Alzheimer-like plaques in several brain regions, which are accompanied in middle age by cognitive deficits. A group of these mice was given BB supplementation beginning at 4 months of age and continuing for 8 months until they were 12 months of age, at which time their performance was tested in a Y-maze. The results indicated that mice supplemented with BB demonstrated Y-maze performance that was similar to that observed in the nontransgenic mice and significantly greater than that seen in the nonsupplemented transgenic mice. Interestingly, there was a dichotomy between the plaque burden and behavior in the BB-supplemented transgenic mice. No differences in the number of plaques were observed between the supplemented and non-supplemented APP/PS1 mice, even though behavioral deficits were pre-vented in the BB-supplemented animals.20 One possible reason that the behavior did not reflect the morphology may be that there was enhanced signaling present in the BB-supplemented transgenic mice that acted to prevent or circumvent any putative deleterious effects of the amyloid plaques on behavior. The evidence for this possibility is provided by data showing that the BB-supplemented APP/PS1 mice exhibited greater levels of hippocampal extracellular signal regulated kinases (ERK1/2 also known as MAPK p42/p44) as well as striatal and hippocampal protein kinase C (PKC) than that seen in the transgenic mice maintained on the control diet. The ERK and PKC kinases have been shown to be important in mediating cognitive function, especially in conversion of short-term to long-term memory.186 Since the cerebellar noradrenergic system (NAS) shows age-related alterations in β -adrenergic function, it has been proposed that the cerebellar NAS may underlie certain age-related deficits in motor learning.157 Investigation of cerebellar β -adrenergic receptor increase of γ -amino butyric acid (GABA) response and motor learning has shown that, in the young rats, as high as 80% of the recorded cerebellar Purkinje cells exhibited β -adrenergic potentiation, while in the aged rats only 30% of the recorded cerebellar Purkinje cells showed β -adrenergic potentiation. However, this decline in the aged rats was restored to the level seen in the young animals by BB supplementation.187 Short-term BB supplementation has also been proven to benefit age-related declines in behavioral parameters such as balance, coordination, working memory, and reference memory seen in F344 rats.185 Furthermore, OX-induced DA release was increased in striatal slices188 isolated from rats on a BB-supplemented diet. It

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has been suggested that the loss in sensitivity of the DA neurotransmitter, as well as the adrenergic,189 the muscarinic,190, 191 and the opioid192 system, is in part due to OS, and that the loss induces impairments in motor functions150,151 and cognitive behaviors.158,160 Therefore, the observed enhancement in DA release may be directly linked to the improvements seen in the rats on a BB-supplemented diet.185 Blueberry supplementation has also been found to reduce ischemia-induced brain damage.193 In this study, rats were fed AIN93G diets for 6 weeks prior to stroke-induction followed by hypoxia. The total neuronal damage in the ischemic hippocampus was found to be reduced by more than half in rats on the BB-supplemented diet, although BB appeared to selectively protect C1 and C2.193 The effects of BB supplementation on object recognition memory (ORM) and the levels of NF-κ B in aged rats have been examined recently.29 The results showed that young rats had significantly higher ORM scores than aged rats on the control diet. This observation showed that ORM declines during normal aging. However, BB supplementation was able to eliminate the ORM deficit seen in the control aged rats. The study also revealed that the expression of NF-κβ (activated by OS and inflammation; see Section 22.3.1) increases with age and that BB supplementation was able to restore this increase in the frontal cortex, hippocampus, and the striatum. Although BB supplementation was able to reduce the cerebellar expression of NF-κβ seen in the control aged rats, it could not restore the expression level to that of the young control.29 Experiments have shown that there is a loss of sensitivity in the muscarinic receptors (MAChRs) as a function of age and AD.194, 195 We have shown that this loss is sensitive both to aging and OS.196 Moreover, there is also a subsequent downstream reduction in inositol-tris-phosphate (IP3) levels.194 Subsequent experiments indicated that the decreases in MAChR sensitivity are reflected upstream as reductions in carbachol-stimulated GTPase activity and deficits in the MAChR–G protein coupling/uncoupling197 in the aged rodent that are exacerbated in age-sensitive AD,194,195 and are sensitive to OS.133 There is also strong evidence suggesting that MAChRs are intimately involved in various aspects of both neuronal APP processing198 and vascular functioning,199 and that the various MAChR subtypes may show differential sensitivity to OS. In fact, COS-7 cells transfected with subtypes M1, M2, and M4 show greater OS susceptibility than M3.200 However, an experiment using a chimeric MAChR (M1M3i3), in which the variable i3 loop of the M3 receptor (M3i3) was replaced with the i3 loop of the M1 receptor, showed that the variable domain of M3 receptor (M3i3) conferred OS protection on Ca2⫹ regulation in COS-7 cells, indicating that the i3 domain of M3 may decrease OS susceptibility by recruiting antioxidants to specific receptor sites that impart OS susceptibility.200 Data from our research have shown that BB treatment antagonized the Aβ and DA-induced deficits in Ca2⫹ flux in MAChR M1 subtype-transfected COS-7 cells.30 The results indicate that BB may mitigate the toxic effects of Aβ and DA seen in the AD patients. In addition, recent findings have shown that the ability of the hippocampus to regenerate new neurons, a process known as neurogenesis, was enhanced by

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short-term BB supplementation.28 There were also cognitive improvements owing to BB supplementation. The protein expression of the insulin growth factor-1 (IGF-1) and the activation of ERK were enhanced significantly by BB supplementation.28 Therefore, BB supplementaion appears to positively modulate hippocampal plasticity via enhanced neurogenesis. Recently, we investigated whether BB supplementation could reduce the deleterious effects of KA-induced inflammation, perhaps by increasing anti-inflammatory protection in the brain and reducing brain susceptibility to inflammatory insults, and regulating inflammatory gene expression.175,176 In this study, 4-monthold male F344 rats were put on a control, 2% BB, or 0.015% piroxicam (PX, a nonsteroidal anti-inflammatory drug, NSAID) diet for 8 weeks before Ringers saline or KA (300 ng in 0.5 µL Ringers saline) was injected bilaterally into the hippocampus. Ten days later, rats were tested for 4 days in the MWM. After behavioral tests, rats were divided into two groups. One group was perfused with saline followed by formalin. The fixed brains were used in immunohistochemistry analysis of OX-6, a glial inflammatory marker. The other group was sacrificed by decapitation. The brains were removed and total RNA was extracted from specific regions. Real-time quantitative RT-PCR was performed on the expression of IL-1β, TNF-α, NF-κ B, and IGF-1 in the hippocampus. The positive effects of the BB-supplemented diet on behavioral performance were seen primarily on days 2 and 3, while the rats on the PX diet showed improved performance on day 4, that is on the reversal day. Therefore, it is possible that BB and PX have differential effects on cognition, and that if both compounds had been fed to the rats in the same diet they may have had a synergistic effect to improve memory better than either of the compounds alone.176 Interestingly, the BB- and PX-supplemented diets were able to improve performance in the animals administered Ringers solution, and presumably, vulnerability to OS and inflammation are not enhanced, as in rats administered KA.176 The phenomenon of this enhanced effect of the BB-supplemented diet has occurred in previous studies. It is postulated that BBs may also have direct effects, i.e., not mediated through OS or inflammatory pathways, on the brain by directly increasing signaling and neurogenesis. In fact, gene expression analysis showed that BB supplementation was able to reduce the expression of IL-1β though not to the control level, and restore the expression of NF-κ b to the control level in the hippocampus. However, BB supplementation did not reduce the expression of TNF-α. Moreover, BB supplementation in KA-injected animals elicited an increase in the expression of the neuroprotective trophic factor IGF-1 indicating that BB exerts its effect through different cascades with respect to inflammation and neurotrophic events.175 Kainic acid produced an inflammatory response, as shown by increased OX-6 activation176 in the hippocampus. The BB-supplemented diet reduced this inflammatory response; however it did not restore OX-6 activation to control levels. The PX diet had no effect on OX-6 activation. It appeared that the significant effects of BBs on cognitive behavior are due to a multiplicity of direct and indirect actions, the former involving effects on

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TABLE 22.1 Reports from Various Studies Showing the Neuroprotective Effects of Blueberry Supplementation in Rodents Improvements

Model Systems

References

Motor functions Cognitive behaviors

Aging

[24]

Cognitive memory GTPase activity Hippocampal ERK expression pPKC-α expression

Alzheimer’s disease

[20]

Motor functions Cognitive behaviors OX-induced DA release

Aging

[185]

Ischemia-induced neuronal damage

Stroke

[193]

Cognitive memory NF-κ B expression

Aging

[29]

Ca2⫹ regulation

Muscarinic receptors Alzheimer’s disease

[30]

Cerebellar β -adrenergic receptor function

Aging

[187]

Hippocampal neurogenesis ERK, IGF-1, IGF-1R expression Cognitive behaviors

Aging

[28]

Motor functions Cognitive behaviors OX-6 expression

Neuroinflammation

[176]

IL-1β, NF-κ b, IGF-1 expression

Neuroinflammation

[175]

neuronal communication and the latter involving antioxidant and anti-inflammatory activities.175,176

22.5 CONCLUSIONS Oxidative stress and inflammation are the major sources contributing to neuronal and behavioral deficits observed in the aging process and age-related neurodegenerative diseases.80,101,124,201–204 A growing interest in dietary antioxidants has been heightened by the revelation that these natural antioxidants possess neuroprotective, cardioprotective, and chemoprotective properties.6,205–207

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Blueberries are among those fruits that contain the highest ORAC index.182–184 More importantly, it appears that BBs may also directly enhance neurogenesis28 and neuronal signaling to increase MAP kinases20 and that might ultimately affect motor and cognitive behavior. In this respect, research from our laboratory has shown that BB supplementation can effectively reverse or forestall some of the neuronal and behavioral deficits inflicted by aging (see Table 22.1). Thus, nutritional interventions may prove to be a valuable asset in the retardation and prevention of inflammation and OS in aging and perhaps in age-related neurodegenerative diseases.

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β Peptide, 23 aAmyloidTherapeutic Target for Alzheimer’s Disease? Yuan Luo

University of Maryland Baltimore, Maryland

Peter Butko

University of Southern Mississippi Hattiesburg, Mississippi

CONTENTS Abstract..............................................................................................................395 23.1 Introduction ............................................................................................396 23.2 The Amyloid β Cascade Hypothesis of Alzheimer’s Disease................396 23.3 Regulating Amyloid β Species In Vitro..................................................397 23.3.1 Supramolecular Assemblies of Amyloid β ..............................397 23.3.2 Inhibition of Amyloid β Oligomerization ................................398 23.4 Modulating Amyloid β Species In Vivo .................................................399 23.4.1 Animal Models of Alzheimer's Disease...................................399 23.4.2 Conventional Pharmacology Targeting Amyloid β Species ....400 23.4.3 Reverse Pharmacology Supporting Amyloid β as a Target .....400 23.5 Summary.................................................................................................402 Acknowledgments .............................................................................................402 References .........................................................................................................403

ABSTRACT The well-known “amyloid cascade hypothesis” associates amyloid beta peptide (Aβ ) with the pathological process of Alzheimer’s disease (AD). Conventional pharmacology targeting Aβ provided evidence in support of this hypothesis. Reverse pharmacology has identified compounds that alleviate symptoms of AD and also target Aβ. While the amyloid cascade hypothesis has been revised and improved in the past decade, it still awaits universal acceptance. To achieve that, the hypothesis must successfully address several issues. For example, Aβ self-assembles into 395

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complexes of various sizes. The species or forms responsible for Aβ neurotoxicity have not yet been unequivocally identified, and the causal relationship between Aβ toxicity and oxidative stress has not been fully established. It has proved difficult to separate various forms of Aβ assemblies and monitor their effects in vivo. Solutions to these problems are critical for determining the mechanism of AD pathology and for designing specific therapeutic strategies. With the help of integrative approaches and emerging applications of molecular biology, immunology, brain imaging, biophysics, and computer simulations, we may be on the verge of understanding and, importantly, preventing AD.

23.1 INTRODUCTION Alzheimer’s disease (AD) is an age-related neurodegenerative disorder widely recognized as a serious public health problem.1 Currently, more than 5 million Americans are affected. Half of the population over 80 years of age suffers from this disorder. Memory impairment progressing to dementia is the main clinical symptom of AD, which is thought to be the consequence of the selective degeneration of nerve cells in the brain regions critical for memory, cognitive performance, and personality.2 A common feature AD shares with other neurodegenerative diseases is the characteristic senile plaques observed in the brain tissues of the cortex, hippocampus, and amygdala. The primary constituents of the plaques are aggregates of amyloid β (Αβ ), a 4-kDa peptide (39 to 43 amino acids) cleaved by β - and γ -secretases from the amyloid precursor protein (APP).3 Amyloid β occurs normally in plasma and cerebrospinal fluid,4 but elevated levels were found in cases of familial AD (fAD), which is characterized by mutated APP.5,6 Other risk factors are also thought to trigger overproduction of Aβ.7 Amyloid β occurs predominantly in two forms, Aβ40 and Aβ42. The longer peptide is increased in fAD, is only accumulated in Down’s syndrome, is more neurotoxic, and forms fibers much faster. With time, Aβ monomers oligomerize into polymers, which assemble into protofilaments and then fibrils.8,9 It was shown that the Aβ -induced cell death requires peptide self-association,10,11 but identification of the toxic species and elucidating the mechanism of their formation has proved difficult. Nevertheless, these two issues are critical for determining the underlying pathology of AD and for developing specific therapeutic strategies.

23.2 THE AMYLOID β CASCADE HYPOTHESIS OF ALZHEIMER’S DISEASE The “amyloid cascade” hypothesis states that accumulation of Aβ deposits initiates a series of downstream neurotoxic events that result in neuronal dysfunction and death.7 The strongest evidence in support of this hypothesis comes from molecular genetic studies. Patients with Down’s syndrome, who have an extra copy of chromosome 21, which contains the APP gene, inevitably develop AD, and increased Aβ deposits are the early signs of brain lesions.12 All forms of fAD-linked mutations in the APP gene or the two presenilin genes (PS1 and PS2) result in an

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increased production of Aβ42, which is the most amyloidogenic form of the Aβ peptide.13 Furthermore, transgenic mice overexpressing the mutant APP develop Aβ −containing amyloid plaques similar to those found in AD. Other factors, such as the intracellular neurofibrillary tangles and apoE4 allele, may further contribute to the imbalance between Aβ production and clearance.7 Therefore, modulation of Aβ production and clearance in the brain is a promising and rational approach to the treatment of AD. Environmental factors, interacting with genetic ones, make some individuals more prone to developing the disease. General risk factors for AD are very diverse and include age, high cholesterol, abnormal endocrine function, lack of education, and mutations of APP (chromosome 21) and PS1/PS2 (chromosome 14/1). Recurrent experimental evidence suggests that oxidative stress is directly or indirectly involved in Aβ toxicity.14,15 Despite a large body of evidence supporting the notion that Aβ deposition is critical in the pathogenesis of AD, the hypothesis has not been universally accepted. One of the intriguing but unresolved questions is the nature of the Aβ species that is toxic to the neurons in the AD brain: Is it the long fibrils or rather the small oligomers?16 There are three competing opinions: (1) Neurotoxicity of Aβ is directly linked to the peptide’s aggregation state in that only the fibrillar Aβ is toxic.10,17,18 (2) Fibrils are not necessary for neurotoxicity, but rather the small intracellular aggregates or oligomers of the soluble Aβ19–21 are the neurotoxic species.22–25 (3) None of the Aβ species is directly neurotoxic, but the Aβ -induced oxidative stress is the cause of the neurodegeneration in AD.26–28 Each of these competing hypotheses enjoys some level of experimental support, but none is able to answer all the questions and explain all the observations.

23.3 REGULATING AMYLOID β SPECIES IN VITRO 23.3.1 SUPRAMOLECULAR ASSEMBLIES

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AMYLOID β

Apparently, all fAD mutants are associated with Aβ overproduction and it was originally thought that Aβ was a pathological product. However, in the early 1990s, the discovery of Aβ in the normal brain and cerebrospinal fluid29,30 caused a paradigm shift: Aβ is now considered a normal part of cell metabolism in the brain, although its function is still a mystery. Identification of the harmful Aβ species and elucidating their molecular structure is critical for the development of therapeutic agents, but it has proved technically difficult. The use of X-ray crystallography to determine the molecular structure of Aβ fibrils is hampered by the difficulty of obtaining well-diffracting crystals, although some recent advances have been reported. For example, Makin et al.31 found that the forces that hold together the antiparallel β sheets of Aβ monomers include pi-bonding between the aromatic amino acid residues and the salt bridges between charged residues. In addition to hydrophobic interactions, these forces are likely to be important in the formation and stability of Aβ fibrils. Amyloid β monomers and oligomers appear to be, at least initially, in dynamic equilibrium and precipitate out of solution at high concentrations, which makes it difficult to study Aβ structure by solution NMR without resorting to

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nonphysiological conditions.32 Solid-state NMR has been used to at least define some structural constraints in Aβ supramolecular assemblies.33 Cryoelectron34 and atomic-force35 microscopies are good for visualizing the fibrils and other types of supramolecular assemblies. Although the images only reveal the overall organization of the assemblies, far above the atomic resolution, these techniques can provide new and interesting information. For instance, Chromy et al.36 used atomic-force microscopy to detect several kinds of subfibrillar assemblies such as rings, protofibrils, and the oligomeric Aβ-derived diffusible ligands (ADDL). The authors concluded that the soluble, globular, Aβ42 oligomers were toxic to PC12 cells. The affected cell-signaling pathway included ERK and Rac, which directly link the adverse effects of Aβ with learning and memory. Inhibition of oligomer formation by the Ginkgo biloba extract EGb 761 was also observed in this study. Computer modeling and simulations might overcome technical difficulties of the experimental methods. The in silico approach has started to bring new — and thus far perhaps tentative — insights into Aβ oligomerization.37 The technical difficulties notwithstanding, our knowledge of Aβ behavior is advancing. We now know many details of Aβ oligomerization, especially in vitro. A recent finding of Bitan et al.38 indicates that Aβ40 and Aβ42 oligomerize via distinct pathways. Using photo-induced cross-linking of unmodified proteins (PICUP), the authors discovered that Aβ40 existed as monomers, dimers, trimers, and tetramers in rapid equilibrum, while Aβ42 preferentially formed higher oligomers (pentamers and hexamers) that assembled into protofilbrils via several transient structures.

23.3.2 INHIBITION

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AMYLOID β OLIGOMERIZATION

The search for effective inhibitors of Aβ oligomerization has been vigorous and its path is marked by many discoveries, some fortuitous and some less so. The known small-molecule inhibitors of Aβ oligomerization have a varied chemical nature ranging from aspirin and other simple nonsteroidal anti-inflamatory agents39 to structures with fused aromatic rings, such as Congo red, thioflavins10 or dianilinophthalimide,18 to whole peptides.40 A unified underlying structure (if there is any) that is responsible for antiamyloidogenesis has not been found. For a more complete picture about molecules that prevent Aβ oligomerization or break down already preformed Aβ complexes, the reader is referred to recently published reviews.41–44 Despite the fact that none of the compounds are presently used in therapeutic practice, some promising pharmacological leads are being tested in clinical trials. One of the compounds of interest is the Ginkgo biloba extract EGb 761. The Ginkgo extract has been the focus of our laboratory for several years. Using electron microscopy, thioflavin T fluorescence assay, and Western blotting, we were able to show that some components of the Ginkgo extract directly interact with Aβ and inhibit its oligomerization.45 In cells, one downstream effect of the EGb 761-induced decrease in amyloidogenesis manifested itself in a decreased activity of the apoptosis signaling pathway. In that study, we did not concentrate on the possible role of EGb 761 as antioxidant, but we noted that vitamin E could not mimic the effects of EGb 761 in vitro. Combining data from

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experiments in vitro with those obtained in cell culture, we concluded that neuronal damage in AD is probably the result of two factors: a direct Aβ toxicity and the apoptosis initiated by the mitochondria. Our results suggested that multiple cellular and molecular neuroprotective mechanisms underlie the neuroprotective effects of EGb 761, which include attenuation of apoptosis and direct inhibition of Aβ aggregation. The multiplicity of the extract’s targets and mechanisms combined with the apparent lack of side effects make EGb 761 one of the most promising candidates for the treatment of AD.

23.4 MODULATING AMYLOID β SPECIES IN VIVO 23.4.1 ANIMAL MODELS

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ALZHEIMER’S DISEASE

Many attempts have been made to generate transgenic mouse models of AD for a mechanistic approach to Aβ toxicity in vivo.2,46 The double-transgenic mice coexpressing the Swedish mutation APPswe and PS147 exhibit three features: (1) an enhanced production of Aβ when compared with the transgenic mice carrying just the APP mutation,5 (2) an accelerated early deposition of Aβ in the hippocampus and cortex regions,47,48 and (3) a sex-specific learning deficiency (at 18 months) in the Morris water maze test. Although these mice do not display the full phenotype of AD,49 in conjunction with a newly developed triple transgenic mice model of AD,21 they represent a useful tool for the investigation of Aβ toxicity and of cognitive impairment.50 Invertebrate models of AD, such as the round worm Caenorhabditis elegans51 and the fruit fly Drosophila melanogaster,52 have been developed. Owing to the fact that the transgenic C. elegans expresses Aβ in the muscle rather than the neurons, the worms carrying the human gene for Aβ42 display a concomitant progressive paralysis phenotype (CL4176)53 instead of cognitive impairment as observed in the transgenic mice54 and the fruit fly.52 The worms live only for 20 days, allowing for rapid evaluation of the sequence of events during their entire lifetime. The Aβ deposits in the transgenic C. elegans are intracellular51,55 and the animals exhibit increased protein carbonyl contents, a biomarker for protein oxidation.56 Increased levels of protein carbonylation were observed in the AD brain.57 DNA microarray assay of the transgenic strain indicated that several stress-related genes were upregulated, particularly two genes homologous to human αB-crystalline and tumor-necrosis-factor-induced protein, which were also found upregulated in the postmortem AD brain.58 A relationship among Aβ amino acid sequence, amyloid formation, and oxidative damage was established using this model. Yatin et al.56 showed both in vitro and in the C. elegans model that methionine (Met35) is critical for free-radical production by Aβ1–42, and it is also critical for β-sheet formation in the transgenic C. elegans lines.59 A correlation between the progressive paralysis phenotype and the increased levels of protein carbonyls in CL417653 supports the advanced amyloid hypothesis.60 It appears that the temporal sequence of events manifested in the transgenic worms is shared with the Drosophila model of AD,52 and that the accumulation of Aβ42

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in the brain is sufficient to cause cognitive impairment and neurodegeneration. Although the invertebrate systems may not be universally accepted as having direct relevance for AD pathology, they are well-suited for correlating Aβ expression and toxicity in vivo, i.e., in model organisms with a short life span.

23.4.2 CONVENTIONAL PHARMACOLOGY TARGETING AMYLOID β SPECIES Within the paradigm of the amyloid hypothesis, many attempts have been made to use amyloid-binding ligands as therapeutic tools in AD. Congo red and the fluorescent dye thioflavin S, both preferentially binding to stacked β sheets, were the first agents known to reduce Aβ toxicity in primary hippocampal culture10 and in mice.61 These molecules do not cross the blood-brain barrier, but derivatives were generated that were shown to inhibit Aβ fibrillization in vitro and in cultured cells.62 In the search for compounds that can disrupt the Aβ42 fibrils and protofibrils, Ingram and co-workers18 have employed high-throughput screening of small molecules of known activity in relation to Aβ. Six compounds out of more than 3000 candidate molecules effectively reduced the β-sheet content and eliminated neuronal toxicity in cultured cells.18 Alternatively, extensive efforts have been made to develop an Aβ vaccine and inhibitors of amyloidogenic secretases. Both active and passive immunization have shown promising results in mice63,64 and in small human trials.65 There is an ongoing debate in the scientific community on the pros and cons of immunotherapy for AD. Although it currently seems premature for clinical applications in humans, immunotherapy is valuable for research on pathological mechanisms of AD in animals. For example, a recent paper published in Nature Medicine reported that passive immunization against Aβ oligomers that disrupted synaptic plasticity in mice66 was very effective.

23.4.3 REVERSE PHARMACOLOGY SUPPORTING AMYLOID β AS A TARGET Revealing the molecular mechanisms of drug action is referred to by Christen as an example of “reverse pharmacology.” 98 The approach is being successfully applied to the Ginkgo biloba extract EGb 761. The extract was introduced on the market in France by IPSEN many years before cellular and molecular tools became available.67 Recent technology advances have led to renewed interest in the therapeutic targets of this natural product. EGb 761 is a popular dietary supplement taken by the general public to “enhance mental focus” and by the elderly to delay onset of age-related loss of cognitive function. EGb 761 has been used for treatment of certain cerebral dysfunctions and dementias associated with aging and AD. Substantial evidence indicates that EGb 761 has neuroprotective effects. However, the mechanisms of action of the components of the extract are, unfortunately, poorly understood. During the past decade, in vivo and in vitro experiments in mammalian systems and clinical studies in humans demonstrated that EGb 761 exhibits a range of biochemical and pharmacological effects.68 Major biochemical and pharmacological activities of EGb 761 include free-radical scavenging,69 inhibition of membrane lipid peroxidation,70 cognition enhancement and stress

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alleviation in the experimental animals,71–73 anti-platelet-activating factor (PAF) activity that contributes to improvements in cerebral insufficiency,74 enhancement of neuronal plasticity,75 anti-inflammatory effects,76 and antiapoptotic effects in neuronal cells.45,77,78 In human studies, more than a dozen clinical trials have supported the efficacy of EGb 761 in primary degenerative dementia of Alzheimer’s type.79–81 While the evidence for EGb 761-induced enhancement of learning in healthy humans is inconclusive,82–84 an NIH-supported Ginkgo Evaluation of Memory (GEM) study in the United States is currently underway to test the efficacy of EGb 761 as a potential preventive treatment for dementia in the normally aging population and in AD.85 Other clinical effects of EGb 761 include improvements in peripheral arterial insufficiency, in cerebral disorders including cognitive decline, tinnitus, acute cochlear deafness, disturbance in equilibrium,86 and cognitive deficits that follow stress or traumatic brain injury.70 Taken together, it can be concluded that EGb 761 has a beneficial effect on brain functions.87 Accumulating evidence suggests that many of the actions of EGb 761 are so-called “polyvalent” actions, i.e., the therapeutic activity of EGb 761 is the synergistic effect of interactions between various biological activities of the individual substances in the extract. Presumably, this is one of the advantages of using natural products for the prevention and treatment of infirmity as well as the maintenance of health.88,89 Our work has focused on understanding mechanisms of action of the herbal extract EGb 761. We have demonstrated that EGb 761 inhibited Aβ aggregation in vitro and attenuated reactive oxidative species (ROS) in C. elegans. Furthermore, EGb 761 eased Aβ toxicity in the transgenic worm. We also found that only a certain size of the Aβ aggregates is toxic to the worms. Elucidating the mechanisms of neuroprotection by EGb 761 will be important for designing therapeutic strategies, for providing a basic understanding of the underlying neurodegenerative processes, and also for providing a better understanding of the effectiveness and complexity of this herbal medicine. While searching for the specific neurotoxic species of Aβ and the nature of its effects, we found that EGb 761 inhibited the formation of an extracellular, sodium dodecyl sulfate (SDS)-stable Aβ species in an Aβ -expressing neuronal cell line.45 The SDS-stable Aβ oligomers (Mr ~ 8–12 kDa) that inhibit hippocampal longterm potentiation11 have been detected in the soluble fraction of Alzheimer’s diseased cortex90 and in cultured cells.91,92 To further verify the inhibitory effect of EGb 761 in vivo, we fed the extract to the C. elegans strain CL2006, which expresses Aβ ,61 and analyzed tissue samples by Western blotting. Our data show that multiple Aβ immunoreactive bands were detected in the Aβ-expressing C. elegans, and that the Aβ species with molecular weight at around 14 kDa (oligomers) were decreased in EGb 761-fed C. elegans (unpublished data). The 14-kDa species inhibited by EGb 761 are very similar, in terms of size, to the small, neurotoxic, diffusible Aβ oligomers referred to as ADDLs, which were found to kill mature neurons in cultured hippocampal slices at nanomolar concentrations22 and were inhibited in vitro by EGb 761 in a dose-dependent manner.93

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To establish a temporal relationship between the onset of Aβ oligomerization and Aβ -induced toxicity, we measured the temperature-inducible paralysis in CL4176 strain. We observed a convincing delay of paralysis in the worms fed with EGb 761. Congo red, which was shown to inhibit the Aβ oligomers in CL2006, did not generate significant decrease in Aβ -induced paralysis in CL 4176 (unpublished data). We reasoned that EGb 761 offers more protective activities than anti-Aβ aggregation alone. Our findings strongly support the current theory that the small Aβ aggregates are more risky than the larger aggregates, which were originally thought to be the main cause of AD. Interestingly, even a known antioxidant, vitamin C, was not sufficient to ease the toxicity in the paralyzed worms by itself. We assume that it is the combined properties (antioxidative and antiamyloidogenic) of the Ginkgo biloba extract that protect the brain against cognitive dysfunction.

