Metal Ions and Neurodegenerative Disorders

Metal Ions and Neurodegenerative

Editor

Paolo Zatta

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Picture on cover Metallothionein-l-ll positive astrocytes in mouse cerebral cortex (Photo: Dr. Pamela Zambenedetti, Brain Bank, General Hospital, Dolo-Venice, Italy)

Metal Ions and Neurodegenerative Disorders

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Metal Ions and Neurodegenerative Disorders

Editor

Paolo Zatta CNR - National Research Council, Italy

'~world Scientific NEW JERSEY· LONDON· SINGAPORE· SHANGHAI· HONG KONG· TAIPEI· BANGALORE

Published by World Scientific Publishing Co. Pte. Ltd. 5 Toh Tuck Link, Singapore 596224 USA office: Suite 202, 1060 Main Street, River Edge, NJ 07661 UK office: 57 Shelton Street, Covent Garden, London WC2H 9HE

British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library.

METAL IONS AND NEURODEGENERATIVE DISORDERS Copyright © 2003 by World Scientific Publishing Co. Pte. Ltd. All rights reserved. This book, or parts thereof, may not be reproduced in any form or by any means, electronic or mechanical, including photocopying, recording or any information storage and retrieval system now known or to be invented, without written permission from the Publisher.

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ISBN 981-238-398-0

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Preface Many metal ions are as essential to life (Williams and Fausto da Silva, 1996) as other constitutive elements within living matter, such as fats, carbohydrates, and proteins. Today, research into the role of metal ions is of particular interest to nutritional sciences, which have discovered that a wide range of common pathologies are caused by a lack of metalloions. Physiologists maintain that the constitutive and functional roles of some metal ions (such as Na, K, Ca, Mg, Fe, Co, Mn, Cu, and Zn) are vital to the smooth functioning of cells, as well as various organs and tissues. Toxicologists have performed indepth research into the harmful properties of some metal ions (such as Al, Cd, Hg, and Pb), and environmental disasters, such as Camelot and Minamata, have enabled them to be studied on a vast scale. However, until recently, little attention had been paid to the role of metal ions as etiopathogenic agents for some neurodegenerative diseases such as Alzheimer's, Parkinson's, and Lateral Amyotrophic Sclerosis, to name but three. Today, a new field, metalloneurochemistry, is breaking fresh ground on account of the burgeoning number of studies which show the importance of metal ions as etiopathogenic factors or co-factors (Bush, 2000; Zatta, 2001). From this point of view, the concept of metallochaperones is comparatively new in that molecules that could be used for this function were only discovered in the mid-1990s (Halloran and Culotta, 2000). Theories that suggest metal ions are vital etiological agents in neurodegenerative diseases are finding it hard to break into mainstream thought not only on account of the technical and conceptual difficulties, but also of the financial complexities of investing in both basic and applied research in order to discover more about the molecular mechanisms that account for the physiopathological action of metalloions. Chelation therapy is still in the early days of its research into new products that could revolutionize treatment. Two international conferences, Metals and the Brain: From Neurochemistry to Neurodegeneration (University of Padova, Italy, 2000 and University of Fez, Morocco, 2002;

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see also Zatta, 2001), were held on metals and their role in physiopathology as a sign of their importance. This book follows the same train of thought as these conferences in order to highlight the unquestionable importance of metal ions to research into the neurochemistry of neurodegenerative diseases. The excellent reputation of the scientists who have contributed to this project bears testimony to the quality of these studies, which we hope can be developed further with a whole host of new research within this field; one which is still in its infancy. PAOLO ZATTA

REFERENCES Bush AI. Metals and neuroscience. Curr Op Chem Biol 2000; 4:184-191. Halloran TV, Culotta VC. Metallochaperones: An intracellular shuttle service for metal ions. J Biol Chem 2000; 275:25047-25060. William RJP, Frausto da Silva JJR. The Natural Selection of the Chemical Elements. Oxford: Clarendon Press, 1996. Zatta P, editor. Metals and the brain. A special issue. Brain Res Bull 2001; 55:123-325.

Contents Preface

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List of Contributors

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Chapter 1. Metal-Catalyzed Redox Activity in Neurodegenerative Disease Marta A Taddeo, Mark A Smith, Quart Liu, Craigh S Atwood, Lawrence M Say re, George Perry Chapter 2. Metals Distribution and Regionalization in the Brain Margherita Speziali, Edoardo Orvini

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Chapter 3. The Olfactory Pathway as a Route of Entry of Metals into the Brain Hans Tjalve, Jonas Tallkvist

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Chapter 4. Aluminum in Neurological Disorders and Systemic Chelation Therapy Theo P Kruck, Walter J Lukiw

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Chapter 5. Alumium and Central Nervous System Morphology in Hemodialysis Erich Reusche

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Chapter 6. Zinc, Brain, and Aging Eugenio Mocchegiani, Mario Muzzioli, Robertina Giacconi, Tiziana Casoli, Giuseppina DiStefano, Patrizia Fattoretti

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Chapter 7. Transition Metals, Oxidation, Lipoproteins, and Amyloid-3: Major Players in Alzheimer's Disease Anatol Kontush

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Chapter 8. Molecular Basis of Copper Transport: Cellular and Physiological Functions of Menkes and Wilson Disease Proteins (ATP7A and ATP7B). David R Kramer, Roxana M Llanos, Julian F B Mercer

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Chapter 9.

Importance of Copper and Zinc in Alzheimer's Disease and the Biology of Amyloid-(3 Protein and Amyloid-(3 Protein Precursor Avi L Friedlich, Xudong Huang, Seiichi Nagano, Jack T Rogers, Lee E Goldstein, Ashley I Bush, Gerd Multhaup, Konrad Beyreuther, Wolfgang Stremmel, Thomas Bayer

Chapter 10. Copper-Zinc Superoxide Dismutase and Familial Amyotrophic Lateral Sclerosis Moon B Yim, P Boon Chock, Earl R Stadtman

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Chapter 11. Copper and Prion Disease Judyth Sasson, David R Brown

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Chapter 12. Metallothioneins in Neurodegeneration Michael Aschner, William F Silverman, Israel Sekler, Paolo Zatta

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Chapter 13. Iron and Neurodegeneration Stacey L Grab, James R Connor

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Chapter 14. Iron, Neuromelanin, and a-Synuclein in Neuropathogenesis of Parkinson's Disease Kay L Double, Kurt Jellinger, Luigi Zecca, Moussa B H Youdim, Peter Riederer, Manfred Gerlach

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Chapter 15. Iron and Epilepsy Wie-Yi Ong, Benjamin Kian-Chung Ong, Akhlaq A Farooqui, Chuang-Chin Chiueh, James R Connor

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Chapter 16. Role of Iron Metabolism in Multiple Sclerosis Maritha J Kotze, J Nico P De Villiers, Monique G Zaahl, Kathryn J H Robson

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Chapter 17. Manganese Toxicity: A Critical Reappraisal Patrizia Vernole, Maria Morello, Giuseppe Sancesario, Alessandro Martorana, Giorgio Bernardi, Antonella Canini, Palma Mattioli

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Chapter 18. Cupric and Mercuric Ions Affect the Structure and Functions of Cell Membranes Mario Suwalsky, Fernando Villena, Hernan Cardenas,Beryl Norris, Carlos Patricio Sotomayor, Paolo Zatta

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Chapter 19. Influence of Lead Exposure on Brainstem Functions Ombretta Mameli, Marcello Alessandro Caria

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Chapter 20. Neuroprotective Effects of Lithium Sophie Ermidiou-Pollet, Serge Pollet

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Chapter 21. Histopathological Changes in Brain of Uremic Patients on Chronic Hemodialysis Pamela Zambenedetti, Mario Andriani, Maurizio Nordio, Paolo Zatta

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Chapter 22. Clinical Neurotoxicity of Metals and Neurodegenerative Disorders Marcello Lotti

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Index

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List of Contributors Michael ASCHNER Departments of Physiology and Pharmacology Wake Forest University School of Medicine Winston-Salem, NC, USA Email: [email protected] Craigh S ATWOOD Institute of Pathology Case Western Reserve University 2085 Adelbert Road Cleveland, OH 44106, USA Thomas BAYER Department of Psychiatry University of Bonn Medical Center Bonn, Germany Giorgio BERNARDI Department of Neuroscience University of Rome Tor Vergata and IRCCS S Lucia 00100 Rome, Italy Konrad BEYREUTHER ZMBH-Center for Molecular Biology University of Heidelberg Heidelberg, Germany David BROWN Department of Biology and Biochemistry University of Bath Bath BA2 7AY, UK Email: [email protected] XI

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Ashley I BUSH Laboratory of Oxidation Biology Genetics and Aging Research Unit Massachusetts General Hospital Building 114, 16th Street Charlestown, MA 02129, USA Email: [email protected] Antonella CANINI Department of Biology University of Rome Tor Vergata 00100 Rome, Italy Hernan CARDENAS Faculty of Biological Sciences University of Concepcion Casilla 160-C Concepcion, Chile Marcello Alessandro CARIA Department of Biomedical Sciences Human Physiology Division University of Sassari Viale San Pietro 43/B 07100 Sassari, Italy Email: [email protected] Tiziana CASOLI Neurobiology Center Research Department Italian National Research Center on Aging (INRCA) Via Birarelli 89 60121 Ancona, Italy Email: [email protected] Chuang-Chin CHIUEH National Institute of Mental Health LCS, NIH 10/3D-41 Bethesda, MD 20892-1264, USA

List of Contributors

List of Contributors

P Boon CHOCK Laboratory of Biochemistry National Heart, Lung and Blood Institute National Institutes of Health Building 50, Room 2152 50 South Drive, MSC-8012 Bethesda, MD 20892-8012, USA James R CONNOR Department of Neuroscience and Anatomy Pennsylvania State University Hershey, PA 17033-0850, USA Email: [email protected] J Nico P DE VILLIERS Division of Human Genetics Faculty of Health Sciences University of Stellenbosch Tygerberg 7500, South Africa Giuseppina DiSTEFANO Neurobiology Center Research Department Italian National Research Centers on Aging (INRCA) Via Birarelli 89 60121 Ancona, Italy Kay L DOUBLE Prince of Wales Medical Research Institute Sydney, Australia Email: [email protected] Sophie ERMIDIOU-POLLET Department of Biochemistry Medical School, University of Athens Athens, Greece Email: [email protected]

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Akhlaq A FAROOQUI Department of Molecular and Cellular Biochemistry The Ohio State University Columbus, Ohio OH 43210, USA Patrizia FATTORETTI Neurobiology Center Research Department Italian National Research Centers on Aging (INCRA) Via Birarelli 89 60121 Ancona, Italy Email: [email protected] Avi L FRIEDLICH Laboratory of Oxidation Biology Genetics and Aging Research Unit Massachusetts General Hospital Building 114, 16th Street Charlestown, MA 02129, USA Manfred GERLACH Clinical Neurochemistry Department of Child and Youth Psychiatry and Psychotherapy University of Wurzburg Wiirzburg, Germany Robertina GIACCONI Immunology Center Section Nutrition, Immunity and Aging Research Department Italian National Research Center on Aging (INRCA) Via Birarelli 8 60121 Ancona, Italy Lee E GOLDSTEIN Laboratory of Oxidation Biology Genetics and Aging Research Unit Massachusetts General Hospital Building 114, 16th Street Charlestown, MA 02129, USA

List of Contributors

Stacy L GRAB Department of Neuroscience Pennsylvania State University College of Medicine/Milton S. Hershey Medical Center Hershey, PA 17033, USA Email: [email protected] Xudong HUANG Laboratory of Oxidation Biology Genetics and Aging Research Unit Massachusetts General Hospital Building 114, 16th Street Charlestown, MA 02129, USA Kurt JELLINGER Institute of Clinical Neurobiology Vienna, Austria Email: [email protected] Anatol KONTUSH INSERM Unite 551 Papillon Benjamin Delessert Hospital de la Pitie, 83 Boulevard de l'Hopital 75651 Paris Cedex 13, France Email: [email protected] Maritha J KOTZE Division of Human Genetics Faculty of Health Sciences University of Stellenbosch Tygerberg 7500, South Africa Email: [email protected] David K KRAMER Center for Cellular and Molecular Biology School of Biological and Chemical Sciences Deakin University, 221 Burwood Hwy Melbourne 3125, Australia Email: [email protected]

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Theo P KRUCK Surrey Place Center and Department of Physiology University of Toronto Ontario M5S 2C2, Canada Email: [email protected] Quan LIU Institute of Pathology Case Western Reserve University 2085 Adelbert Road Cleveland, OH 44106, USA Roxana M LLANOS Center for Cellular and Molecular Biology School of Biological and Chemical Sciences Deakin University 221 Burwood Hwy Melbourne 3125, Australia Email: [email protected] Marcello LOTTI Dipartimento di Medicina Ambientale e Sanita Pubblica Universita di Padova, Azienda Ospedaliera di Padova Via Giustiniani 2 25128 Padova, Italy Email: [email protected] Walter J LUKIW LSU Neuroscience Center and Department of Ophthalmology Louisiana State University Health Science Center New Orleans, LA 70112, USA Email: [email protected] Ombretta MAMELI Department of Biomedical Sciences, Human Physiology Division University of Sassari Viale San Pietro 43/B 07100 Sassari, Italy Email: [email protected]

List of Contributors

Alessandro MARTORANA Department of Neuroscience University of Rome Tor Vergata and IRCCS S Lucia 00100 Rome, Italy Palma MATTIOLI Department of Biology University of Rome Tor Vergata 00100 Rome, Italy Julian F B MERCER Center for Cellular and Molecular Biology School of Biological and Chemical Sciences Deakin University 221 Burwood Hwy Melbourne 3125, Australia Email: [email protected] Eugenio MOCCHEGIANI Immunology Center Section Nutrition, Immunity and Aging Research Department Italian National Research Center on Aging (INRCA) Via Birarelli 8 60121 Ancona, Italy Email: [email protected] Maria MORELLO Department of Neuroscience University of Rome Tor Vergata 00100 Rome, Italy Gerd MULTHAUP Institut fuer Chemie/Biochemie Freie Universitat Berlin Universitat Berlin Thielallee 63 14195 Berlin, Germany Email: multhaup @ chemie.fu-berlin.de

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Mario MUZZIOLI Immunology Center Section Nutrition, Immunity and Aging Research Department Italian National Research Center on Aging (INRCA) Via Birarelli 8 60121 Ancona, Italy Seiichi NAGANO Laboratory of Oxidation Biology Genetics and Aging Research Unit Massachusetts General Hospital Building 114, 16th Street Charlestown, MA 02129, USA Maurizio NORDIO Nephrology Division General Hospital Venice, Italy Email: [email protected] Beryl NORRIS Faculty of Biological Sciences University of Concepcion Casilla 160-C Concepcion, Chile Benjamin Kian-Chung ONG Department of Medicine National University of Singapore Lower Kent Ridge Road Singapore 119260 Wei-Yi ONG Department of Anatomy National University of Singapore Lower Kent Ridge Road, Singapore 119260 Email: [email protected]

List of Contributors

Edoardo ORVINI Department of General Chemistry University of Pavia, Viale Taramelli 12 27100 Padova, Italy Email: [email protected] George PERRY Institute of Pathology Case Western Reserve University 2085 Adelbert Road Cleveland, OH 44106, USA Email: [email protected] Serge POLLET Department of Biochemistry Medical School, University of Athens Athens, Greece Email: [email protected] Erich REUSCHE Institute of Pathology and Neuropathology University of Lubeck Ratzeburgerallee 160 23538 Lubeck, Germany Email: [email protected] Peter RIEDERER Clinical Neurochemistry Department of Psychiatry and Psychotherapy University of Wurzburg 97080 Wurzburg, Germany Email: [email protected] Kathryn J H ROBSON MRC Unit of Molecular Hematology Weatherall Institute of Molecular Medicine John Radcliffe Hospital, Headington Oxford OX3 9DS, UK

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Jack T ROGERS Laboratory of Oxidation Biology Genetics and Aging Research Unit Massachusetts General Hospital Building 114, 16th Street Charlestown, MA 02129, USA Giuseppe SANCESARIO Department of Neuroscience University of Rome Tor Vergata 00100 Rome, Italy Email: [email protected] Judyth SASSOON Department of Biology and Biochemistry University of Bath Bath BA2 7AY, UK. Email: [email protected] Lawrence M SAYRE Department of Chemistry Case Western Reserve University 2085 Adelbert Road Cleveland, OH 44106, USA Israel SEKLER Zlotowski Center for Neuroscience Faculty of Health Science Ben-Gurion University Beer Sheva, Israel William F SILVERMAN Zlotowski Center for Neuroscience Faculty of Health Science Ben-Gurion University Beer Sheva, Israel

List of Contributors

List of Contributors

Mark A SMITH Institute of Pathology Case Western Reserve University 2085 Adelbert Road Cleveland, OH 44106, USA Carlos Patricio SOTOMAYOR Faculty of Basic Sciences Catholic University of Valparaiso Casilla 4059 Valparaiso, Chile Margherita SPEZIALI CNR-Institute for Energetics and Interphases Department of General Chemistry, University of Pavia Via Taramelli 12 27100 Pavia, Italy Email: [email protected] Earl R STADTMAN Laboratory of Biochemistry National Heart, Lung and Blood Institute National Institutes of Health Building 50, Room 2152 50 South Drive, MSC-8012 Bethesda, MD 20892-8012, USA Wolfgang STREMMEL Department of Medicine Division of Gastroenterology University of Heidelberg, Germany Heidelberg, Germany Mario SUWALSKY Faculty of Chemical Sciences University of Concepcion Casilla 160-C, Concepcion, Chile Email: [email protected]

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Marta A TADDEO Institute of Pathology Case Western Reserve University 2085 Adelbert Road Cleveland, OH 44106, USA Jonas TALLKVIST Department of Pharmacology and Toxicology Faculty of Veterinary Medicine Swedish University of Agricultural Sciences Biomedicum, Box 573 Uppsala 751 23, Sweden Hans TJALVE Department of Pharmacology and Toxicology Faculty of Veterinary Medicine Swedish University of Agricultural Sciences Biomedicum, Box 573 Uppsala 751 23, Sweden Email: Hans .Tj alve @ farmatoxslu. se Patrizia VERNOLE Department of Public Health University of Rome Tor Vergata 00100 Rome, Italy Fernando VILLENA Faculty of Biological Sciences University of Concepcion Casilla 160-C Concepcion, Chile Moon B YIM Laboratory of Biochemistry National Heart, Lung, and Blood Institute National Institutes of Health Building 50, Room 2152, 50 South Drive, MSC-8012 Bethesda, MD 20892-8012, USA Email: [email protected]

List of Contributors

List of

Contributors

Moussa B H YOUDIM Department of Pharmacology B Rappaport Faculty of Medicine Eve Topf Neurodegenerative and National Parkinson Foundation Centers Technicon, Haifa, Israel Email: [email protected] Monique G ZAAHL MRC Unit of Molecular Hematology Weatherall Institute of Molecular Medicine John Radcliffe Hospital, Headington Oxford 0X3 9DS, UK Pamela ZAMBENEDETTI Anatomopathology Division and Brain Bank Dolo General Hospital Dolo-Venice, Italy Email: [email protected] Paolo ZATTA CNR-Institute for Biomedical Biotechnologies Padova Unit "Metalloproteins" Department of Biology, University of Padova Viale G. Colombo 3 35121 Padova, Italy Email: [email protected] Luigi ZECCA CNR-Institute for Biomedical Technologies Via Fratelli Cervi 93 20090 Segrate, Italy Email: [email protected]

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CHAPTER 1

Metal-Catalyzed Redox Activity in Neurodegenerative Disease Marta A Taddeo, Mark A Smith, Quan Liu, Craigh S Atwood, Lawrence M Sayre, George Perry

ABSTRACT Oxidative damage and response are major features of Alzheimer's disease and other neurodegenerative diseases. Metal-catalyzed oxidative events are at the center of radical formation and abnormalities in iron and copper have been noted. In this review, we consider the evidence implicating the central role of metals. Keywords: Amyloid-(3; copper; iron; metal-binding protein; oxidative stress; tau.

1. INTRODUCTION There has been considerable investigation into the roles of redox-active transition metals in the pathogenesis of neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), and prion diseases. Iron, copper, manganese, and other such metals are usually found in essential normal biological processes, and are also involved in enzymatic activities such as those related to respiration. In these functions, deficiency of these metals can be hazardous to normal organ function, including central nervous system activities. However, inappropriate accumulation of excessive metal deposits can also be cytotoxic. In short, disruption of the normal homeostatic metal balance in either direction can result in cellular disturbance. In the latter case, these changes are characterized by oxidative 1

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stress and increased production of free radicals. Oxidative stress results from excessive levels of reactive oxygen species (ROS) generated through imbalance of normal biochemical processes. These oxygen radicals can then damage all types of macromolecules, disrupting normal cellular functions and, ultimately, causing cell death. ROS can normally be found at some level in all aerobic organisms, usually arising from the secondary production of superoxide by mitochondrial reduction of molecular oxygen. Increased levels of ROS during oxidative stress point to increased oxygen processing by mitochondria as a possible source. In addition, greater oxidative stress susceptibility can be caused by production of H 2 0 2 by oxidases such as monoamine oxidase. Examination of nitric oxide (NO) has also gained recognition through the term "nitrosative stress", a form of oxidative stress characterized by the reaction of superoxide with NO to yield peroxynitrite, which is further capable of both oxidation chemistry and nitration reactions. The primary ROS implicated in oxidative stress is the hydroxyl radical, whose damage is dependent upon its diffusion capacity over nanometer distances from its generation site. Although peroxynitrite also appears capable of hydroxyl-like activity, most hydroxyl radicals reflect the Fenton reaction between reduced transition metals (usually iron[II] or copper[I]) and H 2 0 2 . Re-reduction of iron[III] and copper[II] can be performed by superoxide or other cellular reductants such as ascorbate. In addition to their role in redox processes, transition metals may contribute to neurodegeneration, along with redox-inactive metal ions, through their deleterious effects on protein and peptide structure, such as a pathological aggregation phenomenon. In such cases, transition metals can sometimes exert dual neurotoxic effects. In contrast, there are also a number of normal antioxidant defenses to combat the effects of ROS through both enzymatic and nonenzymatic pathways in mammalian cells. Cytosolic copper-zinc superoxide dismutase (CuZnSOD) and mitochondrial manganese superoxide dismutase (MnSOD) are two such important players in antioxidant defenses. These enzymes convert reactive superoxide to harmless 0 2 and H 2 0 2 . The latter is then removed by catalase and peroxidases, which are abundant throughout tissues. In addition, there are a number of proteins closely tied to metalinduced redox activity. These include proteins involved in metal transport

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and those which do not normally have a metal-binding function, but are able to bind metals for neuroprotection. This review explores the interplay between these various factors and evaluates recent work concerning the role of redox-active metals in neurodegenerative diseases.

1.1. Redox-Active Transition Metals in AD AD is pathologically diagnosed by the presence of neurofibrillary tangles (NFTs), senile plaques, neuropil threads, amyloid-beta (A(3) deposits, and selective loss of neurons (Perry and Smith, 1999). Though a number of hypotheses have been presented describing the origins of AD, the one receiving the most recent attention involves a role for oxidative stress (reviewed in Markesbery, 1999; Smith et al., 2000a; Perry et al., 2001). Past studies have already implicated a number of trace metals whose imbalances can contribute to AD, including aluminum, silicon, lead, mercury, zinc, copper, and iron (Lovell et al., 1998). Disruptions in iron and copper levels, in particular, have quite a substantial effect on levels of oxidative stress markers such as lipid peroxidation, as well as on damage related to NFT, senile plaque deposition, and nucleic acid oxidation (Smith et al., 2000a). It was recently found through microparticle-induced X-ray emission that Zn(II), Fe(III), and Cu(II) are significantly elevated in the AD neuropil, and that these metals are further concentrated in the core and periphery of senile plaques (Lovell et al., 1998). These results build on earlier studies reporting increased levels of iron, transferrin receptors and ferritin in AD. Using an in situ iron detection method, we found a marked association of redox-active iron with both NFT and senile plaques in AD (Smith et al., 1997). The association with NFT may partly involve iron binding to the primary NFT protein component, tau (Perez et al., 1998). At the same time, iron regulatory protein (IRP)-2 was found to co-localize with redox-active iron in NFT, senile plaque neurites, and neuropil threads (Smith et al., 1998). IRP-1, in contrast, was found to be present at similar levels in both AD and control brain tissues. These latter results suggest that alterations in IRP-2 may be linked to impaired iron homeostasis in AD (Smith et al., 1998). We have found that redox activity in AD lesions can be directly detected in tissue sections and homogenates using cytochemical methods

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(Smith et al., 1997; Sayre et al., 2000). This activity is inhibited by preexposure to copper- and iron-selective chelators, with incubation in metal solutions post-chelation restoring activity. These studies indicating the presence of iron, and probably also copper related to AD pathology, suggest that accumulations of these metals are major generators of ROS. This production is responsible for the numerous oxidative stress markers apparent in NFT and senile plaques, as well as the more global markers observed in AD (Sayre et al., 2001).

1.2. Aggregation of Amyloid-p and Amyloid |3 Protein Precursor/Amyloid-p-Linked ROS Production Evidence for a disruption of trace metal homeostasis in AD has sparked off efforts to identify possible associations between metal ions and A3 aggregation. Under neutral conditions, it was found that aluminum, iron, and zinc accelerated Ap aggregation, while calcium, cobalt, manganese, copper, magnesium, sodium, and potassium did not (Mantyh et al., 1993). More recent studies, however, suggest that this aggregation due to metals depends more critically on pH. For example, it was found that Cu(II) was able to induce A p ^ o aggregation when the pH was lowered from 7.4 to 6.8, and that this result was unique to this particular metal (Atwood et al., 1998). A mildly acidic environment, as well as increased levels of Zn(II) and Cu(II), is a common feature of inflammation associated with increased oxidative damage due to microglial-derived peroxynitrite. The association of Cu(II), Zn(II), and Fe(II) with amyloid-pi seen in vitro could explain the deposition of these metals in senile plaques in AD. It is interesting to note in these studies on A|3 aggregation that the effect of the transition metal does not involve a redox role. This contrasts with results which suggest a synergistic action of A(3 and copper or iron in mediating ROS production (Bondy et al., 1998). Also, Cu(II) binds to amyloid-|3 protein precursor (ApPP) and appears to be reduced to Cu(I) accompanied by production of a disulfide linkage (Multhaup et al., 1998). Subsequent exposure to H 2 0 2 results in reoxidation of Cu(I) along with site-specific cleavage of ApSPP. Redox chemistry associated with Ap- or ApPP-bound metals could, therefore, contribute to a perturbation of free radical homeostasis.

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Recent genetic studies have revealed a number of mutations in the genes encoding the A(3PP and the presenilin proteins (Perry and Smith, 1999). Alterations in these genes lead to overproduction and/or altered proteolytic processing of A(3PP, in both cases leading to increased levels of A(3. Transgenic mouse models expressing these mutations can develop senile plaques and some neurotoxicity, but the model is not complete for familial AD, and the mechanism of this demonstrated neurotoxicity may be unrelated to the neuronal loss present in AD (Perry et al., 2000; Smith et al., 2000b). Furthermore, even though genetic mutations in these genes account for the majority of familial AD cases, they only comprise a small percentage of total AD cases since the predominance of AD is sporadic.

1.3. Iron in Neurodegenerative Disease Free iron has long been implicated in neurodegenerative disease through its redox transitions in vivo. The consequential generation of oxygen free radicals can further induce oxidative stress in tissues. Abnormally high levels of iron and oxidative stress have been found in neurodegenerative disorders such as AD, PD, multiple system atrophy, and progressive supranuclear palsy (Smith et al., 1997; Sayre et al., 2000). Oxidative stress, under these conditions, has been associated with levels of free iron. An increased level of total iron does not necessarily signify increased oxidative stress if it is accompanied by a concomitant increase in iron storage proteins, which keep iron in a redox-inert state. One such iron storage protein, ferritin, contains a core of insoluble and unreactive ferrihydrate. The binding and release of iron to and from ferritin, however, occurs through the ferrous iron state, which is quite active in the generation of hydoxyl radicals by Fenton reactions. Microglia are the main locations of ferritin-bound iron and are thought to be somewhat responsible for oxidative damage in PD and other neurodegenerative diseases (Smith et al., 1994; Premkumar et al., 1995; Kakimura et al., 2002; Castellani et al., 1995; Schipper et al., 1995; Ham and Schipper, 2000). Microglia stimulated in vivo using phorbol ester demonstrate increased lipid peroxidation due to superoxide-dependent release of iron from ferritin (Yoshida et al., 1998). This release can also be induced by 6-hydroxydopamine, a neurotoxin already implicated in PD, and other easily oxidized catechols (Double et al., 1998).

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These studies support the hypothesis that release of iron from ferritin contributes to free radical-induced cellular damage in vivo by Fenton production of hydroxyl radicals. The interplay between oxidative stress and iron metabolism in the brain is further demonstrated by the finding of abnormal iron deposition accompanied by lipid peroxidation in a transgenic mouse model of progressive neurodegeneration (Castelnau et al., 1998). In addition to primary management by the transferrin receptor and ferritin, it has been shown more recently that the handling of iron at the cellular level can be regulated by the lactotransferrin receptor, melanotransferrin, ceruloplasmin (Castellani et al., 1999), and divalent cation transporter 1. Disruptions in the expression of these latter proteins in brain tissues probably contribute to altered iron metabolism in disorders such as AD and PD (Qian and Wang, 1998). Overall, regulation of cellular iron metabolism relies on the actions of two known iron regulatory proteins, IRP-1 and IRP-2. IRPs undergo significant alterations in AD patients, supporting a role for redox-active iron in this disease (Smith et al., 1998). Levels of extracellular H 2 0 2 rapidly activate IRP-1, suggesting a regulatory connection between iron regulation and oxidative stress. This activation has been recapitulated in vitro and shown to require a cellular membrane-associated component which is sensitive to temperature and alkaline phosphatase (Pantopoulos and Hentze, 1998). Future work in this area should result in a clearer understanding of the causative link between iron-induced oxidative stress and neuronal death.

1.4. Iron-Mediated Oxidative Stress in Parkinson's Disease Even though the etiology of PD remains undefined, several biochemical abnormalities in PD brain tissue have been identified, including a mitochondrial complex I deficiency, oxidative stress, and excess iron (Jenner and Olanow, 1996). In addition, the somewhat recent identification of a mutation in the a-synuclein gene in cases of familial Parkinsonism may ultimately lead to an understanding of the biochemical mechanisms of selective dopaminergic cell death in these cases. Iron accumulations have been found in astrocytes in the substantia nigra of old rats (Schipper, 1996; Schipper et al, 1998), along with an increase in

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the Fe(III), to Fe(II) ratio and a decrease in reduced glutathione (Riederer et al., 1989). One interpretation is that mitochondrial sequestration of redoxactive iron in aging nigral astroglia may predispose the senescent nervous system to Parkinsonism and other neurodegenerative disorders. In fact, a persistent condition of elevated oxidative stress associated with greatly perturbed intracellular redox equilibria is widely recognized as a pathogenic factor underlying neurodegeneration. Moreover, there is circumstantial evidence that the intracellular redox imbalance results in aberrant oxidation of dopamine to 6-hydroxydopamine, which in turn can undergo autoxidation to the corresponding quinone accompanied by the generation of superoxide. This reaction cascade, either by itself or amplified by redox cycling of the quinone, which leads to further generation of ROS while depleting cellular reductants, can serve to explain the ultimate demise of these neurons. Studies to clarify the mechanism of dopamine oxidation in vitro have demonstrated conversion to 6-hydroxydopamine in the presence of Fe(II) and either H 2 0 2 or alkyl peroxides (Pezzella et al., 1997).

1.5. Manganese and Parkinson's Disease Chronic exposure to manganese results in extrapyrimidal syndromes resembling PD. Manganese has, therefore, been labeled as an environmentally toxic factor that induces brain dysfunction. One hypothesis to explain these results suggests that manganese acts as a dopaminergic neurotoxin, in the same manner as iron, by promoting the generation of ROS and the subsequent nonenzymatic autoxidation of dopamine to the neurotoxin 6-hydroxydopamine. There is, however, no convincing in vivo evidence for a pro-oxidant role of manganese in the brain. Also, the clinical picture of manganese-induced Parkinsonism is unclear because there is conflicting evidence regarding whether it is selectively toxic to dopaminergic neurons (Calne et al., 1994). In addition, more recent findings suggest that manganese actually may act as an antioxidant rather than as a pro-oxidant (Sziraki et al., 1998). In this study using rats given Fe(II) intranigrally, Mn(II) was found to protect against the toxicity of Fe(II) in a dose-dependent fashion. This protection could result from the fact that Mn(II), while itself inactive in Fenton chemistry, competes with Fe(II) in oxidative cascades. Thus, it appears that the Parkinsonian-like condition induced by chronic

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manganese exposure has little connection to nigrostriatal damage occurring in idiopathic PD.

1.6. Antioxidant and Transition Metal Homeostasis Many studies of oxidative stress in AD have focused on the inducible mitochondrial MnSOD and the constitutive cytoplasmic CuZnSOD enzymes as possible targets for therapy. The CuZnSOD gene has been associated with AD neuropathology, and levels of both MnSOD mRNA and CuZnSOD were found to be increased in AD, whereas the total antioxidant status was decreased. However, since superoxide dismutase (SOD) enzymes are key cellular antioxidant components, any pro-oxidant mechanism linked to SOD must derive from the balance in the local concentrations of superoxide and H 2 0 2 , which together can produce hydroxyl radicals by the Haber-Weiss process. The development of transgenic mouse "knockout" and overexpression models have allowed a range of studies to critically evaluate the extent to which selected biological processes affect cell viability. For example, if one of the SOD enzymes serves a crucial antioxidant function, then knockout animals may exhibit increased oxidative stress parameters, either on a global level or localized to the specific cellular compartment normally protected by that SOD enzyme. In support of this notion, MnSOD knockout mice, which suffer a 50% drop in mitochondrial SOD activity with no reduction in CuZnSOD or glutathione peroxidase activity, were found to exhibit increased oxidative damage to mitochondria. This was shown by increased mitochondrial protein carbonyls and 8-hydroxydeoxyguanosine in mitochondrial DNA (Williams et al., 1998). In contrast, no damage to cytosolic proteins or to nuclear DNA was observed. Analysis of homozygote knockouts showed mitochondrial degeneration throughout the two weeks that the mice lived (Melov et al., 1998). These in vivo results suggest that decreases in MnSOD activity can account for increased oxidative damage in mitochondria and alterations in essential mitochondrial function. In other studies, a tenfold higher expression level of human CuZnSOD in both myocytes and endothelial cells of mice was able to quench a burst of superoxide (in electron paramagnetic resonance detection) and reduce functional damage following 30-minute global ischemia (Wang et al., 1998).

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These results suggest that superoxide is an important factor in protecting against post-ischemic injury, and it is apparent that decreases in CuZnSOD activity can lead to a perturbation of cellular antioxidant defense mechanisms, thereby promoting a pro-oxidant condition. It is surprising, however, that CuZnSOD knockout mice show little, if any, significant neurodegenerative phenotype (Bruijn et al., 1998). Hence, while absences of CuZnSOD have little effect, enzyme inactivation by metal-catalyzed oxidation can promote oxidative damage (Kwon et al., 1998). These differences, while seemingly paradoxical, may be particularly relevant to human diseases. Ceruloplasmin (CP) is an important copper storage protein, the major Fe(II)-oxidizing enzyme in the central nervous system, and one of the key proteins that responds to oxidative stress. A related inherited metabolic disorder, aceruloplasminemia, is associated with impairment in iron homeostasis and consequent neurodegeneration (Harris et al., 1998). Studies directed at clarifying the relationship between oxidative stress and tissue metal-ion levels indicate that both the ratio of copper to zinc and the levels of CP are significantly higher with increasing age, and higher yet in cases with neurodegeneration. Interestingly, while CP is increased in brain tissue and cerebrospinal fluid in AD, PD and Huntington's disease patients (Loeffler et al., 1996), neuronal levels of CP remain unchanged (Castellani et al., 1999). Therefore, while increased CP may indicate a compensatory response to increased oxidative stress in AD, its lack of increase in neurons may play an important role in metal-catalyzed damage. Since the copper to zinc ratio is significantly correlated with systemic oxidative stress (that is, lipid peroxidation), it is probable that an increased oxidative stress burden in aging and neurodegeneration may reflect, in large part, copper-mediated ROS production. In this case, redox-inert zinc may serve as an antioxidant by preventing binding of pro-oxidant copper at tissue sites. A novel glycolipid-anchored membrane-bound form of CP expressed by astrocytes in the mammalian CNS has been identified, and any possible role for this CP in the neurodegenerative process will be investigated. The escalating interest in prion diseases (such as Creutzfeldt-Jakob disease), characterized by infection with an altered prion protein conformation that is transmissible through endogenous protein, continues to question the role of this cell-surface glycoprotein in healthy individuals. Thus far,

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several lines of evidence indicate that the prion protein binds Cu(II) and, thus, as with CP, may serve a cytoprotective role (Brown et al., 1997, 1998). One possibility, consistent with the finding in cell cultures that copper stimulates endocytosis of prion protein from the cell surface, is that prion protein controls copper metabolism by serving as a recycling receptor for uptake of extracellular copper (Pauly and Harris, 1998).