23.5 SUMMARY The development of a drug for treatment of AD still remains a challenging task. Recent technological advances have led to new interest in drug discovery from natural products. It is fundamentally important to determine the temporal sequence of events leading to neurodegeneration in order to understand the early events in the onset of AD and to develop successful therapeutic strategies. C. elegans has proved to be a suitable tool for mechanistic examination of transgene products as well as for pharmacological kinetics analysis.94 We demonstrated that EGb 761 exerts an anti-Aβ aggregation effect in a neuroblastoma cell line expressing Aβ,45 exhibits antistress effects in wild-type C. elegans,95 reduces intracellular free-radical production in the transgenic C. elegans,96 and significantly attenuates expression of stress-response protein hsp16-2 in C. elegans.97 We have recently observed that EGb 761 decreased Aβ oligomerization and Aβ − induced paralysis in transgenic C. elegans (unpublished data). These findings suggest that Aβ oligomers and Aβ-induced oxidative stress are crucial for Aβ toxicity, and that EGb 761 has a clear therapeutic potential for prevention and treatment of AD.

ACKNOWLEDGMENTS This work has been supported by NIH grants R21AT00293-01A2 and R01AT001928-01A1 from the National Center for Complementary and Alternative Medicine (NCCAM), and by IPSEN, Paris, France. Special thanks to our collaborators Chris Link of the University of Colorado, Boulder, CO, and Ikhlas Khan of National Center for Natural Products Research, at the University of Mississippi, School of Pharmacy, Oxford, MS. Further thanks are extended to the past and current members of our laboratory who contributed to this work: Marishka Brown, Adam Burdick, Astrid Gutierrez-Zepeda, Julie Smith, Amy Strayer, Yanjue Wu, Zhixin Wu, and Yanan Xu. Thanks are also due to Laura Causey for her critical reading of the manuscript.

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Oxidative Stress and Age-Related Neurodegeneration brains of transgenic mice coexpressing mutant presenilin 1 and amyloid precursor proteins. Neuron, 1997, 19: 939–945. Borchelt, DR, Thinakaran, G, Eckman, CB, Lee, MK, Davenport, F, Ratovitsky, T, Prada, CM, Kim, G, Seekins, S, Yager, D, Slunt, HH, Wang, R, Seeger, M, Levey, AI, Gandy, SE, Copeland, NG, Jenkins, NA, Price, DL, Younkin, SG, and Sisodia, SS. Familial Alzheimer’s disease-linked presenilin 1 variants elevate Abeta1-42/1-40 ratio in vitro and in vivo. Neuron, 1996, 17: 1005–1013. Sommer, B, Sturchler-Pierrat, C, Abramowski, D, Wiederhold, KH, Calhoun, M, Jucker, M, Kelly, P, and Staufenbiel, M. Transgenic approaches to model Alzheimer’s disease. Rev Neurosci, 2000, 11: 47–51. van Leuven, F. Single and multiple transgenic mice as models for Alzheimer’s disease. Prog Neurobiol, 2000, 61: 305–312. Link, CD, Johnson, CJ, Fonte, V, Paupard, M, Hall, DH, Styren, S, Mathis, CA, and Klunk, WE. Visualization of fibrillar amyloid deposits in living, transgenic Caenorhabditis elegans animals using the sensitive amyloid dye, X-34. Neurobiol Aging, 2001, 22: 217–226. Iijima, K, Liu, HP, Chiang, AS, Hearn, SA, Konsolaki, M, and Zhong, Y. Dissecting the pathological effects of human Abeta40 and Abeta42 in Drosophila: a potential model for Alzheimer’s disease. Proc Natl Acad Sci USA, 2004, 101: 6623–6628. Drake, J, Link, CD, and Butterfield, DA. Oxidative stress precedes fibrillar deposition of Alzheimer’s disease amyloid beta-peptide (1-42) in a transgenic Caenorhabditis elegans model. Neurobiol Aging, 2003, 24: 415–420. Hsiao, K, Chapman, P, Nilsen, S, Eckman, C, Harigaya, Y, Younkin, S, Yang, F, and Cole, G. Correlative memory deficits, Abeta elevation, and amyloid plaques in transgenic mice. Science, 1996, 274: 99–102. Link, CD. Expression of human beta-amyloid peptide in transgenic Caenorhabditis elegans. Proc Natl Acad Sci USA, 1995, 92: 9368–9372. Yatin, SM, Varadarajan, S, Link, CD, and Butterfield, DA. In vitro and in vivo oxidative stress associated with Alzheimer’s amyloid beta-peptide (1-42). Neurobiol Aging, 1999, 20: 325–330; discussion 339–342. Hensley, K, Hall, N, Subramaniam, R, Cole, P, Harris, M, Aksenov, M, Aksenova, M, Gabbita, SP, Wu, JF, Carney, JM. Brain regional correspondence between Alzheimer’s disease histopathology and biomarkers of protein oxidation. J Neurochem, 1995, 65: 2146–2156. Link, CD, Taft, A, Kapulkin, V, Duke, K, Kim, S, Fei, Q, Wood, DE, and Sahagan, BG. Gene expression analysis in a transgenic Caenorhabditis elegans Alzheimer’s disease model. Neurobiol Aging, 2003, 24: 397–413. Fay, DS, Fluet, A, Johnson, CJ, and Link, CD. In vivo aggregation of beta-amyloid peptide variants. J Neurochem, 1998, 71: 1616–1625. Butterfield, DA. Beta-amyloid-associated free radical oxidative stress and neurotoxicity: implications for Alzheimer’s disease. Chem Res Toxicol, 1997, 10: 495–506. Lim, GP, Yang, F, Chu, T, Gahtan, E, Ubeda, O, Beech, W, Overmier, JB, HsiaoAshec, K, Frautschy, SA, and Cole, GM. Ibuprofen effects on Alzheimer pathology and open field activity in APPsw transgenic mice. Neurobiol Aging, 2001, 22: 983–991. Lee, VM. Amyloid binding ligands as Alzheimer’s disease therapies. Neurobiol Aging, 2002, 23: 1039–1042. Schenk, D, Barbour, R, Dunn, W, Gordon, G, Grajeda, H, Guido, T, Hu, K, Huang, J, Johnson-Wood, K, Khan, K, Kholodenko, D, Lee, M, Liao, Z, Lieberburg, I,

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80. Le Bars, PL, Kieser, M, and Itil, KZ. A 26-week analysis of a double-blind, placebo-controlled trial of the Ginkgo biloba extract EGb 761 in dementia. Dement Geriatr Cogn Disord, 2000, 11: 230–237. 81. Oken, BS, Storzzbach, DM, and Kaye, JA. The efficacy of Ginkgo biloba on cognitive function in Alzheiner disease. Arch Neurol, 1998, 55: 1409–1415. 82. Curtis-Prior, P, Vere, D, and Fray, P. Therapeutic value of Ginkgo biloba in reducing symptoms of decline in mental function. J. Pharm Pharmacol, 1999, 51: 535–541. 83. Solomon, PR, Adams, F, Silver, A, Zimmer, J, and DeVeaux, R. Ginkgo for memory enhancement: a randomized controlled trial. JAMA, 2002, 288: 835–840. 84. Mix, JA and David Crews, W, Jr. A double-blind, placebo-controlled, randomized trial of Ginkgo biloba extract EGb 761(R) in a sample of cognitively intact older adults: neuropsychological findings. Hum Psychopharmacol, 2002, 17: 267–277. 85. Christen, Y, Olano-Martin, E, and Packer, L. EGb 761 in the postgenomic era: new tools from molecular biology for the study of complex products such as Ginkgo biloba extract. Cell Mol Biol, 2002, 48: 593–599. 86. Meyer, B. A multicenter study of tinnitus. Epidemiology and therapy. Ann Otolaryngol Chir Cervicofac, 1986, 103: 185–188. 87. DeFeudis, FV. Effects of Ginkgo biloba extract (EGb761) on gene expression: possible relevance to neurological disorders and age-associated cognitive impairment. Drug Dev Res, 2002, 57:214–235. 88. NIH. Basic and preclinical research on complementary and alternative medicine. PA-02-124. 2002. 89. Normile, D. Asian medicine. The new face of traditional Chinese medicine. Science, 2003, 299: 188–190. 90. McLean, CA, Cherny, RA, Fraser, FW, Fuller, SJ, Smith, MJ, Beyreuther, K, Bush, AI, and Masters, CL. Soluble pool of Abeta amyloid as a determinant of severity of neurodegeneration in Alzheimer’s disease. Ann Neurol, 1999, 46: 860–866. 91. Morishima-Kawashima, M and Ihara, Y. The presence of amyloid beta-protein in the detergent-insoluble membrane compartment of human neuroblastoma cells. Biochemistry, 1998, 37: 15247–15253. 92. Walsh, DM, Tseng, BP, Rydel, RE, Podlisny, MB, and Selkoe, DJ. The oligomerization of amyloid beta-protein begins intracellularly in cells derived from human brain. Biochemistry, 2000, 39: 10831–10839. 93. Yao, Z, Drieu, K, and Papadopoulos, V. The Ginkgo biloba extract EGb 761 rescues the PC12 neuronal cells from beta-amyloid-induced cell death by inhibiting the formation of beta-amyloid-derived diffusible neurotoxic ligands. Brain Res, 2001, 889: 181–190. 94. Driscoll, M and Gerstbrein, B. Dying for a cause: invertebrate genetics takes on human neurodegeneration. Nat Rev Genet, 2003, 4: 181–194. 95. Wu, Z, Smith, JV, Paramasivam, V, Butko, P, Khan, I, Cypser, JR, and Luo, Y. Ginkgo biloba extract EGb 761 increases stress resistance and extends life span of Caenoraibditis elegans. Cell Mol Biol, 2002, 48: 725–731. 96. Smith, JV and Luo, Y. Elevation of oxidative free radicals in Alzheimer’s disease models can be attenuated by Ginkgo biloba extract EGb 761. J Alzheimers Disease, 2003, 5: 287–300. 97. Strayer, A, Wu, Z-X, Christen, Y, Link, CD, and Luo, Y. Expression of small heatshock protein Hsp16-2 in Caenorhabditis elegans is suppressed by Ginkgo biloba extract EGb 761. FASEB J, 2003, 17: 376. 98. Christen, Y. Current Topics in Nutraceutical Research, 2003, 1: 41–49.

Brain Aging, 24 Nutrition, and Alzheimer’s Disease Yafei Zhang and Rena Li

L.J. Roberts Center for Alzheimer’s Research Sun City, Arizona

CONTENTS Abstract ............................................................................................................410 24.1 Introduction .............................................................................................410 24.2 Nutrition and Brain Aging ......................................................................411 24.2.1 Caloric Restriction ......................................................................411 24.2.2 Essential Nutrients ......................................................................413 24.2.2.1 Fatty Acids ...................................................................413 24.2.2.2 Vitamins .......................................................................414 24.2.2.3 Amino Acids ................................................................416 24.2.2.4 Minerals .......................................................................418 24.2.3 Nonessential Nutrients ................................................................419 24.2.3.1 Phosphatidylserine .......................................................419 24.2.3.2 Phytoestrogens .............................................................419 24.2.3.3 Antioxidants ................................................................420 24.3 Nutrition and Alzheimer’s Disease .........................................................422 24.3.1 Caloric Restriction ......................................................................422 24.3.1.1 Human Studies ............................................................422 24.3.1.2 Animal Studies ............................................................423 24.3.2 Fatty Acids ..................................................................................423 24.3.2.1 Human Studies ............................................................423 24.3.2.2 Animal Studies ............................................................424 24.3.3 Vitamins and Homocystiene .......................................................424 24.3.3.1 Human Studies ............................................................424 24.3.3.2 Animal Studies ............................................................426 24.3.4 Minerals ......................................................................................426 24.3.4.1 Human Studies ............................................................426 24.3.4.2 Animal Studies ............................................................427 24.3.5 Phosphatidylserine ......................................................................427

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24.3.5.1 Human Studies ............................................................427 24.3.5.2 Animal Studies ............................................................428 24.3.6 Phytoestrogen ..............................................................................428 24.3.6.1 Human Studies ............................................................428 24.3.6.2 Animal Studies ............................................................429 24.4 Recommendations ...................................................................................429 24.5 Conclusion ..............................................................................................430 References ........................................................................................................431

ABSTRACT Neurodegenerative diseases are increasing as life expectancy increases. During the past century, human life expectancy has almost doubled. Alzheimer’s disease (AD) is one of the neurodegenerative diseases and is the most common cause of dementia in patients over 60 years of age. It is characterized by progressive degradation of cognitive function. It is a disease that affects one of every ten individuals over the age of 65 and 50% of individuals over the age of 85. The estimated number of individuals with AD could be over 14 million by the year 2050 unless a clear understanding of AD pathogenesis along with new methods of prevention and treatment is developed. Although aging is a natural process and associated with many variables, such as genetics, the environment, stress, lifestyle factors, many aging processes can be controlled by diet. It is a well-known fact that reduced amounts of certain dietary nutrients are associated with memory loss and other thinking problems, especially in older individuals. Also reduced levels of vitamins C and E have been associated with increased severity of AD. High intake of cholesterol and saturated fats is also associated with an increased risk of AD. In this chapter, we review general dietary essential and non-essential nutrients, including various resources and their biological functions. Further, we discuss how dose nutrition affects brain function and aging. Lastly, we gather evidences on how to “eat right” to prevent aging and AD by adopting a nutritional approach. We believe that this exciting and intriguing information will stimulate great interest in healthy diet research.

24.1 INTRODUCTION During the past century, human life expectancy has almost doubled, leading to a burgeoning aged population in virtually all parts of the world. An immediate need associated with this increase is to maintain a high quality of life and a healthy body and mind, and treat and ultimately prevent aging-related diseases. There are both physical and mental changes that occur normally with aging. For example, overall cognitive function, learning and memory in particular, show a major decline with increasing age, representing just a few of the major complaints of the aging population. In addition, it has been well documented that

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aged individuals are at an increased risk of developing cardiovascular disease, stroke, osteoporosis, and Alzheimer’s disease (AD).1,2 Although the fundamental causes of human aging and aging-related diseases remain unknown, a strong consensus is building that the interaction of genetics and environment is the foundation for aging and, indeed, for human health and disease. Nutrition is a key environmental factor, and gene-nutrient interaction and its role in preventing diseases and the process of aging is one of the most attractive research areas in the 21st century.3,4 It is imperative that nutrition scientists cooperate with scientists in other disciplines to determine the relationships among dietary factors, the process of aging, and the development of diseases.5,6 In this chapter, we will discuss a few major age-related topics, including caloric restriction (CR), some essential nutrients and nonessential nutrients, and their roles in the process of normal brain aging and in AD.

24.2 NUTRITION AND BRAIN AGING Nutrients are substances in food that provide structural and functional components or energy to the body. They are grouped into three classes: substances that generate calories from food intake and metabolism; essential substances that must be obtained from the diet, because the body cannot make them in sufficient quantity to meet basic growth and/or maintenance needs (e.g., fatty acid and vitamins); and nonessential nutrients that are not required for human life, but are important for maintaining good health (e.g., fiber and phytonutrients). Every nutrient and dietary factor has its own complex function and association with human health and disease. In this chapter, we will focus on the three major classes of nutrients and their functional association with normal brain aging and AD.

24.2.1 CALORIC RESTRICTION Calorie intake greatly impacts metabolism and even longevity. Disruption of normal calorie intake is involved in many diseases, in particular, some agingrelated diseases.7 For example, epidemiological findings suggest that high-calorie diets increase the risk of AD and Parkinson disease. Interestingly, findings from animal models of these disorders have shown that dietary restriction (reduced calorie intake or intermittent fasting) can reduce neuronal damage and improve behavioral outcome.8 The observation that reducing calorie intake can positively change the brain and behavior has led to the idea of caloric restriction as a simple “life extender.” Caloric restriction refers to a diet high in nutrients but very low in calories, typically about 30% lower than the accepted maintenance level of calories. Caloric restriction is by far the most studied “anti-aging” method and clearly extends longevity in many species. It has been well documented that chronic CR delays the onset of many age-related diseases by suppressing oxidative damage to proteins, lipids, and DNA in several organ systems. Caloric restriction attenuates the development of many

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physiological expressions of aging.9 Current research on CR suggests that its use may extend the maximum life span in rodents and nonhuman primates.10–12 It is clear that the effects of CR depend mainly on the limitation of calorie intake. Recent findings indicate that CR may have a profound effect on brain function and vulnerability to injury and diseases by stimulating the production of new neurons and enhancing synaptic plasticity.13–17 Although recent studies using rodents have revealed cellular and molecular mechanisms underlying the beneficial effects of dietary restriction on the brain (see Table 24.1), a long-term investigation of CR and health to further advance our understanding of the effectiveness of CR in aging is needed. The benefits of CR have been assumed to result from a reduction in cumulative calorie intake, but recent studies suggest that periodic fasting (alternate-day feeding) is more effective than limited daily feeding (fast daily).18,19 These studies tested various physiological parameters and neuronal vulnerability to excitotoxicity in two groups of C57BL/6 mice, one group undergoing alternate-day feeding and the other undergoing limited daily feeding. Periodic fasting was more effective than limited daily feeding in protecting hippocampal neurons against excitotoxic injury. Future studies will need to focus on two outcomes of CR: reduction of oxidative stress and improved glucoregulation. These outcomes of CR will need to be examined in nonhuman primates prior to any future studies involving humans.20

TABLE 24.1 Caloric Restriction and Brain Aging Subjects Rats

Rats Presenilin 1 mutant knocking mice

Mice Male Fischer 344 rats

Findings CR exhibited increased resistance of hippocampal neurons to degeneration. It increased the production of neurotrophic factors [brain-derived neurotrophic factors] in many regions of brain Neuroprotective effect, stimulated neural stem cells in brain to reproduce new nerve cells CR decreased the vulnerability of hippocampal and cortical neurons to excitotoxicity and apoptosis. It exhibited reduced excitotoxic damage to hippocampal CA1 and CA3 neurons compared to mice fed ad libitum. The increase in levels of the lipid peroxidation product 4-hydroxynonenal following the excitotoxic insult was lower in CR mice CR induced the production of protein chaperones, heat-shock protein 70, glucose-regulation protein 78 Attenuated the increase in specific DNA fragmentation indicative of apoptosis with age and the increases in cytosolic cytochrome C and caspase-2 activity with age, suppressed the age associated rise in cleaved caspase-9 and cleaved caspase-3

Reference(s) [13–15]

[16] [122]

[106] [105]

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It was recommended that future studies should also focus on the long-term effects of compounds known to lower circulating glucose and insulin concentrations or should increase insulin sensitivity.20 One study on aged rhesus monkeys showed that chronic dietary restriction increased insulin-stimulated glucose uptake.21 The authors concluded that dietary restriction may be able to prevent Type II diabetes. Other studies of CR using rats as subjects concluded similarly. In rats, 8 months of CR did not cause a decrease in glucose uptake in certain tissues such as the kidney, lung, spleen, and brain. Interestingly, insulin-sensitive tissues such as white adipose did show increased glucose uptake after 8 months of CR.22

24.2.2 ESSENTIAL NUTRIENTS Essential nutrients are classically defined as those substances that must be obtained from the diet because the body cannot produce them in sufficient quantities to meet its needs. There are over 40 different types of essential nutrients, which can be categorized into six groups: water, vitamins, minerals, amino acids, fatty acids, and some carbohydrates. The dramatic difference in the required amounts of certain nutrients gives rise to the concept of macronutrient (many grams of nutrients required daily) and micronutrient (milligrams of nutrients required daily). Macronutrients include carbohydrates, proteins, fats, macrominerals, and water. Micronutrients include vitamins and trace minerals. Essential nutrients play a very important role in human life, including aging and agingrelated diseases. In both healthy and disease states, individuals must take in adequate quantities of all essential nutrients. 24.2.2.1 Fatty Acids Fatty acids are believed to be disproportionately involved in healthy aging and aging-related diseases. For example, decosahexaenoic acid (DHA) is a docosahexaenoic acid, commonly known as ω -3 fatty acid. The best sources of DHA are seafood, algae, and especially cold water fish. Popular sources of DHA are salmon, mackerel, herring, sardines, and tuna, although chicken, eggs, and organ meats also have small amounts of DHA.23 Decosahexaenoic acid participates in numerous cellular functions, including membrane fluidity, membrane enzyme activities, and eicosanoid syntheses;23 eicosanoid synthesesare essential for brain development in infants and is also required for maintaining normal brain function in adults.24 Decosahexaenoic acid can also improve vascular endothelial function and plays a role in reducing blood pressure, platelet sensitivity, and serum triglyceride level. Although DHA can be synthesized in the body from α-lenolenic acid, the capacity for synthesis through this route declines with age. The brain is rich in lipids and fatty acids. It contains roughly 25% DHA, 25% stearic acid, 14% arachidonic acid, and 12% oleic acid. Experimental research on DHA (ω -3 fatty acids) in both animals and humans has demonstrated the effect of fatty acids on the structure and function of the brain. Decosahexaenoic acid is essential for brain development and maintenance of normal functions, specifically by altering membrane fluidity, membrane enzyme activities, and eicosanoid

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syntheses.25,26 In aging rats, a decline of DHA concentration in hippocampus has been reported.27,28 This age-related DHA deficiency can be restored by DHAenriched supplement.29 Furthermore, feeding aged rats fish oil with 11 to 27% DHA content induced alterations of 23 different gene expressions, one of which is called transthyretin (TTR), a known amyloid β (Aβ ) protein scavenger.27,30,31 Fish oil also alters brain phospholipid composition and shows neurochemical and structural marker changes in frontal cortex.27,32,33 Aging also affects behavioral and neurological function, manifesting as memory loss, depression, and impairment of hearing and vision. These changes are linked with DHA deficiency or low levels of DHA intake.34,35 In addition, low DHA levels might be associated with hostility and aggression,23,24 two behaviors sometimes seen in AD patients. Sujimoto and colleagues36 examined the effect of DHA-fortified oil on memory performance in aged mice and found that high brain DHA content in the DHA-treated mice was significantly associated with improvement in working memory. 24.2.2.2 Vitamins Vitamin C, also known as ascorbic acid, is a water-soluble vitamin found mainly in fresh fruits and vegetables. The best-characterized function of vitamin C is its participation in the synthesis of collagen connective tissue protein, specifically the hydroxylation of prolyl and lysyl residues of procollagen.37,38 Vitamin C also plays important roles in the synthesis of neurotransmitters, steroid hormones, carnitine, and conversion of cholesterol to bile acids, tyrosine degradation, and metal ion metabolism.37,39 This vitamin may enhance iron bioavailability.37,39 As a highly effective antioxidant, it has long been accepted that a diet rich in vitamin C from fruits and vegetables provides protection against cancer, heart disease, eye diseases, and neurodegenerative conditions.38 Vitamin E is a fat-soluble vitamin that exists in eight different forms, alpha-tocopherol (α-tocopherol) being the most active form in humans. Vitamin E is found in margarine and vegetable oil (soybean, corn, safflower, and cottonseed), wheat germ, and green leafy vegetables. As a powerful biological antioxidant, vitamin E boosts the immune system, and may help reduce the risk of some of the most common diseases, such as heart disease and AD40,41 when included in the diet. Vitamin E is also vital for healthy skin and hair, and may reduce the risk of cataracts.42,43 In contrast to vitamin E, vitamin B6 is a water-soluble vitamin that exists in three major chemical forms: pyridoxine, pyridoxal, and pyridoxamine.44 It participates in a wide variety of functions in the body and is required as a cofactor for more than 100 enzymes involved in protein metabolism. It is essential for red blood cell metabolism and the nervous and immune systems. For instance, patients with mild cognitive impairment have higher levels of homocysteine in serum, and vitamin B6 can normalize the serum homocysteine levels and reduce the ratio of tau protein and the albumin, a vascular risk factor for dementia.45,46 A wide variety of foods are rich in vitamin B6, including fortified cereals, beans, meat, poultry, fish, and some fruits and vegetables.47