1.7. CuZnSOD Mutations and Familial Amyotrophic Lateral Sclerosis A crucial breakthrough in our understanding of amyotrophic lateral sclerosis (ALS) comes from the finding that many cases of familial ALS (FALS) are associated with mutations in the CuZnSOD gene. The protein products of these mutations retain nearly identical SOD activity, but they take on altered properties related to oxidative stress, possibly involving a gain-of-function peroxidase activity (Wiedau-Pazus et al., 1996). Consistent with these findings, transgenic mice overproducing a human FALS CuZnSOD mutant display increases in protein carbonyls, suggestive of increased hydroxyl radical production or lipoxidation-derived radicals (Andrus et al., 1998). Also, using in vivo microdialysis, increased hydroxyl radical production in the striatum was seen for mice overexpressing mutant CuZnSOD relative to mice overexpressing the wildtype human enzyme (Bogdanov et al., 1998), as determined by conversion of 4-hydroxybenzoic acid to 3,4-dihydroxybenzoic acid. The hypothesis that mutant SOD-induced neurodegeneration is associated with disturbances in neuronal free radical homeostasis is further supported by observations made on several neuronal cell cultures expressing the mutant SOD (Ghadge et al., 1998). The link between the SOD mutations and oxidative stress indicators appears not to be simply due to increased production of hydroxyl radicals, however, since there are no increases in hemeoxygenase. Adducts are seen in vitro for the Gly93 to Ala and Ala4 to Val mutant enzymes relative to the wild-type enzyme (Singh et al., 1998). Recent studies have shown that the mutant and wild-type SODs differ neither in their rates of superoxide dismutation nor in H202-mediated inactivation (Liochev et al., 1998; Goto et al., 1998). Structural analysis of the mutant enzymes did reveal, in some cases, alterations such as subunit asymmetry that suggest aberrant copper-mediated redox chemistry due to

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less tight folding and, thus, more open "active sites" (Corson et al., 1998; Hart et al., 1998). Therefore, the gain-of-function activity may reflect a "rechanneling" of the enzyme to oxidize biomolecules that would usually never gain access to the SOD oxidative half-reaction. Alternatively, the mutant SOD may possess weakened affinities for zinc or copper, leading to a fraction of enzyme with abnormal activity (Singh et al., 1998) or oxidative reactions associated with copper leakage (Goto et al., 1998). For the predominant, sporadic form of ALS, an imbalance of trace metal ions, possibly tied to increased oxidative stress, has been considered for some time. Recent studies provide evidence for decreases in copper in cerebrospinal fluid and serum, as well as increases in manganese in the serum of affected individuals versus age-matched controls.

2. CONCLUSIONS There has been an increasing awareness of the role that redox-active transition metals play in a variety of neurodegenerative diseases. The next step is to critically examine the importance of these basic research findings as they are translated into therapeutic modalities, such as antioxidants and chelating agents, to be used clinically. Hopefully, these treatments will yield promising results.

REFERENCES Andrus PK, Fleck TJ, Gumey ME, Hall ED. Protein oxidative damage in a transgenic mouse model of familial amytrophic lateral sclerosis. J Neurochem 1998; 71:2041-2048. Atwood CS, Moir RD, Huang X, Scarpa RC, Bacarra NME, Romano DM, Hartshorn MA, Tanzi RE, Bush Al. Dramatic aggregation of Alzheimer Afi by Cu(II) is induced by conditions representing physiological acidosis. J Biol Chem 1998; 273:12817-12826. Bogdanov MB, Ramos LE, Xu Z, Beal MR Elevated "hydroxyl radical" generation in vivo in an animal model of amyotrophic lateral sclerosis. J Neurochem 1998; 71:1321-1324. Bondy SC, Guo-Ross SX, Truong AT. Promotion of transition metal-induced reactive oxygen species formation by P-amyloid. Brain Res 1998; 799:91-96. Brown DR, Qin K, Herms JW, Madlung A, Manson J, Strome R, Fraser PE, Kruck T, von Bohlen A, Schulz-Schaeffer W, Giese A, Westaway D, Kretzschmar H. The cellular prion protein binds copper in vivo. Nature 1997; 390:684-687. Brown DR, Schmidt B, Kretzschmar HA. Effects of copper on survival of prion protein knockout neurons and glia. J Neurochem 1998; 70:1686-1693.

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Bruijn LI, Houseweart MK, Kato S, Anderson KL, Anderson SD, Ohama E, Reaume AG, Scott RW, Cleveland DW. Aggregation and motor neuron toxicity of an ALS-linked SOD1 mutant independent from wild-type SOD1. Science 1998; 281:1851-1854. Calne DB, Chu NS, Huang CC, Lu CS, Olanow W. Manganism and idiopathic Parkinsonism: similarities and differences. Neurology 1994; 44:1583-1586. 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. Castellani RJ, Smith MA, Nunomura A, Harris PLR, Perry G. Is increased redox-active iron in Alzheimer disease a failure of the copper-binding protein ceruloplasmin? Free Radic Biol Med 1999; 26:1508-1512. Castelnau PA, Garrett RS, Palinski W, Witztum JL, Campbell IL, Powell HC. Abnormal iron deposition associated with lipid peroxidation in transgenic mice expressing interleukin-6 in the brain. J Neuropathol Exp Neurol 1998; 57:268-282. Corson LB, Strain JJ, Culotta VC, Cleveland DW. Chaperone-facilitated copper binding is a property common to several classes of familial amyotrophic lateral sclerosis-linked superoxide dismutase mutants. Proc Natl Acad Sci USA 1998; 95:6361-6366. Double KL, Maywald M, Schmittel M, Riederer P, Gerlach M. In vitro studies of ferritin iron release and neurotoxicity. J Neurochem 1998; 70:2492-2499. Ghadge GD, Lee JP, Bindokas VP, Jordan J, Ma L, Miller RJ, Roos RP. Mutant superoxide dismutase-1-linked familial amyotrophic lateral sclerosis: Molecular mechanisms of neuronal death and protection. JNeuwsci 1998; 17:8756-8766. Goto JJ, Gralla EB, Valentine JS, Cabelli DE. Reactions of hydrogen peroxide with familial amyotrophic lateral sclerosis mutant human copper-zinc superoxide dismutases studies by pulse radiolysis. J Biol Chem 1998; 273:30104-30109. Ham D, Schipper HM. Heme oxygenase-1 induction and mitochondrial iron sequestration in astroglia exposed to amyloid peptides. Cell Mol Biol 2000; 46:587-596. Harris ZL, Klomp LW, Gitlin JD. Aceruloplasminemia: An inherited neurodegenerative disease with impairment of iron homeostasis. Am J Clin Nutr 1998; 67:S972-S977. Hart PJ, Liu H, Pellegrini M, Nersissian AM, Gralla EB, Valentine JS, Eisenberg D. Subunit asymmetry in the three-dimensional structure of a human CuZnSOD mutant found in familial amyotrophic lateral sclerosis. Protein Sci 1998; 7:545-555. Jenner P, Olanow CW. Oxidative stress and the pathogenesis of Parkinson's disease. Neurology 1996; 47:S161-S170. Kakimura J-I, Kitamura Y, Takata K, Umeki M, Suzuki S, Shibagaki K, Taniguchi T, Nomura Y, Gebicke-Haerter PJ, Smith MA, Perry G, Shimohama S. Microglial activation and amyloid-|3 clearance induced by exogenous heat-shock proteins. FASEB J 2002; 10.1096/fj.01-0530fje. Kwon OJ, Lee SM, Floyd RA, Park JW. Thiol-dependent metal-catalyzed oxidation of copper, zinc superoxide dismutase. Biochim Biophys Acta 1998; 1387:249-256. Liochev SI, Chen LL, Hallewell RA, Fridovich I. The familial amyotrophic lateral sclerosisassociated amino acid substitutions E100G, G93A, and G93R do not influence the rate of inactivation of copper- and zinc-containing superoxide dismutase by H 2 0 2 . Arch Biochem Biophys 1998; 352:237-239.

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Loeffler DA, LeWitt PA, Juneau PL, Sima AA, Nguyen HU, DeMaggio AJ, Brickman CM, Brewer GJ, Dick RD, Troyer MD, Kanaley L. Increased regional brain concentrations of ceruloplasmin in neurodegenerative disorders. Brain Res 1996; 738:265-274. Lovell MA, Robertson JD, Teesdale WJ, Campbell JL, Markesbery WR. Copper, iron and zinc in Alzheimer's disease senile plaques. J Neurol Sci 1998; 158:47-52. Mantyh PW, Ghilardi JR, Rogers S, DeMaster E, Allen CJ, Stimson ER, Maggio JE. Aluminum, iron, and zinc ions promote aggregation of physiological concentrations of beta-amyloid peptide. J Neurochem 1993; 61:1171-1174. Markesbery WR. The role of oxidative stress in Alzheimer's disease. Arch Neurol 1999; 56:1449-1452. Melov S, Schneider JA, Day BJ, Hinerfeld D, Coskun P, Mirra SS, Crapo JD, Wallace DC. A novel neurological phenotype in mice lacking mitochondrial manganese superoxide dismutase. Nature Genetics 1998; 18:159-163. Multhaup G, Ruppert T, Schlicksupp A, Hesse L, Bill E, Pipkorn R, Masters CL, Beyreuther K. Copper-binding amyloid precursor undergoes a site-specific fragmentation in the reduction of hydrogen peroxide. Biochemistry 1998; 37:7224-7230. Pantopoulos K, Hentze MW. Activation of iron regulatory protein-1 by oxidative stress in vitro. Proc Natl Acad Sci USA 1998; 95:10559-10563. Pauly PC, Harris DA. Copper stimulates endocytosis of the prion protein. J Biol Chem 1998;273:33107-33110. Perez M, Valpuesta JM, de Garcini EM, Quintana C, Arrasate M, Lopez Carrascosa JL, Rabano A, Garcia de Yebenes J, Avila J. Ferritin is associated with the aberrant tau filaments present in progressive supranuclear palsy. Am J Pathol 1998; 152:1531-1539. Perry G, Smith MA. Alzheimer's disease. In: Adelman G, Smith BH, editors. Encyclopedia ofNeuroscience, 2nd edition. Amsterdam: Elsevier Science BV, 1999: 59-61. Perry G, Nunomura A, Avila J, Perez M, Rottkamp CA, Atwood CS, Zhu X, Aliev G, Cash AD, Smith MA. Oxidative damage and antioxidant responses in Alzheimer's disease. In: Iqbal K, Sisodia SS, Winblad B, editors. Alzheimer's Disease: Advances in Etiology, Pathogenesis and Therapeutics. Chichester: John Wiley & Sons, 2001: 371-378. Perry G, Nunomura A, Raina AK, Smith MA. Amyloid-P junkies. Lancet 2000; 355:757. Pezzella A, d'Ischia M, Napolitano A, Misuraca G, Prota G. Iron-mediated generation of the neurotoxin 6-hydroxydopamine quinone by reaction of fatty acid hydroperoxides with dopamine: A possible contributory mechanism for neuronal degeneration in Parkinson's disease. J Med Chem 1997; 40:2211-2216. Premkumar DRD, 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. Qian ZM, Wang Q. Expression of iron transport proteins and excessive iron accumulation in the brain in neurodegenerative disorders. Brain Res Rev 1998; 27:257-267. Riederer P, Sofic E, Rausch W-D, Schmidt B, Reynold GP, Jellinger K, Youdim MBH. Transition metals, ferritin, glutathione, and ascorbic acid in Parkinsonian brains. J Neurochem 1989; 52:515-520.

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Sayre LM, Perry G, Harris PLR, Liu Y, Schubert KA, Smith MA. In situ oxidative catalysis by neurofibrillary tangles and senile plaques in Alzheimer's disease: a central role for bound transition metals. J Neurochem 2000; 74:270-279. Sayre LM, Smith MA, Perry G. Chemistry and biochemistry of oxidative stress in neurodegenerative disease. Curr Med Chem 2001; 8:721-738. Schipper HM, Cisse S, Stopa EG. Expression of heme oxygenase-1 in the senescent and Alzheimer-diseased brain. Ann Neurol 1995; 37:758-768. Schipper HM. Astrocytes, brain aging, and neurodegeneration. Neurobiol Aging 1996; 17:467^180. Schipper HM, Vininsky R, Brull R, Small L, Brawer JR. Astroctye mitochondria: A substrate for iron deposition in the aging rat substantia nigra. Exp Neurol 1998; 152:188-196. Singh RJ, Karoui H, Gunther MR, Beckman JS, Mason RR Kalyanaraman B. Re-examination of the mechanism of hydroxyl radical adducts formed from the reaction between familial amyotrophic lateral sclerosis-associated CuZn superoxide dismutase mutants and H 2 0 2 . Proc Natl Acad Sci USA 1998; 95:6675-6680. Smith MA, Taneda S, Richey PL, Miyata S, Yan S-D, Stern D, Sayre LM, Monnier VM, Perry G. Advanced Maillard reaction end products are associated with Alzheimer's disease pathology. Proc Natl Acad Sci USA 1994; 91:5710-5714. Smith MA, Harris PLR, Sayre LM, Perry G. Iron accumulation in Alzheimer's disease is a source of redox-generated free radicals. Proc Natl Acad Sci USA 1997; 94:9866-9868. Smith MA, Wehr K, Harris PLR, Siedlak SL, Connor JR, Perry G. Abnormal localization of iron regulatory protein (IRP) in Alzheimer's disease. Brain Res 1998; 788:232-236. Smith MA, Nunomura A, Zhu X, Takeda A, Perry G. Metabolic, metallic, and mitotic sources of oxidative stress in Alzheimer's disease. Antiox Redox Signal 2000a; 2:413^120. Smith MA, Joseph JA, Perry G. Arson: tracking the culprit in Alzheimer's disease. Ann NY Acad Sci 2000b; 924:35-38. Sziraki I, Mohanakumar KP, Rauhala P, Kim HG, Yeh KJ, Chiueh CC. Manganese: A transition metal protects nigrostriatal neurons from oxidative stress in the iron-induced animal model of Parkinsonism. Neuroscience 1998; 85:1101-1111. Wang P, Chen H, Qin H, Sankarapandi S, Becher MW, Wong PC, Zweier JL. Overexpression of human copper, zinc-superoxide dismutase (SOD1) prevents postishemic injury. Proc Natl Acad Sci USA 1998; 95:4556-4560. Wiedau-Pazus M, Goto JJ, Rabizadeh S, Gralla EB, Roe JA, Lee MK, Valentine JS, Bredesen DE. Altered reactivity of superoxide dismutase in familial amyotrophic lateral sclerosis. Science 1996; 271:515-518. Williams MD, Van Remmen H, Conrad CC, Huang TT, Epstein CJ, Richardon A. Increased oxidative damage is correlated to altered mitochondrial function in heterozygous manganese superoxide dismutase knockout mice. J Biol Chem 1998; 273: 28510-28515. Yoshida T, Tanaka M, Sotomatsu A, Hirai S, Okamoto K. Activated microglia cause irondependent lipid peroxidation in the presence of ferritin. Neuro Report 1998; 9:1929-1933.

CHAPTER 2

Metals Distribution and Regionalization in the Brain Margherita Speziali, Edoardo Orvini

ABSTRACT A review of the literature on trace element levels in normal and diseased human brain is undertaken in an attempt to recognize possible distribution patterns of aluminum, iron, copper, and zinc. Trace element concentration changes are known to occur in brain areas of subjects affected with neurodegenerative diseases. We have considered the levels of the four metals in Alzheimer's disease, Parkinson's disease, and Western Pacific Parkinsonism-dementia along with amyotrophic lateral sclerosis. For each disease, we selected articles that consider well-defined brain sites for which the element values observed in controls were found to be significantly different from those determined in patients. For a more complete picture statistically not significant differences of the same element concentrations in the same regions, whenever available, were also taken into consideration. Keywords: Trace elements; human brain; brain areas; brain regions; Alzheimer's disease; Parkinson's disease; amyotrophic lateral sclerosis.

1. TRACE ELEMENT DISTRIBUTION IN NORMAL HUMAN BRAIN Minor and trace element levels appear to be unevenly distributed throughout the human central nervous system. Several studies have reported on different metal concentrations in various areas of the brain (encephalon) in the same subjects; sometimes, they also focus on possible changes in relation to age. We have considered aluminum (Al), iron (Fe), copper (Cu), 15

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and zinc (Zn). Generally, the authors found the amounts of these four elements to be higher in gray than in white matter. When comparing the metal content of various sites, the relative composition of both matters could not be negligible.

1.1. Aluminum, Iron, Copper and Zinc Distribution in Normal Human Brain Sites in Relation to Age We summarize the data on autoptic samples taken from individuals with no signs of neurologically related diseases ("controls") in an attempt to identify possible distribution patterns of Al, Fe, Cu and Zn in "normal" human brain regions. We consider experiments in which the number of sites examined were not very low, as our purpose could be attained whether or not the areas taken into account in the different experiments were mostly the same. In trace element determination, every scientific team analyzed brain samples in different number, areas, and gray/white matter ratio. Most Authors published tables with all the values obtained, while others pulished selective information.

1.2. Aluminum McDermott et al. (1977) determined Al concentration in the frontal cortex/ hippocampus of three younger controls and six elderly controls (age not reported). They found a double Al level in the elderly compared to the younger subjects. McDermott et al. (1979) analyzed Al concentration in the lobe cortices, cerebellum and corpus callosum of three controls (25-65 y) and in another six controls (75-99 y). Considered together, the highest Al concentrations in the nine subjects were found in the hippocampus, the lowest in corpus callosum (white matter). In bulk brain, the mean Al content in the elder group was found to be considerably higher than in the younger one. Ehmann et al. (1980) analyzed Al levels in the brains of 20 controls in five different age groups. They observed a positive correlation between an abundance of Al and age. Markesbery et al. (1981), in their study of 28 controls (0-85 y) derived the same results. Ehmann et al. (1982) and Markesbery et al. (1984) published the results of a systematic study on minor and trace element concentrations over the

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whole human life span. Aluminum levels were determined in a very high number of brain regions in 29 adult controls (age 19-85) and seven infants (premature - 6 months). They found that the mean Al levels were highest in globus pallidus (GP), putamen, and middle temporal lobe, and lowest in the superior parietal lobule. No data on other areas were reported. Aluminum exhibited a significant increase in the brain with advancing age. Roider and Drasch (1999) evaluated Al concentration in brain gray matter and white matter (sulcus lateralis), nucleus lentiformis, brainstem (pons) and cerebellum (arbor vitae) of 140 adults in dependence of age. The highest Al concentrations were found in gray matter, the lowest in white matter, with a statistically high significance difference. The differences in Al contents of gray matter, nucleus lentiformis and cerebellum were also shown to be highly significant statistically. The dependence of Al values on age appeared biphasic: there was an increase with age up to a first maximum at approximately 40 years followed by a plateau or even a small decrease at up to 70 years. A second increase then followed in the eighth and ninth decades of life. No significant gender differences in Al concentrations in the brain were noticed. Taking into account papers not related to Al changes in the different periods of life, Yoshimasu et al. (1976 and 1980) published Al determination in 10 brain sites of three Japanese controls (28^-0 y); the highest value was in gyrus praecentralis, the lowest in capsula interna (CI). Yoshimasu et al. (1982) analyzed Al in 20 brain areas of three Chamorro (from Guam island) controls (57-87 y); gyri praecentralis and postcentralis showed the highest contents, followed by parietal and temporal cortices; CI, putamen and cerebellar white matter had the lowest levels. Rajput et al. (1985) and Uitti et al. (1989) determined Al in frontal cortex, nucleus caudatus (NC), substantia nigra (SN), and cerebellum in nine (no age given) and 12 individuals (average age = 70 y), respectively. The highest amounts were found in NC. Yasui (1991a and 1991b) evaluated Al contents in three areas of five control brains (65-75 y); gyrus praecentralis showed higher levels than CI and cms cerebri. Yasui et al. (1992) reported Al values in 10 areas of the same brains; the highest contents were found in SN, nucleus ruber (NR), pons and cerebellar cortex; the lowest was found in GR Xu et al. (1992) analyzed Al levels in the middle frontal gyrus, hippocampus, inferior parietal lobule, and superior and middle temporal gyri

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of 10 controls (57-93 y). Middle frontal gyrus and inferior parietal lobule contained higher values than the other two areas. Andrasi et al. (1989) studied Al in 10 regions of nine controls (65-75 y). The highest amounts were found in GP and cerebellar cortex, the lowest in putamen and pulvinar thalami. In another study (1994), they evaluated Al contents in 12 sites of 11 (no age given) subjects; they found the highest Al levels in NR and SN, the lowest in pulvinar thalami, putamen and NC. In a later study (1995), they determined Al concentrations in 20 (average age = 70 y) individuals. Samples from the cortices, thalamus and GP showed the maximum levels, while NC, Ammon's horn and other cortical specimens showed the minimum levels. Rajan et al. (1997) determined Al contents in 12 regions of eight individuals (50-60 y); they found the highest amounts in the temporal cerebrum and thalamus, the lowest in pons and cerebellum. In the case of aluminum, only GP, SN, and NR were analyzed and found to be the richest in Al by different teams. A positive correlation between Al amounts in the brain and age was observed.

1.3. Iron Hallgren and Sourander (1958) published the levels of nonhemin Fe in 16 brain sites of 37-59 individuals (30-100 y). The highest Fe values were found in the structures of the extrapyramidal system: the maximum amount occurred in the GP, while somewhat lower levels were obtained for NR and SN, followed by putamen, nucleus dentatus (ND) and NC. As for the cerebral cortex, the highest concentrations were observed in the motor cortex followed by the occipital, sensory and parietal cortices; the lowest levels were found for the prefrontal and temporal cortices. An increase in nonhamin Fe occurred with advancing age in all regions examined, except in the medulla oblongata. The rise in Fe values was rapid during the first two decades of life, and then became more gradual. Sundermann and Kempf (1961) analyzed Fe content in the brainstem ganglia of 100 human brains divided into 10 age groups. The Fe value was richest in GP followed by, in order of decreasing content, putamen, NC, thalamus, cerebral cortex, NR and cerebral medulla. While the increase in iron content with increasing age could be proven statistically for GP, putamen, and NC, the thalamus and cerebral cortex showed a clear increase in

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iron content only up to the fourth or fifth decade of life. In old age, however, a sharp tendency towards reduction appeares. Schicha et al. (1971) analyzed Fe in 16 brain sites of six subjects (newborn - 74 y). In four adults (34-74 y), the highest Fe concentration was found in GP and the lowest in NR. NC and putamen showed the same intermediate concentration. In cortical areas considerably lower Fe levels than in the basal ganglia were observed. The average Fe concentration in the brain of a newborn is only 20% that of an adult and about 60% that of a 4-year-old child. The Fe values in adults appeared to be reached between seven and nine years of age. The Fe concentration in Broca's center of speech and the motor cerebral cortex was, in all cases, higher in the left hemisphere than in the right. Volkl and Ule (1972 and 1974) determined the Fe concentration in 13 defined areas of the brain in 33 subjects (newborns - 80 y). The highest values were found in GP, putamen, and NC; the lowest in the oliva inferior and corpus callosum (CC), with intermediate values in the frontal and occipital cortices. The Fe content, in relation to age, seemed to differ considerably among the various areas examined. There was a sharp rise in the basal ganglia during the first year of life followed by a gradual leveling off. The increase in Fe content in the cerebral cortex was observed to be much less marked. Both the thalamus and hippocampus showed little change in their Fe content during life, while the olives tended to lose iron. Volkl and Ule (1974) analyzed Fe contents in 13 regions of six infant (two were born premature, three were born at full term, and one infant was 21 months old) and juvenile (3 children of 6-11 y) brains, and compared these levels to those of adult brains. Fe rose in the juvenile years in gray matter, exhibiting intermediate values; in white matter, Fe rose by approximately 50% compared to perinatal levels. Along with the increase in Fe, typical regional distributions developed until the juvenile years thereby resembling the distribution in an adult brain. Ehmann et al. (1982) and Markesbery et al. (1984) observed the highest mean Fe values in the GP and putamen; the cerebellar vermis and hippocampus contained the lowest concentrations. Iron appeared to peak between 40 and 79 years of age before declining, although this trend was not significant. Connor et al. (1992) studied Fe contents in the superior temporal gyrus, motor cortex, and occipital cortex (both white and gray matters in each site) of nine normal adults (mean age, 55 years; range, 44 to 64 years) and 11 elderly (mean age, 71.6 years; range, 65 to 80 years).

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In both groups, Fe was highest in the motor cortex. In the gray matter in the occipital cortex, there was a 130% increase in Fe with age; in the white matter, no statistically significant increase occurred with age. Loeffler et al. (1995) determined Fe concentration in eight young adult controls (<65 years) and eight elderly controls (3=65 years). In the former, Fe concentration was greatest in GP, whereas it was in SN in the later. For both groups, Fe content in the frontal cortex was less than that of other regions. Fe concentration increased by >80% with age in SN, NC, and putamen, but decreased by 46% in GP; these differences were not statistically significant. Hebbrecht et al. (1999) determined Fe levels in the cerebral and cerebellar cortex, white matter, basal ganglia, and brainstem of 18 individuals (7-79 y) to assess any changes related to age. They observed an age-related increase in Fe concentration. Zecca et al. (2001) measured the concentration of Fe in SN from normal subjects aged one to 90 years old. Fe levels in SN appeared to increase by the fourth decade of life and to remain stable until 90 years of age. Hill (1988) reviewed the distribution of iron in the brain in relation to age. Dealing with papers not related to Fe changes in aging, Harrison et al. (1968) analyzed Fe contents in 10 regions of 35 individuals (no age given). The highest Fe amounts were found in the GP, putamen, and NC; the lowest in the corpus callosum and cerebellar white matter. Hock et al. (1975) determined Fe amounts in 27 sites of seven brains (23-66 y). The maximum level of Fe was detected in the GP, followed by the putamen, NC, and SN; the minimum in the cms fornicis, corpus callosum, corpus geniculatus lateralis, and corpus semiovale (white matter). Goldberg and Allen (1979 and 1981) studied Fe amounts in six sites of three control subjects (no age given). They observed the maximum Fe level in SN and elevated contents in GP, NC, and putamen, while the minimum level was found in the hippocampus and cerebral cortex. Duflou et al. (1989) analyzed Fe contents in about 50 brain sites of 12 subjects (7-69 y). They found the highest Fe concentrations in the plexus choroideus, GP, SN, and putamen. The lowest concentrations were observed in the pyramid, tractus corticospinalis, and nucleus olivaris inferior. Kornhuber et al. (1994) measured Fe in seven brain regions of 12 individuals (41-91 y). The highest amounts were detected in NC and the corpus mamillare, the lowest in the hypothalamus and gyrus cinguli. Bush et al. (1995) analyzed gray and white matters of the lobes along with

Metals Distribution and Regionalization in the Brain

21

six other areas of 30 subjects (18-85 y). The highest Fe values were found in the putamen, SN, GP, and NC, while the lowest values were found in the white matter of the occipital and parietal lobes. Rajan et al. (1997) observed the highest Fe concentrations in the frontal, temporal, and parietal cerebrum, and the lowest in the medulla oblongata and pons. Andrasi et al. (1999) determined Fe amounts in 20 sites of five control subjects (>65 years). The highest concentrations were found in the putamen, followed by GP, NC, and SN; the lowest concentrations were found in NR and gyrus cinguli. Most authors found the highest Fe concentrations in GP, followed by the putamen, NC (caput), and SN. The metal showed a different increase with age in various areas.

1.4. Copper Ule et al. (1974) and Volkl and Ule (1974) determined Cu amounts in 13 brain areas of 29 and 33 subjects (age 0-80 y), respectively. In adults, the highest concentrations of Cu appeared to occur in SN, which was significantly different from the frontal cortex. High levels were also observed in the occipital cortex, neostriatum, and nucleus dentatus, while the lowest levels were seen in the thalamus and centrum semiovale. The Cu level of newborns was found to be somewhat lower than that of adults, while between the fourth and seventh decades of life these values remained independent of age. Volkl et al. (1974) observed that Cu rose during the juvenile years in the gray matter; in white matter, Cu changed little during maturation. Along with the increase in Cu, typical regional distributions developed until the juvenile years, thereby resembling the distribution in an adult brain. Saito et al. (1993 and 1994) analyzed Cu levels in the brains of 14 subjects (4-54 y) in five brain regions (lobes and cerebellum). The concentration of Cu in the cerebellum appeared to be significantly higher than that in the temporal lobe. No relation to age was observed. Tandon et al. (1994) examined 17 controls (age 33-85) and observed a significant positive correlation between Cu content in bulk brain and age. Considering experiments not related to age, Warren et al. (1960) and Thompson (1961) reported the results of analyses of Cu contents in 26 areas of nine brains (no age given). The highest amounts were found in the

22

Speziali M & Orvini E

locus coeruleus, followed by SN, ND, and parietal cortex (all samples were gray matter); the lowest amounts were found in the optic chiasma, base of pons, and corpus callosum (all made of white matter). SmeyersVerbeke et al. (1974) evaluated the distribution of Cu in 13 brain areas of 11 subjects (no age given). The maximum values were observed in the cerebellar cortex followed by the occipital cortex and basal ganglia, while the lowest values were found in the brainstem and corpus callosum. Harrison et al. (1968) detected the highest Cu amounts in the cerebellar gray matter, putamen, and NC; the lowest amounts were found in the corpus callosum, thalamus, and frontal and cerebellar white matter. Smeyers-Verbeke et al. (1974) verified the distribution of Cu in the brain. The highest values were observed in the cerebellar and occipital cortices and basal ganglia; the lowest values were found in the corpus callosum and brainstem. Goldberg and Allen (1981) observed maximum Cu level in SN and elevated contents in the putamen and NC, while the minimum levels were found in the hippocampus and cerebral cortex. Bonilla et al. (1984) determined Cu concentrations in 38 brain areas of seven subjects (11-75) y. The highest Cu levels were detected in the olfactory bulb, gyrus postcentralis superior, NC (tail), and calcarine cortex (cuneus); the lowest levels were found in the frontal, occipital, and parietal lobes' white matter along with the optic chiasma and thalamus. Duflou et al. (1989) found the highest Cu concentrations in SN, ND, lobulus flocculo-nodularis cerebelli, paraflocculus ventralis cerebelli, and putamen. The lowest concentrations were observed in chiasma opticum, tractus corticospinalis, and corpus fornicis. In Kornhuber et al. (1994), the maximum levels were detected in NC, followed by the hypothalamus and cortex; the lowest levels were found in the hippocampus and corpus mamillare. Deibel et al. (1997) analyzed Cu contents in 13 regions of five subjects (no age given). They found the highest levels in the amygdala and cerebellum, with the lowest levels in the cerebellum and hippocampus. In Rajan et al. (1997), the highest Cu concentrations were determined in the cerebellum and midbrain, with the lowest levels in the pons and thalamus. Andrasi et al. (1999) found the highest concentrations of Cu in SN, NR, and putamen, and the lowest in the genu corporis callosi. Most authors found the highest concentrations of Cu in SN, ND and other cerebellar regions, putamen, and NC. Copper seemed to increase at least until adult age, possibly by the fourth decade of life.

Metals Distribution and Regionalization in the Brain

23

1.5. Zinc Hu and Friede (1968) determined Zn in 24 regions of five adults (34-87 y). They found the maximum level in Amnion's horn, SN, ND, and amygdala, and the lowest level in the pyramids, optic chiasma, and cerebral peduncle. In 17 areas of two newborns (four to five days' old), Amnion's horn, SN, cerebellar white matter, and cortex showed the highest Zn concentrations; the frontal and occipital white matters showed the lowest concentrations. Ule et al. (1974) and Volkl and Ule (1974) found the highest zinc concentrations in adults in the hippocampus, followed by the frontal cortex, NC, oliva inferior, and SN; in the last three sites, the Zn levels sometimes appeared to increase with age. The lowest values were seen in the centrum semiovale. Between the fourth and seventh decades of life, the Zn values remained independent of age. Volkl et al. (1974) observed that Zn rose during the juvenile years in regions of gray matter; in white matter, Zn changed little during maturation. The perinatal distribution pattern of Zn was already similar to that found in adult brains. Ehmann et al. (1982), Markesbery et al. (1984), and Ehmann et al. (1984) reported on Zn levels in 29 adult controls (age 19-85) and seven infants (=S6 months of age), by considering many regions. For controls aged 20 and above, the highest mean Zn levels were found in the hippocampus, NC, amygdala, and cerebellar vermis, and the lowest in SN, middle frontal lobe, and GP. Among infants, Zn concentrations were observed to be highest in the posterior occipital lobe, middle frontal lobe, and putamen; the lowest concentrations were detected in the thalamus. The difference between the infants and the adults was significant. Zn concentrations in controls aged above one exhibited a highly significant positive correlation with age. However, if the 19-year-old patient was eliminated from the group, the Zn concentration showed no significant correlation with age. In the hippocampus, the Zn level reached a maximum in those aged between 40 and 59, but declined gradually afterwards. This was also true of the putamen and superior parietal lobe. No regular patterns, as a function of age, were observed for any of the other brain regions in the adult control group. Saito et al. (1994) found that Zn concentration in the frontal lobe was significantly related to age. Hebbrecht et al. (1999) observed an age-related increase in Zn concentration.

24

Speziali M & Orvini E

Regarding works in which Zn possible changes in aging are not considered, Kornhuber et al. (1994) found the highest Zn values in the hippocampus and cortex, and the lowest in the corpus mamillare and hypothalamus. Rajan et al. (1997) observed the highest levels in parietal cerebrum and medulla oblongata, and the lowest in the hippocampus, hypothalamus, and pons. In Smeyers-Verbeke et al. (1974), the highest Zn values were seen in the temporal, frontal, and cerebellar cortices, while the lowest levels were seen in the corpus callosum centrum ovale, and CI. Hock et al. (1975) found the highest Zn levels in the epiphysis, gyrus dentatus, hippocampus, and insula, and the lowest in the corpus callosum, crus fornicis, and centrum semiovale (white matter). In Harrison et al. (1968), the hippocampus and frontal gray matter showed the highest Zn amounts, while the frontal and cerebellar white matter, together with the corpus callosum, showed the lowest. Duflou et al. (1989) found the highest Zn concentrations in the epiphysis, uncus hippocampi, plexus choroideus, and amygdala. The lowest concentrations were observed in the radiatio optica, pedunculus cerebri, CI, and genu corporis callosi (all samples were of white matter). Andrasi et al. (1999) found the highest Zn concentrations in the gyrus frontalis medius, putamen, and NC (caput), and the lowest amount in the genu corporis callosi. From the papers examined, it appears that several authors found the highest concentrations of Zn in the hippocampus, amygdala, NC, and epiphysis. Zn seems to increase until adult age, depending on the areas being considered.