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Vitamin B12 contains cobalt, and so is also known as cobalamin. It is exclusively synthesized by bacteria and is found primarily in meat, eggs, and dairy products. Vitamin B12 is necessary for the synthesis of red blood cells, the maintenance of the nervous system, and growth and development in children.48 A deficiency of vitamin B12 is commonly found in the elderly population.49 Folic acid is a water-soluble vitamin of the B-complex group and is necessary for the production of red blood cells and for the synthesis of DNA.50 Its main food sources are beans, citrus fruits, whole grains, and dark green leafy vegetables. Vitamins B6 and B12 and folic acid are all involved in methionine and then homocysteine metabolism. Vitamin B6 is a cofactor for cystathionine β synthase, which mediates the transformation of homocysteine to cystathionine. About 50% of homocysteine is remethylated to methionine via steps that require folic acid and vitamin B12. A deficiency of any of these three vitamins leads to modest homocysteine elevation, as well as to diminished renal function, both of which are common in the elderly.51 Among all the vitamins, vitamins E and C may be the most investigated, and the most confusing nutritional supplements for aging, age-related diseases, and for helping the memory and mental decline of aging. Researchers from the National Institute on Aging (NIA) report that elderly people who take vitamin C and E supplements have a 50% lower risk of dying prematurely from disease than do people who do not supplement their diets with these vitamins.52 A study, using the data from the third National Health and Nutrition Examination Survey in the United States, which included 4809 people aged 60 or older between 1988 and 1994, found a connection between poor memory and low blood levels of vitamin E in this elderly population. Overall, 7% of the group had poor memory, and people who ate inadequately or skipped meals also had greater memory loss than those who ate regularly. The study findings link vitamin E from the diet, rather than from supplements, to memory. The researchers report that they found no connection between other antioxidants and memory loss.53 Similar reports can be found in a review of recent literature on the sources of tissue levels and roles of vitamins E and C in cognitive performance and pathologic processes of the central nervous system in the elderly.54 The status of the B-vitamin folic acid and vitamins B12 and B6 is frequently inadequate in the elderly and therefore has been subject to investigation. Evidence of the importance of these vitamins for maintaining the well-being of normal brain function comes from studies of vitamin deficiency states and hyperhomocysteinemia.55 At least two decades ago, it was claimed that elevated levels of homocysteine are an important risk factor for heart disease as is elevated serum cholesterol. A majority of the scientific articles confirmed the dangers of elevated homocysteine levels.55 In addition to contributing to cardiovascular disease, homocysteine may also be detrimental to the brain, since it can act as a toxin to brain cells.56,57 Elevated plasma homocysteine levels have been associated with poor cognition and dementia in cross-sectional and longitudinal studies.58–60 Vitamin B6 is a cofactor in the metabolism of homocysteine through the transsulfuration pathway. The impact of vitamin B6 treatment on homocysteine levels is

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controversial.61 In a case-control study, Kelly and his associates61 found a significant independent association between low levels of vitamin B6 and stroke or transient ischemic attack but not between elevated homocysteine levels and vascular events. A more recent study by Miller and associates62 report elevated plasma levels of homocysteine in AD patients may be more related to vascular disease than AD pathology. Agreement on a possible relationship between serum folate and vitamin B12 status and their impact on cognitive impairment has been mixed. A review by Malouf reports that there seems to be a connection between elevated homocysteine levels and vitamin B deficiency, but there is no evidence that vitamin B supplementation improves cognition.63 This is consistent with the findings from recent systematic reviews of randomized double-blind trials, which have found no evidence of potential benefits of vitamin supplementation64,65 (see Table 24.2). Further research is required to establish whether raised serum total homocysteine is a cause or consequence of disease. 24.2.2.3 Amino Acids Amino acids are also essential nutrients that are important for body development and tissue regeneration. If amino acid intake is insufficient, structural proteins cannot be renewed and aging will occur more rapidly.66,67 Carotenoids are a class of natural, fat-soluble pigments found principally in plants. Some 600 different carotenoids are known to occur naturally, but the best studied and understood carotenoids are α-carotene, lycopene, lutein, zeaxanthine, crytoxanthin, and β -carotene. α-carotene is plentiful in carrots and pumpkin. Lycopene is found in red fruits, such as watermelon, guava, red grapefruit, and especially processed tomatoes. Lutein and zeaxanthin are present in red peppers, pumpkins and dark green vegetables (the yellow-orange pigments are masked by green chlorophyll). Cryptoxanthin is abundant in peaches, mangoes and oranges. β -Carotene is present in all these foods, as well as in sweet potatoes and cantaloupes.68 Evidence from past clinical trials suggests that β -carotene supplementation does not decrease the risk for lung, prostate, colon, breast, and nonmelanoma skin cancers and myocardial infarction.69–79 However, a recent study from Harvard researchers found that higher specific caroteniod intake from the diet may reduce the risk of lung cancer and coronary artery diseases (CAD).80 Using the primary data from seven cohort studies in North America and Europe, Mannisto and his colleagues from the Harvard School of Public Health analyzed the association between lung cancer risk and dietary intake of specific carotenoids. Carotenoid intake was estimated from dietary questionnaires administered at baseline in each study. Study-specific multivariate relative risks (RRs) were calculated and combined with a random-effects model. The multivariate models included smoking history and other potential risk factors. During follow-up of up to 7 to 16 years across studies, 3,155 incident lung cancer cases were diagnosed among 399,765 participants. β -Cryptoxanthin intake was inversely associated with lung cancer risk (RR ⫽ 0.76; 95% confidence interval, 0.67 to 0.86; highest vs lowest quintile), but other

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TABLE 24.2 Essential Nutrients and Brain Aging Nutrients DHA

Vitamin C Vitamin E

Carotenoids

Selenium

Vitamin B6

Vitamin B12

Folic acid

Food Sources Seafood, algae, water fish

Findings

Impairment of memory is accompanied by low brain DHA levels in aged rats. DHA reduced in brains of old rats, but was restored by administration of fish or DHA. DHA-fortified oil improved working memory in mice Fresh fruits and As antioxidants to scavenge free radicals vegetables and may prevent oxidative damage. Vegetable oils, Supplementation of vitamins C and E nuts, green together might yield additional benefits. leafy vegetables Human trials of varying doses of vitamins E and C have found that they may improve immunity, vascular function, and brain performance. Chemicals present in fruits and vegetables, in addition to antioxidants, may be important for aging and brain function Carrots, pumpkin, Beta-carotene supplementation does not red fruits, darkdecrease the risk for some cancers or for green vegetables cardiovascular events, but food rich in carotenoids may have beneficial effects Cereal grains, Antioxidant. Low level of selenium is linked grassland legumes with age and age-related diseases, but garlic, onions, none of the therapeutic studies showed broccoli benefits in environment-associated health disorders to date beans, nuts, A deficiency of any of these three vitamins legumes, eggs, leads to modest homocysteine elevation, meats, fish, while elevated plasma homocysteine whole grains levels have been associated with poor Eggs, meat, poultry, cognition and dementia in cross-sectional shellfish, and and longitudinal studies, in addition to milk and contributing to cardiovascular conditions. milk products There is little evidence to justify treating Leafy greens, dry cognitive impairment with these vitamins; beans and peas, whether these B vitamin inadequacies fortified cereals contribute to brain dysfunction or result and grain products from aging and disease are still in question

References [27–36]

[54,129,131]

[80,81,133]

[89,91]

[55,58,59]

carotenoids (β - and α-carotene, lutein/zeaxanthin, and lycopene) were not. They suggested that although smoking was the strongest risk factor for lung cancer, greater intake of β -cryptoxanthin-rich foods, such as citrus fruit, may modestly lower the risk.80 Harvard researchers also found that a high dietary intake of foods

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rich in β - and α-carotene, but not other carotenoids, reduced the risk of coronary artery disease in women.81 In 1984, using a modified food questionnaire for carotenoid intake, they studied the eating habits and health of more than 73,000 female nurses. During 12 years of follow-up, nonfatal heart attacks or fatal heart disease occurred in 998 of the subjects. Taking into account the possible effects of weight, smoking, and other risk factors, those with the highest intake of β -carotene were 26% less likely to have CAD than those with the lowest intake, and those with the highest intake of α-carotene were 20% less likely to experience CAD. Although high cholesterol and cardiovascular disease are risk factors for dementia in the elderly, there are few studies on carotenoids and age-related dementia. In an attempt to determine the mechanism of β -carotene’s effect on the brain, Santos and his colleagues82 studied Boston-area participants in the Physicians’ Health Study (men aged 65 to 88 years; the mean age was 73 years) who had supplemented their diet with β -carotene (50 mg on alternate days) for an average of 12 years. Participants were enrolled in a randomized, placebo-controlled, double-blind study. The results suggest that elderly men receiving β -carotene supplements had significantly greater natural killer (NK) cell activity than did elderly men receiving placebo. Watzl et al.83 investigated the effects of tomato juice (47.1 mg/d lycopene) consumption on cell-mediated immunity in well-nourished, healthy elderly German persons aged 63 to 86 years. The number and lytic activity of NK cells, secretion of cytokines by activated peripheral blood mononuclear cells, lymphocyte proliferation, and delayed-type hypersensitivity skin responses were used to assess immune status. Consumption of tomato juice for 8 weeks increased plasma lycopene concentrations without significantly affecting cell-mediated immunity in these participants. Corridan et al.84 assessed the immune function of 52 well-nourished, independently living, healthy elderly Irish individuals. After 12 weeks of receiving lycopenen (13.3 mg) or β -carotene (8.2 mg), no significant differences were observed in any parameters among treatments and controls. They concluded that in well-nourished, independently living, healthy elderly individuals, supplementation with relatively low levels of β -carotene or lycopene is not associated with either a beneficial or detrimental effect on several aspects of cell-mediated immunity. From a biochemical viewpoint, the activity of dietary carotenoids in biological systems depends on a number of factors, such as nutritional and peroxide status. While the structure of carotenoids, especially the conjugated double-bond system, gives rise to many of the fundamental properties of these molecules, it also affects how these molecules are incorporated into biological membranes. The effectiveness of carotenoids as antioxidants also depends on their interaction with other coantioxidants, especially vitamins E and C. Thus, carotenoids may lose their effectiveness as antioxidants at high concentrations or at high partial pressures of oxygen.85 24.2.2.4 Minerals Selenium has been intensively investigated as an antioxidant trace element, and for over three decades now has been repeatedly implicated in some human

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diseases.86–88 The selenocompounds in foods can be inorganic or organic. Selenate is the major inorganic selenocompound found in both animal and plant tissues (leaves, stems). Selenomethionine is the major organic selenocompound in cereal grains, grassland legumes and soybeans, as well as in selenium-enriched yeast used for selenium supplementation. Selenium deficiency has been linked to a number of disorders, such as heart disease, diabetes, and diseases of the liver. It is also required for sperm motility and may reduce the risk of miscarriage.89 In 239 healthy elder people, 108 males and 131 females with mean age 73.7 years, plasma selenium decreased significantly with age, but a significant trend was not observed for erythrocyte selenium or for the activity of oxygen-metabolizing enzymes.90 Therefore, Se supplementation has been promoted from nutrition to pharmacological intervention, especially for cancer chemoprevention.89 However, a recent review reported that among 1290 studies only 12 met the inclusion criteria. After being evaluated individually, none of the studies included in the analysis provided evidence of the therapeutic benefits of Se supplementation in environment-associated health disorders.91

24.2.3 NONESSENTIAL NUTRIENTS 24.2.3.1 Phosphatidylserine Phosphatidylserine (PS) is a phospholipid, is hydrophobic, and is an essential component of cell membranes. Phosphatidylserine is highly concentrated in animal brain as well as in foods such as soy and egg.88 For decades, PS has been gaining recognition as a brain function-enhancing supplement. Specifically, PS helps prevent chemical-induced amnesia and improves cognitive and brain chemical functions in young, middle-aged, and aged animals.92,93 Brain-derived PS also produces mild but lasting improvements in the dementia and cognitive decline of AD. Numerous studies have explored the effects of PS on cognition. However, a vast majority of these studies have used injectable PS derived from cow brains, rather than that extracted from soy and egg.94 Owing to concerns about the possibility of humans contracting infectious diseases (such as Creutzfeld–Jakob or “mad cow” disease), the interest to develop plant-derived PS has accelerated. Experimental data suggest that injectable PS derived from soy is as efficacious in its cognitive-producing effects as is PS derived from cow brain, despite being much lower in fatty acid content (brain PS contains the ω -3 fatty acid DHA, which is not found in the soy form).94 However, there is still not enough evidence to support the hypothesis that PS derived from plants can delay brain aging or boost human brain functions (see Table 24.3). 24.2.3.2 Phytoestrogens Phytoestrogens are plant-derived nonsteroidal compounds that possess estrogenlike biological activity. Soy isoflavones are one class of phytoestrogens found in soybeans, and they have both weak estrogenic and weak anti-estrogenic effects. They bind to estrogen receptors α (ER-α) and β (ER-β ).92 Soy isoflavones

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comprise three main glycosylated forms: genistein (~50%), daidzein (~40%), and glycitein (~5 to 10%),95 which are aglycones. Isoflavones have been studied for their potential beneficial effects in treating/preventing hormone-dependent cancers and disease. However, little is known about their influence on brain function and neurodegenerative diseases.94 Recently, Pan et al.96 reported that maintaining retired breeder, ovariectomized rats on an isoflavone-rich diet for 10 months improves cognitive function. There was a specific dose-dependent benefit of soy on working memory as assessed by radial arm maze performance. Lephart et al.97 examined the influence of dietary soy isoflavones on certain aspects of brain structure, learning, memory, and anxiety in perinatal, maternal, and both female and male adult rats; they also assessed the brain estrogen synthesis enzyme aromatase. The volume of the sexually dimorphic nucleus of the preoptic area (SDN-POA) was significantly lesser in male rats on an isoflavone-free diet administered during adulthood compared with the males on an isoflavone-rich diet. Additionally, the isoflavone-rich diet produced enhanced anxiolytic effects in both male and female adult rats. Visualspatial learning and memory in the radial arm maze was better in adult females maintained on an isoflavone-rich diet compared with females on an isoflavonefree diets; male rats showed the opposite pattern of maze performance.97 Another group of researchers reported similar negative effects of dietary soy isoflavone on male rats. Compared with males fed an isoflavone-free diet, males on an isoflavone-rich diet spent less time in active social interactions, performed worse in a maze, and had elevated stress-induced corticosterone and vasopressin concentrations.98 The mechanism of isoflavone action was studied in the context of sulfotransferase and sulfatase enzymes, brain-derived neurotrophic factors, and specific gene expression.99,100 Since results from animal studies indicate that soy may have bidirectional effects that depend on the soy concentration and duration of the diet, it is very important to study the supplement duration and administration level for isoflavone benefits (see Table 24.3). 24.2.3.3 Antioxidants The free radical theory of aging might be one of the most accepted of all aging theories. To date, compelling evidence supports the notion that oxidative and nutritive stresses play important roles in the aging process as well as in the pathophysiology of age-related illnesses.101–103 Some have suggested that oxidation of lipids, nucleic acids, and proteins is involved in the etiology of several chronic diseases, including cancer, cardiovascular disease, cataracts, age-related macular degeneration, and, indeed, aging in general.104 In the normal aging brain, oxidative stress may activate mitochondrial-mediated apoptotic pathways.105 It is known that mitochondria in neurons of aged brain produce more oxidants, accumulate more calcium, and exhibit more overall oxidative damage; all of these are known to be stimuli for apoptosis.105 Furthermore, oxidative stress has been implicated in mechanisms leading to

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TABLE 24.3 Nonessential Nutrients and Brain Aging Nutrients Phosphatidylserine (PS)

Isoflavones

Subjects Rats with memory deficits caused by reserpine Middle-aged rats

120 elderly individuals (>57 years) of both sexes with ageassociated memory impairment Older adults with moderate cognitive impairment Ovariectomized female rats Prenatal, maternal female and male adult rats

Adult male rats

Postmenopausal women (50–65 years), 60 mg daily for 12 weeks (n⫽33) Postmenopausal women (55–74 years), 110 mg daily for 12 months (n⫽56) Postmenopausal women (60–75 years), 99 mg daily for 12 months (n⫽202) Japanese-American men (71–93 years) Cohort study (n⫽3734)

Findings

Reference(s)

Help prevent chemical-induced amnesia, improve cognitive and brain function

[92]

Mild and lasting improvements in brain function, metabolism and behavior in animals 300–600 mg soybean-derived PS supplement for 6–12 weeks does not affect memory or other cognitive function in old individuals with memory complaints Only one small sample of older adults out of six studies seemed to improve memory Beneficial effect on cognitive function

[94,101]

The brain volumes of sexually dimorphic regions of male rats on isoflavonesfree diet were smaller; isoflavone-rich diet produced elevated anxiolytic effects on both male and female rats. Learning and memory better in females on isoflavone-rich diet, but opposite effect in males Less time iteration, lower performing in maze, elevated stress-induced corticosterone and vasopressin in male rats with isoflavone-rich diet Improvements in learning and planning tasks

[97]

[154]

[153]

[96]

[98]

[155]

Improved some brain functions

[157]

No effect on cognitive function, BMD, or plasma lipids

[158]

Poor cognitive performance, ventricles enlarged, and low brain weight was associated with higher midlife tofu consumption

[160]

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Oxidative Stress and Age-Related Neurodegeneration

various pathological states of the brain.106 Therefore, reduction of oxidative stress is a reasonable target for anti-aging strategies and for age-related diseases. Nutrients, both vitamins and trace elements, comprise an important aspect of the antioxidant defense system. In recent years, there has been an increasing interest in dietary intervention for prevention or treatment of various disorders, including dementia.107–109 Many compounds with antioxidant properties, such as vitamins C and E, carotenoids, and the trace element selenium, can supplement the diet to defend against oxidant damage.107,109 Epidemiological data indicate that an inadequate dietary supply, unbalanced/poor nutrition, and increased oxidative stress in the elderly are correlated with increased risk for neurodegenerative disease.108 However, little is known about the underlying mechanisms of nutrient action on aging brain and neurodegenerative disease because the oxidative theory of pathological brain aging is supported primarily by animal laboratory experiments. Further studies are needed to identify the specific role of various nutrients in aging, such as the interactions between different nutrients, as well as the influence of genetic factors and living habits on cerebral aging and dementia. This should be undertaken before a nutritional intervention is recommended. Such an intervention must be based on evidence of benefit, since high doses of some vitamins, like vitamin E, and trace elements could be seriously harmful.110,111

24.3 NUTRITION AND ALZHEIMER’S DISEASE Alzheimer’s disease is responsible for the most common dementia observed in the elderly and is characterized by impaired cognitive function and severe brain pathology. According to a new study published in the August 2003 issue of the Archives of Neurology, the number of people who will suffer from AD is projected to triple in the next 50 years. While the prevalence of AD is increasing with longer life expectancy, no environmental risk factors have been identified with certainty.112 In this section, we will present evidence — both from current animal and human studies — on how nutrition affects the progression of AD, and will discuss possible dietary recommendations for preventing AD.

24.3.1 CALORIC RESTRICTION 24.3.1.1 Human Studies Overeating is not only a major modifiable risk factor for several age-related diseases, including cardiovascular disease, type 2 diabetes mellitus, and some cancers, but might also be a risk factor for AD.113–116 According to a recent Swedish study, overweight women in their 70s were significantly more likely to develop AD between the ages of 79 and 88. The women who developed AD were much heavier and had a greater body mass index than did normal controls.117 Although this study did not directly examine calorie intake, it is known that type 2 diabetes is strongly associated with AD, and the resulting high blood sugar is a

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major calorie resource in CR.118 Another study followed 980 subjects over 4 years in the context of AD incidence; the mean age of the subjects was 75.3 years. Hazard ratios for AD in each quartile of caloric intake were calculated. After adjusting for age, sex, APOE genotypes, years of education, and ethnic background, those subjects in the highest quartile of caloric intake had a significantly increased risk of being diagnosed with AD compared to subjects in the lowest quartile. The study also reported that the higher the intake of calories and fats, the higher the risk that AD subjects carried the APOE e4 allele.119 A longitudinal study of a total of 4606 individuals with high- or low-calorie intake showed thatthe former commonly had higher age-adjusted incidence of dementia.119 Another prospective study of a large cohort of New York residents revealed that the individuals with low-calorie intake had significantly lower risk for AD and Parkinson’s disease than those with higher calorie intake. In addition, the risk is more directly related to calorie intake than to body mass index.119–121 24.3.1.2 Animal Studies Calorie or dietary restrictions have been shown to affect the risk of developing neurological diseases such as Parkinson’s disease and AD. While there is emerging data showing that CR modifies the pathology in a variety of certain animal models of disease, few studies have been done using animal models designed to mimic AD. Recently, transgenic mice expressing the disease causing mutations related to AD have been used to assess how CR might affect the progression of AD. One group of investigators assessed the effects of CR in transgenic mice with a presenilin-1 (PS1) mutation. Presenilin-1 is an integral membrane protein in the endoplasmic reticulum, and mutation of PS1 has been linked to AD. When mutated, this protein can cause increased neuronal vulnerability to apoptosis (programmed cell death); however, caloric restriction may reduce or protect against the neurodegenerative effects of this mutation.122 With further studies to confirm these results in other AD-related animal models, these results show promise as a simple way to treat AD.

24.3.2 FATTY ACIDS 24.3.2.1 Human Studies A link has been established between low levels of DHA and certain behavioral and neurological conditions, specifically AD, depression, memory loss, and attention-deficit hyperactivity disorder (ADHD). Morris and colleagues123 from the Rush Institute for Healthy Aging in Chicago examined 815 elderly individuals to determine whether an association exists between consuming fish and developing AD. Using a model adjusted for age and other risk factors, they showed that individuals who consumed fish once per week or more had a 60% lower risk of developing AD compared with those who rarely or never ate fish (RR, 0.4; 95% confidence interval, 0.2 to 0.9). A reduced risk of developing AD was also associated with the intake of n–3 polyunsaturated fatty acids as well as DHA.123

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Kalmijn et al. of the University Medical Center Utrecht, the Netherlands, found in a population of middle-aged Dutch subjects that consumption of fatty fish and marine ω -3 polyunsaturated fatty acids reduced the risk of developing impaired cognitive function.124 This conclusion was derived from data obtained from a cross-sectional population-based study of 1613 subjects, aged 45 to 70 years, who completed a self-administered food-frequency questionnaire. 24.3.2.2 Animal Studies Research on DHA is now focused on its effects on age-induced alteration in gene expression and molecular composition of the brain.27,30 Dr. Cole and his team found that elevated dietary DHA protects neurons in transgenic mice expressing the human mutant amyloid precursor protein (APP).125 Transgenic APP mice aged 17 months that were fed a DHA-poor diet had decreased levels of DHA in frontal cortex compared to wild-type (WT) control mice. The increased oxidative stress in APP mice related to depleted DHA is accompanied by several kinds of neuronal changes that are similar to the changes found in AD brains. These include postsynaptic caspase-mediated actin cleavage, loss of the actin-regulating dendritic spine protein drebin, and the postsynaptic density protein-95 (PSD-95). In addition, APP mice on the DHA-poor diet had memory performance deficits compared to APP mice on a standard diet. These performance deficits were prevented by dietary DHA supplementation. Their findings provide evidence for the idea that the combination of genetic and environmental risk factors, for AD such as DHA deficiency, can act synergistically to cause loss of synaptic proteins required for cognition. Another study using an AD rat model showed that DHA may prevent cognitive deficits that are usually associated with AD.126 These results suggest that patients with a genetic risk of AD may be more vulnerable than others to a lack of DHA, and increasing DHA intake should be considered as a potential neuroprotective strategy for AD.125,126

24.3.3 VITAMINS AND HOMOCYSTIENE 24.3.3.1 Human Studies The use of vitamin supplementation, especially vitamin E, has been under intense investigation, as reports have noted high mortality rates when supplementation exceeds 400 IU/d.110 One study supporting the use of vitamin supplementation examined 5383 subjects aged at least 55 years and free of dementia. Dosages of vitamins C and E exceeding 15.5 mg/d decreased AD incidence by 18% when comparedwith dosages of less than 10.5 mg/d.127 Another study corroborated this finding in 815 people aged 65 years or older who were free of dementia. This study showed a consistent reduction in AD incidence with increasing intake of dietary vitamin E; dosages up to 50 IU/d were assessed.128 Those subjects taking greater than 10.4 IU/day showed a remarkable 70% reduction in AD incidence when compared with those subjects taking less than 7.9 IU/d.129 However, intake

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of vitamins E and C and β -carotene supplements was not significantly associated with AD risk in this study.129 The association between total intake of vitamin E and reduced cognitive decline with age was confirmed by another report, while no evidence of beneficial effects of vitamin C or β -carotene intake was found.129 Masaki et al.54 found that elderly men who took vitamins E and C supplements at least once a week for over more than 1 year were protected from vascular or mixed dementia, but not the dementia associated with AD, although some improvements were noted in cognitive function. This same group observed the inconsistent result from the data of the Honolulu-Asia Aging Study. After adjustment for sociodemographic and lifestyle factors, cardiovascular risk factors, other dietary constituents, and APOE e4, the dietary intake of β -carotene, flavonoids, and vitamins E and C at midlife did not modify the risk of late-life dementia nor its most prevalent subtypes.130 Dr. Zandi and his colleague131 found that the combination of vitamins E and C reduced the prevalence and incidence of AD. A similar finding was reported by Grodstein et al.132 who reported that in women, the long-term, combined use of vitamins E and C resulted in significantly better cognitive performance than did the use of vitamins E or C alone. However, other researchers did not find a relationship between antioxidant vitamin intake and risk of AD. For 4 years Luchinger et al. followed 980 elderly subjects who did not have evidence of dementia at the beginning of the study. With the use of a semiquantitative food-frequency questionnaire and Cox proportional hazards regression model, they reported that there were 242 incident cases of AD in 4023 person-years of follow-up (6 per 100 person-years). Intake of carotenes and vitamin C, or vitamin E in supplemental form or through the normal dietary (nonsupplemental) route or through both, was not related to a decreased risk of AD. Trend tests for the association between quartiles of total intake of vitamins C and E were also not significant.133 Vitamin B is another vitamin that has implications for AD. Lower concentrations of vitamin B12 and folate and higher concentrations of homocysteine are associated with poor memory. Furthermore, people with low levels of vitamin B12 or folic acid may have a higher risk of developing AD.134 On the basis of this research that demonstrated associations between folate, B12, and B6 and cognition and mood, the effects of vitamin B supplementation were investigated. Elevated homocysteine levels may precede the onset of AD. Seshadri et al.135 examined the relationship of plasma total homocysteine level and AD using samples from the Framingham Study; 1092 subjects without dementia initially were studied. Over a median follow-up period of 8 years, dementia developed in 111 subjects, with 83 being diagnosed with probable AD. After adjusting for age, sex, apolipoprotein E genotype, and vascular risk factors, they found that with a plasma homocysteine level greater than 12 µmol/L, the risk of AD was nearly doubled and the relative risk of AD was 1.6 per increase of 1 SD of homocysteine level.136 Folic acid deficiency could cause hyperhomocysteine and a study in AD found a reduction of folic acid level in serum.135 A study of 211 healthy women receiving daily 750 µg of folate, 15 µg of vitamin B12, and 75 mg of vitamin B6 or a placebo daily for 35 days found that the short-term supplementation had a

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Oxidative Stress and Age-Related Neurodegeneration

significant positive effect of some measures of memory performance only, and no effect on mood.137 A controversial result was reported by Deijen and his colleagues. One of each of 38 pairs of healthy men aged 70 to 79 years received 20 mg of vitamin B6 per day for 12 weeks and the other was given placebo. No statistically significant differences in cognition or mood between treatment and placebo were found.138 In contrast, evidence of the efficacy of vitamin B12 in improving cognitive function of people with dementia is limited and was restricted to a small number of patients.139 A review that examined all doubleblind, placebo-controlled, randomized trials found no benefit from folic acid with or without vitamin B12 on any measures of cognition or mood for healthy or cognitively impaired or demented people.139 Folic acid plus vitamin B12 has been reported to be effective in reducing serum homocysteine concentrations, and no adverse effects were noted in this report.63 24.3.3.2 Animal Studies Research has indicated that oxidative stress may play a role in the pathogenesis of AD. Research has begun to intensely focus on the effect of antioxidants in foods and supplements on AD animal models.140–142 A study involving vitamin E and transgenic mice overexpressing human APP (Tg2576 mice) demonstrated a possible use of vitamin E supplementation for altering AD pathology.143 Tg2576 mice administered an aluminum-rich diet had higher oxidative stress levels and more Aβ deposition than Tg2576 administered a standard diet. Vitamin E supplements added into the chow of Tg2576 mice on the aluminum-rich diet reduced levels of oxidative stress as well as Aβ formation, comparable with that measured in the Tg2576 mice on the standard diet. These findings support the notion that oxidative stress plays a role in producing the pathology in AD mouse models and point to the need for further investigations of antioxidant supplementation. Homocysteine may also play a role in the pathogenesis of AD. While the mechanisms of elevated homocysteine and impaired one-carbon metabolism related to cognitive decline are unclear, it is becoming increasingly clear that its neurotoxicity may involve multiple, divergent routes. For example, homocysteine may produce more DNA damage and increase insult-induced cytotoxicity to neurons. These insults include high levels of glutamate, kinase hyperactivation, and Aβ, one of the major peptides found in AD brains.144,145 Further animal studies are needed to clarify the role of homocysteine in AD pathogenesis.

24.3.4 MINERALS 24.3.4.1 Human Studies Although variations in Se levels in the blood and brain have been reported in individuals with AD and brain tumors, few indications of the protective or therapeutic effects of this trace element have been found.146 Researchers in France examined Se levels along with seleno-dependent glutathione peroxidase (GSHPx) and glutathione reductase activities in 40 patients with dementia of the

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Alzheimer-type (DAT) and in 34 aged control subjects with normal cognitive function. They found a negative association between age and plasma GSH-Px activity and plasma Se levels only in the DAT group (P ⬍ 0.01).147 In a study comparing cerebrospinal fluid (CSF) and serum Se levels in 27 AD patients (mean age 73.4 years) and 34 age matched controls, no correlation was found between CSF and serum Se levels in AD patients and age, age of onset, disease duration, or performance on the MiniMental State Examination.148 Moreover, no significant differences were found among the AD patients and age-match controls.148 Se appears to play some kind of role in AD, but more studies are needed to better understand the role of Se in AD pathology and its mechanism of action. Other minerals that may be associated with AD are Zn, Al, Fe, and Cu.149–150 Previous studies have established the role of each of these metals in AD pathogenesis; however, data from these studies have been based mainly on cell cultures and animal models.150 Further studies are needed to elucidate the roles of these minerals prior to examining them in human trials. 24.3.4.2 Animal Studies Zn, Al, Fe, and Cu have not only been detected in AD patients, but have been found to colocalize in AD amyloid plaques.151 Investigations of the roles that each of these metals plays in the pathogenesis of AD have been based on AD cell and animal models. One study investigated the fibrillization of Aβ 42 in the presence or absence of these metals.151 Aβ 42 formed β -pleated sheets of plaque-like amyloid in the absence or presence of Fe and Al; however, Zn and Cu appeared to inhibit or prevent this action. Interestingly, this study also showed that adding chelating reagents like desferrioxamine (DFO) and ethylenediaminetetraacetic acid (EDTA) dissolved the plaque-like amyloid formed in the presence of Fe and Al. This additional information may lead to possible AD therapeutics involving chelation.151 Se has also been linked to AD, but little is known about its role in AD pathology.152 Future studies involving both AD animal models and Se could help elucidate the mechanisms of how this mineral is involved in AD pathology.

24.3.5 PHOSPHATIDYLSERINE 24.3.5.1 Human Studies Although modest memory increases have been reported after treatment with phosphatidylserine (PS), there is still not enough evidence to support the premise that PS can boost brain function in humans.153 Jorissen et al.154 reported the results of a clinical trial examining the effects of soybean-derived PS (S-PS) in aged subjects with memory complaints. In this trial, 180 elderly individuals (aged ⬎57 years; both male and female) with age-associated memory impairment (AAMI) or ageassociated cognitive decline were treated with a placebo or 300 or 600 mg of S-PS daily, and then tested for learning and memory function (e.g., choice reaction time test, planning and attention function test). No significant differences were found in

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any of the outcome variables among the treatment groups, indicating that a daily supplement of S-PS does not affect memory or other cognitive functions in older individuals with memory complaints.154 24.3.5.2 Animal Studies Studies have found that PS administration results in mild and lasting improvements in various parameters of brain function, metabolism, and behavior.92 There are currently a limited number of studies showing PS effects in an AD animal model.

24.3.6 PHYTOESTROGEN 24.3.6.1 Human Studies The benefits of isoflavones on cognitive function in women have been shown in a few randomized control trials. The finding that young volunteers on a high soy diet displayed improved memory and frontal lobe function prompted File and colleagues155 to investigate the effect of isoflavone supplements (60 mg isoflavones/day for 12 weeks) on 33 postmenopausal women (50 to 65 years). The isoflavone group showed significantly greater improvements in learning rule reversals and greater improvement in a planning task compared with the placebo group.156 The beneficial action of isoflavones on learning and memory was further confirmed by Kritz–Silverstein’s group. They found a significantly greater improvement in category fluency as well as greater improvements in story recall, and in a planning task in women (aged 55 to 74 years) who received soy supplements (110 mg isoflavones/day for 6 months) compared with those who received placebos.157 However, no improved effects were observed in a long-term, relatively large, double-blind, placebo-controlled, randomized trial. In the Netherlands, Kreijkamp-Kaspers and colleagues158 examined the effect of dietary isoflavones on 202 healthy postmenopausal women (aged 60 to 75 years). The women were randomly assigned to receive either 25.6 g of soy protein containing 99 mg of isoflavones or the placebo for 12 months. The soy protein supplements had no effect on cognitive function, bone mineral density (BMD), or plasma lipids of these aged women. These findings do not support the hypothesis that, when started in women aged 60 years or older, dietary, soy protein supplements containing isoflavones improve cognitive function, bone mineral density, or plasma lipids in healthy postmenopausal women. In contrast to findings in women, a double-blind, placebo-controlled study showed that men on a high-soy (100 mg/day isoflavones) diet for 10 weeks displayed improved cognitive function. In this study, both young (25.5 years) men and women were randomly allocated to either high (100 mg/day isoflavones)-or low (0.5 mg/day)-soy-content supervised diets. The groups did not differ in age, IQ, education, anxiety, or depression. Both the men and women allocated to the high-soy diet showed significantly greater improvements in episodic memory (immediate recall of a story, short-term recognition of patterns, delayed picture

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recall) and in a task measuring frontal lobe function (rule shifting and reversal).158 In a randomized, placebo-controlled, cross-over trial of 20 men on wheat or soya flour (120 mg/day for 6 weeks), decreased total serum testosterone and improved oxidative stress markers were found.159 Tofu is a soybean food rich in isoflavones and is mostly consumed by Asian people. White et al.160 identified an association between brain aging and midlife tofu consumption by analyzing data from the Honolulu-Asia Aging Study. The subjects of this study were the surviving Japanese-American men of a longevity study. Their tofu consumption was assessed with a dietary intake questionnaire, which was completed by the subjects 25 and 20 years prior to cognitive testing. Poor cognitive test performance, enlargement of ventricles, and low brain weight were found to be significantly and independently associated with higher midlife tofu consumption, although the actual intake level of soy-related isoflavones was unknown. The tofu intake of the subjects’ wives was assumed to be similar to that of their husbands. Interestingly, in cases of presumed high tofu intake by wives, no significant association was found with cognitive impairment in these women. The authors of this study concluded that, in this population, higher midlife tofu consumption was independently associated with indicators of cognitive impairment and brain atrophy late in life. Although the study by White et al. was not controlled and was plagued by many confusing factors, it raised the important possibility that the cognitive effects of a soy diet may depend on the levels of soy isoflavones present in that diet, the duration and timing of the diet, other chemicals in the diet, and, perhaps, gender. 24.3.6.2 Animal Studies The influence of soy isoflavones on certain aspects of brain structure, learning, memory, and anxiety has been examined in rodents, along with the brain aromatase. Researchers from The Neuroscience Center of Brigham Young University (USA) reported significant changes in sexually dimorphic brain regions, and in anxiety, learning and memory between male and female rats after soy isoflavones treatment.152 The mechanism of isoflavone action was also studied in the context of sulfotransferase and sulfatase enzymes, brain derived neurotrophic factors, and specific gene expression.99,100 However, as with PS, soy has not been directly studied in AD animal models.

24.4 RECOMMENDATIONS Although the American Medical Association has officially recognized the benefits of dietary supplements in preventing disease, much more work is needed before we can confidently recommend nutritional interventions for human diseases to the general public. The U.S. Preventive Services Task Force (USPSTF) concludes that there is not enough evidence to recommend or to contest the use of supplements — vitamins A, C, or E; multivitamins with folic acid; or antioxidant combinations — for

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the prevention of cancer or cardiovascular disease. This is a grade I recommendation.161 The American Academy of Family Physicians states, “The decision to provide special dietary intervention or nutrient supplementation must be on an individual basis using the family physician’s best judgment based on evidence of benefit as well as lack of harmful effects. Megadoses of certain vitamins and minerals have been proven to be harmful”.162

The Canadian Task Force on Preventive Health Care is reviewing the role of vitamin E supplements in the prevention of cardiovascular disease and cancer.163 The American Cancer Society recommends a well-balanced diet and does not recommend the use of vitamin and mineral supplements as a preventive or therapeutic intervention.164 The American Heart Association Dietary Guidelines: Revision 2000 recommends that vitamin and mineral supplements should not be considered to be a substitute for a balanced and nutritious diet that is designed to emphasize intake of fruits, vegetables, and grains.165 To help determine whether antioxidants might play a role in preventing AD, or at least in delaying its onset, a number of clinical trials are now being supported by the . These include the Memory Impairment Study, Prevention of AD By Vitamin E and Selenium (Preadvise), Women’s Antioxidant Cardiovascular Study (WACS), Women’s Health Study (WHS), and Physician’s Health Study II (PHS II) (from the NIA’s general information Web site www.nia.nih.gov). In the absence of evidence proving the benefits of essential and nonessential nutrients to brain function and AD, specific dietary interventions cannot be recommended to prevent AD at this time.

24.5 CONCLUSION Searching for approaches to prevent or delay age-related cognitive decline and age-related diseases has become more and more important and urgent. Alzheimer’s disease is the most common age-related dementia. While the genesis of aging and AD still remains unclear, multiple lines of evidence demonstrate a role for dietary factors in extending life span, delaying age-related cognitive decline, and increasing the resistance of neurons to degeneration. For example, calorie intake restriction and periodic fasting have been proven to protect against brain aging Antioxidant nutrients, such as vitamins E and C, carotenoids, and Se, have been found to comprise an important aspect of the antioxidant defense system and may have the specific protective influence on brain functions. Furthermore, dietary folate and vitamins B12 and B6 might help prevent cardiovascular disease, cognitive decline, and dementia, including AD, by regulating the metabolism of homocysteine. Finally, fish oil, rich in DHA, is suggest to lower the risk of middle-aged memory loss and AD. In addition to these essential nutrients, recent studies have suggested that some nonessential nutrients in diet, such as PS and isoflavones, may have beneficial effects in preventing brain aging and AD, but more evidences are needed. In this chapter, we have reviewed major dietray factors, including calorie intake, essential nutrients (antioxidant nutrients,

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B vitamins, unsaturated fatty acid), and nonessential nutrients (phosphatidylserine, isoflavones), and their roles in prevention of aging and AD.

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between 25 Interaction Dietary and Genetic Deficiencies in the Modulation of Homocysteine Elimination Flaubert Tchantchou University of Maryland Baltimore, Maryland

Thomas B. Shea

University of Massachusetts Lowell, Massachusetts

CONTENTS 25.1 Introduction..............................................................................................443 25.2 The Transmethylation Pathway — Folate Metabolism and Homocysteine Elimination ......................................................................444 25.3 The Transsulfuration Pathway — Homocysteine Elimination and Glutathione Metabolism................................................448 References .........................................................................................................451

25.1 INTRODUCTION Homocysteine (HCY) is a neurotoxic nonprotein amino acid that is derived from the metabolism of the essential protein amino acid methionine. Hyperhomocysteinemia represents an independent risk factor in several pathological conditions including cardiovascular diseases, neural tube defects, and dementia of which, Alzheimer’s disease (AD) is the most prominent cause.1,2 Homocysteine levels predict the incidence of AD and other dementias despite other known risk factors such as age, sex, and genetic form of apolipoprotein E (ApoE). Deleterious mechanisms 443

444

Oxidative Stress and Age-Related Neurodegeneration

involving HCY were first studied in the context of cardiovascular disease.3 Because of a growing recognition that cerebrovascular disease may promote AD, ideas taken from studies of HCY and heart disease research are being extended to the brain. This correlation is based on the fact that plasma HCY maybe directly toxic to vascular endothelial cells or induce their dysfunction, leading to loss of blood-brain barrier function and altered production of nitric oxide. In addition, HCY crossing the blood-brain barrier or being released by cells within the brain, could act as a potent neurotoxin.4 Such neurotoxic effects may be due to direct interaction of HCY with plasma membrane components, or due to intracellular accumulation of S-adenosyl-homocysteine (SAH). The latter metabolite inhibits methylation of catechol substrates, resulting in the generation of oxyradicals and other chemically reactive products that are cytotoxic.2,5 Overall, HCY may damage brain tissue through multiple pathways. One of the considerations is the ability of HCY to mobilize, in vitro, the storage of iron from ferritin.6,7 Moreover, sulfhydryl compounds, like HCY, are electron donors in mixed-function oxidation systems, acting with the transition metal ions, iron and copper. In this process, HCY generates hydrogen peroxide within cultured endothelial cells.8 Thus, elevated HCY, by interfering with iron sequestration by ferritin in vivo, would explain the increase in redox-active iron in AD neurons and concomitant oxidative stress,9 subsequently triggering deposition of amyloid plaques in the AD brain. Dietary, genetic, and environmental approaches have been used to experimentally induce oxidative stress and hyperhomocysteinemia in mice. Homocysteine transitionally exists at the intersection of the transmethylation and the transsulfuration pathways, which regulate its elimination.5

25.2 THE TRANSMETHYLATION PATHWAY — FOLATE METABOLISM AND HOMOCYSTEINE ELIMINATION The transmethylation pathway is derived from the intersection of two biochemical pathways, and results in the transfer of a methyl group (CH3) to HCY by either methylcobalamin or betaine to form methionine. These biochemical pathways are the folate metabolic pathway and the methionine metabolic pathway. Folate (folic acid or vitamin B9) is a water-soluble member of the B-vitamin family. It derives its name from the Latin word “folium” for leaf10 and is a carrier of one-carbon fragments, which it transfers to various biochemical targets.11 Folate metabolism starts with its deconjugation in the cells of the intestinal wall to the monoglutamate form. This form is further reduced to dihydrofolate and then to tetrahydrofolate (THF) via the catalytic actions of folate and dihydrofolate reductase enzymes, respectively (Figure 25.1; Reaction 1 and Reaction 2, respectively). Both of these enzymes require NADPH as a cofactor. In the next step in the folate metabolic pathway, a serine molecule combines with pyridoxal5⬘-phosphate to transfer a hydroxymethyl group to THF to form 5, 10-methylene tetrahydrofolate (methylene THF) and glycine in a reaction catalyzed by the enzyme serine hydroxymethyltransferase (Figure 25.1, Reaction 3). Methylene THF is of central importance. It is the precursor of the metabolically active

Interaction between Dietary and Genetic Deficiencies

445 Folate

Diet intake

NADPH + H+

FR; Rx-1

Acetylcholine

NADP+

Dihydrofolate DR; Rx-2

Acetyl-CoA

NADPH + H+ NADP+

ATP PPi + Pi

THF

Methionine

Serine HMT; Rx-3 Glycine

MAT; Rx-6 CH3-Ac CH3

ChaT,Rx15

SAM

5, 10 Methylene SAMT; Rx-7

Betaine

MS/Vit B12; Rx-5 MTHFR; Rx-4

SAH Choline SAHH; Rx-8 Homocysteine Adenosyl

5-MeTHF (Circulating folate)

CBS/Vit B6; Rx-9

Serine Cystathionine

CL; Rx-10 Cysteine GS; Rx-11
-KB H2O2

2H2O GPx; Rx-12

E+P

GSH-E-CDNB

GsT(E); Rx-14 CDNB

GS-SG

GSH GR; Rx-13

NADP+

NADPH + H+

FIGURE 25.1 Pathway regulating homocysteine elimination and glutathione metabolism in the brain.

5-methyltetrahydrofolate (5-MTHF), which is involved in HCY metabolism, and methylidynetetrahydrofolate, involved in purine synthesis and in the generation of thymine side chains for incorporation into DNA.5,12,13. With relevance to the transmethylation pathway, methylene THF produces 5-MTHF in a reaction catalyzed by methylene tetrahydrofolate reductase (MTHFR) (Figure 25.1, Reaction 4). Variations in the gene encoding for that enzyme can result in decreased folate metabolism, which increases HCY levels and the risk of neural tube defects. Patients with congenital MTHFR deficiency have reduced levels of several important

446

Oxidative Stress and Age-Related Neurodegeneration

biological metabolites, such as methionine and S-adenosylmethionine (SAM), in the cerebrospinal fluid (CSF), and show demyelination in the brain, which might be due to decreased methylation.13 Polymorphisms in 5,10-MTHFR that exhibit decreased activity are present in as much as 20% of some populations.14 Diminished activity of this enzyme also reduces production of THF (required for DNA synthesis) and reduced adenosylmethionine (required for DNA methylation;15). Notably, a 36% increase in MTHFR polymorphisms has recently been reported among young people. Although no direct evidence exists to date indicating that a deficiency in MTHFR activity is a risk factor for AD in isolation,16,17 MTHFR deficiency and the presence of ApoE4 may represent synergistic risk factors for AD. This is a plausible extension of the known association of diminished folate with AD, in that individuals homozygous for the deficient MTHFR polymorphisms are at particular risk for other folate-related neural defects when plasma folate is at the “low end” of the normal range.18,19 Moreover, individuals affected with AD who are also homozygous for the deficient MTHFR polymorphisms show elevated HCY compared with nonhomozygous AD patients despite the presence of equal levels in other considerable biological compounds such as folate and B12 levels in both groups.17 APOE⫹/⫹, APOE⫹/⫺, and APOE⫺/⫺ mice exposed to an oxidative stress-inducing diet deficient in folate show increased transcription and activity levels of MTHFR when compared with APOE⫹/⫹ mice maintained on folate-supplemented diet. This is indicative of the need for folate, for which metabolic product 5-MTHF is the principal methyl donor to the remethylation pathway. Significant intake of dietary methionine has been shown to induce increase in intracellular SAM concentration, which consequently inhibited N-5-MTHF synthesis and hence depressed use of HCY through remethylation.5 The mechanism via which SAM inhibits N-5MTHF synthesis is its ability to act as an allosteric inhibitor of methylenetetrahydrofolate reductase.20 The decrease SAM observed in the brain of APOE⫹/⫹, APOE⫺/⫹ and APOE⫺/⫺ mice maintained on an oxidative stress-inducing diet,21 could in a feedback process induce the activation of MTHFR gene and therefore justify the increased transcription and activity present under oxidative stress. The end product of folate metabolic pathway is 5-MTHF, which is a methyl donor. It donates a methyl group to HCY, necessary for its reconversion to L-methionine in a reaction catalyzed by methionine synthase (MS) also known as 5-MTHF–HCY S-methyltransferase (Figure 25.1, Reaction 5). Tetrahydrofolate can be regenerated as the second product of the reaction. This reaction is precursor to the regeneration of 5-MTHF via the methylene THF.7,22,23 Folate deficiency mediates neurotoxicity in part by increasing levels of HCY.24 This nonprotein amino acid overstimulates N-methyl-D-asparate (NMDA) receptors, potentiates glutamate and amyloid-β aggregation and neurotoxicity, and induces DNA breakage and lipid peroxidation.25,26,27 Mouse models of AD and Parkinson’s disease as well as wild-type mice subjected to folate deficiency show elevated HCY and place neurons at risk of degeneration.28,29,30 Oxidative damage due to folate deficiency is potentiated by lack of ApoE gene and iron supplementation as pro-oxidant. The extent of damage to brain cells can be determined by the measure of Thiobarbituric acid reactive species (TBARS) levels, which is an index of

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oxidative damage induced by lipid peroxidation.31,32,33,34 The expression status of MS can be easily altered under oxidative stress. The transcription levels and the activity of the enzyme it encodes for is significantly decreased under different oxidizing conditions. This alteration can be induced experimentally via the elimination of one (APOE⫹/⫺) or both copies (APOE⫺/⫺) of ApoE alleles in animal models such as mice, or by subjecting them to a folate-deficient diet, supplemented with iron as pro-oxidant,35 or can be affected by the lability of the reactive cofactor intermediate, cob(I)alamin. Vitamin B12 is an indispensable cofactor in the transmethylation reaction in the brain. This reaction is of great importance in the regulation of serum HCY levels and is the only reaction in the body in which folate and vitamin B12 are coparticipants.36,37 The decrease in MS transcription and activity observed in APOE⫹/⫹ mice under oxidizing conditions can be viewed as a natural, down-regulating compensatory process in the attempt to avoid the regeneration of methionine from HCY, which demethylation would continuously come down to further increased HCY formation. Consistent with this line of thought, other investigations have shown that the increased HCY flux through the transsulfuration pathway could, in principle, result from an increase in the levels of methionine adenosyl transferase, and cystathionine β -synthase (CBS), or a decrease in MS activity.35,38 The compound commonly referred to as vitamin B12 has a cyanide molecule at the metal-carbon position and the oxidation state of the cobalt is ⫹3 instead of the biologically active ⫹1. In order to be utilized in the body, the cyanide molecule must be removed. It is thought that glutathione (GSH) may be the compound that performs this function. The two active forms of vitamin B12 are adenosylcobalamin (cobamamide) and methylcobalamin. Methylcobalamin predominates in blood plasma and certain other body fluids, and in cells, it is found in the cytosol.37 The up-regulation of MTHFR and down-regulation of MS is suggestive of a “methyl trap.” Folate, through its metabolite 5⬘-MTHF, is, as mentioned above, the main provider of the methyl group for the reconversion of HCY to methionine. This methyl transfer contributes to the formation of SAM in a one-step reaction in which an adenosine triphosphate (ATP) molecule is involved (Figure 25.1, Reaction 6). S-adenosyl Methionine formed in the reaction is an essential ubiquitous metabolite well known as the major methyl donor in biological systems for most methyltransferase reactions in biological systems.13,39 The involvement of ATP, which contributes to the S-adenosyl group that binds to the essential amino acid methionine to form SAM, indicates that an active compound is generated.40 It is very important to know that in the remethylation of HCY to methionine, a methyl group is transferred to MS cofactor cob(I)alamin, which is then activated by forming methylcobalamin. That is reportedly the only biological function of methylcobalamin in humans. But in order to originally form methylcobalamin from cobalamin’s precursors, SAM must be available to supply a methyl group. Once methylcobalamin is formed, it functions in the regeneration of methionine by transferring its methyl group to HCY. Methylcobalamin can then be regenerated by 5-MTHF.37

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The central nervous system (CNS) has no alternative for the remethylation of HCY, such as the betaine pathway present in other organs; therefore, if MS is inactivated, the CNS has a greatly reduced methylation capacity.41 Other causes of reduced MS activity include folic acid deficiency.42 Folate deprivation and increased HCY completely inhibit transmethylation reactions by reducing SAM.43 Considering that the action of vitamin B12 plays a role in HCY metabolism that is similar to that of folate,39 and because folate and vitamin B12 deficiencies retard methionine regeneration, SAM levels are also reduced as a consequence of lack of folate or deficiency in vitamin B12 action.44 S-adenosyl Methionine levels are low in the CSF and brains of individuals with several neurological disorders, including AD.45 Decreased levels of SAM are also present in the brains of wild-type mice maintained on an oxidative stressinducing diet and among APOE⫺/⫺ mice maintained on a folate-supplemented diet, as well as those maintained on folate-deprived diets.21 Oxidative stress induced by either folate deprivation or deficiency in ApoE function46,47 could be the leading cause of this reduction in SAM levels. This might in part explain the decrease in transcription and activity of MS. These impaired methylation pathways have been implicated in many neurological and psychological disorders, including dementia, depression, and psychosis. Decreased intracellular methylation reactions can also result in an increase of SAH.48 This line of reasoning is supported by the recent demonstration that HCY induces DNA breakage and resultant apoptosis,49 and that co-treatment with SAM prevented HCY-induced apoptosis.50 Some other reactions in which SAM plays an important role include (1) methylation of DNA and RNA via the enzymes DNA- and RNA-methylases, and (2) the conversion of epinephrine to norepinephrine, which is also catalyzed by an N-methyl transferase that uses SAM.10,51,52 The participation of SAM in those biochemical reactions results in its demethylation to produce SAH under the catalysis of SAH methyltransferase (Figure 25.1, Reaction 7) .The hydrolysis of SAH catalyzed by SAH hydrolase produces HCY and adenosine (Figure 25.1, Reaction 8).39 The compensatory down-regulation of the reconversion of HCY to methionine and the absence of the betaine metabolic pathway in the brain further exclude the possibility of regenerating methionine from HCY. Choline, precursor of betaine in other organs, could therefore condense with acetyl-coA to optimally produce the neurotransmitter acetylcholine in the brain, in a reaction catalyzed by choline acetyltransferase (ChaT) (Figure 25.1, Reaction 15).53

25.3 THE TRANSSULFURATION PATHWAY — HOMOCYSTEINE ELIMINATION AND GLUTATHIONE METABOLISM The transsulfuration pathway comprises several reaction sequences, which lead to the formation of cystathionine and, subsequently, cysteine and GSH. The first reaction of this pathway, in which product cystathionine is derived from the condensation of L-serine with HCY, is catalyzed by the heme and vitamin B6-dependent CBS. Cystathionine is subsequently cleaved to yield cysteine and 2-ketobutyrate in a reaction catalyzed by cystathionine γ -lyase.13,38 Cystathionine γ -lyase is absent or