2. TRACE ELEMENT IMBALANCES IN NEURODEGENERATIVE DISEASES The biochemical role that the redox-active transition metals, especially Fe, Cu, and Zn, play as mediators of oxidative stress in a variety of neurodegenerative diseases has recently been reviewed by Sayre et al. (1999 and 2000), Bush (2000), and Campbell et al. (2001). The last paper also considers Al. The role of metal ions (mainly Fe and possibly Cu) in oxidative processes and aging was reviewed by Floyd and Carney (1993). In single elements, the role of Al in human brain disease was explained by Yase (1980), Crapper McLachlan and De Boni (1980a and

Metals Distribution and Regionalization in the Brain

25

1980b), Crapper McLachlan et al. (1983), Crapper McLachlan and Farnell (1985), Wisniewski et al. (1985), Wisniewski and Sturman (1989), Savory et al. (1991), Youdim (1994 and 2001), and Yokel (1997). The function of Cu in neurodegenerative diseases was described in Scheinberg (1988), Waggoner et al. (1999), Rotilio et al. (2000), Brown (2001), and Strausak et al. (2001). The implication of Fe in neurodegenerative diseases was elucidated by Beard et al. (1993), Gelman (1995), Qian et al. (1997), Pinero and Connor (2000), and Thompson et al. (2001). Fe and Al homeostases in neural disorders were treated by Joshi et al. (1994). A book on Fe metabolism in Central Nervous System disorders was published by Riederer and Youdim in 1993. Gerlach et al. (1994 and 2000) also reviewed, besides metabolic studies, papers on Fe evaluation in different brain regions in several neurodegenerative diseases. The role of Zn in the same disorders was discussed by Wallwork (1987), Cuajungco and Lees (1997a), and Yasui et al. (1997). Sandyk (1991) has examined the relationship between Zn deficiency and cerebellar diseases, and a review on Zn, human diseases, and aging was published by Fabris and Moccheghiani in 1995. Cu and Zn functions and levels in the brain in health and neurological disease were reviewed by Cumings (1965). Analytical studies on trace element imbalances in agerelated neurological diseases were published by Ehmann et al. in 1987 and 1993. Markesbery and Ehmann (1988) have reviewed the possible trace element (especially Al and Zn) concentration changes in dementing disorders; while Ehmann and Vance (1996) have reviewed studies of trace element involvement in neurodegenerative diseases using activation analysis. For this overview, we have taken into consideration literature data on the tissue level, with concentrations expressed as the amount of element per gram of tissue or protein. Concentrations as a ratio between a couple of elements were not considered, as well as observations at the cellular or subcellular level. For each neurological disease, original papers with analytical data on both control subjects and patients were retrieved from the literature. Of these, those reporting on Al, Fe, Cu, and Zn concentration values in bulk samples, in wide regions such as lobes, lobe cortices, cerebellum, or in not so clearly delimited sites were excluded. We selected only articles that consider well-defined brain sites in which the element

26

Speziali M & Orvini E

values were significantly or highly significantly different between the control and patient groups. To provide a more complete picture, statistically not significant (NS) differences of the same element concentrations in the same regions, whenever available, were reported in the tables. Captions valuable for all tables are shown in Fig. 1.

SD = standard deviation; M = male, F = female; S* = significant or highly significant difference; pS = possibly significant difference;

SEM = standard error of the mean; av = average; mS = marginally significant difference; NS = not significant difference.

w = concentration expressed on a wet weight basis. To allow a comparison among all the values, the data were converted to a dry weight basis; the values obtained are reported, in parentheses, beside. When possible (Thompson et al., 1988 and Cornett et al., 1998b) conversion was made using the FD/WET weight ratio provided by the authors; in the other cases, the data were calculated using the ratio 0.21. v subjects from Eastern Canada; * combined data for olfactory bulb, tract and olfactory trigone; A data converted from nmol/g;

A subjects from United Kingdom; • combined data for olfactory bulb, tract and nucleus; 0 data expressed as mg Fe/g of protein.

E in Griffits et al., 1989, the site is named Substantia Nigra Pars Reticulata. ® precise data are reported in Galazka-Friedman and Friedman, 1997. C = Controls, not better specified; JC = Japanese Controls; ChC = Chamorro Controls; AD = Alzheimer's Disease patients; PD = Parkinson's Disease patients; PD<*i = Mild or moderate stage of PD patients; PDV = Severe grade of PD patients; GPD = Parkinsonism-Dementia patients from Guam; PD1 = Severe stage of PD with Dementia of Alzheimer's Type patients (Sofic et al., 1991); ALS = Amyotrophic Lateral Sclerosis patients, nor better specified; GALS = Amyotrophic Lateral Sclerosis patients from Guam island; KALS = Amyotrophic Lateral Sclerosis patients from the Kii Peninsula of Honshu island, Japan. INAA = Instrumental Neutron Activation Analysis; CNAA = Pre-irradiation chemical separation NAA; MS = Mossbauer Spectrometry; ICP= Inductively Coupled Plasma; ICP-AES = ICP- Atomic Emission Spectrometry;

Fig. 1. Captions for all tables.

RNAA = Radiochemical NAA; AAS = Atomic Absorption Spectrometry; PIXE = Proton Induced X-ray Emission; ICP-MS = ICP- Mass Spectrometry; Spectroph. = Spectrophotomertry.

Metals Distribution and Regionalization in the Brain

27

2.1. Aluminum, Iron, Copper and Zinc in Alzheimer's Disease Alzheimer's Disease (AD) is the most common cause of senile dementia. Etiologically, it is a heterogeneous condition in which several factors control the expression of the disease. The first structure to show pathological changes is the cerebral cortex (Braak and Braak, 1994). Al plays a major role in the AD pathogenesis (McLachlan et al., 1996). The role of Al, Fe, Cu, and Zn in the pathogenesis of AD was recently reviewed by Atwood et al. (1999). The relationship between Al and AD was described by Edwardson et al. (1986 and 1991), Crapper McLachlan (1986 and 1990), Birchall and Chappell (1988a and 1988b), Kruck and Crapper McLachlan (1989), Sturman and Wisniewski (1988), Wisniewski (1991), Wisniewski and Wen (1992), McLachlan (1994 and 1996), Lukiw (1997), and Campbell (2002). Al toxicity and content in AD were described by Crapper McLachlan et al. (1978); the evidence for a possible link between Al and AD was reviewed by Markesbery and Ehmann (1993). Chazot and Broussolle (1993) reviewed Al and Zn alterations in aging and in AD. Ehmann and Markesbery (1994) presented a summary of Al results determined in their laboratory and explored the reasons for the discrepancies among data obtained in various studies carried out in theirs and in other laboratories. Al and Zn alterations in AD were reviewed by Markesbery and Ehmann in 1988; Al, Fe, and Zn changes by Markesbery and Ehmann (1994). The implication of Fe in AD was suggested by Youdim (1988); Fe levels in AD were reviewed by Gerlach et al. in 1994 and 2000. Cu and Zn involvement in AD was described by Moir et al. in 1999. The role of Zn in AD was discussed by Constantinidis and Tissot (1982), as well as by Constantinidis in 1990 and 1997. Nachev and Larner (1996), besides describing the role of Zn in AD, also reviewed the concentration levels. Several original papers were retrieved on Al, Fe, Cu, and Zn determination in brain samples of both AD patients and non-AD controls at the tissue level: Hallgren and Sourander (1958, controls; 1960, patients), Crapper McLachlan et al. (1973, 1974, 1976, and 1980c), Constantinidis et al. (1977), McDermott et al. (1977 and 1979), Trapp et al. (1978), Yoshimasu et al. (1980), Traub et al. (1981), Markesbery et al. (1981 and 1983), Hershey et al. (1984), Ehmann et al. (1984 and 1986), Yoshimasu

28

Speziali M & Orvini E

(1985), Thompson et al. (1988), Plantin et al. (1987), Ward and Mason (1987), Jacobs et al. (1989), Corrigan et al. (1993), Xu et al. (1992), Dedman et al. (1992), Griffiths and Crossman (1993), Tandon et al. (1994), Andrasi et al. (1994 and 1995), Samudralwar et al. (1995), Deibel et al. (1996), Cornett et al. (1996, 1998a, and 1998b), Kala and Hasinoff (1996), Bjertness et al. (1996), Danscher et al. (1997), Stedman and Spyrou (1997), Rao et al. (1999 and 2000), Rulon et al. (2000), Panayi et al. (2000 and 2001). Two original works with only data on patients, VanDalsem et al. (1995) and Ishihara et al. (1999), were also found. Connor et al. (1992) and Loeffler et al. (1995) reported on Fe levels in both control and AD subjects as the amount of Fe per gram of protein. Corrigan et al. (1991) considered patients affected with senile dementia of Alzheimer's type. Tables 1 to 4 list the concentration values of Al, Fe, Cu, and Zn in brain sites of both control and AD subjects. The data show the significant difference between the two groups in some brain areas, along with other data that do not show a significant difference for the same elements in the same regions. In analytical studies on AD, the hippocampus and amygdala were sites where the concentrations of each element were most frequently found to be significantly different between the control and AD groups. Table 1 shows the data for Al. The amount of Al in the hippocampus of AD subjects was found to be increased in most papers, significantly or not; only one paper (McDermott et al., 1979) showed the same values. A significant enrichment in patients was reported in the inferior parietal lobule and in the superior and middle temporal gyrus, while a marginally significant increase was found in the gyrus postcentralis. Other results in the same sites were not available in the literature. Table 2 shows data on Fe. Fe values in the hippocampus are conflicting. In some experiments (Deibel et al, 1996; Cornett et al., 1998b), a significant elevation was detected in the AD group. In Thompson et al. (1988), the higher amount was only marginally significant and in GalazkaFriedman et al. (1996), not significant. Plantin et al. (1987) observed the same data in both the control and patient groups. On the other hand, Corrigan et al. (1993) found a significant Fe decrease in the AD group. Ward and Mason (1987) observed a possibly significant reduction in subjects from the United Kingdom and a not significant reduction in those from Eastern Canada. In the amygdala, either significantly (Thompson

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et al., 1988; Samudralwar et al., 1995; Cornett et al., 1998b) and not significantly (Deibel et al., 1996) higher amounts were observed. In the inferior parietal lobule and olfactory region, a similar situation was found. GP, considered in toto, showed a significant Fe enrichment in AD patients, while in the lateral and medial parts, analyzed separately, a not significant elevation was found. The unique experiment where the frontal pole and temporal pole were analyzed demonstrated a significant Fe enrichment in the AD group in both sites. In the piriform cortex, a significant Fe increase in patients was reported by Samudralwar et al. (1995). Table 3 reports on the Cu levels. The amount of Cu in the hippocampus appeared to decrease, significantly or not, except for Canadian subjects examined by Ward and Mason (1987). In the amygdala, the unique datum (Deibel et al., 1996) indicated a significant decrease. For Zn (Table 4), the data published in the literature on the hippocampus are conflicting. Ward and Mason (1987), who examined two populations, and Corrigan et al. (1993) found a significant decrease in the patient groups. In contrast, Deibel et al. (1996) and Cornett et al. (1998b) observed a significant elevation, while Plantin et al. (1987) and Rulon et al. (2000) evaluated the higher amount as not significant. Thompson et al. (1988) found the same results in both control and AD subjects. In the amygdala, according to four papers, significantly higher Zn contents were observed in patients; only Rulon et al. (2000) considered the increase as not significant. In the olfactory region, the enrichment of Zn was found to be significant by Samudralwar et al. (1995) and not significant by Cornett et al. (1998b). A significant Zn increase in AD patients was reported in the frontal pole and temporal pole (Cornett et al., 1998b), as well as in the inferior parietal lobule (Deibel et al., 1996; Cornett et al., 1998b). Thompson et al. (1988) found a marginally significant Zn increase in the nucleus basalis of Meynert.

2.2. Factors Affecting the Final Results Several factors can influence the final results of trace element determination in humans: biological/environmental factors of variation, such as age, gender, genetic factors, possible environmental exposure to specific elements, lifestyle, diseases, and medical treatments; preanalytical factors (control of contamination and losses) due to post-mortem changes, sample collection

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and handling, storage, and preanalytical chemical treatments; analytical techniques (choice of suitable, specific, and sensitive techniques); and data quality control (accuracy tested with the use of appropriate standard or certified reference materials; precision assessed by running replicate analyses of splits of the same sample). Statistical data treatments for significance are important to draw conclusions. Dealing with brain Al in AD, problems associated with the abovementioned factors and the possible reasons leading to discordant or conflicting results, already examined in some of the selected original publications, were discussed in great detail by Markesbery and Ehmann (1993) and by Ehmann and Markesbery (1994). In these reviews, special attention was paid to Al determination in brain samples by Instrumental Neutron Activation Analysis (INAA), which can be greatly affected by interference from phosphorus. As the correction factor can exceed 40% of the Al signal, the analytical results, if not corrected, are questionable. Crapper McLachlan et al. (1980c) and Krishnan et al. (1987, 1988a, and 1988b) considered the influence of sample size, case selection, and the technique employed on the results of analyses. Taylor (1994) and Lovell et al. (1996) pointed out the problems and approaches to the measurement of Al in AD studies. Markesbery and Ehmann (1994) critically reviewed data on Al, Fe, and Zn concentrations. Fe levels in AD were also commented on by Gerlach et al. (1994 and 2000); their reviews were mainly on Parkinson's disease (PD). Zn levels were also reviewed by Nachev and Larner (1996) and Cuajungco and Lees (1997b).

2.3. Aluminum, Iron, Copper and Zinc in Parkinson's Disease PD is a common neurodegenerative disorder. The core pathology of PD is the destruction of pigmented brainstem nuclei, particularly the SN pars compacta (Dexter et al., 1993). A central role in this disease is played by Fe, as explained by Youdim (1988, 1994 and 2001), Youdim et al. (1989, 1990, 1991, and 1993), Montgomery (1995), Hirsch and Faucheux 1998), and Berg et.al. (2001). The implications of alterations in trace element in the brain in PD and other neurological diseases were described by Dexter et al. (1993), Dexter

Metals Distribution and Regionalization in the Brain

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and Jenner (1997), and Good et al. (1997). The amount of Fe in the SN in PD, the techniques used, and sampling conditions were debated by Gerlach et al. (1997) and Friedman et al. (1997). Galazka-Friedman and Friedman (1997) reviewed the controversies on Fe in controls and in the SN of PD patients. Gerlach et al. (1994 and 2000), besides describing the role of Fe in neurodegenerative diseases, reviewed the alterations of Fe, Cu, Zn, and Al in these conditions. The papers retrieved on Al, Fe, Cu, and Zn determination in brain samples from both PD patients and non-PD controls were Earle (1968), Traub et al. (1981), Rajput et al. (1985), Dexter et al. (1987a, 1987b, 1989, 1991, and 1992), Sofic et al. (1988 and 1991), Uitti et al. (1989), Riederer et al. (1989), Griffiths et al. (1989 and 1999), Griffiths and Crossman (1993), Yasui et al. (1992), Galazka-Friedman et al. (1996). Loeffler et al. (1995) and Mann et al. (1994) reported Fe levels in both control and PD subjects as milligrams of Fe per gram of protein. Tables 5 to 8 list the concentration values of Al, Fe, Cu, and Zn in brain sites of both control and PD subjects. They report on the significantly different values between the two groups in some brain areas, along with other data that do not show a significant difference for the same elements in the same regions. In the analytical studies on PD, the SN, in toto or in parts (compacta and reticulata, oral and caudal), was the site where each element was most frequently found to show significantly different concentrations between the controls and PD patients. The Al contents are shown in Table 5. Al in the whole SN appeared to increase in patients, significantly or not, in comparison with the controls. In the GP as a whole and in the gyrus hippocampalis, the unique datum retrieved for Al indicated a significant enrichment in PD patients. In NC, the significant Al elevation found by Yasui et al. (1992) was not confirmed. A not significant decrease was shown in two papers by another team. Fe concentrations are listed in Table 6. Fe in SN in patients was found to be significantly increased in some experiments. In others, the difference between the two groups was not significant. Fe in the zona reticulata was significantly increased in the studies by Griffiths and co-workers, but not significantly varied in Sofic et al. (1991). Referring to the zona compacta, a significantly higher level was reported in two papers. In the oral and caudal parts, NS enrichment was observed in the unique paper considering both sites (Riederer et al., 1989). In the total GP, the Fe concentration was

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significantly increased or not varied. The data for Fe in the lateral GP are conflicting. Both a significant elevation and a significant reduction in the PD groups were published by two teams. In the medial part of GP, the experiments carried out by the same two teams showed, however, a significantly lower content in patients. Cu levels are shown in Table 7. Cu concentration in the whole SN is significantly decreased in most papers (not significantly only in one). The unique datum on the zona compacta confirms the significance of the decrease. A significant increase in PD patients was observed in the raphe plus reticular formation (Riederer et al., 1989). The levels for Zn are shown in Table 8. The Zn amount was found to be significantly higher in the whole SN of PD patients by Dexter and associates (1987a and 1991). The increase was found to be not significant by Rajput et al. (1985), while Uitti et al. (1989) and Mann and co-workers (1994) found a not significant decrease. In the SN zona compacta, Dexter et al. (1991) found significant enrichment in the PD group. In the NC, the Zn increase, estimated as significant in one paper, was not significant in two papers. In the total putamen and medial part, no significant changes in the Zn levels were found. On the other hand, in the lateral part significant enrichment was reported. A significantly higher amount of Zn was observed in PD patients in the raphe plus reticular formation (Riederer et al., 1989). The importance of factors affecting the final results (see comments on the tables in the AD section) was examined in several original papers and in Gerlach et al. (1994 and 2000), together with the reasons for the discrepancies in the results obtained in different experiments. For analytical problems in Al determination by INAA, see also the comments in the AD section.