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has a limited presence in the brain, therefore, levels of cystathionine found in brain tissues are putatively due to the action of CBS. As CBS is the rate-limiting enzyme in HCY transsulfuration, it was found that a CBS genetic knockout mice model, first developed by Watanabe and coworkers,54 exhibited increased HCY levels. Among them, CBS⫺/⫺ mice developed significantly elevated levels of plasma total HCY (~200 µM), compared with CBS⫹/⫺ mice, which showed only mildly elevated concentrations of total plasma HCY (6–15 µM), closely similar to concentrations in humans with heterozygous CBS deficiency. These findings suggest that partial impairment in HCY transsulfuration produces similar effects of HCY metabolism in humans and mice. Deficiency in CBS leads to homocysteinuria, a rare autosomal recessive disease of sulfur amino acid metabolism, and hyperhomocysteinemia, which results in multiple organ/system damage, severe vascular disease, and mental retardation.55 The human hepatoma cell line demonstrates increased synthesis of cystathionine under oxidative stress conditions. The increased synthesis of cystathionine is followed by an increase in HCY flux through the transsulfuration pathway, which exhibits a linear dose dependence on the concentrations of two oxidants, namely, H2O2 and tertiary butyl hydroperoxide.38 Increased transcription levels of the CBS gene are present in APOE⫺/⫹ and APOE⫺/⫺ mice in a gene dosage manner. This increase is potentiated by folate deprivation.35 The immediate consequence of this increase in cystathionine levels could be an increase in the concentrations of the downstream metabolites, cysteine and GSH, the former (cysteine) being the precursor of the latter (GSH). The transsulfuration reaction thus provides a direct link between HCY and GSH, the major redox buffer in mammalian cells. It is therefore not surprising that a number of enzymes at this metabolic nexus display sensitivity to redox changes.38 Thus, changes in the levels of expression or functional activity of CBS can affect levels of HCY.39 Such a regulatory switch could be rationalized as representing a self-correcting response to depleted GSH levels in cells faced with an oxidative challenge.56 This highlights the overwhelming importance of the transsulfuration pathway since its up-regulation, under oxidative stress, has a dual beneficial effect which, is expressed by the accelaration of homocysteine elimination and the increase synthesis of the antioxidant GSH in many cell types. In so doing, the transsulfuration pathway contributes, at least indirectly, to preventing or quenching oxidative damage to the brain and other organs. Glutathione systematically called gamma-glutamylcysteinylglycine is a ubiquitous tripeptide, formed from the amino acids glutamate, glycine, and cysteine by two ATP-dependent enzymatic reactions.57,58 Glutathione is a major intracellular antioxidant and its antioxidant activity depends upon the thiol group within its molecules. It is crucial in the free radical scavenging of singlet oxygen59,60 and the OH radical. It also strongly binds heavy metal ions61 that in some cases also produce free radicals, and is extremely susceptible to binding by strong electrophiles because of the sulfhydryl group of cysteine. Glutathione plays this crucial role in detoxifying peroxides and electrophilic toxins as a substrate for GSH peroxidase (GPx) (Figure 25.1, Reaction 12) and GSH S-transferase (Figure 25.1, Reaction 14).62 Nearly all the plasma GSH derived from GSH is synthesized

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in the cytosol of hepatocytes and released by carrier-mediated transport.63 Intracellular GSH is maintained in its thiol form by GSH disulfide (GSSG) reductase, which requires NADPH (Figure 25.1, Reaction 13). The availability of cysteine is critical for the synthesis of GSH in most cells.64 Glutathione levels and enzyme GSH synthase (GS) are increased under oxidative stress conditions induced by dietary deficiency (folate and vitamin E).65,66 This increase in GSH levels is substantiated by ApoE deficiency.46,67 Deficiency in this gene was shown to promote increased oxidative stress.68 Moreover, experimental elevations in GSH in AD brain were capable of reducing oxidative damage69 and therefore represented an attempt to compensate for increased reactive oxygen species (ROS) induced by dietary and genetic deficiency.46,65 Furthermore, this increase in GSH levels in the nervous system is triggered by the up-regulation of the transcription and activity profile of GS. The GS gene and its activity displayed differential-compensatory responses to dietary folate and ApoE deficiency as follows: increased activity of GS was observed in both APOE⫹/⫹ and APOE⫺/⫺ mice when maintained on a folate-deficient diet and this increase is augmented by ApoE deficiency. The overall increase in GS activity among APOE⫺/⫺ mice maintained on the deficient diet beyond that among normal mice on the deficient diet is likely to derive from both increased activity and increased transcription. At least part of the increase in activity was apparently in response to diet-induced oxidative stress rather than oxidative stress resulting from the lack of APOE, in that (1) no change in activity is observed in APOE⫺/⫺ mice maintained on the complete diet and (2) normal mice maintained on the deficient diet also show an increase in activity, albeit less than that of APOE⫺/⫺ mice on the deficient diet. Increased transcription was not a response to diet-induced oxidative stress, insofar as APOE⫹/⫹ mice maintained on the deficient diet do not demonstrate increased transcription. By contrast, a significant increase in transcription of GS is observed only in APOE⫺/⫺ mice and only when they were maintained on folate-deficient diet, suggesting that the combined impact of diet-induced and genetically induced oxidative stress is required to induce an increase in transcription. The magnitude of this combined impact is reflected by a synergistic increase in TBARS, a marker of lipid peroxidation that is indicative of the magnitude of the ongoing oxidative damage levels in brain tissue of APOE⫺/⫺ mice maintained under folate deficiency. Increase in oxidative damage to brain tissue results from either dietaryinduced or genetically induced oxidative stress. However, there is a synergistic increase in oxidative damage resulting from the combined impact of both dietaryinduced and genetically induced oxidative stress. Maintenance of normal mice on dietary folate deficiency induces an ~40% increase in the combined impact of the absence of APOE, and the dietary folate deficiency results in a 300% increase in TBARS in brain tissue,47 indicating a synergistic deleterious impact of these dietary and genetic deficiencies. Therefore the further increase in GS transcription and activity in APOE⫺/⫺ mice subjected to oxidative stress-inducing diet correlates with the synergistic increase in TBARs. But these increases in both

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activity (30%) and transcription (48%) of GS in the brain of APOE⫺/⫺ mice maintained on a folate-deficient diet are unable to compensate fully for the synergistic increase in oxidative damage. This observation underscores the extent of oxidative damage that diet-induced and genetically induced oxidative stress could cause to brain tissue.70 These findings highlight the fact that distinct compensatory responses (i.e., increased expression and, independently, increased activity) in an antioxidantgenerating enzyme can be invoked depending on the nature and extent of oxidative stress. The combined efficacy of these responses was reflected by steady-state levels of GSH, in that both diet-induced and genetically induced oxidative stress individually elevated GSH levels, whereas the combined impact of both induced an apparent additive increase.47,70 The cumulative increase of GSH levels in brain tissues under oxidative damage is suggestive of a possible alteration of the activity of enzymes that help GSH to quench ROS and toxins that induce oxidative damage to the brain. The activity and the transcription profile of GPx, which catalyzes the reaction in which GSH is used to eliminate hydrogen peroxide and results in the formation of the oxidized form of GSH (GSSG) and that of GSH reductase (GR), which catalyzes the reconversion of GSSG to GSH, are elevated in the hippocampus and inferior parietal lobule of AD patients.69,71,72 This might reflect the protective gene response to the increased peroxidation in the brain regions showing severe AD pathology.70,72 The levels of GSH S transferase (GST), a protective enzyme against aldehydes and especially 4-hydroxynonenal (HNE, a marker of lipid peroxidation), are decreased in the brain and ventricular CSF of autopsied AD.71 APOE⫺/⫺ mice maintained on a folate-deficient diet demonstrated 70% and 60% increase in the activity of GPx and GSH reductase, respectively, compared with APOE⫹/⫹ mice on the complete diet. By contrast, but consistent with observations made in AD patient brains, APOE⫺/⫺ mice display a significant decrease (67%) in GSH S transferase activity. The decrease might be due to the methylation status of this enzyme. The similar increase in GPx and GR activity, which contributes in recycling the oxidized GSH (GSSG) back to the reduced form (GSH), combined with the significant decrease in GST activity, constitutes a justification for the increase in GSH levels in mice brain under oxidative damage.47,70 The supplementation of APOE⫺/⫺ mice with a potent methyl donor, SAM, when maintained on folatedeficient diet, restores GST, GPx, and GR activity.35 This highlights the importance of potent methyl donors in the regulation of enzymes that catalyze reactions that involvethe utilization of GSH.

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response to deficiencies to deficiencies in folate, vitamin E and apolipoprotein E, J Neurosci Res, 75: 508–515, 2004. 71. Lovell M.A., Xie C., and Markesbery W.R., Decreased glutathione transferase activity in brain and ventricular fluid in Alzheimer’s disease, Neurology, 51, 1562–1566, 1998. 72. Aksenov M.Y., Tucker H.M., Nair P., Aksenova M.V., Butterfield D.A., Estus S., and Markesbery WR., The expression of key oxidative stress-handling genes in different brain regions in Alzheimer’s disease, J Mol Neurosci, 11: 151–164, 1998.

26 Nutriproteomics Approach to Understanding Dementia-Relevant Brain Protein Changes in Response to Grape Seed Extract, a Dietary Antioxidant Helen Kim, Shannon Eliuk, Jessy Deshane, Stephen Barnes, and Sreelatha Meleth University of Alabama at Birmingham Birmingham, Alabama

CONTENTS Abstract..............................................................................................................459 26.1 Introduction..............................................................................................459 26.2 Application of Proteomics Technologies in the Identification of Targets of Antioxidants in Normal and Diseased Mammalian Brain......461 26.2.1 Normal Young Adult Rat Brain ...............................................461 26.2.2 Transgenic Model of Dementia, the Tg2576 Mouse...............461 26.2.3 Rodent Brain Tissue Preparation for Two-Dimensional Electrophoresis........................................................................461 26.2.4 Derivatization of Protein Carbonyls with 2,4-Dinitrophenylhydrazine ....................................................462 457

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26.2.5

Protein Extraction and Concentration with Methanol-Chloroform .............................................................462 26.2.6 Two-Dimensional Gel Electrophoresis....................................462 26.2.7 Western-Blot Analysis .............................................................463 26.2.8 Image Analysis.........................................................................463 26.2.9 Mass Spectrometry Methods to Identify Proteins Following Image Analysis.......................................................463 26.2.9.1 Peptide Mass Fingerprint Analysis Using Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry.........................463 26.2.9.2 Liquid Chromatography–Tandem–Mass Spectroscopy.....................................................................464 26.2.10 Statistical Analysis...................................................................464 26.3 Findings from Proteomics Investigations of Actions of Grape Seed Extract .............................................................................................464 26.3.1 Proteins that Discriminated 100% between Grape Seed Extract and Control Dietary Groups ..............................464 26.3.2 Validation of Putative Protein Identifications Generated by Peptide Mass Fingerprint Analysis .........................................465 26.3.3 Western-Blot Analysis for Protein Oxidations Affected by Grape Seed Extract in the Tg2576 Mouse Brain....................465 26.4 Initial Conclusions ...................................................................................467 26.4.1 Proteins in Normal Young Adult Mammalian Brain are Affected by Grape Seed Extract .............................................467 26.4.2 Attenuation of Protein Oxidations in Tg2576 Brains by Grape Seed Extract .................................................................469 26.4.3 Challenges for the Future in Analysis of Neurodegeneration-Relevant Protein Oxidations ...................470 26.4.3.1 Rigorous Identification of DNP-Modified Proteins ..470 26.4.3.2 Identifying the DNP-Modified Peptides and the Modified Amino Acid Residues........................471 26.4.3.3 How Do We Know if the Specific Protein Oxidations are Functionally Related to the Disease?...................471 26.4.4 Role of Proteomics in Nutrition-Related Research ................471 26.4.4.1 2D Gel Patterns are Informative even without Protein Identification ..............................................471 26.4.4.2 Rationale for Analysis of Intact vs. Proteolytically Cleaved Proteins.............................472 26.4.4.3 Identification of Intact Proteins Affected by Potentially Pathologic Modifications is Essential for Understanding the Disease Process ..................472 26.5 Summary ..................................................................................................473 26.5.1 Timing of the Baby Boomers Coincident with Emerging Technologies Enables Concerted Efforts in Analysis of Chronic Diseases.....................................................................473

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26.5.2 26.5.3

Elements of Productive Proteomics.........................................473 Proteomics Identification of Effects/Targets of Dietary Antioxidants in Chronic Conditions Such as Neurodegeneration: Value-Added Research ...........................475 Acknowledgments .............................................................................................476 References .........................................................................................................477

ABSTRACT The question of the extent to which food or food supplements can protect us from either age- or disease-induced neuro-oxidative stress is an important research issue today. We showed previously, using proteomics technologies, that specific proteins were changed in abundance in normal adult rat brain following ingestion of grape seed extract (GSE), a popular dietary supplement. Since GSE is enriched in polyphenols with documented antioxidant activity, a current focus in our laboratory is determining whether protein oxidations are directly affected by GSE. In this chapter, we review the information obtained in the previous proteomics study of the actions of GSE in normal rat brain and present initial results from ongoing studies on protein oxidations in rodent brain that may be modulated by GSE. These initial results were obtained from a study in which a transgenic mouse model of dementia, the Tg2576 mouse, was fed GSE, and the protein oxidations in its brain homogenate compared with the protein oxidations in the brains of animals that did not receive a GSE-supplemented diet. Following other investigators, we detected oxidized proteins in our brain samples by derivatizing the oxidized proteins with 2,4-dinitrophenylhydrazine (DNPH), and then carried out Western-blot analysis for the derivatized epitope. Initial proteomics results indicate that when fed a diet supplemented with GSE, the anti-dinitrophenyl (DNP) reactivity in the transgenic mouse brain was quantitatively attenuated. Thus, GSE and possibly other antioxidants may have multiple health benefits, including the inhibition of pathology-relevant brain protein oxidations.

26.1 INTRODUCTION The adage “we are what we eat” is increasingly proving to be true; we have understood for many years that we eat to maintain our bodies and provide them with sources of amino acids and carbohydrates, to obtain essential nourishment during normal growth, and to fulfill caloric requirements. Evidence of the past several years is, however, pointing to the concept that we can eat to protect ourselves from disease as well as possibly to treat disease, either by what we eat as food or in the form of dietary supplements. By defining dietary supplements as “food” and therefore not subjecting them to regulation by the Food and Drug Administration (FDA), the Dietary Supplement Health and Education Act (1994) enabled a rush of vendors to market a plethora of dietary supplements that have multiple purported benefits. One of the categories of benefits targeted by many supplements is antioxidant activity. Because of aging, disease, or

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some kind of insult such as ischemia-reperfusion injury, which involves momentary oxygen deprivation, various reactive oxygen and nitrogen species can cause accumulative damage to tissues. These include peroxynitrite, hydrogen peroxide, and free radicals. In the presence of these reactive species, proteins can be chemically modified in a number of different ways under an umbrella of “oxidation,” including carbonyl formation, S-nitrosylation, S-gluthiolation, and tyrosine nitration. In Alzheimer disease (AD) brain, initial Western-blot analysis suggested that a number of protein carbonyls were increased relative to age-matched control brain, implicating this modification in AD pathology.1 The etiology of AD is thought to involve oxidative stress, because of evidence that the amyloidogenic fragment amyloid-β (Aβ ) has pro-oxidant activity,2 which may be the basis for some of the molecular hallmarks of oxidative stress detected in neurons when Aβ was introduced in neuronal cell culture.3 The reversal of memory deficits following vaccination against Aβ 4–6 strongly suggested that if Aβ is a pro-oxidant, protecting against its actions has to be a goal in the development of treatments or preventives against AD. If allowed to occur without any intervention, AD will affect 1 in 45 Americans by the year 2050.7 The rationale is clear for (1) the identification of the protein oxidations and other protein changes that are really causal to AD or any neurodegenerative disease, and (2) determining whether we can attenuate or prevent critical oxidations and other protein changes implicated in AD with the “right” diet or dietary supplements enriched in “antioxidants.” This chapter describes studies in our lab that utilized proteomics technologies to identify specific proteins in mammalian brain that were affected by dietary supplements enriched in the polyphenols that have antioxidant activity, and whether these have relevance to neurodegenerative conditions involving oxidative stress. Our initial efforts focused on the actions of one dietary supplement enriched in procyanidins, grape seed extract (GSE), which has been shown to have high antioxidant activity.8 We utilized two-dimensional (2D) gels and statistical analysis, and determined that dietary supplementation with a high but nontoxic level of GSE resulted in differences in abundance and in modifications of specific proteins in the brains of young adult female rats.9 Because the directions of change for the majority of proteins in the study were opposite to those detected for many of the same proteins in either AD tissues10,11 or brains of transgenic models of dementia,12,13 this study was the first to identify neurodegenerationrelevant proteins affected by a dietary supplement without having hypothesized specific proteins prior to the analysis. Because of the antioxidant activity previously attributed to GSE components,14 we hypothesized that in addition to the changes in protein abundance resulting from ingestion of GSE, there might be differences in protein oxidations, and that these might be disease related. We analyzed protein oxidations affected by GSE in the Tg2576 mouse, which is a documented transgenic mouse model of dementia, caused by deposition of amyloid plaques resulting from aberrant cleavage of the amyloid precursor protein.15 For detection of protein oxidations, we

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incubated mouse brain homogenates with 2,4-dinitrophenylhydrazine (DNPH),16 which derivatized the protein carbonyls, and then carried out 2D gel electrophoresis and Western blots of replicate 2D gels using an anti-DNP antibody. Initial results from these ongoing efforts indicated that ingestion of GSE by the transgenic mice results in lowered extent of protein oxidations. The results are discussed in the context of the rationale for application of proteomics technology to the identification of targets of dietary antioxidants, and the value this approach can have in the identification of pathways implicated as targets of oxidative stress in the brain.

26.2 APPLICATION OF PROTEOMICS TECHNOLOGIES IN THE IDENTIFICATION OF TARGETS OF ANTIOXIDANTS IN NORMAL AND DISEASED MAMMALIAN BRAIN 26.2.1 NORMAL YOUNG ADULT RAT BRAIN The proteomic differences in normal, relatively young rat brain induced by GSE were studied in normal, 35-day-old female Sprague–Dawley rats; these were segregated into two dietary groups, one that received AIN-76A diet (Teklad Industries, Madison, WI) (control [CT] diet), and one that received AIN-76A diet supplemented with 5% powdered GSE preparation (Gravinol™, Kikkoman Corporation, Chiba, Japan).14 The animals were maintained in these two groups for 6 weeks, after which they were sacrificed by carbon dioxide suffocation, the brains dissected out above the brain stem, and snap-frozen in liquid nitrogen prior to storage at –80°C.

26.2.2 TRANSGENIC MODEL

OF

DEMENTIA, THE Tg2576 MOUSE

Initial experiments have been carried out to study potentially dementia-relevant protein oxidations in the Tg2576 mouse, previously described by Hsiao et al.15 Twentyseven-week-old transgene-positive (tg⫹) female mice were segregated into two dietary groups, one that received CT diet and one that received GSE-supplemented diet. The animals were maintained in these dietary groups for 9 weeks, after which they were sacrificed by cervical dislocation, their whole brains above the brainstem removed, weighed, snap-frozen in liquid nitrogen, and stored at ⫺80°C. All animal protocols were reviewed and approved by the University of Alabama, Birmingham (UAB) Institutional Animal Care and Use Committee (IACUC).

26.2.3 RODENT BRAIN TISSUE PREPARATION ELECTROPHORESIS

FOR

TWO-DIMENSIONAL

Brains were removed from storage at ⫺80°C and homogenized using the Sample Grinding Kit (Amersham Biosciences, Piscataway, NJ, U.S.A.) in isoelectric focusing (IEF) buffer (7 M urea, 2 M thiourea, 4% 3-[(3-cholamidopropyl) demethyl-ammonio]-1-propanesulfonate (CHAPS), 50 mM dithiothreitol [DTT])

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at a tissue:buffer ratio of 1:5 (w/v). The IEF buffer was adjusted with a tablet of Complete Mini, EDTA-Free Protease Inhibitor Cocktail (Roche, Mannheim, Germany) (1 tablet/10 mL IEF buffer) within 30 min of use. After clarification by ultracentrifugation in an Optima Beckman tabletop ultracentrifuge at 55,000 rpm at 22°C for 30 min, the supernatant was removed and its protein concentration was determined using the Bradford Reagent (BioRad Laboratories, Hercules, CA, U.S.A.).

26.2.4 DERIVATIZATION OF PROTEIN CARBONYLS WITH 2,4-DINITROPHENYLHYDRAZINE Thirty micrograms of protein in 10 µL of IEF buffer was adjusted to 6% sodium dodecyl sulfate (SDS), then mixed with 10 µL of DNPH-derivatization solution from the OxyBlot Protein Oxidation Detection Kit (Chemicon International, Temecula, CA, U.S.A.), and incubated for 20 min at 22°C, after which the reaction was terminated by the addition of 7.5 µL of “neutralization” buffer, provided by the Oxyblot Kit.

26.2.5 PROTEIN EXTRACTION AND CONCENTRATION WITH METHANOL-CHLOROFORM To concentrate the proteins and extract them out of the DNPH-derivatization mixture, chloroform–methanol extraction was used according to established procedures.17 Briefly, the sample was mixed sequentially with 4 vols of methanol, 1 vol of chloroform, and then with 3 vols of water, and centrifuged, resulting in the proteins collecting at the interface between the aqueous and organic layers. The aqueous layer was discarded, 3 vols of methanol was added and the mixture was recentrifuged, this time spinning down the protein that had precipitated owing to methanol. After discarding the supernatant and drying the protein precipitate under nitrogen gas, the pellet was resuspended in IEF buffer and the protein concentration was determined.

26.2.6 TWO-DIMENSIONAL GEL ELECTROPHORESIS Immobilized pH gradient (IPG) strips (11 cm) (BioRad Laboratories, Hercules, CA, U.S.A.) containing a 4 to 7 pH gradient were rehydrated overnight over varying amounts of protein in 200 µL of IEF rehydration buffer adjusted to 0.5% ampholytes, 5 mM tributylphosphine (TBP) (BioRad) and 0.1% Bromophenol Blue. Isoelectric focusing was performed using an Ettan IPGphor II (Amersham Biosciences, Piscataway, NJ, U.S.A.) set at 18°C using the following focusing protocol: 500 V for 60 min, gradient to 1000 V over 60 min, gradient to 6000 V over 120 min, then held at 6000 V for 120 min. After IEF, the strips were stored overnight at ⫺80°C. The second dimension consisted of SDS–PAGE, where the IPG strips were thawed, agitated for 2 ⫻ 15 min at room temperature in SDS–PAGE equilibration buffer (50 mM Tris-HCl, pH 8.8; 6 M urea; 30% [v/v] glycerol; 2% SDS; and Bromophenol Blue), and freshly adjusted to 65 mM DTT.

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The strips were finally agitated for 15 min in 80 mM iodoacetamide in equilibration buffer, after which the second dimension separation was performed in 10 to 20% polyacrylamide gradient Criterion gels (BioRad) at 200 V for 70 min. One gel for each sample, loaded with 100 µg of protein, was stained with GelCode Blue Stain Reagent (Pierce, Rockford, IL, U.S.A.).

26.2.7 WESTERN-BLOT ANALYSIS For the Western-blot analysis, 50 µg of protein in IEF buffer was rehydrated into an IPG strip and processed through the same 2D electrophoresis protocol as the strips containing the higher amounts of protein, except that the second dimension SDS–PAGE gel was transblotted overnight to an Immobilon-FL polyvinylidene flouride (PVDF) membrane (Millipore Corporation, Bedford, MA, U.S.A.) at 130 mA, with the tank in an ice bath. The blots were blocked for 1 h at room temperature with Odyssey Blocking Buffer (LiCor Biosciences, Lincoln, Nebraska) and then probed with anti-DNP (DakoCytomation, Denmark) 1:2500 in Odyssey blocking buffer for 1 h. Tris-buffered saline solution containing Tween 20 (TBST) was then used to wash the blots for 6 ⫻ 10 min and the blot was further probed with Alexa Fluor 680 goat–antirabbit IgG antibody (Molecular Probes, Eugene, OR) 1:5000 in Odyssey Blocking Buffer for 1 h in the dark followed by overnight wash in TBST. Images of both the Coomassie Blue-stained gels and the blots were acquired on the LiCor Odyssey Infrared Imaging System.

26.2.8 IMAGE ANALYSIS Images of the Coomassie Blue-stained gels and blots of the mouse brain homogenates were analyzed via Progenesis Discovery image analysis software (Nonlinear Dynamics Ltd, Newcastle, U.K.), which calculated individual spot volumes after subtraction for background. Total gel volumes were calculated by summation of the individual spot volumes for a given gel; differences in antiDNP reactivity between gels were calculated by comparing one total gel volume with another. The 2D gel images were calibrated for pI and molecular weight assuming linearity between the ends of the first-dimension IPG strip and using prestained molecular weight standards co-electrophoresed in the second dimension alongside the first-dimension strip.

26.2.9 MASS SPECTROMETRY METHODS TO IDENTIFY PROTEINS FOLLOWING IMAGE ANALYSIS 26.2.9.1 Peptide Mass Fingerprint Analysis Using Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry For putative identification of the rat brain proteins, appropriate spots indicated by the image and statistical analysis were excised from the stained 2D gels. Trypsin

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digestion, solvent exchange, and peptide mass fingerprint (PMF) analysis by MALDI–TOF–MS were carried out using established procedures9,18,19 with a Voyager DePro MALDI–TOF–MS (Perseptive Biosystems, Foster City, CA, U.S.A.). Calibration was accomplished internally using trypsin autolysis peaks. 26.2.9.2 Liquid Chromatography–Tandem–Mass Spectroscopy Protein identities indicated by PMF were confirmed by reverse-phase liquid chromatography–electrospray–tandem–MS (LC–ESI–MS/MS) using a Qtof3 hybrid quadrupole orthogonal TOF mass spectrometer (Waters, Manchester, U.K.) as described previously.9 MS/MS data were recorded using automated MS to MS/MS data-dependent scanning, switching at a threshold of six counts.