2.4. Aluminum, Iron, Copper and Zinc in Western Pacific Parkinsonism-Dementia The Mariana island of Guam is the focus of an epidemic of neurodegenerative diseases with features of both amyotrophic lateral sclerosis (ALS; see the next section) and Parkinsonism, known as ALS/Parkinsonismdementia complex (ALS/PDC) of Guam. There is an extensive overlap among the neuropathological changes found in clinically described cases

46

Speziali M & Orvini E

of ALS and Parkinsonism-dementia, as reported by Good et al. (1997). ALS, Parkinsonism, and dementia may occur individually or, more commonly, in association with the other two (Lilienfeld et al., 1994). Other high-incidence foci of ALS/PDC in the Western Pacific are in the Kii Peninsula of Honshu Island in Japan and in western New Guinea (Auyu and Jakai populations). These areas are geochemically poor in calcium and magnesium, but rich in Al and manganese. In Guam, the disease affects mainly adults of the Chamorro population, male more frequently than female (Chen and Yase, 1985). The influence of metals in Parkinsonism-dementia was reviewed by Lilienfeld et al. (1994); their role and alterations were reviewed by Chen and Yase (1985) and Markesbery and Ehmann (1988). The importance of Fe in this disease was also described by Good et al. (1997), and the role ofZnby Yasuietal. (1997). The original papers on Al, Fe, Cu, and Zn determination in brain samples from both Parkinsonism-dementia and control subjects retrieved were Yoshimasu et al. (1980, 1982, and 1985), Traub (1981), Yoshida (1987 and 1988), and Yasui et al. (1993). Of these, only two papers with metal (Al and Cu) levels in well-defined sites of the encephalon were found, describing experiments carried out by the same team using INAA. For Cu, only data on patients were published by Yoshimasu et al. (1980). Table 9 reports data on Al. Yoshimasu et al. (1980) observed significant enrichment in the GP of diseased patients in Guam compared to Japanese controls. In their experiment in 1982, Al was also significantly higher in the GP of Guam patients than in Chamorro controls. In 1980, they found that the Al increase in the insula of the patient group was marginally significant, whereas the elevation in the capsula interna and pons were considered not significant. Yoshimasu et al. (1982) observed a significant Al increase in the putamen, thalamus, NC (areas not analyzed in the previous experiment) and in capsula interna and pons, whose Al content was previously not significant. For Cu (see Table 10), only one value (in putamen), indicating a significant decrease of the metal in the patient group from Guam, was published in Yoshimasu et al. (1982). For results of Al determination by INAA, see comments in the AD section.

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Table 10. Copper concentration values in brain sites of control and Western Pacific Parkinsonismdementia subjects (ppm, dry weight). Site Putamen

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References Yoshimasu et al., 1982

Comparing the tables on PD and Parkinsonism-dementia, it is evident that the brain sites considered in the literature with significant Al and Cu differences between the control and diseased subjects are not the same, except, in the case of Al, GP and NC. In GP, a significantly increased Al concentration in the patient group was found in two papers on Parkinsonism-dementia (Table 9) and in one paper on PD (Table 5). In NC, Yoshimasu et al. (1982; see Table 9), whilst examining controls and patients from Guam, found a significant Al elevation in the diseased subjects. The same results were observed by Yasui et al. (1992; see Table 5). In PD patients, however, an Al decrease, evaluated as statistically not significant by the authors, was found in two papers published by Rajput et al. (1985) and Uitti et al. (1989).

2.5. Aluminum, Iron, Copper and Zinc in Amyotrophic Lateral Sclerosis ALS, or motor neuron disease (MND), is an adult-onset, progressive, and neurodegenerative disorder. Three clinical variants are recognized, including a classical sporadic variant, an autosomal dominantly inherited variant, and the Western Pacific variant. An overlap with PD may occur, particularly in the Western Pacific variant (Strong and Garruto, 1997; see section on Parkinsonism-dementia). According to Lilienfeld et al. (1994), most cases of ALS in Guam were diagnosed among the Chamorros. The importance of heavy metals and trace elements in ALS was described by Mitchell (1987 and 2000), Kasarskis et al. (1993), Strong and Garruto (1997), and Yase et al. (2001). The involvement of elements in ALS and their levels in brain sites were reviewed by Yanagihara (1982), Kasarskis et al. (1990), Kasarskis (1991), and Ehmann and Vance (1996). The role of Al in ALS was described by Yase (1980, 1987, and

Metals Distribution and Regionalization in the Brain

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1988) and Wisniewsi et al. (1985). The involvement of Fe in ALS was explained by Youdim (1994) and Good et al. (1997), and the importance of Cu by Waggoner et al. (1999). The role of Zn in ALS was elucidated by Cuajungco and Lees (1997a) and Yasui et al. (1997). The original papers retrieved on Al, Fe, Cu, and Zn determination in brain samples from both patients affected by ALS (not better defined) and non-ALS controls were Yoshimasu et al. (1976 and 1980), Yase (1978), Yoshida et al. (1988), Khare et al. (1990), and Tandon et al. (1994). The papers with data on controls and patients affected by the Western Pacific variant of ALS were Yoshimasu et al. (1982), Traub et al. (1981), Yoshida (1987 and 1988), Yasui et al. (1991a, 1991b, and 1993). For Cu, only data on patients were published by Yoshimasu et al. (1980). Due to the scarcity of data and lack of description of the race of the subjects examined, we have listed the values for all the variants in a unique series of tables. Similar to Western Pacific Parkinsonism-dementia, all the papers reporting on metal (Al and Cu) levels in well-defined sites of the encephalon for ALS found in the literature were published by Japanese scientists from the Wakayama Medical College who used the same technique (IN A A). They also examined, besides the controls and ALS patients from Japan, subjects from the Chamorro population of Guam or those from the Kii Peninsula, areas that are the foci of the Western Pacific variant of ALS. The Al concentrations are shown in Table 11. In only one paper was Al determined in the SN; both the controls and patients were from Guam and a significant elevation was observed in the AD group. A particular situation was retrieved for the insula. In fact, in the experiment on subjects from Guam, the mean difference between the two groups was significant. In one experiment on Japanese subjects, the variation was marginally significant while, according to two other papers, it was not significant. For pons, in the work on the Guam subjects, a significant Al increase was observed in the patients; in other experiments on Japanese individuals, the difference was not significant. In gyrus praecentralis, when the controls (their origins were not specified) and two patients in the severe stage of ALS from the Kii Peninsula were compared (Yasui et al., 1991a and 1991b), a significantly higher Al content was found in the diseased women. In the study by Yoshimasu et al. (1982), where the Al levels were evaluated in both groups from Guam, the decrease appeared to be not

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Table 12. Copper concentration values in brain sites of control and amyotrophic lateral sclerosos subjects (ppm, dry weight). Subjects

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References Yoshimasu et al., 1982

significant. Other papers which reported on results obtained in controls and patients from the Japanese population showed no significant increase. In gyrus postcentralis, a significant elevation in the Al amounts of a Japanese ALS group was shown in one experiment, whereas in others no significance was found. Yoshimasu et al. (1982), taking into account the two groups from Guam, observed a nonsignificant Al enrichment in patients. As far as CI is concerned, only the papers which considered controls not better described and the two ALS subjects from the Kii Peninsula showed a significant Al elevation in the patients. In other experiments which examined controls and patients from Japan, as well as one that takes into account normal and diseased people from Guam, the higher Al amounts observed in the ALS groups were not significant. A significant increase was found by Yasui et al. (1991a and 1991b) in the cms cerebri of two ALS patients from the Kii Peninsula; Yoshimasu et al. (1982) considered the Al elevation in the Guam patient group as not significant. An overview of the results published on gyrus praecentralis, gyrus postcentralis, and CI suggests that the significance of the difference in Al levels between the controls and patients appears to be unrelated to the race of the subjects studied. For Cu (Table 12), only one value (in the putamen), indicating a significant decrease of the metal in ALS patients from Guam, was published by Yoshimasu et al. (1982). Al was determined by INAA. Yasui et al. (1991a and 1991b) applied a correction for the interference from phosphorus. (See comments in the AD section.)

3. FINAL REMARKS In normal brains, several authors found the highest Al levels in the GP, SN, and NR. In the studies on AD, a significant increase in Al amounts was found in the hippocampus and, according to a single experiment, in the inferior parietal lobule and superior and middle temporal gyri.

Metals Distribution and Regionalization in the Brain

53

In PD, SN, GP, NC, and gyrus hippocampalis were sites in which at least one experiment found an Al elevation in patients. In Western Pacific Parkinsonism-dementia, GP, putamen, thalamus, NC, capsula interna, and pons, at least in one work each, showed an Al enrichment in samples taken from diseased individuals. For ALS, the Japanese detected significantly higher Al amounts in the patient group in at least one experiment in several sites (SN, insula, pons, gyrus praecentralis, gyrus postcentralis, CI, and cms cerebri). In the controls, most authors found the highest Fe concentrations in GP, putamen, NC, and SN. Some scientists observed significant Fe changes in AD subjects in the hippocampus (with conflicting results) and an increase in the amygdala; in at least one paper, a significant elevation of the metal in the frontal pole, temporal pole, inferior parietal lobule, piriform cortex, olfactory region, and GP of patients was reported. As far as PD is concerned, some authors found a significant Fe enrichment in total SN in afflicted subjects. In two papers, Fe elevation resulted also in SN zona compacta and, in another experiment, in SN zona reticulata. In total GP, Fe appeared to be significantly higher in patients in one paper; for lateral GP, conflicting data were found, whilst for medial GP a significant reduction was discovered by two different teams. In the normal brain, most authors found the highest Cu concentrations in SN, ND, putamen, and NC. A significant decrease in Cu in AD subjects was found in the hippocampus by two teams, and by one in the amygdala. In PD, a decrease was observed, according to some papers, in SN whereas an increase was found, in a single work, in the raphe plus reticular formation. Only one paper reported a significant decrease in Cu in the putamen from patients with both Parkinsonism-dementia and ALS. In the controls, several teams detected the highest Zn concentrations in the hippocampus, amygdala, NC, and epiphysis. Significant changes in Zn in AD patients were detected in the hippocampus, with conflicting results; in amygdala, higher amounts were determined in different experiments. An elevation in Zn in diseased subjects was observed in two works in the inferior parietal lobule and, at least in one paper, in the olfactory tract, frontal pole, and temporal pole. In PD, the total SN, SN zona compacta, NC, lateral putamen, and raphe plus reticular formation from the patient group contained, according to at least one experiment, significantly higher Zn amounts than the

54

Speziali M & Orvini E

corresponding sites in the controls. The authors had analyzed a wide number of regions. These areas were chosen according to those parts of the brain known to be as prominently affected by the specific disease being studied.

ACKNOWLEDGMENT The authors are grateful to Stelio Locchi, Department of General Chemistry, University of Pavia, Italy, for valuable suggestions.

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Campbell A, et al. Mechanisms by which metals promote events connected to neurodegenerative diseases. Brain Res Bull 2001; 55:125-132. Chazot G, Broussolle E. Alterations in trace elements during brain aging and in Alzheimer's disease. In Prasad AS, editor. Essential and Toxic Trace Elements in Human Health and Disease: An Update, Progress in Clinical and Biological Research, Vol. 380. New York: Wiley-Liss, 1993: 269-281. Chen KW, Yase Y. Parkinsonism-dementia, neurofibrillary tangles and trace elements in the Western Pacific. In Hutton and Kenny, editors. Senile Dementia of the Alzheimer Type. New York: Alan R Liss, Inc., 1985: 153-173. Connor JR et al. Regional distribution of iron and iron-regulatory proteins in the brain in aging and Alzheimer's disease. J Neurosci Res 1992; 31:327-335. Constantinidis J. Zinc toxicity in Alzheimer's disease: An emerging hypothesis. In: Yasui M, Strong MJ, Ota K, Verity MA, editors. Mineral and Metal Neurotoxicology. Boca Raton: CRC Press, 1997: 147-156. Constantinidis J. Maladie d'Alzheimer et la theorie du zinc. U Encephale 1990; 16:231-239. Constantinidis J, Tissot R. Degenerative encephalopathies in old age: Neurotransmitters and zinc metabolism. In: Terry RD, Bolis CL, Toffano G, editors. Neural Aging and Its Implications in Human Neurological Pathology, Vol. 18. New York: Raven Press, 1982: 53-59. Constantinidis J, et al. Demences degeneratives de Pick et d'Alzheimer et metabolisme du zinc. Medecine et Hygiene 1977; 35:3706-3710. Cornett CR, et al. Trace elements in Alzheimer's disease pituitary glands. Biol Trace Element Res 1998a; 62:107-114. Cornett CR, et al. Imbalances of trace elements related to oxidative damage in Alzheimer's disease brain. Neurotoxicology 1998b; 19:339-346. Cornett CR, et al. Pituitary gland levels of mercury, selenium, iron, and zinc in an Alzheimer's disease study. Transactions 1996; 74:6. Corrigan FM, et al. Hippocampal tin, aluminum and zinc in Alzheimer's disease. Biometals 1993; 6:149-154. Corrigan FM, et al. Reductions of zinc and selenium in brain in Alzheimer's disease. Trace Elements in Medicine 1991; 8:1-5. Crapper McLachlan DR, et al. Aluminum, altered transcription, and the pathogenesis of Alzheimer's disease. Environmental Geochem Health 1990; 12:103-114. Crapper McLachlan DR. Aluminum and Alzheimer's disease. Neurobiol Aging 1986; 7:525-532. Crapper McLachlan DR, Farnell BJ. Aluminum and neuronal degeneration. In: Gabay S, Harris J, Ho BT, editors. Metal Ions in Neurology and Psychiatry. New York: Alan R Liss, Inc., 1985: 69-87. Crapper McLachlan, et al. Aluminum in human brain disease. In: Sarkar B, editor. Biological Aspects of Metals and Metal-related Diseases. New York: Raven Press, 1983: 209-218. Crapper McLachlan DR, De Boni U. Aluminum in human brain disease: An overview. Neurotoxicology 1980a; 1:3-16.

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Crapper McLachlan DR, De Boni U. Aluminum. In: Spencer PS, Schaumburg HH, editors. Experimental and Clinical Neurotoxicology. Baltimore: Williams and Wilkins, 1980b: 326-335. Crapper McLachlan, et al. Brain aluminum in Alzheimer's disease: Influence of sample size and case selection. Neurotoxicology 1980c; 1:25-32. Crapper McLachlan DR, et al. Aluminum and other metals in senile (Alzheimer) dementia. In: Katzman R, Terry RD, Bick KL, editors. Alzheimer's Disease: Senile Dementia and Related Disorders. Vol. 7. New York: Raven Press, 1978: 471^189. Crapper McLachlan DR, et al. Aluminium, neurofibrillary degeneration and Alzheimer's disease. Brain 1976; 9:67-80. Crapper McLachlan DR, et al. Brain aluminum distribution in Alzheimer's disease and experimental neurofibrillary degeneration. Trans Amer Neurol Assoc 1974; 98:17-20. Crapper McLachlan DR, et al. Brain aluminium distribution in Alzheaimer's disease and experimental neurofibrillary degeneration. Science 1973; 180:511-513. Cuajungco MP, Lees GJ. Zinc metabolism in the brain: Relevance to human neurodegenative disorders. Neurobiol Disease 1997a; 4:137-169. Cuajungco MP, Lees GJ. Zinc and Alzheimer's disease: Is there a direct link? Brain Res Rev 1997b; 23:219-236. Cumings JN. Trace elements in the brain in health and in neurological disease. Scientific Basis Med Ann Rev 1965: 37-57. Danscher G, et al. Increased amount of zinc in hippocampus and amygdala of Alzheimer's diseased brains: A PIXE spectroscopic analysis of cryostat sections from autopsy material. JNeurosci Methods 1997; 76:53-39. Dedman DJ, et al. Iron and aluminium in relation to brain ferritin in normal individuals and Alzheimer's disease and chronic renal dialysis patients. Biochem J 1992; 287:509-514. Deibel MA, et al. Copper, iron, and zinc imbalances in severely degenerated brain regions in Alzheimer's disease: Possible relation to oxidative stress. J Neurol Sci 1996; 143:137-142. Deibel MA, et al. Non-destructive analysis of Cu in human brain tissue by neutron activation analysis using coincidence and anti-coincidence techniques. J Radioanalytical Nucl Chem 1997; 217:153-155. Dexter DT, Jenner P. Alterations in iron in neurodegenerative disorders: Implications and possible therapeutic agents. In: Yasui M, Strong MJ, Ota K, Verity MA, editors. Mineral and Metal Neurotoxicology. Boca Raton: CRC Press, 1997: 365-378. Dexter DT, et al. Implications of alterations in trace element levels in brain in Parkinson's disease and other neurological disorders affecting the basal ganglia. In Narabayashi H, Nagatsu T, Yanagisawa N, Mizuno Y, editors. Advances in Neurology, Vol. 60. New York: Raven Press, 1993: 273-281. Dexter DT, et al. Alterations in levels of iron, ferritin, and other trace metals in neurodegenerative diseases affecting the basal ganglia. Ann Neurol 1992; 32:S94-S100. Dexter DT, et al. Alterations in the levels of iron, ferritin and other trace metals in Parkinson's disease and other neurodegenerative diseases affecting the basal ganglia. Brain 1991;114:1953-1975.

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CHAPTER 3

The Olfactory Pathway as a Route of Entry of Metals into the Brain Hans Tjalve, Jonas Tallkvist

ABSTRACT The central nervous system (CNS) is partially protected from circulating toxicants by the blood-brain barrier. However, for xenobiotics which are excluded from the CNS the olfactory pathway provides an alternative route of passage into the brain. Thus, in the olfactory epithelium the primary olfactory neurons have dendrites in contact with the nasal lumen and axons which project to the olfactory bulbs. Materials which come into contact with the olfactory epithelium may be taken up in the primary olfactory neurons and transported to the olfactory bulbs and even further into other areas of the brain. The article deals with the uptake and transport of metals in the olfactory system. Metals discussed are mainly manganese, cadmium, nickel, aluminum, zinc, cobalt and mercury. Among these metals manganese has a unique capacity to be taken up via the olfactory pathway. Thus, following transport along the primary olfactory neurons to the olfactory bulbs manganese continues via secondary and tertiary olfactory neurons and further connections to all parts of the brain and even into the spinal cord. Cadmium is transported along the axons of the primary olfactory neurons to the olfactory bulb, but this metal appears unable to leave the terminal arborizations of the axons in the glomeruli of the bulb. Studies with nickel, zinc, cobalt and mercury indicate that these metals are transported along the primary olfactory neurons and accumulated in the olfactory nerve layer and the glomerulular layer of the bulbs. In addition, low levels of these metals leave the terminations of the primary olfactory neurons. There is evidence that mercury also may undergo axonal transport in the primary olfactory neurons following uptake in these neurons from the systemic circulation. This may be a part of a more general ability of mercury to be taken up in neurons with access to the systemic circulation and reach the CNS via transport in these neurons. There is evidence 67

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Tjdlve H & Tallkvist J that aluminum is transported along the olfactory pathway, but the fate of this metal in the olfactory system is not yet known in detail. The olfactory pathway is a route by which metals may circumvent the blood-brain barrier and reach the central nervous system. The possibility that uptake and transport of metals in the olfactory pathways may induce olfactory dysfunction and neurotoxicity should be taken into account in risk assessments of occupational metal exposure via inhalation. Keywords: Alzheimer's disease; Parkinson's disease; olfactory; neuronal transport; manganese; cadmium; nickel; aluminum; zinc; cobalt; mercury.