26.2.10 STATISTICAL ANALYSIS In presenting some of the rat brain data here as a prelude to the protein oxidation analysis, it should be emphasized that the determination of spots that discriminated 100% between the two dietary groups was only possible by exporting the data generated by PDQuest image analysis software out of PDQuest. The data were processed through various statistical algorithms in the Statistical Analysis System (SAS, v. 09, SAS Institute, Cary, NC, U.S.A.) starting with transformation of the intensity values to enable analysis of normally distributed data.9 A complete description of the algorithms to determine significant differences in variability of spot intensities was given by Meleth et al.20

26.3 FINDINGS FROM PROTEOMICS INVESTIGATIONS OF ACTIONS OF GRAPE SEED EXTRACT 26.3.1 PROTEINS THAT DISCRIMINATED 100% BETWEEN GRAPE SEED EXTRACT AND CONTROL DIETARY GROUPS Initial 2D gel proteomics analysis identified multiple proteins in rat brain that were significantly different in abundance and in isoform complexity following ingestion of GSE-supplemented diet.9 Figure 26.1 shows a 2D gel of rat brain from that database where only the subset of the protein spots that discriminated 100% between the two dietary groups, as determined by Stepwise Discriminant Analysis,21 are shown; in other words, the differences for this subset of proteins were sufficiently significant to segregate the two dietary groups. Only the protein identities are given to emphasize the different functions represented by these proteins. It can be seen that only a few functional categories are represented by these proteins, namely protein folding (chaperone proteins: hps60, hsc70, 71), energy utilization and metabolism (creatine kinase [CK], enolases), and cytoskeletal proteins (neurofilament proteins, actin). It should be pointed out that none of the differences between GSE and CT groups was greater than twofold for the proteins that discriminated 100%, or for the other proteins that were significantly different.9

Nutriproteomics Approach to Dementia-Relevant Brain Changes hsc70 NFL-L 14-3-3-


-Enolase

465

hsp60 GFAP -Enolase CK-BB

RIKEN cDN A NM 025994

Actin

FIGURE 26.1 2D gel proteomics identification of rat brain proteins affected by GSE that discriminated 100% between GSE- and control-diet groups. Following dietary supplementation with 5% GSE for 6 weeks, rats were sacrificed, and homogenates of sagittal sections of their brains were processed through 2D gel electrophoresis as described previously,9 using 4–7 pH gradients and 10–20% acrylamide gradients for the first and second dimensions, respectively. Shown are representative images of the colloidal Coomassie Brilliant Blue-stained 2D gels. “5% GSE” indicates brain homogenate from an animal that ingested the GSE-supplemented diet, whereas “control” indicates brain homogenate from an animal that ingested “control” (CT) (nonsupplemented) diet. The indicated spots were determined by Stepwise Discriminant Analysis to discriminate 100% between the two dietary groups.10

26.3.2 VALIDATION OF PUTATIVE PROTEIN IDENTIFICATIONS GENERATED BY PEPTIDE MASS FINGERPRINT ANALYSIS For the rat brain proteins indicated to be affected by GSE, the protein identities generated by PMF analysis using MALDI–TOF–MS were confirmed by either LC–MS/MS, by Western-blot analysis, or both, for all but two proteins (Table 26.1). It should be noted that the Western-blot analysis also confirmed the quantitative nature of the data in all cases examined.

26.3.3 WESTERN-BLOT ANALYSIS FOR PROTEIN OXIDATIONS AFFECTED BY GRAPE SEED EXTRACT IN THE TG2576 MOUSE BRAIN Since polyphenols in general, and the grape polyphenols in particular, have been documented to have antioxidant activity,8 it was logical to follow up the proteomics studies described above in the rat brain with an analysis of whether protein oxidations in the brains were affected by the dietary GSE. To address this, we examined the transgenic mouse, Tg2576, a documented model of dementia,15 hypothesizing that its brain would contain readily detected protein oxidations owing to the pro-oxidant status of Aβ.1 Analysis of protein carbonyls (the most common form of protein oxidation) was accomplished by reacting the brain homogenates with DNPH using the OxyBlot kit according to manufacturer’s instructions; the reaction is shown schematically in Figure 26.2. This method and

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TABLE 26.1 Validation of Proteomics Results Using Complementary Methods Protein CK-BB Hsp60 GFAP Actin NFL-M α-Enolase γ -Enolase Hsc70 Hsc71 14-3-3ε NFL-L

LC-MS/MS

Western Blot

⫹ ⫹ ⫺ ⫺ ⫹ ⫹ ⫹ ⫺ ⫺ ⫺ ⫺

⫹ ⫹ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫹

Note: The proteins in this list were determined in earlier proteomics studies to be significantly different in abundance or isoform complexity in the brains of rats that were given 5% GSE, relative to rats that were given nonsupplemented diet.9 Following initial identification by peptide mass fingerprinting by MALDI–TOF–MS, the protein identities were confirmed by either LC–MS/MS, by Western blot, or both, for all but GFAP and the heat shock proteins 70 and 71. In all cases, the Western-blot analyses confirmed the quantitative nature of the difference originally detected by image analysis.9

approach has been used by others to study protein oxidations.1,22–34 The resulting moiety is indicated by the brace; this DNP moiety or adduct is recognized by antiDNP antibody (DakoCytomation, Denmark). Female, tg⫹, 27-week-old Tg2576 mice were administered either a CT diet or one supplemented with GSE for 9 weeks, after which their brain homogenates were reacted with DNPH and the derivatized proteins analyzed by 2D Western blots. Figure 26.3A shows that equal amounts of protein were loaded on the 2D gels of the tg⫹ mouse brains; Figure 26.3B shows that equal amounts of proteins from the brains of tg⫹ mice that ate GSE diet vs CT diet gave Western-blot signals for anti-DNP reactivity that were quantitatively different, although qualitatively similar. The densitometric analysis of the Western blots (Figure 26.3C) confirmed that the tg⫹ mice that ingested GSE diet apparently had lower anti-DNP reactivity overall in their brain proteins than did the tg⫹ animals that ingested CT diet. At this level of detection, there were no obvious protein spots that were initially reactive with the anti-DNP antibody that were not affected by the GSE diet. These results are preliminary, and experiments are ongoing to confirm these initial findings as well as to validate them using complementary methods.

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Protein carbonyl R1

NH2

O +

NH

C R2 O2N

NO2 R1 C R2

N

N H

NO2

NO2 DNPH

FIGURE 26.2 DNPH derivatization of protein carbonyls generates the DNP-modified epitope that indicates protein oxidation. To generate an epitope to facilitate detection and quantitation of protein oxidations, the Oxyblot kit (Chemicon International, Temecula, CA) was used to react protein carbonyls in the mouse brain homogenates studied here with 2,4-dinitrophenylhydrazine, as described in section 26.2.4. The resultant moiety in the modified protein indicated by the brace was recognized by an anti-DNP antibody (DakoCytomation, Denmark).

26.4 INITIAL CONCLUSIONS 26.4.1 PROTEINS IN NORMAL YOUNG ADULT MAMMALIAN BRAIN ARE AFFECTED BY GRAPE SEED EXTRACT The simple conclusion from the proteomics analysis of the rat brain proteins affected by GSE is that specific proteins are affected in reproducible and quantitative ways by ingestion of GSE, a complex dietary supplement enriched in procyanidins. While there is controversy as to the bioavailability of procyanidins and their metabolism, our data showed undeniably that ingestion of these complex chemicals resulted in molecular changes in mammalian brain. This is consistent with and extends the evidence that related polyphenols in green tea have been detected in the brain following oral ingestion,35 and that these and other related, polyphenol-enriched dietary additions may have beneficial actions in the brain milieu.36,37 Of the number of proteins that underwent statistically significant changes in either abundance or isoform complexity in response to GSE, a subset (Figure 26.1) discriminated 100% between the two dietary groups. Future experiments may demonstrate that the functions or pathways represented by these proteins are involved in the neuroprotective actions of GSE. It is noteworthy that of the proteins in this list (Table 26.1), several that were detected at higher abundance, including CK-BB, both enolase isoforms, and selected heat shock proteins, were detected at lower levels in AD10 as well as at higher levels of oxidation than in age-matched non-AD human brain.27,28 It could be that dietary GSE enables a slightly higher and stabilized expression of those proteins to compensate for a functional impairment resulting from oxidation occurring because

468

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(a)

CT

GSE Western blots with anti-DNP

(b)

CT

GSE

(c)

% anti-DNP signal, relative to tg −, CT diet

120 100

80

60 40 20 0 tg+, CT

tg+, GSE

FIGURE 26.3 Western-blot analysis showing that GSE attenuates anti-DNP reactivity in Tg2576 mouse brain. (A) Coomassie Blue-stained 2D gels of 100 µg of protein from Tg2576 mouse brain homogenate following ingestion of control (CT) or GSE-supplemented diet. (B) Western-blot analysis of 50 µg of protein. (C) Densitometric analysis of the total spot intensities in the Western blots shown in B indicated that the anti-DNP reactivity was higher in the tg⫹ brains relative to the tg⫺ brains, and that this increase in total reactivity in the brain from the tg⫹ animal was reduced significantly (40%) with dietary GSE supplementation relative to the reactivity measured in the brain from a tg⫺ animal that did not ingest GSE.

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of aging or as part of a disease process involving increased oxidative stress, as in the Tg2576 brain. The identification of the proteins affected by GSE thus has face value in identifying proteins affected by a dietary supplement. Where disease relevance is documented for targets of dietary compounds, the latter, such as GSE, can also be thought of as molecular “tools” that in conjunction with proteomics can reveal proteins that may be implicated in disease, and therefore be candidates for protection before onset of disease. Additionally, once changes in a protein or set of proteins are correlated with a beneficial phenotype in response to dietary administration of a complex mixture, those proteins become markers of that phenotype, against which purified fractions of the complex mixture can be tested, to enrich for activity. It should be pointed out, however, that the experiment described previously captured a single time point, 6 weeks, at a single dose, 5% GSE. To fully understand the roles of the different proteins in a disease process such as dementia, and the potentially protective role that GSE might have against the development of dementia, additional time points will have to be studied as well as different doses of GSE, to determine the timing of events, and the optimal as well as minimal dose for achieving desirable effects. Having made the case for the discriminating spots being predictive of proteins involved in the health benefits of GSE, it is also possible that some of these differences, for all the tightness of their statistical significance, are benign differences; in other words, they are “real” differences, but they do not contribute to the primary physiological effect. One way to determine whether this might be the case with the proteins affected by GSE is to carry out behavior analysis of rats following ingestion of GSE, determine that cognitive functions are enhanced by the GSE, then fractionate the GSE, test the different fractions in the same behavior analysis, and determine by proteomics analysis the subset of protein changes induced by the fraction that had behavioral benefit vs those protein changes associated with the fractions that had no behavioral benefit. Thus, a smaller and smaller proteome can be identified that is associated with phenotypic differences. In this regard, initial studies showed in a spontaneously hypertensive rat (SHR) model that dietary supplementation with a very low dose of GSE, 0.1%, was sufficient to attenuate the cognitive impairment that resulted from the ovariectomy in the animals that did not receive GSE in their diet.38 Experiments are ongoing to determine whether higher doses have greater effect, and whether subfractions of GSE have similar protection.

26.4.2 ATTENUATION OF PROTEIN OXIDATIONS GRAPE SEED EXTRACT

IN

Tg2576 BRAINS

BY

Given that the Aβ peptide fragment of the parent protein has been shown to have pro-oxidant activity, it was reasonable to hypothesize that the Tg2576 mouse brain, where it has been shown that Aβ accumulates, would contain readily detected protein oxidations. The 2D Western-blot analysis with the anti-DNP antibody (Figure 26.3B) suggested that indeed there was readily detected protein

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oxidation in the tg⫹ brain. Comparison of the stained gel images with the Western-blot images before and after reaction with DNPH by image analysis software indicated that several of the protein spots that were reactive with antiDNP corresponded to the more abundant proteins detected on the stained gels (Figures 26.3A and B), consistent with the concept that the oxidations suggested by the anti-DNP reactivity, while specific, would affect the more abundant sites. Nitrative oxidation of actin, an abundant cytoskeletal protein, has been detected and characterized in disease tissue.39 The specificity of the DNP derivatization on the other hand, is indicated by the fact that not all protein spots readily detected on the Coomassie Blue-stained gel were derivatized with DNPH. Conversely, some readily detected anti-DNP-reactive spots were barely detected in the stained gel (examine Figure 26.3 A and B closely), indicating a higher level of oxidation for these proteins than for the more abundant proteins. Experiments are underway to identify the proteins indicated on the Oxyblots and to determine the functional significance of the oxidations.

26.4.3 CHALLENGES FOR THE FUTURE IN ANALYSIS OF NEURODEGENERATION-RELEVANT PROTEIN OXIDATIONS 26.4.3.1 Rigorous Identification of DNP-Modified Proteins Ongoing experiments are in progress to identify the proteins that reacted with the anti-DNP antibody. Since minor proteins, completely different from abundant proteins such as the cytoskeletal proteins tubulin or actin, can comigrate on 2D gels within the large spots or strings of isoforms that comprise the cytoskeletal proteins, it is not sufficient to conclude that an anti-DNP-reactive spot is the protein whose spot comigrates with the former. Fortunately, this can be addressed in straightforward ways; an immunoprecipitation can be carried out using the anti-DNP antibody, and the proteins brought down resolved on 2D gels (of the same parameters as the original) that are stained to visualize the captured proteins. Comparison of the image of the resultant gels with the initial 2D Western blot will confirm that the immunoprecipitation was productive; it will be informative to note whether any spots on the original Western blots were not precipitated by this procedure. Alternatively, especially if a protein spot that is reactive with the anti-DNP antibody is a minor spot relative to others (such as the cytoskeletal protein spots), it would be more productive to identify the protein first by MALDI–TOF–MS, then use antibody to that protein to immunoprecipitate it, re-run it on a 2D gel, separate oxidized from nonoxidized forms, and determine whether any of the spots is reactive with the anti-DNP antibody. For truly abundant proteins, where a modification may affect a minor portion of the whole population of that protein, it may be necessary to release it from the first immunoprecitation, then reimmunoprecipitate with the antibody to the modification, and detect and quantify the modified protein by running either an SDS–PAGE gel or 2D gel, and Western blotting for the epitope. Such an approach was used successfully by Aslan et al.39 to show that actin was nitrated in sickle-cell disease.

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26.4.3.2 Identifying the DNP-Modified Peptides and the Modified Amino Acid Residues Once the protein that reacts with the anti-DNP has been identified, rigorous analysis will include identifying the peptide that is modified as well as the determining the exact mass change induced by the modification. These can be accomplished using appropriate MS techniques. Different residues on cytochrome c that can be modified by the lipid oxidant 4-hydroxy-nonenal (4-HNE), and the different mass changes associated with the oxidations, depending on the exact chemistries involved, were identified by a combination of MALDI–TOF–MS and LC–MS/MS by Isom et al.40 It should be noted that while a number of protein spots on 2D gels of healthy and diseased brains have been shown by others to react with anti-DNP antibody in similar Western blots as described here,26–28 conclusive demonstration of DNP-modification or identification of the oxidized amino acid residue has not been accomplished for most of the AD-relevant proteins, as has been shown with immunoreagents for actin by Aslan et al.,39 and by MS for the specific residues in cytochrome c by Isom et al.40 26.4.3.3 How Do We Know if the Specific Protein Oxidations are Functionally Related to the Disease? Once the specificity of the anti-DNP reactivity has been demonstrated, the real challenge will be to demonstrate the physiological significance of the modification for any given protein. This will involve considering (1) whether the function of the protein is affected and (2) whether this causes, or affects the progression of, the disease in question. It is outside the scope of this chapter to delve into how functional issues should be addressed, but clearly, the question of the relevance to disease of specific modifications such as oxidations is the central one that ultimately has to be answered, since it will distinguish those proteins whose modifications are relevant from those that are indeed modified but are not relevant to the disease.

26.4.4 ROLE

OF

PROTEOMICS

IN

NUTRITION-RELATED RESEARCH

26.4.4.1 2D Gel Patterns are Informative even without Protein Identification The data shown in this chapter demonstrate that analysis of patterns of protein spots, resolved on 2D gels, can quantitatively suggest protein changes that reflect the actions of GSE. It should be pointed out that this pattern information, without protein identifications, can be an end in itself. Ultimately, of course, the identification of the proteins that are suggested to be different between two groups has to be carried out in order to understand the physiology. But, an entire analysis could conceivably be carried out, comparing 2D gel patterns of brain proteins following exposure of the animals to different nutrients or different dietary supplements, for example, GSE vs a single grape polyphenol resveratrol, or GSE vs Ginkgo biloba,

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which has been documented to have neuroprotective actions.41–45 At the end of the image analysis, a small subset of protein spots would be excised and analyzed by MS to obtain their identities. In this way, 2D gel pattern analysis could be used to determine whether the health benefits of different nutrients/dietary supplements involve common proteins, or common modifications. 26.4.4.2 Rationale for Analysis of Intact vs. Proteolytically Cleaved Proteins While analysis of proteolytically cleaved protein mixtures in concept appears faster, since the entire separation and analysis can be done “online” by LC–MS/MS methods,46 it is also true that valuable information is lost the minute a polypeptide is cleaved into pieces.47 The position of an intact polypeptide on a 2D gel is deceptively simple, but instantly yields the pI as well as the mass of the polypeptide. These are valuable parameters that assist in the identification of that protein, particularly if posttranslational modifications that affect charge are involved. The same protein, however, may become confused with several others, once cleaved, if it belongs, for example, to a family of enzymes with highly conserved domains, such as the catalytic domain in the protein kinase family. Moreover, the peptides of a protein that is not abundant might get “lost in the shuffle,” whereas the intact protein might be readily detected on a 2D gel. If there is a question of the reproducibility of the amount in that protein spot owing to insufficient protein, the 2D gel can be re-run with higher or lower amounts of protein to enable enhanced analysis of the image. This particular type of follow-up decision making is more difficult in peptide-based analyses, since the abundance of the protein in question is less straightforward as neither MALDI nor LC–MS/MS is quantitative. Finally, if only a subset of the polypeptide is modified or undergoes change in modification, this might be readily detected on a 2D gel or by other separation technologies that separate intact proteins,47 but could be difficult to detect, if at all, once the whole mixture of proteins is digested. For these reasons, it seems clear that 2D gel separations, alone or in conjunction with other types of separations of intact proteins,47 will continue to have an important role in proteomics analysis of biological samples. 26.4.4.3 Identification of Intact Proteins Affected by Potentially Pathologic Modifications is Essential for Understanding the Disease Process Recent experiments identified oxidized amino acid adducts and free oxidation adducts in the CSF of AD patients, suggesting that adducts of protein oxidation as well as glycation and nitration might be useful as diagnostic markers of AD.48 While the identification of these adducts has value in determining the nature and extent of the adducts that might be involved in the disease, it should be kept in mind that identifying the parent protein that undergoes the modification will be critical for determining the specificity of the markers and the disease process

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itself. For example, it could be that certain proteins are oxidized but do not undergo proteolysis to release the adducts; these proteins might be candidates for pathology-relevant oxidations, but their fragments would be underrepresented in CSF or serum adduct analysis.

26.5 SUMMARY 26.5.1 TIMING OF THE BABY BOOMERS COINCIDENT WITH EMERGING TECHNOLOGIES ENABLES CONCERTED EFFORTS IN ANALYSIS OF CHRONIC DISEASES The American baby boomers now constitute a substantial portion of the American population; the baby boomers are now aging, but at the same time, are in a unique position to control and influence research agendas. Thus, we can suffer from agerelated conditions and diseases if we fail to focus research in directions that can prevent or treat age-related conditions. If we can make fundamental progress in identifying the molecular basis of the role of oxidative stress in neurodegeneration, and in particular identify dietary factors that can attenuate this oxidative stress, we leave a legacy for our children that will be unappreciated by them (as many of preventive measures are), but that will nonetheless enhance the health of their and future generations, at home and in the world. Currently available proteomics technologies have the capacity to dramatically enhance the bodies of knowledge in the areas of human dementias. In combination with experimental approaches that include the use of nutrients or dietary supplements, proteomics can identify proteins that undergo abundance or modification changes in disease brain, and which of these are amenable to prevention or attenuation by dietary intervention. The specificity of available immunoreagents such as the anti-DNP antibodies in combination with proteomics technologies allows the neuroscientist and the nutrition researcher interested in neurodegenerative diseases and the role of modifications such as oxidations unparalleled analytical opportunities.

26.5.2 ELEMENTS

OF

PRODUCTIVE PROTEOMICS

Figure 26.4 outlines a generalized workflow in proteomics analysis of biological samples, with the goal of identifying protein markers of disease (neurodegeneration) or of identifying proteins affected by a dietary component or supplement (such as GSE). Several aspects of this schematic have been demonstrated and discussed in this chapter. Although the upfront separation method specified in this diagram is 2D gel electrophoresis, the same issues apply to other separation technologies, such as LC-based separations, or LC inline with MS. The essential elements in a proteomics strategy for analysis of an experiment are the following. After an experiment, the proteome in the biological samples is resolved by 2D electrophoresis, or any other multidimensional method. Images of the resultant gels are captured and “analyzed” (A) by image analysis and then by statistics. The (statistically) significantly different spots (in the case of 2D gels) are then analyzed

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Western blots for: oxidations, other modifications

C

Image, statistical analysis

Transgenic or treated (GSE?) Wild-type or control

Pattern for A or for C: significant spots

A

MS protein identification/ identification & mapping of modifications Pattern for B: ID protein partners

B. Back to samples: immunoprecipitation of significant protein

FIGURE 26.4 Elements of successful proteomics analysis. Proteomics encompassesseveral different analytical aspects. In this scheme, 2D gels are used to separate the proteins. Proteins are identified by PMF by MALDI–TOF–MS, with follow-up protein identifications by LC-tandem MS. “A” shows an initial round of 2D gel analysis of a set of samples, either control and diseased, or control and treated (e.g., with grape seed). Gels are processed through image and statistical analysis, and spots are deemed to be significantly different between groups, based on quantitative differences in intensity or position. This portion of proteomics, represented by the arrows surrounding “A,” is often underestimated; successful image and statistical analysis that extracts information in the gel images can involve several iterations between the images and the statistical analysis. Ultimately, gel spots are cut out, and digested (usually with trypsin, but could be any one of several proteases), and putative identifications are generated by PMF analysis and confirmed by LC-tandem MS of the dominant peptides. These data can then be used (B) to design immunoprecipitation experiments with one of the proteins, to determine, for example, its binding partners, to determine how they are affected by the changes in the antigen of interest. This begins another iteration through the 2D gel analysis, but this time, the proteome is reduced in complexity. The image analysis may involve 10 or 30 total proteins on the gels, and the “significant” spots — those that are affected in extent of binding, for example — may be only reduced to one. “C” represents another iterative round of analysis, where replicate 2D gels can be Western blotted and probed with antibodies to quantify differences in oxidation of proteins between normal and disease or control and GSE-supplemented dietary groups. Thus, meaningful proteomics is an iterative process, with reduction in proteome complexity and increase in specificity with increasing iterations; it begins as discovery and ends with functional information.

by MS to identify the protein and its modifications. These data then generate a follow-up experiment (B), such as an immunoprecipitation experiment, which will bring down proteins that interact with the original protein. These immunoprecipitates are then processed through the same analytical process as the original

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samples were. Alternatively, Western blots can be run in parallel with or immediately after the first set of 2D gels (C) to probe for a modification such as oxidation, or phosphorylation. Depending on the results from C, a follow-up experiment might be to immunoprecipitate a protein in its oxidized and unoxidized forms, then determine by 2D gels whether its protein-binding partners are affected by the oxidation. Thus, productive proteomics involves several elements: (1) it should be an iterative process, where the same analytical loops may be applied several times; (2) validation of data by complementary methods such as Western blot, or LC–tandem MS, is important wherever possible; and (3) steps subsequent to the first analysis should either reduce proteome complexity or increase specificity of the analysis. In the best scenario, proteomics takes the investigator from a discovery phase to functional information. A final important element when analyzing multiple samples or conditions is quality control. Simple steps such as randomization of samples to be analyzed, analyzing replicates of a sample on different days, and ensuring use of the same batch of gels or membranes, make a qualitative difference in the reproducibility of the data.

26.5.3 PROTEOMICS IDENTIFICATION OF EFFECTS/TARGETS OF DIETARY ANTIOXIDANTS IN CHRONIC CONDITIONS SUCH AS NEURODEGENERATION: VALUE-ADDED RESEARCH One final statement should be made about the role of nutrition in experiments addressing chronic age-related conditions such as neurodegeneration and oxidative stress. As evidence accumulates, it is becoming clear that nonnutrient components in food, particularly the flavonoids, have health benefits that justify being marketed as dietary supplements. It is reasonable to think that their beneficial effects may derive from their capacity as antioxidants; this is consistent with the evidence that oxidative environments or molecules such as Aβ can be pathologic, and various polyphenols with antioxidant activity can directly inhibit Aβ fibrillar formation in vitro.49 But it is also clear that not everyone who eats a diet high in antioxidants stays healthy, and (probably) not everyone who suffers oxidative stress gets sick or becomes demented. So there is complexity in the oxidative stress paradigm of disease. Proteomics technology allows us to analyze patterns of protein expression and change, and to decipher which ones change the most in a disease. The same technology applied to the actions of dietary antioxidants, as demonstrated in the earlier experiments with GSE,9 allows identification of patterns of protein change also, as part of the response to the dietary component. Experiments addressing what happens in mammalian tissues in response to dietary components are “value-added” research, because while the identification of the proteins affected by the particular dietary component is an end in itself, the data also predicts proteins and pathways that may be at risk for oxidative damage. In this way, the dietary component(s) are molecular “tools” that can be used to understand a disease process, even while studying protein changes in normal tissues. Ironically, such understanding of a disease process and of the protection afforded by mixtures of

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antioxidants may lead to the dissection of the mixture, to identify a more potent bioactive component that could ultimately be purified and be taken outside of the diet to prevent or treat the disease. Frautschy and co-workers50 have shown that curcumin has both antioxidant and anti-inflammatory activity in both normal aged rats and in transgenic models of dementia as well as inducing an apparent inhibition of Aβ oligomers,51 suggesting that curcumin, taken outside of food and perhaps at different doses than when taken in food, could protect against the development of AD pathology. In this regard, however, the epidemiologic data is controversial; there is evidence that dietary intake of vitamin E, a known antioxidant, is correlated with reduced incidence of AD,52 but other evidence suggests that simple intake of one antioxidant may not be sufficient for disease protection, that it may require a combination of dietary components that enable or enhance the antioxidant activity, such as vitamin C.53 Indeed, in view of the current understanding of the protective role of vitamin C in regenerating the antioxidant activity of many polyphenols,54 experiments testing single antioxidants, in animals, or in clinical trials may give erroneous results, or nonresults.55 Moreover, the composition of the diet itself may affect the bioactivity of a compound, as was shown for the chemopreventive activity of GSE in an established model of breast cancer — it had efficacy in a plant-based diet, but not in a milk casein protein-based diet.56 The current availability of animal models of neurodegenerative disease that better mimic human AD,57 the number of dietary antioxidants already identified to be bioavailable,36 and the accessibility of proteomics technologies58 affords investigators interested in the complexities of oxidative stress in the brain timely opportunities for valuable research.

ACKNOWLEDGMENTS The studies presented here were supported in part by a grant from the NIH National Center for Complementary and Alternative Medicine to the Purdue/UAB Botanicals Center for Age-Related Diseases (1-P50-AT-00477, C. Weaver, PI), by an IDEA award from the Department of Defense (DAMD1701-0469, HK, PI), and by a grant from the United Soybean Board (HK, PI). The purchase of integrated proteomics instrumentation used in this work was enabled by NIH/NCRR Shared Instrumentation Grant (S10-RR16849 to HK) and by grants from the UAB Health Services Foundation General Endowment Funds to HK; the mass spectrometry instrumentation was purchased by NIH/NCRR Shared Instrumentation Grant (S10 RR06487 to SB). Ongoing support for the operation of the Shared Facility is provided by grant (P30 CA13148) from the National Cancer Institute to the UAB Comprehensive Cancer Center (P. Emanual, PI). HK, SB and SM efforts were also supported in part by the UAB Center for Nutrient-Gene Interaction in Cancer Prevention grant (U54 CA100949, SB, PI), Finally, the grape seed extract was generously contributed by Kikkoman Corporation (Chiba, Japan); HK especially acknowledges Dr. Takuro Koga (Kikkoman Corporation) for his sustained efforts at being a liason between our laboratory and Kikkoman Corporation.

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45. Strayer, A., Wu, Z., Christen, Y., Link, C.D., and Luo, Y., Expression of the small heat-shock protein Hsp16-2 in Caenorhabditis elegans is suppressed by Ginkgo biloba extract EGb 761, FASEB J, 17(15), 2305–2307, 2003. 46. Wolters, D.A., Washburn, M.P., and Yates, J.R., III, An automated multidimensional protein identification technology for shotgun proteomics, Anal Chem, 73(23), 5683–5690, 2001. 47. Wang, H. and Hanash, S., Intact-protein based sample preparation strategies for proteome analysis in combination with mass spectrometry, Mass Spectrom Rev, 24(3), 413–426, 2005. 48. Ahmed, N., Ahmed, U., Thornalley, P.J., Hager, K., Fleischer, G., and Munch, G., Protein glycation, oxidation and nitration adduct residues and free adducts of cerebrospinal fluid in Alzheimer’s disease and link to cognitive impairment, J Neurochem, 92(2), 255–263, 2005. 49. Ono, K., Yoshiike, Y., Takashima, A., Hasegawa, K., Naiki, H., and Yamada, M., Potent anti-amyloidogenic and fibril-destabilizing effects of polyphenols in vitro: implications for the prevention and therapeutics of Alzheimer’s disease, J Neurochem, 87(1), 172–181, 2003. 50 Frautschy, S.A., Hu, W., Kim, P., Miller, S.A., Chu, T., Harris-White, M.E., and Cole, G.M., Phenolic anti-inflammatory antioxidant reversal of Abeta-induced cognitive deficits and neuropathology, Neurobiol Aging, 22(6), 993–1005, 2001. 51. Yang, F., Lim, G.P., Begum, A.N., Ubeda, O.J., Simmons, M.R., Ambegaokar, S.S., Chen, P.P., Kayed, R., Glabe, C.G., Frautschy, S.A., and Cole, G.M., Curcumin inhibits formation of amyloid beta oligomers and fibrils, binds plaques, and reduces amyloid in vivo, J Biol Chem, 280(7), 5892–5901, 2005. 52. Morris, M.C., Evans, D.A., Bienias, J.L., Tangney, C.C., Bennett, D.A., Aggarwal, N., Wilson, R.S., and Scherr, P.A., Dietary intake of antioxidant nutrients and the risk of incident Alzheimer disease in a biracial community study, JAMA, 287(24), 3230–3237, 2002. 53. Engelhart, M.J., Geerlings, M.I., Ruitenberg, A., van Swieten, J.C., Hofman, A., Witteman, J.C., and Breteler, M.M., Dietary intake of antioxidants and risk of Alzheimer disease, JAMA, 287(24), 3223–3229, 2002. 54. Patel, R.P., Boersma, B.J., Crawford, J.H., Hogg, N., Kirk, M., Kalyanaraman, B., Parks, D.A., Barnes, S., and Darley-Usmar, V., Antioxidant mechanisms of isoflavones in lipid systems: paradoxical effects of peroxyl radical scavenging, Free Radic Biol Med, 31(12), 1570–1581, 2001. 55. Barnes, S. and Prasain, J., Current progress in the use of traditional medicines and nutraceuticals, Curr Opin Plant Biol, 8(3), 324–328, 2005. 56. Kim, H., Hall, P., Smith, M., Kirk, M., Prasain, J.K., Barnes, S., and Grubbs, C., Chemoprevention by grape seed extract and genistein in carcinogen-induced mammary cancer in rats is diet dependent, J Nutr, 134(Suppl. 12), 3445S–3452S, 2004. 57. Oddo, S., Billings, L., Kesslak, J.P., Cribbs, D.H., and LaFerla, F.M., Abeta immunotherapy leads to clearance of early, but not late, hyperphosphorylated tau aggregates via the proteasome, Neuron, 43(3), 321–332, 2004. 58. Kim, H., Page, G.P., and Barnes, S., Proteomics and mass spectrometry in nutrition research, Nutrition, 20(1), 155–165, 2004.

Index A ACAT inhibitors, see Acyl-coenzyme A:cholesterol acyltransferase inhibitors Acetyl-L-carnitine (ALC), 83, 135 Acetylcholine receptor, 65 Acetylcholinesterase inhibitors, 136 Acetyl-coenzyme A, 75 Acrolein, 183, 184, 189 Activator protein-1 (AP-1), 51 Acyl-coenzyme A:cholesterol acyltransferase (ACAT) inhibitors, 46 S-Adenosyl-homocysteine (SAH), 444 AD, see Alzheimer’s disease ADAS, see Alzheimer Disease Assessment Scale Adenosine triphosphate (ATP), 447 Adenylate cyclase, 65 ADHD, see Attention-deficit hyperactivity disorder Advanced glycation endproducts (AGEs), 64, 107, 248 AGEs, see Advanced glycation endproducts Aging canine model of human, 215 normal, 124 AIDS, 46 ALC, see Acetyl-L-carnitine ALCAR supplementation, 84 Aldehyde dehydrogenases (ALDHs), 186, 187 ALDHs, see Aldehyde dehydrogenases Alzheimer Disease Assessment Scale (ADAS), 136 Alzheimer’s disease (AD), 60 Americans affected by, 2, 460 animal models of, 30 Assessment Scale-Cognitive subscale, 252

atherosclerosis and, 164 cellular insult in, 302 characteristics of, 109 cholesterol and, 410 cognitive decline and, 164 definition of, 27, 132, 328, 396 dementia and, 410 diagnosis of, 132 familial, 204, 396 free radical-mediated damage in, 149 hippocampal neuronal cell death in, 270 incidence, prediction of, 443 invertebrate models of, 399 late onset, 147 major hallmark of, 27 mitochondrial decay and, 78 models, neuronal loss in transgenic, 124 neurodegeneration in, 277 neuropathological hallmark of, 328 oxidative stress and, 132 pathogenesis, homocysteine and, 426 pathogenetic steps of, 239 pathogenic phenotype in, 109 plasma, oxidized proteins in, 11 progression, ALCAR and, 88 reactive oxygen species and, 303 risk of idiopathic, 250 role of Aβ in pathogenesis of, 329 thiamine effect in, 81 toxicity, proteins involved with, 201 transition metals in, 29 vitamin B and, 425 vitamin E and, 108 Alzheimer’s disease brain and models, proteomics identification of oxidatively modified proteins in, 1–25 future of proteomics in Alzheimer’s disease, 16 methodology, 3–8 Alzheimer’s disease brain tissue, 3–4 Caenorhabditis elegans, 4 481

482 cholinergic animal model, 4 mass spectrometry and database searching, 6–8 senescence-accelerated mice prone 8, 4 two-dimensional polyacrylamide gel electrophoresis, 4–6 oxidized proteins in Alzheimer’s disease plasma, 11 proteomic studies on oxidatively modified proteins in animal models of Alzheimer’s disease, 11–15 Caenorhabditis elegans, 14–15 cholinergic dysfunction-related animal model, 12–13 SAMP8 mice, 13–14 proteomics studies on oxidatively modified proteins in Alzheimer’s disease brain, 8–11 altered synaptic function, 9 cell cycle, 10–11 lipid abnormalities and cholinergic failure, 10 neuritic abnormalities, 10 pH maintenance, 11 proteasomal dysfunction, 9–10 proteins involved in energy metabolism, 8–9 proteins involved in glutamate reuptake or conversion, 9 Alzheimer’s disease-specific oxidative stress with multiphoton microscopy, direct evaluation of, 27–42 mechanisms of oxidative stress-related toxicity, 28–30 reactive oxygen species, 29 transition metals, 29–30 multiphoton microscopy, 31–36 effect of antioxidants on reactive oxygen species, 33–36 imaging reactive oxygen species, 32–33 natural antioxidants, 30–31 oxidative stress markers, 31 American Cancer Society, recommendation of, 430 American diet, antioxidant contribution of apple to, 252

Index American Medical Association, dietary supplements and, 429 Amino acid(s) definition of, 416 derivatives of branched-chain, 75 oxidative modification of, 63 residues, modified, 471 Amyloid-β and τ in Alzheimer’s disease, 121–129 amyloid-β, 122–124 tau, 124–125 Amyloid cascade hypothesis, 395, 396 Amyloid deposits, 166 Amyloid-β peptide, 395–408 amyloid β cascade hypothesis of Alzheimer’s disease, 396–397 modulating amyloid β species in vivo, 399–402 animal models of Alzheimer’s disease, 399–400 conventional pharmacology targeting amyloid β species, 400 reverse pharmacology supporting amyloid β as target, 400–402 regulating amyloid β species in vitro, 397–399 inhibition of amyloid β oligomerization, 398–399 supramolecular assemblies of amyloid β, 397–398 Amyloid precursor protein (APP), 2, 166, 314, 328, 396, 424 altered proteolytic processing of, 235 β -amyloid peptide generation and, 329 apple phenolics and, 247 chromaffin vesicles and, 333 gene, mutation of, 147 holoprotein, 271 mRNA, 283 mutations, 397 soluble, 270, 348 Swedish mutation, 285 Amyloid β protein precursor (Aβ PP) mutations, 123 scavenger, 414 Amyotrophic lateral sclerosis, 28, 61, 163 Analgesic regimes, prevalence of various, 170

Index Anti-aging method, caloric restriction as, 411 Anticholesterol treatment, nicotinic acid as, 81 Anti-DNP antibody, 461, 466, 470, 473 Anti-DNP reactivity, 471 Anti-inflammatory drugs, 45 Antioxidant(s), 420 benchmark, 50 capacity, apple, 252 carotenoids as, 418 combinations, 429–430 defense systems, strengthening of, 85 diet, 217 ideal, 309 importance of in fight against AD, 44 lipid-soluble, 305 major chain-breaking, 304 medicines, attractiveness of, 46 metabolic, 135 natural, 30 response elements (AREs), 189 supplementation, effect of on cognition, 214 vitamin C as, 414 Antioxidant therapies in prevention and treatment of Alzheimer disease, 131–145 antioxidant therapies, 135, 304 direct antioxidants, 137–139 indirect antioxidants, 139–140 metabolic antioxidants, 135–137 mitochondria, 133–135 oxidative stress, 132–133 Anti-Parkinson drug, 347, 348 see also Neuroprotective action of antioxidants compared with rasagiline, molecular mechanism of AP-1, see Activator protein-1 apoE, see Apolipoprotein E Apolipoprotein E (apoE), 2, 268, 443 Apoptosis, neurodegenerative diseases and, 311 APP, see Amyloid precursor protein Apple phenolics and Alzheimer’s disease, 247–263 Alzheimer’s disease and amyloid β protein, 247–248 apple phenolics, 252–257

483 action of quercetin, 253–257 composition and antioxidant capacity in apple, 252–253 phytochemicals, 250–252 action of antioxidants in Alzheimer’s disease, 251–252 health benefits, 250–251 production of oxidative stress in Alzheimer’s disease, 248–250 Aβ and oxidative stress, 248–249 high fragility of brain on oxidative stress, 250 naturally occurring oxidative stress, 249 Aβ PP mutations, see Amyloid β protein precursor mutations Arachidonic acid, 161, 182, 249, 256, 413 AREs, see Antioxidant response elements L-Ascorbate, 50 Ascorbic acid, 414 Aspirin and Alzheimer’s disease protection, 159–180 aspirin and modern successors, 160–162 aspirin and non-aspirin NSAIDs in modern pharmacotherapy, 163–164 cardiovascular diseases, 163 diabetes, 163–164 malignancy, 164 clinical trials, 167–168 cognitive decline and Alzheimer’s disease, 164–167 amyloid deposits, 166–167 brain insulin, 166 circulation, 164–165 glutamate excitoxicity, 167 inflammation, 165–166 oxidative stress, 165 effects of salicylic compounds in plants, 159–160 OCTO-twin study, 168–169 results, 169–173 side effects and risks of aspirin, 162–163 Atherosclerosis, AD and, 164 ATP, see Adenosine triphosphate Attention-deficit hyperactivity disorder (ADHD), 77, 423

484 B Baby boomers, aging of, 473 BACE 1, 239, 328, 337 BAD phosphorylation, see Bcl-xL/Bcl2-Associated Death Promoter phosphorylation Bax immunoreactivity, 282 Bax protein expression, 352 Bayer Pharmaceutical Industry, 161 BBB, see Blood–brain barrier BCA, see Bicinchoninic acid Bcl-xL/Bcl-2-Associated Death Promoter (BAD) phosphorylation, 83 BDNF, see Brain-derived neurotrophic factor Behavioral deficits, 377 Behavioral and psychological symptoms of dementia (BPSD), 47 Bicinchoninic acid (BCA), 4 Biotin deficiency, 75 Blood–brain barrier (BBB) cytidine and, 83 DFO penetration across, 283 quercetin and, 257 Blueberry supplementation, age-related neuronal and behavioral deficits improved by polyphenol-rich, 373–393 neuronal and behavioral changes in aging, 377–378 behavioral deficits, 377–378 neuronal changes, 377 neuroprotective effects of blueberries, 378–382 oxidative stress, 374–377 age-related increase in oxidative stress, 375–376 inflammation, 376–377 oxidant–antioxidant balance, 375 reactive oxygen species and reactive nitrogen species, 374–375 BMD, see Bone mineral density Bone mineral density (BMD), 428 BPSD, see Behavioral and psychological symptoms of dementia Brain -derived neurotrophic factor (BDNF), 271, 353

Index function-enhancing supplement, 419 MPM imaging, 32 BSO, see Buthionine sulfoximine Buthionine sulfoximine (BSO), 378 C CAD, see Coronary artery diseases Caenorhabditis elegans, 4, 14 Aβ oligomerization in, 48 EGb 761 protection and, 313 mutants, ROS production and, 316 reactive oxygen species and, 314 Calcium homeostasis, dysregulation of, 248 Calmodulin, 65 Caloric restriction (CR), 411 animal studies, 423 benefits of, 412 brain aging and, 412 human studies, 422 Camellia sinensis, catechins in, 279 Cancer, natural antioxidants and, 30 Carbonyls, lipid-derived, 182, 184 Carboxylases, biotin-dependent, 75 Cardiac autonomic neuropathy, treatment of, 85 Cardiolipin, age-related loss of, 62 Cardiovascular disease, dementia and, 418 Carotene(s) relationship between AD and intake of, 138 -rich fruits, 251 Carotenoids, 416, 418 Catechins, Camellia sinensis, 279 Catechol-O-methyltransferase (COMT), 280, 347 Cell cycle machinery, altered, 10 Cell death antioxidant-attenuated, 349 Bad-mediated, 352 dopaminergic, 112 Cell signaling pathways, effect of tea catechins on, 289 Cell survival/death gene expression, 351 Cellular-function regulator, EGb 761 as, 52 Central nervous system (CNS), 11, 254, 307 disease, 153 HCY methylation and, 448

Index inflammation in, 377 neurotransmission, 308 polyunsaturated fatty acids and, 182 Cerebrospinal fluid (CSF), 11, 151, 446 Chaotropic agents, 5 Chloride channel protein 3, 49 Chloroform–methanol extraction, 462 Chlorogenic acid, 252, 254 Cholesterol AD and, 410 conversion of to bile acids, 414 Choline deficiency, 77 supplementation, 80, 83 Cholinergic function, deficits in, 80 Cholinergic receptor stimulation, 331 Cholinesterase inhibitors, 45 Chromaffin cells, 330–331 vesicles, APP and, 333 Cigarette smoke, 265 CJD, see Creutzfeldt-Jakob disease CK, see Creatine kinase Clioquinol, 140, 279 CNS, see Central nervous system CoA, see Coenzyme A Cobalamin, 415 Coenzyme A (CoA), 74 Coenzyme Q, 84, 214 Coenzyme Q10, 78, 86, 278 Cognition effect of antioxidant supplementation on, 214 enhancement, 400–401 Cognitive dysfunction mitochondrial decay and, 61 mt-nutrient deficiency and, 70 Cognitive function, preservation of, 213 Cognitive impairment, ALCAR supplementation and, 84 Collapsin, 13 COMT, see Catechol-O-methyltransferase Copper deficiency, 74 AD and, 30 Coronary artery diseases (CAD), 416 COX, see Cyclooxygenase CR, see Caloric restriction

485 Creatine kinase (CK), 8, 87, 464 Creatine supplementation, 86 Creutzfeldt-Jakob disease (CJD), 200, 201, 206, 419 Cryptoxanthin, 416 CSF, see Cerebrospinal fluid Curcumin, 139, 476 Cyclooxygenase (COX), 161, 228 enzymes, oxidative reactions and, 165 inhibition, 162 -mediated oxidation, 183 Cystathionine, 415, 448–449 Cysteine proteases as β-secretases for Aβ production in major regulated secretory pathway of neurons, 327–342 β-amyloid involved in development of Alzheimer’s disease, 329 β-amyloid peptide generated by proteolytic processing of amyloid precursor protein, 329 β-amyloid production and secretion in major regulated secretory pathway of neurons, 330 BACE 1 in minor constitutive secretory pathway for basal secretion of β-amyloid, 337–339 chromaffin vesicles for identification of β-secretase, 332–333 cysteine proteases for β-secretase activity in regulated secretory pathway of neurons, 333–336 kinetic studies of cysteine β-secretases demonstrate preference for cleavage of wild-type β-secretase site, 336–337 regulated cosecretion of β-amyloid with neurotransmitters from neuronal chromaffin cells in primary culture, 330–332 unifying hypothesis for distinct proteases in regulated pathways for β-amyloid production and secretion, 339 Cytidine 5⬘-diphosphocholine, 83 Cytochrome oxidase, immunocytochemistry of, 133 Cytosol proteins, 65

486 D DAT, see Dementia of the Alzheimer-type Database searching, 6 DCF assay, see Dichlorofluorescein assay Decosahexaenoic acid (DHA), 413 cellular functions and, 413 deficiency, 414 fatty acids and, 413 -poor diet, 424 supplementation, 82 Dehydroepiandrosterone (DHEA), 240 Dementia AD and, 410 of the Alzheimer-type (DAT), 136, 426–427 behavioral and psychological symptoms of, 47 common form of, 266 with Lewy bodies (DLB), 149 progression to Alzheimer’s, 148 risk factors for, 418 transgenic model of, 461 vascular, 168 Desferrioxamine (DFO), 139, 283, 427 Detoxification, mechanisms of, 185 Dextropropoxyphene, 170 DFO, see Desferrioxamine DHA, see Docosahexaenoic acid DHEA, see Dehydroepiandrosterone DHFR, see Dihydrofolate reductase DHLA, see Dihydrolipoic acid Diabetes, increased risk of, 166 Diabetic glucosuria, salicylate intake and, 163 Diabetic peripheral neuropathy, treatment of, 85 Dichlorofluorescein (DCF) assay, 228 Dietary reference intakes (DRIs), 66, 70 Dietary supplement(s) American Medical Association and, 429 herbal, 309 marketing of, 459 Dietary Supplement Health and Education Act, 459 Dihydrofolate reductase (DHFR), 281 Dihydrolipoic acid (DHLA), 84 Dinitrophenyl (DNP), 459

Index 2,4-Dinitrophenylhydrazine (DNPH), 459, 461 Direct antioxidants, 137 Disease pathogenesis, central mediators of, 121 DLB, see Dementia with Lewy bodies DNA damage, zinc deficiency and, 74 fragmentation, 269, 270, 348 microarray analysis, EGb 761 neuroprotective effects and, 312 microarray assay, 399 mitochondrial, 49, 71, 81, 282 oxidation, 28, 61, 248 DNP, see Dinitrophenyl DNPH, see 2,4-Dinitrophenylhydrazine Donepezil, 45, 174 L-DOPA, 345 Dopamine levels, regulation of, 111 oxidation, 111 receptor, 65 Down’s syndrome (DS), 13, 123, 147, 205, 396 DRIs, see Dietary reference intakes DS, see Down’s syndrome E EAE, see Experimental autoimmune encephalomyelitis EGb 761 advantage of, 310 biological effects of, 43 cognitive symptoms and, 47 first marketing of, 44 inhibition of oligomer formation by, 398 neuroprotective effects of, 311, 312 polyvalent activity, 311–312 protection, model organism of, 313 senile plaques and, 48 EGCG, see (-)-Epigallocatechin-3-gallate EGF, see Epidermal growth factor Eicosapentaenoic acid (EPA), 76 Electron paramagnetic resonance, 248 Electron-transport system (ETS), 214 Electrospray ionization (ESI), 6, 150 ELISAs, see Enzyme-linked immunosorbent assays

Index Encephalopathic symptoms, riboflavin plus nicotinamide to improve, 87 Energy metabolism, proteins involved in, 8, 14 Enrichment Factor values, 269 Enzyme(s) anabolic, 66 COX, 165 dysfunction, reversible, 62 insulin-degrading, 167 -linked immunosorbent assays (ELISAs), 150 membrane, 413 oxidative, 186, 205 reductive, 186 tetrahydrofolate-related, 76 thiamine-dependent, 80 EPA, see Eicosapentaenoic acid Epidermal growth factor (EGF), 164 (-)-Epigallocatechin-3-gallate (EGCG), 225, 278, 284, 291, 350 Equine estrogen, 139 ERE, see Estrogen-responsive element ERK, see Extracellular signal regulated kinase ESI, see Electrospray ionization Essential nutrients brain aging and, 417 definition of, 413 Estrogen receptors, 419 -responsive element (ERE), 227 N-Ethylmaleimide-sensitive factor (NSF), 9 ETS, see Electron-transport system Experimental autoimmune encephalomyelitis (EAE), 281 Extracellular signal regulated kinase (ERK), 226, 241 F FAD, see Flavin adenine dinucleotide Familial AD (FAD) mutations, 248 Fat-soluble, 414 Fatty acid(s) animal studies, 424 -binding protein, 15

487 degradation of, 249 human studies, 423 Fenton chemistry, 344–345 Fenton reaction, 133, 303 Ferulic acid, 139 F2-isoprostanes (F2-IsoPs), 149, 150 F2-isoprostanes as biomarkers of late onset Alzheimer’s disease, 147–157 Alzheimer’s syndrome, disease, and dementia, 147–149 assays for F2-IsoPs, 149–150 F2-IsoPs in brain, 150 F2-IsoPs in lumbar cerebrospinal fluid, 151–152 F2-IsoPs in plasma and urine, 152–153 F2-IsoPs in ventricular cerebrospinal fluid, 151 free radical-mediated damage in Alzheimer’s disease, 149 F2-IsoPs, see F2-isoprostanes Flavin adenine dinucleotide (FAD), 62, 76 Flavin mononucleotide (FMN), 62 Flavonoid(s) glycosides, 306 green tea, 281 phenolic hydroxyl groups of, 307 use of plants containing, 306 FMN, see Flavin mononucleotide Folate deficiency, 76, 446, 447 metabolism, 444 Folic acid, 415 deficiency, 425 supplementation, 82 Folk medicine, 161 Framingham Study, 425 Free radical(s) damage, vitamin E and, 304 dense-core plaques and, 34 excess production of, 52 production, excess in, 302 scavenger(s) EGb 761 as, 44 neuroprotective effect of, 50–51 transcription factors and, 51 theory of aging, 303, 420 Fursultiamine, 81

488 G GAD mouse model, see Gracile axonal dystrophy mouse model GAP, see Growth-associated protein Gas chromatography-mass spectrometry (GC-MS), 150 Gas-phase of cigarette smoking (GPCS), 267 GC-MS, see Gas chromatography-mass spectrometry GDNF, see Glial cell line-derived neurotrophic factor GEM, see Ginkgo Evaluation of Memory Gene expression, effect of EGb 761 on, 49 Geriatric Evaluation by Relative’s Rating Instrument (GERRI) score, 137 GERRI score, see Geriatric Evaluation by Relative’s Rating Instrument score GFP, see Green fluorescent protein Gingko biloba, 36 Aspirin-like effect of, 160 extract biological effects of, 43 first marketing of, 44, 48 inhibition of oligomer formation by, 398 standardized, 305 flavonoid components of, 317 natural antioxidant properties of, 310 Ginkgo biloba extract and Alzheimer’s disease, 43–58 effects of EGb 761 on amyloid-β , 48–49 effects of EGb 761 on gene expression, 49–50 effects of EGb 761 on human subjects, 47–48 cognitive symptoms in Alzheimer’s disease, 47 other cognitive effects, 48 prevention of Alzheimer’s disease, 47–48 effects of EGb 761 possibly linked to antioxidant action, 50 EGb 761 as free-radical scavenger, 48 Ginkgo biloba extracts, 46–47 hypothesis, 50–52 importance of antioxidants in fight against Alzheimer’s disease, 44–46

Index Ginkgo biloba extract EGb 761 in C. elegans model of Alzheimer’s disease, 301–326 C. elegans, 313–316 Alzheimer’s disease-associated C. elegans mutants, 316–317 Alzheimer’s disease-associated mutant models exhibit higher reactive oxygen species levels, 314–316 Alzheimer’s disease-associated transgenic C. elegans mutant, 313–314 analysis of reactive oxygen species levels in C. elegans, 314 EGb 761 components, 317–318 advancement in recent methodology for reactive oxygen species detection, 317–318 kaempferol and quercetin, 317 Ginkgo biloba extract, 305–313 natural antioxidant properties of Ginkgo biloba, 310–311 neuroprotective effects of EGb 761, 311–313 standardized Ginkgo biloba extract, 305–310 reactive oxygen species, aging, and Alzheimer’s disease, 303–305 supplemental antioxidant therapies, 304 vitamin C, 305 vitamin E, 304–305 stress resistance and longevity, 318–319 enhanced life span in C. elegans treated with EGb 761, 318–319 oxidative stress resistance in C. elegans fed with EGb 761, 318 Ginkgo Evaluation of Memory (GEM), 47, 401 Glial cell line-derived neurotrophic factor (GDNF), 353 Glucosuria, provoked, 166 Glutamate excitoxicity, 167 reuptake, proteins involved in, 9 synthase (GS), 8

Index Glutathione (GSH), 282 activity, reduced, 344 antioxidant activity of, 449 loss, 256 metabolism, 448 pathway metabolites, formation of, 188 reductase (GR), 451 reduction of, 378 Glutathione S-transferases (GSTs), 15, 182 GPCS, see Gas-phase of cigarette smoking GR, see Glutathione reductase Gracile axonal dystrophy (GAD) mouse model, 12 Grape seed extract (GSE), 459 Grape seed extract, dementia-relevant brain protein changes in response to, 457–480 elements of productive proteomics, 473–475 findings from proteomics investigations, 464–466 proteins discriminated between grape seed extract and control dietary groups, 464 putative protein identifications generated by peptide mass fingerprint analysis, 465 Western-blot analysis for protein oxidations, 465–466 initial conclusions, 467–473 attenuation of protein oxidations in tg2576 brains, 469–470 future challenges in analysis of neurodegeneration-relevant protein oxidations, 470–471 proteins in normal young adult mammalian brain affected by grape seed extract, 467–469 role of proteomics in nutrition-related research, 471–473 proteomics identification of effects/targets of dietary antioxidants in chronic conditions, 475–476 proteomics technologies in identification of targets of antioxidants, 461–464