1. INTRODUCTION This article is built upon observations that metals can be efficiently transported via olfactory pathways to the brain. This area of research is important since it suggests that the nasal cavity and the olfactory pathways may serve as a critical route for deposition, absorption and transport of metals into the central nervous system (CNS). Thus, the role of the olfactory pathway must be taken into account in future risk assessments of occupational metal exposure via inhalation. Traditionally researchers have focussed on uptake of metals via the lung and it is considered that the lung may serve as a long-term reservoir of metals deposited in the lower airways. The CNS is partially protected from uptake of metals present in the circulation by the blood-brain barrier. Therefore, metals taken up in the blood from the lung may have a limited ability to pass to the brain. The olfactory epithelium is a unique tissue in which the olfactory neurons have dendrites in contact with the environment in the nasal cavity and axons, which reach the glomeruli of the olfactory bulbs. In addition to metals several other materials may enter the CNS via this route. This applies to organic xenobiotics, such as dyes (Holl, 1980), organic solvents (Ghantous et al., 1990), polychlorinated biphenyls (Apfelbach et al., 1998), benzo(a)pyrene (Persson et al., 2002) and aflatoxin Bj (Larsson and Tjalve, 2000), protein tracers, such as albumin labelled with Evans blue, horseradish peroxidase and the conjugate between horseradish peroxidase and wheat-germ agglutinin (Kristensson and Olsson, 1971; Shipley, 1985; Stewart, 1985; Baker and Spencer, 1986; Itaya, 1987; Thome et al., 1995), several neurovirulent viruses (Johnson, 1964; Monath et al., 1983; Charles et al., 1995) and some endogenous materials, such as carnosine, taurine, amino acids and proteins (Gross and Kreutzberg, 1978; Burd et al., 1982; Lindquist et al., 1983).

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In this article metal transport into the brain via the olfactory route is reviewed. The outline of the article is as follows: (i) anatomy of the olfactory system; (ii) deposition of inhaled materials on the olfactory epithelium; (iii) uptake mechanisms into olfactory neurons; (iv) mechanisms of neuronal transport; (v) transport of metals in the olfactory system (manganese, cadmium, nickel, aluminum, zinc, cobalt, mercury); (vi) abundance of metals in the olfactory bulb; (vii) metal transfer into the cerebrospinal fluid via the olfactory pathway; (viii) concluding remarks.

2. ANATOMY OF THE OLFACTORY SYSTEM The dorsal and caudal portions of the olfactory cavity are lined with olfactory epithelium. The major cells in the olfactory epithelium are the primary olfactory neurons (or receptor cells) and the sustentacular cells (or supporting cells) (Fig. 1). The primary olfactory neuron is a bipolar cell in which a single dendrite extends to the olfactory mucosal surface, where it enlarges to a dendritic knob. From the knob fine olfactory cilia extend into a layer of mucus, which covers the epithelium. The receptor cells and the supporting cells form typical tight junctions with one another (Farbman, 1992). The basal cell constitutes a third cellular component in the olfactory epithelium. Bowman's glands, situated in the lamina propria mucousa, provide most of the mucus which covers the olfactory epithelium (Getchell and Mellert, 1991). These cells and the supporting cells contain high levels of xenobiotic metabolizing enzymes, including cytochromes P450 (Dahl and Hadley, 1991). The axons of the primary olfactory neurons pass through the lamina propria of the olfactory mucosa and the cribriform plate of the ethmoid bone to the glomeruli of the olfactory bulb, in which they synapse onto the dendrites of the mitral and tufted cells. The axons of these cells, which constitute the secondary olfactory neurons, project to the olfactory cortex. This area includes the anterior olfactory nucleus, the olfactory tubercle and the pyriform, amygdaloid and entorhinal cortices (Allison, 1953; Heimer, 1968; Scott, 1986; Haberly, 1990; Greer, 1991; Farbman, 1992). In the olfactory cortex synapses are formed with dendrites of pyramidal cells, which constitute the tertiary olfactory neurons. The axons of these cells distribute to multiple subcortical and cortical regions (Allison, 1953;

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Heimer, 1968; Haberly, 1990; Price, 1985; Scott, 1986; Greer, 1991; Farbman, 1992) (Fig. 1).

3. DEPOSITION OF INHALED MATERIALS ON THE OLFACTORY EPITHELIUM There are indications that in the rat about 15% of the inspired air pass over the olfactory mucosa during normal breathing (Jaillardon et al., 1992). Similar values have been obtained in human models (Hahn et al., 1993). In the rat nasal mucosa deposition efficiency of particles follows a bimodal distribution: For larger particles (>l|jim) the deposition efficiency increases with increasing particle size, whereas for particles ranging from 0.2 to 0.005 |xm deposition fraction increases with decreasing particle size (Cheng et al., 1990). Only a proportion of the inhaled particles can be expected to reach the olfactory mucosa, but the degree of regional uptake in the olfactory area is not known at present (Lewis et al., 1994). The nasal mucociliary apparatus may remove particles from the respiratory mucosa, but such a clearance does not appear to occur from the olfactory mucosa (Morgan et al., 1984). When extrapolations are made from experiments in animals to the human situation interspecies differences in nasal anatomy should be considered. In rodents and several other mammals the olfactory mucosa covers a larger proportion of the nasal cavity than in humans and the olfactory route of brain delivery may therefore be less important in humans than in some animals.

4. UPTAKE MECHANISMS INTO OLFACTORY NEURONS An important component of the olfactory mucosa is the mucus layer, which covers the epithelium. Inhaled odor molecules are trapped in this layer as they pass the olfactory mucosa. Toxic materials, which reach the olfactory mucosa, may also be trapped in the mucus. The mucus contains mucopolysaccharides (Getchell and Mellert, 1991) and can be expected to bind metal cations. However, the degree of binding of different metals and the potential effect this may have on the penetration to the underlying cell-surfaces are not known at present.

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Fig. 1. Overview of the anatomy of the olfactory mucosa, the olfactory bulb and the efferent neuronal projections from the olfactory bulb. The figure indicates the essential morphology of the olfactory mucosa and the main neuronal elements in the olfactory bulb as well as most of the main efferent connections from the olfactory cortex. 1, mucus layer; 2, dendritic knob; 3, cilia containing the olfactory receptors; 4, olfactory receptor cell; 5, sustentacular cell; 6, basal cell; 7, basement membrane; 8, Bowman's gland; 9, blood vessel; 10, Schwann cell; 11, cribiform plate of the ethmoid bone; AC, amygdaloid cortex; AON, anterior olfactory nucleus; EC, entorhinal cortex; OT, olfactory tubercle; PC, pyriform cortex; epl, external plexiform layer; g, glomerulus; gel, granular cell layer; gl, glomerular layer; mc, mitral cell; mcl, mitral cell layer and internal plexiform layer; onl, olfactory nerve layer; pgc, periglomerular cell; tc, tufted cell. The asterisc given at the neocortex indicates that the neocortical areas are localized in the dorsal bank of the sulcus rhinalis (from Tjalve and Henriksson, 1999; with permission).

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The uptake of materials in neurons may occur by different mechanisms. Thus, the uptake may involve a binding of the materials to specific binding sites at the cell surface. This mechanism is termed adsorptive endocytosis. Alternatively materials may also be passively trapped in an endocytotic vesicle along with extracellular fluid in a process termed fluid phase endocytosis. Some metals, such as cadmium and mercury, have a strong affinity for SH-groups and other negatively charged groups on proteins (Berlin, 1986; Friberg et al., 1986). Conceivably, a binding to such groups on the olfactory receptor cell membrane may result in internalization of these metals, according to the mechanism of adsorptive endocytosis. Other metals may be taken up in the olfactory receptor cells by fluid phase endocytosis. However, even a weak electrostatic interaction with the cell surface may facilitate the uptake. This has been shown to be the case in axonal terminals. Thus, studies by Olsson and Kristensson (1981) have shown that, following injection in the vibrissae muscles of mice, cationized ferritin is more avidly accumulated in the facial nerve nucleus as compared to native ferritin. It was considered that the positive charge of the cationized ferritin interacts with negatively charged macromolecules on the neuronal surface, in turn facilitating uptake in the axonal terminal. A similar facilitated uptake of positively charged metal cations may take place in the olfactory receptor cells. The knobs of the dendritic processes of the olfactory receptor cells, which extend to the surface of the olfactory epithelium, are rich in endocytic vesicles, indicating that they are actively engaged in the uptake of exogenous materials from the environment (de Lorenzo, 1970; Bannister and Dodson, 1992). Particulate materials, such as the silver-coated colloid gold particles examined by de Lorenzo (1970), appear to be taken up from the overlying mucus by internalization together with the surface membrane (de Lorenzo, 1970; Bannister and Dodson, 1992). There may also be specific transport processes, which mediate the uptake of some metals. These processes may vary depending on the metal in question. As concerns manganese, it has been shown that this metal can enter nerve terminals via calcium channels during nerve action potentials (Narita et al., 1990). The metal also passes through calcium channels in myoepithelial cells (Anderson, 1979). Manganese permeates presynaptic calcium channels and induces dopamine release from depolarized nerve endings (Drapeau and Nachsen, 1984). Conceivably, manganese may be

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taken up in the olfactory receptor cells via calcium channels. Mercury might enter these cells via sodium or calcium channels, as has been proposed as the mode of uptake of this metal in motor nerve terminals in frog muscle (Miyamato, 1983). Recent studies have revealed the presence of metal transporters for iron (Divalent Metal Transporter 1 /DMT1/) (Gunshin et al., 1997) and zinc (Zrt, Irt-like Proteins /ZIP/) (Gaither and Eide, 2001) in various tissues. These transporters have also been shown to act on other metals, such as manganese, cadmium and nickel (Gunshin et al., 1997; Gaither and Eide, 2001). Conceivably, these transporters may be involved in the uptake of metals in the olfactory neurons.

5. MECHANISMS OF NEURONAL TRANSPORT Since biosynthetic processes are largely restricted to the perikaryal regions of nerve cells there is a need for transport of materials along the axons and the dendrites to their terminals. In the anterograde direction — from the cell body to the periphery — there appears to be at least two components of transport: Fast and slow. In the retrograde direction — from the periphery to the cell body — the transport occurs at a rate, which is similar to that of fast anterograde transport. The anterograde transport is necessary for synaptic function and maintenance of neuronal constituents, whereas the retrograde transport may serve the purpose of shipping constituents back to the cell body for re-use or final degradation. Biochemical research during the last decades has revealed that the fast anterograde transport and the retrograde transport are microtubule-associated processes, in which kinesin generates the movement in the antero-grade direction and cytoplasmic dynein is the motor protein in the retrograde direction (Okabe and Hirokawa, 1989; Cyr and Brady, 1992; Hammerschlag et al., 1994). The molecular mechanism of the slow transport is still not known. The calculated values for the rate of the fast anterograde transport varies in different experimental models (rate -20-400 mm/day). This involves predominantly particulate materials, such as membrane proteins and lipids and neurotransmitter-containing synaptic vesicles. There is also evidence for fast neuronal transport of low-molecular-weight materials, such as amino acids and calcium (Hammerschlag et al, 1975; Knull and Wells, 1975; Gross and Kreutzberg, 1978). The slow anterograde transport (rate -0.1-4 mm/day)

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involves cytoskeletal proteins and various soluble enzymes, including cytoplasmic enzymes of intermediary metabolism (Vallee and Bloom, 1991; Nixon, 1992). Materials transported in the retrograde direction consist of pinocytotic vesicles and lysosomal organelles, as well as transmittercontaining vesicles and mitochondria. Neuronal transport of metals is a phenomenon, which in addition to the olfactory system, is known to occur in other areas of the CNS and also in the peripheral nervous system. It can be assumed that the metals are bound to some endogenous neuronal constituent(s) during this transport. The binding of metals to components in the olfactory neurons will be discussed below when the olfactory transport of the various metals is described.

6. TRANSPORT OF METALS IN THE OLFACTORY SYSTEM Transport of metals in the olfactory system was first observed for cadmium and has then been shown for several other metals, such as manganese, nickel, aluminum, zinc, cobalt and mercury. Among these metals manganese is the only one that so far has been shown to readily distribute in the entire brain following uptake via the olfactory pathways.

6.1. Manganese We have examined the uptake of 54Mn2+ in the brain via olfactory pathways in pike, brown trout and rat (Tjalve et al., 1995, 1996; Rouleau et al., 1995). The results showed that manganese is taken up from the olfactory mucosa to the olfactory bulbs and then passes transneuronally to various parts of the brain. In our study in the rat, in which 54MnCl2 was applied in the nasal cavity, it was possible to follow the transfer of 54 Mn 2+ in the brain. Thus, initially the metal passed from the olfactory bulbs to the basal forebrain areas, which constitute the terminal regions of the secondary olfactory neurons (the olfactory cortex; see Fig. 1). The results indicated that from these areas the 54 Mn 2+ continued along tertiary olfactory neurons — as shown in Fig. 1 — in the anterior commissure to areas reached via this pathway and to the hypothalamus, thalamus, habenula, hippocampus and the neocortex dorsal to the fissura rhinalis. At long survival intervals the 54 Mn 2+ was seen to be spread to various grey matters at the base of the cerebral hemisphere, including the basal ganglia, and also to the

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grey matter in various areas of the cerebral cortex, and eventually to the cerebellum and the spinal cord (Fig. 2). Other authors have shown transport of manganese in the olfactory system following topical application of MnCl2 on the olfactory mucosa of rats (Gianutsos et al., 1997) and mice (Pautler et al., 1998). Brenneman et al., (2000) demonstrated uptake of manganese in the olfactory system of rats following inhalation exposure to 54MnCl2. Vitarella et al. (2000) showed uptake of manganese in the olfactory bulb of rats exposed to manganese phosphate via inhalation. Manganese phosphate is the primary respirable manganese compound emitted from automobile exhaust-pipes following combustion of methylcyclopentadienyl manganese tricarbonyl (MMT), which is added to gasoline as an octane enhancer. Normandin et al. (2002) showed a dose-dependent increase of the manganese concentration in the olfactory bulb of rats following subchronic inhalation of manganese phosphate. In this study a dose-dependent increase in manganese concentration was also seen in the caudate/putamen. These authors concluded that the olfactory bulb is the brain target for manganese accumulation and that uptake of manganese into the brain may occur via the olfactory pathway. Studies by Dorman et al. (2001) showed higher brain manganese in rats exposed via inhalation to soluble MnS0 4 compared to insoluble Mn 3 0 4 . Studies by Fechter et al. (2002) showed uptake of manganese into the brain via the olfactory pathway in rats exposed via inhalation to small-particle aerosols (up to 1.3 |xm) of Mn0 2 , but not to large-particle aerosols (up to 18 |jim) of Mn0 2 . Studies in the California ground squirrel, which is a fossorial animal, indicate that manganese, and also cadmium, present in soil are taken up in the olfactory bulb via the olfactory pathway (Bench et al., 2001). The application of 54 Mn 2+ (as 54MnCl2) in the olfactory chambers of pike resulted in transport of the metal in the olfactory neurons towards the olfactory bulbs at a rate of about 2.9 mm/hour at 10 °C (Tjalve et al., 1995). This rate falls into the category of fast axonal transport. Sloot and Gramsbergen (1994) injected Mn 2+ in the substantia nigra or the striatum of rats and showed an anterograde transport of the metal, both in dopaminergic nigral neurons and in -y-aminobutyric acidergic striatal neurons. They reported that the manganese-transport was inhibited by injections of colchicine, which is an inhibitor of cellular functions dependent on microtubules.

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It can be assumed that manganese adheres to and moves along with some protein(s), which undergo fast axonal transport. Cell fractionations and gel chromatography of the olfactory nerve and bulb and the telencephalon of the pike given manganese in the olfactory chambers showed that the metal was present both in the soluble cell fraction (cytosol) and in association with various particulate cell constituents (Tjalve et al., 1995). A similar subcellular distribution of manganese was seen in the olfactory epithelium and various parts of the brain in rats in which the metal was applied in the nasal cavity (Henriksson et al., 1999). Unlike many other metals manganese does not possess a high affinity for any endogenous ligand, such as e.g., SH-groups, and it is possible that manganese relatively easily can move between different tissue constituents within the olfactory system, as well as in the brain. One also has to consider that the administered Mn 2+ partly may undergo oxidation to Mn 3+ . Mn 3+ appears to have a slower elimination rate than Mn 2+ , which may be related to a higher affinity of the trivalent manganese than the divalent species to endogeneous ligands (Gibbons et al., 1976). Mn 3+ is bound to transferrin. Sloot and Gramsbergen (1994) proposed that manganese may be transported from the substantia nigra to the striatum and vice versa bound to transferrin. However, our study in the rat indicated that the major proportion of the metal is not bound to transferrin at the transport along olfactory-related pathways of the brain (Henriksson et al., 1999). There is also a possibility that the binding pattern of the manganese in the neurons may change over time. It is well known that chronic exposure to manganese can cause neurologic symptoms, usually manifested by an extrapyramidal syndrome resembling Parkinson's disease. Neuropathology is induced in the basal ganglia with lesions being localized in the substantia nigra and, in addition, at other sites, such as the globus pallidus, nucleus caudatus and putamen and also the thalamus. Lesions may, in addition, be found throughout the cerebrum, the brain stem and the cerebellum (Donaldson, 1987; Mergler, 1996). The results discussed above indicate that the olfactory route may be an important pathway through which inhaled manganese gains access to the CNS. Manganese intoxications have been observed primarily at occupational exposure among workers involved in steel manufacturing and welding and in mining and processing of manganese ores, and have been related exclusively to inhalation of manganese-containing dusts or fumes (Barbeau, 1984; Donaldson, 1987). Manganese is also ubiquitously

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present in ambient air due to industrial activities and release from crustal sources. There is, in addition, an increasing interest in the neurotoxicity of this metal because of the use of MMT as a gasoline octane enhancer. The risk posed to the general population by chronic environmental manganese exposure is unknown, but aging may be a susceptibility factor, and there may be individuals which are especially sensitive (Mergler, 1996). The main cellular target for the damage in neurological manganism has not been identified with certainty, and the crucial biochemical events which cause the neurotoxicity remain unknown. Several authors have proposed that the mechanism of manganese toxicity may involve formation of free radicals in the presence of catecholamines (Donaldson et al., 1982; Graham, 1984; Halliwell, 1984). Recently several authors have proposed that the astrocytes may be the primary targets of manganese toxicity in the brain. Manganese has been shown to affect processes in the astrocytes such glutamate uptake, expression of glyceraldehyde-3-phosphate dehydrogenase and nitric oxide synthesis (Hazell and Norenberg, 1997; Spranger et al., 1998; Hazell et al., 1999). Astrocytes play an important role in normal brain functions, such as regulation of ion homeostasis, uptake of transmittors and contribution to the CNS immune system (Aschner, 1998). Manganese is avidly taken up in astrocytes in vitro (Aschner et al., 1992). Sloot and Gramsbergen (1994) showed that striatal injection of manganese resulted in a higher uptake of the metal into a quinolinic acidlesioned striatum, which is depleted of intrinsic nerve cells and contains an abundant amount of glia cells, than into a non-lesioned striatum. We have recently shown that intranasal administration of manganese in rats results in decreased levels of the astrocytic proteins GFAP (glial fibrillary acidic protein) and S-lOOb (a low-molecular-weight calcium binding protein) in the olfactory cortex, the hypothalamus, the thalamus and the hippocampus — brain regions with prominent connections to the olfactory bulb (Henriksson and Tjalve, 2000). We assume that the decreased levels of GFAP andS-lOOb are due to adverse effects of manganese on the astrocytes.