489 -blot analysis, 463 derivatization of protein carbonyls with 2,4-dinitrophenylhydrazine, 462 image analysis, 463 mass spectrometry methods to identify proteins, 463–464 normal young adult rat brain, 461 protein extraction and concentration with methanol-chloroform, 462 rodent brain tissue preparation for two-dimensional electrophoresis, 461–462 statistical analysis, 464 transgenic model of dementia, 461 two-dimensional gel electrophoresis, 462–463 timing of baby boomers coincident with emerging technologies, 473 Green fluorescent protein (GFP), 318 Green tea catechins, see Iron chelation, essentiality of in neuroprotection flavonoids, 281 polyphenol, 225, 345 Green tea and resveratrol as protective agents against neurotoxins, 225–234 in vitro neuroprotective effects, 227–228 epigallocatechin gallate, 228 resveratrol, 227–228 in vivo neuroprotective effects, 228–231 epigallocatechin gallate, 230–231 resveratrol, 228–230 Growth-associated protein (GAP), 281 GS, see Glutamine synthase GSE, see Grape seed extract GSH, see Glutathione GSTs, see Glutathione S-transferases H Haber–Weiss reaction, 133 HCY, see Homocysteine Heat shock proteins, 318 Heme deficiency, causes of, 73 Heme-oxygenase-1 (HO-1), 134

490

Index

Herbal dietary supplements, 309 Hesperitin, 35 HIF, see Hypoxia-inducible factor Hippocrates, 160 HNE, see 4-Hydroxy-2-nonenal HO-1, see Heme-oxygenase-1 Homocysteine (HCY), 443 animal studies, 426 elimination, 443–456 transmethylation pathway, 444–448 transsulfuration pathway, 448–451 human studies, 424 metabolism, 415 transsulfuration, 449 Honolulu-Asia Aging Study, 138, 425 Hormone replacement therapy (HRT), 139 HRT, see Hormone replacement therapy HSP60, 13 HSP70, 49 Huntington’s disease, 61, 125 6-Hydroxydopamine (6-OHDA), 345 4-Hydroxy-2-nonenal (HNE), 2, 63 Hyperglycemia, 167 Hyperhomocysteinemia, 82, 415 Hyperuricemia, 162 Hypoxia -inducible factor (HIF), 287 -regulated genes, tea catechins and, 287

Insulin -degrading enzyme (IDE), 167 growth factor-1 (IGF1), 164, 381 receptors, 65, 166 Iodochlorhydroxyquin, 279 Iron chelators, 283, 286 deficiency, 71 -responsive element, 355 Iron chelation, essentiality of in neuroprotection, 277–299 effects of tea catechins on cell signaling pathways, 289–291 neuroprotective effects in vitro, 281–282 neuroprotective effects in vivo, 280–281 tea catechins as brain-permeable, nontoxic iron chelators to “iron out” iron from brain, 282–289 Islet amyloid polypeptide (IAPP), 167 Isoelectric focusing (IEF), 5, 461 Isoflavone(s), 420 action, mechanism of, 420 soybean food and, 429 Isoketals, 183 Isolevuglandins, 183 Isotopically coded affinity tags (ICAT), 5

I

Krebs cycle, 75

IAPP, see Islet amyloid polypeptide ICAT, see Isotopically coded affinity tags IDE, see Insulin-degrading enzyme Ideal antioxidant, 309 IEF, see Isoelectric focusing IGF-1, see Insulin growth factor-1 Indirect antioxidants, 139 Indoleamine hormone, 347 Inducible nitric oxide synthase (iNOS), 65, 280 Inflammatory gene expression, 381 Inflammatory processes, NSAIDs and, 165 Inflammatory response, 248 iNOS, see Inducible nitric oxide synthase Inositol 1,4,5-trisphosphate receptor, 65 Insoluble protein deposits, accumulation of, 65

L

K

LA, see α-Lipoic acid Late onset AD (LOAD), 147 LCPUFA, see Long-chain polyunsaturated fatty acids LDL, see Low-density lipoprotein Lecithin, Tacrine and, 88 Lewy bodies, 110–111, 113, 149 Life expectancy, 410 Life extender, caloric restriction as, 411 Lipid(s) oxidation of, 420 peroxidation, 110 products generated by, 149 rise of, 216 -soluble antioxidant, 305

Index Lipid-derived carbonyls in Alzheimer’s disease, 181–197 formation of lipid-derived carbonyls, 182–184 α,β -unsaturated aldehydes, 182–183 γ -keto aldehyde prostanoids, 183–184 lipid-derived carbonyls and AD, 184–185 mechanisms of detoxification, 185–189 phase I metabolism, 186–187 phase II metabolism, 187–189 potential therapeutic avenues, 189–190 α-Lipoic acid (LA), 84 GSH and, 86 oxidized, 85 protective effect of, 85 Lipopolysaccharide (LPS), 378 Liquid chromatography–tandem-mass spectroscopy, 464 LOAD, see Late onset AD Long-chain polyunsaturated fatty acids (LCPUFA), 76, 77 Long-term potentiation (LTP), 123 Low-density lipoprotein (LDL), 251 LPS, see Lipopolysaccharide LTP, see Long-term potentiation Lutein, 416 Lysine-serine-proline domains, 125 M Macronutrients, 413 Mad cow disease, 419 Magnetic resonance imaging (MRI), 32 Maillard products, 64 MALDI, see Matrix-assisted laser desorption/ionization Malondialdehyde (MDA), 63, 107 MAO, see Monoamine oxidase MAO-B inhibitor, 344 MAPK, see Mitogen activated protein kinase Mass spectrometry (MS), 3, 6, 7 Matrix-assisted laser desorption/ionization (MALDI), 6 MCI, see Mild cognitive impairment MDA, see Malondialdehyde Mecamylamine, 271

491 Melatonin, 347, 352 Membrane enzyme activities, 413 Menantine, 78 Metabolic antioxidants, 135 Metabolism, calorie intake and, 411 Metals, neuropsychiatric diseases and, 282 Methionine aminopeptidase, 334, 335–336 oxidation, 201, 205 Methionine sulfoxide reductase system, 199–212 methionine oxidation and amyloid-β, 205–206 methionine oxidation and prion, 206–207 methionine oxidation and α-synuclein, 202–204 protein aggregation, 201–202 soluble a αβ levels are closely linked to Alzheimer’s disease pathogenesis, 204–205 N-Methyl-D-aspartate (NMDA), 28, 109, 266, 289 Methylcobalamin, 447 Methylene tetrahydrofolate reductase (MTHFR), 445, 446 N-Methyl-4-phenyl 1,2,3, 6-tetrahydropyridine (MPTP), 50 Michaelis constant, increased, 62 Micronutrients, 413 Microtubule-associated tau, 49 Mild cognitive impairment (MCI), 48, 132, 152, 215 Milk casein protein-based diet, 476 Minerals, 418 animal studies, 427 human studies, 426 Mini Mental State Examination (MMSE), 137, 169 Mitochondria membrane potential, 352 oxidative stress and, 133 Mitochondrial DNA (mtDNA), 71 EGb 761 and, 49 -encoded cytochrome oxidase III subunit, 282 lack of histones in, 133 mutations, 81 oxidative damage to, 376

492 Mitochondrial nutrients, 59–105 definition of, 60 effectiveness of combinations of mt-nutrient supplementation on AD and PD prevention and treatment, 87–89 Km concept and metabolism, 62–63 mitochondrial decay with age, 61–62 mt-nutrient deficiency and cognitive dysfunction, 70–78 acetyl-carnitine, R-α-lipoic acid, coenzyme Q10, and creatine decline, 78 biotin deficiency, 75 choline deficiency, 77–78 copper deficiency, 74 docosahexaenoic acid and eicosapentaenoic acid deficiency, 76–77 folate deficiency, 76 iron deficiency, 71–74 niacin deficiency, 76 pantothenic acid deficiency, 74–75 riboflavin deficiency, 76 thiamine deficiency, 75–76 vitamin B6 deficiency, 75 zinc deficiency, 74 mt-nutrients and mitochondrial decay, 66–70 mt-nutrient supplementation for the prevention and amelioration of AD and PD, 78–87 acetyl-L-carnitine/L-carnitine supplementation, 83–84 choline supplementation, 83 coenzyme Q10 supplementation, 86 creatine supplementation, 86–87 docosahexaenoic acid supplementation, 82–83 folic acid, B6, and B12 supplementation, 82 α-lipoic acid/dihydrolipoic acid supplementation, 84–86 mitochondrial decay due to oxidative damage, 78–80 niacin/NADH supplementation, 81–82 riboflavin supplementation, 81 thiamine supplementation, 80–81

Index perspectives, 89 possible underlying mechanisms for altered enzyme Km, 63–66 aging-associated protein/enzyme oxidation, 65–66 protein degradation/turnover, 64–65 protein glycation and cross-linking, 64 protein nitration and chlorination, 64 protein oxidation and adduction by aldehydes, 63 Mitogen activated protein kinase (MAPK), 226, 280, 349, 377 Mixed-function oxidation reactions, 63 MMSE, see Mini Mental State Examination Model(s) AD animal, 30 chronic disease, 154 neuronal loss in transgenic, 124 canine, of human aging, 215 dementia, 461 Huntington’s, 89 Huntington’s disease, 125 mouse, gracile axonal dystrophy, 12 PD, 89 Molecular markers, oxidative stress and, 109 Monoamine oxidase (MAO), 249, 289, 344 Morris water maze (MWM), 378 Motor deficits, 377 MPM, see Multiphoton microscopy MPTP, see N-Methyl-4-phenyl 1,2,3, 6-tetrahydropyridine MRI, see Magnetic resonance imaging MS, see Mass spectrometry mtDNA, see Mitochondrial DNA MTHFR, see Methylene tetrahydrofolate reductase MTT conversion, 269 Multiphoton microscopy (MPM), 28, 31, 48, see also Alzheimer’s diseasespecific oxidative stress with multiphoton microscopy, direct evaluation of MWM, see Morris water maze

Index N NADH dehydrogenase, 65 NAD(P)H:quinone oxidoreductase, 189 Namenda, 78 Naproxen, 45, 165 NAS, see Noradrenergic system National Health and Nutrition Examination Survey, 415 National Institute on Aging (NIA), 415 Natural antioxidants, 30 Natural killer (NK) cell activity, 418 NB cells, see Neuroblastoma cells NBM, see Nucleus basalis of Meynert Nerve growth factor (NGF), 271, 347 Neuritic abnormalities, 10 Neuritic plaques (NPs), 148 Neuroblastoma (NB) cells, 345 Neurodegeneration, AD and, 237 Neurodegenerative diseases, most common, 374 Neurofibrillary degeneration (NFD), 140 Neurofibrillary tangles (NFT), 2, 121, 148, 204, 235 Neuron(s) cell survival factor, 290 GSH precursors supplied to, 189 nondividing, 213 Neuronal cell death, 108 Neuronal nitric oxide synthase (nNOS), 65 Neuropeptide Y (NPY), 331 Neuroprotective action of antioxidants compared with rasagiline, molecular mechanism of, 343–363 future perspectives, 355 mechanism of action of rasagiline, 352–355 mechanisms underlying antioxidantattenuated cell death, 349–352 effect of antioxidants on cell survival/death gene expression, 351–352 modulation of cell signaling pathways, 349–351 neuroprotective studies in models of Alzheimer’s disease, 348–349 antioxidant agents, 348 rasagiline, 348–349

493 studies of neuroprotective properties in models of Parkinson’s disease, 345–348 antioxidant agents, 345–347 rasagiline, 347–348 Neuroprotective drug, 291 Neurospora crass, pantothenic acid deficiency in, 75 Neurotoxins, see Green tea and resveratrol as protective agents against neurotoxins Neurotransmitters, synthesis of, 414 Neutralization buffer, 462 NFD, see Neurofibrillary degeneration NFT, see Neurofibrillary tangles NGF, see Nerve growth factor NIA, see National Institute on Aging Niacin deficiency, 76 /NADH supplementation, 81 Nicotine cholinergic receptor stimulation by, 331 fibrillar Aβ formation and, 265 treatment, chronic, 271 Nicotine, effects of in models of Alzheimer’s disease, 265–275 antioxidant actions of nicotine and scavenging effect of nicotine on oxygen free radicals, 266–267 chronic nicotine treatment reduces β-amyloidosis in brain of mouse model of Alzheimer’s disease, 271–272 nicotine breaks down preformed Alzheimer’s amyloid β fibrils, 268–269 nicotine inhibits β-amyloidosis, 267–268 nicotine protects hippocampal neuronal cells against Aβ-induced apoptosis, 269–271 Nicotinic acid, 81, 82 Nitration, selective target for, 110 Nitric oxide reactive species derived from, 107–108 synthase (NOS), 109, 133, 375 NK cell activity, see Natural killer cell activity

494 NMDA, see N-Methyl-D-aspartate nNOS, see Neuronal nitric oxide synthase Nonessential nutrients, 419, 421 Nonsteroidal anti-inflammatory drugs (NSAIDs), 45, 87, 381 first, 161 inflammatory process and, 165 preventative effect of, 164 Noradrenergic system (NAS), 379 Nordihydroguaiaretic acid, 269 NOS, see Nitric oxide synthase NPs, see Neuritic plaques NPY, see Neuropeptide Y NSAIDs, see Nonsteroidal antiinflammatory drugs NSF, see N-Ethylmaleimide-sensitive factor Nuclear factor κ B (NFκ B), 51, 226, 353 Nucleic acid(s) oxidation of, 420 synthesis, folate and, 76 Nucleus basalis of Meynert (NBM), 2 Nutritional antioxidant enrichment and improved cognitive function in canines, 213–223 canine model of human aging, 215–216 age-dependent cognitive decline in canines, 215 neuropathology in aged canines, 215–216 treatment with antioxidant-enriched diet improves cognition in aged canines, 216–217 Nutrition, brain aging, and Alzheimer’s disease, 409–441 nutrition and Alzheimer’s disease, 422–429 caloric restriction, 422–423 fatty acids, 423–424 minerals, 426–427 phosphatidylserine, 427–428 phytoestrogen, 428–429 vitamins and homocysteine, 424–426 nutrition and brain aging, 411–422 caloric restriction, 411–413 essential nutrients, 413–419 nonessential nutrients, 419–422 recommendations, 429–430

Index O Obesity, insulin levels and, 167 Object recognition memory (ORM), 380 OCTO-Twin study, 168, 174 6-OHDA, see 6-Hydroxydopamine Oleic acid, 413 ORAC, see Oxygen radical absorbance capacity assay ORM, see Object recognition memory OS, see Oxidative stress Osteoporosis, 411 Oxidant –antioxidant balance, 375 damage, defense against, 422 Oxidation COX-mediated, 183 dopamine, 111 methionine, 201, 205 protein-methionine, 199 Oxidative damage mitochondrial decay due to, 79 PD and, 79 Oxidative enzymes, 186 Oxidative stress (OS), 278, 344 AD and, 238 age-related increase in, 375 cellular dysfunction and, 181 -dependent apoptosis, 345 elevated, 113 genetically induced, 450 -induced injury, 78 markers, 31, 205 molecular markers and, 109 naturally occurring, 249 occurrence of, 107 production of in AD, 248 relevance of in brain, 132–133 senile plaques and, 36 target of, 133 Oxidative stress and Aβ PP processing, 235–246 hypothetical sequence of pathogenetic steps of AD, 239–241 neurodegeneration and AD, 237 oxidative stress and Aβ overproduction, 238–239 oxidative stress and AD, 238

Index Oxidative stress in pathological cascade of Alzheimer disease, 365–372 in autosomal-dominant familial Alzheimer disease and oxidative stress, 366 risk factors for Alzheimer disease and oxidative stress, 366–369 temporal primacy of oxidative stress in pathological cascade of Alzheimer disease, 369 Oxidized proteins, 63, 64 OxyBlot kit, 464, 470 Oxygen deprivation, 460 Oxygen free radicals, scavenging effect of nicotine on, 266 Oxygen radical absorbance capacity assay (ORAC), 378 P PAF, see Platelet-activating factor Paired helical filaments (PHF), 109 Pantothenic acid deficiency, 74 Para-aminosalicylic acid (PAS), 164 Paracetamol, 167–168, 170 Parkinson’s disease (PD), 60 Aspirin and, 163 characteristics of, 110 cigarette smoking and, 266 definition of, 344 mitochondrial decay and, 78 models, 50, 89 morphological hallmark of, 282 neurodegeneration in, 277 nicotinic acid and, 82 oxidative damage and, 79 pathogenesis, genetic factors in, 112 toxicity, proteins involved with, 201 PAS, see Para-aminosalicylic acid PD, see Parkinson’s disease PDQuest image analysis software, 464 PEBP, see Phosphatidylethanolaminebinding protein Pellagra, 76 Peptide(s) mass fingerprint analysis, 463, 465 release of from neurons, 330 Peptidyl-prolyl isomerases, 10

495 Peroxisome proliferator-activated receptors (PPAR), 230 Peroxynitrite, 133, 280 PET, see Positron emission tomography Pharmaceutics, socio-economic worth of newly introduced, 174 PHF, see Paired helical filaments Phosphatidylethanolamine-binding protein (PEBP), 10 Phosphatidylserine (PS), 419 animal studies, 428 human studies, 427 phospholipids, 77 Phospholipids, choline deficiency and, 77 Photo-induced cross-linking of unmodified proteins (PICUP), 398 Phytochemicals action of antioxidants in AD, 251 health benefits, 250 Phytoestrogen(s), 419 animal studies, 429 human studies, 428 PICUP, see Photo-induced cross-linking of unmodified proteins PKA, see Protein kinase A PKC, see Protein kinase C Platelet-activating factor (PAF), 307, 401 PLP, see Pyridoxal 5⬘-phosphate PMIs, see Postmortem intervals Polymorphonuclear leukocytes, rhodamine 123 fluorescence of, 72 Polyphenol(s), see also Blueberry supplementation, age-related neuronal and behavioral deficits improved by polyphenol-rich green tea, 345 investigation of, 225 natural, 30, 35 Polyunsaturated fatty acids (PUFA), 182, 250, 256 Population estimates, 373 Positron emission tomography (PET), 32 Postmortem intervals (PMIs), 3 PPAR, see Peroxisome proliferatoractivated receptors Premarin, 139 Presenilin-1 (PS-1), 2 Presenilin gene, 147 Prion protein, 206

496 Procyanidins, 460 2-Propenal, 183 Prostacyclin, anti-coagulation effect of, 162 Prostaglandin rebuilding synthetases, 161 Protease inhibitors, 333 Proteasomal activity, inhibition of, 15 Proteasome, declining activity of, 64 Protein(s) accumulations, damaged, 199 aggregation, 201, 202 amyloid precursor, 2, 166, 314, 328, 424 antioxidative, 353 Bax/Bad, 282 carbonylation, 200 carbonyl groups, 2–3 carbonyls, 2, 467 cell-survival, 351 cytosol, 65 degradation, mitochondrial nutrients and, 64 dysfunction, mutation-driven, 123 energy metabolism and, 8 expression, Bax, 352 fatty-acid-binding, 15 glial fibrillary acidic, 271 grape seed extract and, 467, 469 growth-associated, 281 GTP-binding, 238 heat shock, 318 identification of, 6 kinase, mitochondrial, 112 low–abundance, 5 mass spectrometry methods to identify, 463 -methionine oxidation, 199 mitochondrial chaperone, 13 modification, reversible, 66 neurofilament, 124 nitration, 64 oxidation(s) aging-associated, 65 aldehydes and, 63 antioxidants and, 420 dementia-related, 461 grape seed extract and, 460 neurodegeneration-relevant, 470 rise of, 216 Western-blot analysis for, 465

Index oxidative damage to, 61 oxidatively modified, 8 oxidized, 64 prion, 206 protease digestion of, 7 proteolytically cleaved, 472 sequence database, 7 tau, 122, 124 tyrosine nitration, detection of, 110 zinc finger, 313 Protein kinase A (PKA), 290 Protein kinase C (PKC), 226, 280, 349, 350 activation, 355 inhibition, 227, 353 –MAPK pathways, 355 pathway blockade, cell death under, 289 Proteomics elements of productive, 473, 474 role of in nutrition-related research, 471 validation of, 466 PS, see Phosphatidylserine PS-1, see Presenilin-1 Psychological disorders, 448 PUFA, see Polyunsaturated fatty acids Purkinje cells, 186, 379 Pyridoxal 5⬘-phosphate (PLP), 75 Pyrodoxamine, 190 Q Quercetin, 253, 255, 257 R RAGE, see Receptor for advanced glycation end products Rasagiline, 289, 344, 347, 348, see also Neuroprotective action of antioxidants compared with rasagiline, molecular mechanism of mechanism of action of, 352 neuroprotective activity of, 354 Reactive microglia, proliferation of surrounding neurons, 344 Reactive nitrogen species (RNS), 250, 374, 375

Index Reactive oxygen and nitrogen species (RONS), 107–119 acute neural injury, 108–109 chronic neurodegenerative diseases, 109–113 Alzheimer’s disease, 109–110 Parkinson’s disease, 110–113 Reactive oxygen species (ROS), 199, 302, 401 accumulation, 227, 238, 270 AD and, 303 buildup of iron gradient in conjunction with, 278 C. elegans and, 314 clearance, antioxidant therapies and, 304 compensation for increased, 450 detection, methodology for, 317 direct antioxidants and, 137 direct scavenging by, 280 generation, 248 imaging of using multiphoton microscopy, 32 increased production of, 29, 132 -induced cellular changes, 308 major source of, 374 peroxidation, 377 production, 315–316 Receptor for advanced glycation end products (RAGE), 29, 237 Redox-active metals, 278 Reductive enzymes, 186 Red wine, polyphenols in, 225 Resveratrol, 139, see also Green tea and resveratrol as protective agents against neurotoxins neuroprotective effects of, 227, 228, 229 reduced incidence of convulsions using, 229 Retinoic acid receptor, 65 Reverse pharmacology, example of, 400 Reverse transcriptase–polymerase chain reaction (RT-PCR), 351 Rheumatism, 160 Riboflavin deficiency, 76 supplementation, 81 RNS, see Reactive nitrogen species ROA, senile plaques and, 33 Rofecoxib, 45

497 RONS, see Reactive oxygen and nitrogen species ROS, see Reactive oxygen species RT–PCR, see Reverse transcriptase–polymerase chain reaction S SAH, see S-Adenosyl-homocysteine Salicylic compounds, effects of in plants, 159 SAMP8, see Senescence-accelerated mice prone 8 Sample biases, 214 SAPKs, see Stress-activated protein kinases sAPP, see Soluble APP SAR, see Systemic acquired resistance SDS, see Sodium dodecyl sulfate SDS-PAGE, 4, 462, 463, 470 Secretases, identification of, 328 SELDI-TOF, see Surface-enhanced laser desorption/ionization time of flight Selenium, 418, 419 Selenomethionine, 419 Semiubiquinone, 375 Senescence-accelerated mice prone 8 (SAMP8), 4, 13 Senile plagues (SP), 2, 27 EGb 761 and, 48 oxidative stress from, 36 reactive oxygen species and, 33 Serotonin receptor, 65 Signaling pathways, activation of, 248 Single-photon emission tomography (SPECT), 32 SNP, see Sodium nitroprusside SOD, see Superoxide dismutase Sodium dodecyl sulfate (SDS), 123, 401 Sodium nitroprusside (SNP), 227 Soluble APP (sAPP), 348 Soybean food, isoflavones and, 429 Soy isoflavones, 419–420 SP, see Senile plagues Spatial memory task, 217 SPECT, see Single-photon emission tomography

498 Stepwise Discriminant Analysis, 464 Steroid hormone(s) receptor, 65 synthesis of, 414 Stress-activated protein kinases (SAPKs), 241 Stroke, 225, 237 Succinyl-coenzyme A, 75 Superoxide dismutase (SOD), 29, 108, 280, 303, 353, 375 Surface-enhanced laser desorption/ionization time of flight (SELDI-TOF), 7 SwissProt database, 7 Synaptic function, altered, 9 α-Synuclein, 202, 282, 286 fibril formation, 111 nitrated, 112 β -Synuclein, 12 Systemic acquired resistance (SAR), 160 T Tacrine, 88 Tamarixetin, 319 Tau accumulation, 124 Tau pathology, 110 Tau protein, 122 TBARS, see Thiobarbituric acid-reacting substrates TBP, see Tributylphosphine Tea catechins, 306 effect of on cell signaling pathways, 289 effect of on hypoxia-regulated genes, 287 Terpene lactones, 307, 309 Tetrahydroaminoacridine, 88 Tetrahydrofolate (THF), 444 Tetrahydrofuran cycle, 307 TfR mRNA, see Transferrin receptor mRNA TGF receptor, see Transforming growth factor-β receptor THF, see Tetrahydrofolate Thiamin deficiency, 75 monophosphate (TMP), 75 supplementation, 80 Thiobarbituric acid-reacting substrates (TBARS), 31, 446

Index Thioflavin, 33, 35, 398 Thioredoxin, 88 Thromboxane, 162 TMP, see Thiamin monophosphate TNF, see Tumor necrosis factor Tofu, 429 Transcription factor signal transducer, 280–281 Transferrin receptor (TfR) mRNA, 289 Transforming growth factor-β (TGF) receptor, 65 Transition metals, 29, 344 Transmethylation pathway, 444 Transsulfuration pathway, 447, 448 Transthyretin (TTR), 414 Tributylphosphine (TBP), 462 TTR, see Transthyretin Tuberculosis, 164 Tumor necrosis factor (TNF), 351 Two-dimensional gel electrophoresis, 4, 462 Tyrosine degradation, 414 hydroxylase, 111, 112, 249 U US Food and Drug Administration, 78 U.S. Preventive Services Task Force (USPSTF), 429 USPSTF, see U.S. Preventive Services Task Force V Value-added research, 475 Vascular dementia, 168 Visuospatial attention, animals tested for, 217 Vitamin(s) animal studies, 426 deficiency states, 415 fat-soluble, 414 human studies, 424 water-soluble, 305, 414 Vitamin B6 deficiency, 75 supplementation, 82 Vitamin C, levels of in brain, 305

Index Vitamin E, 414 lipid peroxidation and, 304 protection against Aβ toxicity by, 50 VK-28 iron chelator, 286

499 X Xanthine oxidase activity, reduced, 230 Z

W Water-soluble vitamin, 305, 414 Wernike-Korsakoff syndrome, 75 Wilson’s disease, 74, 282

Zeaxanthine, 416 Zinc deficiency, 74 finger protein, 313