6.2. Cadmium Transport of cadmium in the olfactory system was first shown in fish exposed to the metal via the water. Thus, whole-body autoradiography in brown trout exposed to 109CdCl2 via aquarial water showed an accumulation of l09 Cd 2+ in the olfactory rosette, the olfactory nerve and the anterior part

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of the olfactory bulb (Tjalve et al., 1986). Studies in pike, in which 109Cd2+ was applied in the olfactory chambers, confirmed that cadmium is transported along the olfactory nerve and is accumulated in the anterior part of the olfactory bulb of the brain (Gottofrey and Tjalve, 1991). The uptake of the metal in the olfactory bulb was confined to the anterior part, whereas in other brain areas the level of the metal remained low, indicating that cadmium accumulates in the terminal parts of the olfactory axons, but is unable to continue along secondary olfactory neurons. The results showed a wave of 109Cd transported along the olfactory nerve with a maximal velocity of about 2.4 mm/hour at the experimental temperature (+ 10°C). This rate falls into the category of fast axonal transport. Hastings and Evans (Hastings and Evans, 1991; Evans and Hastings, 1992) applied 109Cd intranasally in rats and found an accumulation of the metal in the olfactory bulb on the side of the application, whereas the level in the contralateral bulb was low. There was no accumulation of 109Cd2+ in the forebrain of the rats. We performed autoradiography in rats given 109 Cd2+ intranasally and found an accumulation of the metal in the anterior part of the olfactory bulb, whereas the levels in other brain areas remained low (Tjalve et al., 1996) (Fig. 2). Gamma spectrometry confirmed that intranasal administration of 109Cd2+ results in a selective accumulation of the metal in the olfactory bulb (Tjalve et al., 1996). As mentioned studies in the California ground squirrel indicate that cadmium (as well as manganese) present in soil is taken up in the olfactory bulb via the olfactory pathway (Bench et al., 2001). Recent studies in rats and pikes indicate that cadmium administered by intranasal administration induces metallothionein synthesis in the olfactory receptor cells in the olfactory mucosa and that the metal is transported as a cadmium metallothionein complex along the axons of these cells to the olfactory bulbs of the brain (Tallkvist et al., 2002) (Fig. 3). These data also implies that metallothionein is transported in the olfactory neurons at the same rate as cadmium. The affinity for metallothionein may render cadmium prone not to leave the primary olfactory neurons, resulting in an accumulation in the terminal parts of the olfactory axons. It has been reported in several clinical studies that occupational exposure to cadmium results in olfactory dysfunction (Friberg, 1950; Adams and Crabtree, 1961; Rose et al, 1992). Baader (1952) reported that a worker exposed to cadmium oxide dust for 16 years had an atrophic nasal mucosa

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Fig, 2. Details of a whole-body autoradiograms (sagittal sections) of rats killed three weeks after intranasal instillations of (A) 54 Mn 2+ (0.6 |xg) or (C) 109Cd2+ (3,3 fig) in the right nostrils, (B) and (D) are the tissue section corresponding to A and C, respectively. White areas in the autoradiograms denote high levels radioactivity. (From Tjalve et al„ 1996; with permission.)

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rb

lb

rb onl

onl 8

lm

rm

B

ns n nf

b Bg

nf

A Fig. 3. Immunohistochemical staining for metallothionein in sections of the olfactory system of a rat, killed 48 h after intranasal instillation of cadmium (5 \ig) in the right nostril. (A) Section showing the left and right olfactory mucosa, turbinates and bulbs. The immunohistochemical staining is much higher on the right than on the left side. (B) Section showing the right and left olfactory bulb. Higher levels of metallothionein are present in the olfactory nerve layer of the right bulb than of the left bulb. A homogeneously distributed staining is present in the glomeruli of the right olfactory bulb, whereas in the left bulb the glomerular staining shows a mesh-like pattern. (C) Detail of the right olfactory mucosa. Metallothionein-staining is present in neuronal, sustentacular, and basal cells. Nerve fascicles in the lamina propria also show a marked immunoreactivity. The staining in Bowman's glands is low. Legends: b, basal cell; Bg, Bowman's glands g, glomeruli; lb, left olfactory bulb; lm, left olfactory mucosa; n, olfactory nerve cell nf, nerve fascicles; ns, nasal septum; onl, olfactory nerve layer; rb, right olfactory bulb rm, right olfactory mucosa; s, sustentacular cell. Magnification: (A)X12, (B) X60 (C) X180 (from Tallkvist et al., 2002; with permission).

and the olfactory bulbs were stained bright yellow. Although olfactory dysfunction is a common finding in cadmium-exposed workers, no effects on olfaction were observed in an experimental study in rats exposed to cadmium oxide dust for 20 weeks (Sun et al., 1996). One possible explanation for this finding is that a long latency period is required (in man up to 20 years) before the development of anosmia. Cadmium has a noxious effect on the olfactory epithelium and is a strong inhibitor of olfactory

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function in fish (Stromberg et al., 1983; Rehnberg and Schreck, 1986). It is possible, therefore, that low levels of cadmium in the water may be injurious to the olfactory sense in fish.

6.3. Nickel We have studied the uptake of nickel in the olfactory system of rats and pikes (Henriksson et al., 1997; Tallkvist et al, 1998). It was found that intranasal instillation of 63 Ni 2+ (as 63NiCl2) in rats resulted in an uptake of the metal in the olfactory epithelium and a migration along the primary olfactory neurons to the glomeruli of the olfactory bulb. The metal was then seen to pass to the interior of the bulb and further to the olfactory peduncle and tubercle and to the rostral parts of the prepyriform, frontal and cingulate cortices. The experiments in pike, in which 63NiCl2 was applied in the olfactory chambers, showed a transport of the metal in the olfactory neurons at a rate of about 0.13 mm/hour at 10°C. This is about 20 times slower than the transport-rate for cadmium and manganese. Cellular fractionations and gel chromatography showed that nickel in the olfactory system of pikes and rats was present both in the cytosol and in association with various particular cell constituents (Tallkvist et al., 1998). We assume that nickel undergoes slow axonal transport bound to these constituents. There is also a possibility that the movement of nickel in the olfactory system might be explained by diffusion. However, the observation that the transport of nickel in the olfactory nerve of the pike occurs at a constant rate (Tallkvist et al., 1998) does not support this assumption. In addition, it seems less likely that diffusion of the metal would occur over such long parts of the olfactory nerve as was observed in the pike. Exposure to soluble nickel compounds in electrorefining factories has been shown to impair the olfactory sense (Tatarskaya, 1960; Kucharin, 1970). Abnormal olfactory function has also been observed in rats exposed experimentally to this metal (Benson et al., 1987, 1988; Evans et al., 1995).

6.4. Aluminum Rabbits that received nasal implants of gelfoam pads impregnated with soluble aluminum salts developed granulomas in the olfactory bulb and

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cerebral cortex (Perl and Good, 1987). Laser microprobe mass analysis (LAMMA) revealed presence of aluminum in cytoplasmic granules of macrophages in the granulomas, whereas adjacent neurons had no evidence of aluminum. Zatta et al. (1993) showed accumulation of aluminum in the olfactory bulb, hippocampus, cerebellum and pons-medulla in rats exposed via inhalation to aluminum acetylacetonate. Divine et al. (1999) showed accumulation of aluminum in the olfactory bulb of rats exposed via inhalation to aluminum chlorohydrate. It is possible that the disposition of aluminum in the olfactory system may vary depending on the aluminum compound in question. Additional studies on the uptake and distribution of aluminum in the olfactory system would be of interest. Aluminum is a known neurotoxic metal, which is present in high concentration in dust particles. There are indications that aluminum is present in senile plaques and in the neurofibrillary tangles of Alzheimer's disease (Itzhaki, 1994). It has been proposed that aluminum may be taken up the brain via the olfactory route and play a role in the etiology of Alzheimer's disease (Roberts, 1986; Perl and Good, 1991; Zatta, 2000).

6.5. Zinc Zinc (as 65ZnCl2) applied on the olfactory mucosa of rats or pikes resulted in an uptake of the metal in the olfactory epithelium and a transport along the primary olfactory neurons to their terminations in the olfactory bulbs (Persson et al., 1998; and unpublished observations). Low levels of 65 Zn 2+ passed these terminals and continued into the interior of the bulbs. In rats 65 Zn 2+ was also detected in the anterior parts of the olfactory cortex. Studies by Takeda et al. (1997) indicate that zinc injected into the olfactory bulb or the amygdaloid nuclei undergoes anterograde axonal transport in the secondary olfactory neurons. In another study, Takeda etal. (1998) found that injection of zinc into the striatum resulted in a transport of the metal in striato-nigral 7-aminobutyric acid (GAB A)-ergic neurons, whereas injection of zinc in the substantia nigra resulted in transport of the metal in nigro-striatal dopaminergic neurons. Zinc homeostasis in the brain is very complex. Zinc is an essential element and a component of a number of metalloenzymes. In the brain about 90% of the total zinc is bound in zinc proteins (Frederickson, 1989). A separate pool of zinc is present as free or loosely bound ions in some neurons of the brain. These neurons ("zinc-containing neurons") contain

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a pool of histochemically stainable zinc localized in presynaptic vesicles of nerve terminals and are believed to be a subset of glutamatergic neurons (Jo et al., 2000; Frederickson and Bush, 2001). The olfactory bulb contains high concentrations of zinc. Most of this zinc is found in the glomerular layer and the granular cell layer of the olfactory bulb. It is believed that the terminals of the primary olfactory neurons contain vesicles in which glutamate is present together with zinc and that these two constituents are co-released (Joet al., 2000). Palmiter et al. (1996) have identified a zinc transporter (ZnT-3), which is essential for the transport of cytosolic zinc into the glutamate containing synaptic vesicles. ZnT-3 has been reported to be present in presynaptic vesicles of olfactory neurons together with zinc (Jo et al., 2000). Our results showed that there was an accumulation of zinc in the terminals of the primary olfactory neurons in the glomeruli. It is possible that following axonal transport in these neurons the zinc may be taken up in presynaptic vesicles, resulting in cellular sequestration. The zinc may then be released into the synapses in the glomeruli as a response of action potentials. Metallothionein is considered to be involved in intracellular zinc homeostasis. In the brain metallothionein-I and metallothionein-II are induced by heavy metals, such as zinc and copper (Ebadi et al., 1995; Hidalgo and Carrasco, 1998). Metallothionein-III is highly expressed in zinc containing glutamatergic neurons (Masters et al., 1994). This metalloprotein may be an important regulator of neuromodulatory zinc in the brain, including the olfactory system. Occupational exposure to zinc-containing dusts or fumes occurs in some industrial environments, such as in galvanization plants and at welding of galvanized iron or steel (Walsh et al., 1994; Barceloux, 1999). This may result in accumulation of the metal in the nasal pathways and an uptake of the metal in the brain via the olfactory route. It is not known whether this may cause neurotoxicity. However, it can be noted that zinc dysregulation leading to increased extracellular Zn 2+ , has been implied to play a role in Alzheimer's disease by inducing precipitation of amyloid beta (Frederickson and Bush, 2001).

6.6. Cobalt Studies in rats in which 57CoCl2 was applied intranasally showed a similar picture as observed for 65ZnCl2 (Persson et al. 1998; and unpublished observations). Thus, 57 Co 2+ was transported along the olfactory neurons

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and accumulated in the olfactory nerve layer and the neuronal terminals in the glomerular layer of the bulb. In addition, low levels of cobalt were seen in the interior of the bulbs and the anterior parts of the olfactory cortex indicating that cobalt is able to leave the terminals of the primary olfactory neurons. Occupational exposure to cobalt occurs mainly by inhalation and is considerable and widespread. Cobalt is a technically important metal used mainly as a constituent of many alloys and as a binder in hard-metal industry. Its salts are used e.g. in electroplating and electrochemical industries and as colorating agents in ceramics (Jensen and Tuchsen, 1990). It is not known whether uptake of cobalt into the brain via the olfactory route may cause neurotoxicity. However, it has been reported that workers exposed to cobalt-containing dust may suffer from memory deficits (Jordan et al., 1997). The mechanism for this condition is not known, but one possibility is that cobalt taken up into the brain via olfactory pathways may play a role in the etiology.

6.7. Mercury Studies in pike, in which inorganic mercury (as 203HgCl2) was applied in the olfactory chambers, showed a transport along the olfactory neurons to the anterior part of the olfactory bulb (Borg-Neczak and Tjalve, 1996). In rats intranasal instillation of the metal resulted in an uptake of the metal along the olfactory axons to the olfactory nerve-layer and the glomerular layer of the bulb (Henriksson and Tjalve, 1998). Low levels of the metal were also discernible in the external plexiform layer, indicating that a small part of the 203jjg2+ ] e a v e s m e terminal arborizations of the axons in the glomeruli. There is evidence that mercury may undergo axonal transport in the primary olfactory neurons following uptake from the systemic circulation. Thus, autoradiography in rats injected i.p. with inorganic mercury (203HgCl2) showed a labeling of the glomerular layer of the olfactory bulbs which exceeds the labeling of other brain areas (Henriksson and Tjalve, 1998) (Fig. 4). There was also an uptake of the 203 Hg 2+ in the olfactory mucosa, and it was proposed that the metal may be taken up from the blood into the primary olfactory neurons and then move along the axons to their terminations in the bulbs. The capillary bed, which underlies the olfactory neuroepithelium, is composed of conventional fenestrated capillaries.

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Fig. 4. (A) Detail of an autoradiogram of the head (horizontal section), showing the nasal cavity, the olfactory bulbs and the anterior parts of the olfactory cortex, of a rat killed three weeks after i.p. injection of 203 Hg 2+ (6.7 |xg). (B) The corresponding tissue-section. Legends: e, olfactory epithelium; gl, glomerular layer of the olfactory bulbs. White areas denote high levels of 2
Blood-bome mercury may therefore diffuse into the extracellular space and then have direct access to the unmyelinated olfactory neurons. Alternatively, mercury may be taken up from the circulation into the cellbodies of the olfactory receptor cells.

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For most people mercury released from dental amalgam fillings is the main source of exposure to the inorganic mercury (Halbach, 1994). Mercury is released from the amalgam surface mainly as mercury vapor (Hg°). The Hg° will be oxidized to Hg 2+ by the H202-catalase pathway, which is ubiquitously present in various tissues (Sichak et al., 1986; Clarkson, 1989). The fate of the Hg° before it becomes oxidized to Hg 2+ is not known in detail, but it is assumed that the Hg° is distributed throughout the oral and the nasal cavities and also via respiration will be transferred to the lungs, which are considered to be the major sites of uptake of the metal. It is considered that the Hg° partly is oxidized to Hg 2+ in the lungs and the red blood cells, but the oxidation is considered to be slow enough to allow the lipophilic Hg° to reach the brain and diffuse through the blood-brain barrier into the nervous tissues. In the brain the Hg° can be oxidized to Hg 2+ which binds to cellular nucleophiles (Sichak et al., 1986; Clarkson, 1989). In the blood the Hg 2+ will become distributed in roughly equal concentrations between the plasma and the red blood cells (Ekstrand et al., 1998). Little attention has been paid so far to the fate of the Hg° in the nasal tissues. However, there are indications that the Hg° is oxidized to Hg 2+ in the nasal mucosa (Khayat and Dencker, 1984). It has been proposed that Hg° released from amalgam fillings will reach the primary olfactory neurons in the olfactory epithelium and become oxidized to Hg 2+ in these cells (Henriksson and Tjalve, 1998). The intracellular Hg 2+ may then be transported along the axons to the olfactory bulbs. Occupational exposure to Hg° by inhalation occurs in gold mining and is a widespread problem in several countries of South America and Africa and also in Indonesia and the Philippines (Asano et al., 2000). The Hg° is used for amalgamation of gold and mercury is released by evaporation at reburning sites. The tissue disposition of the occupationally inhaled Hg° can be assumed to be similar as for the Hg° released from dental amalgam fillings, as described above. In humans exposure of the nasal olfactory epithelium to Hg°, released from amalgam fillings or related to occupational inhalation, as well as an uptake of Hg 2+ in the olfactory neurons from the blood, may result in considerable concentrations of the metal in the olfactory bulbs. Maas and coworkers (1996) examined the concentration of mercury in the olfactory bulbs of 55 deceased persons and found significantly higher levels of the metal in

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the bulbs than in the occipital lobe cortex (geom. mean 17.4 |Jig/kg w.wt. compared to 9.2 |xg/kg w.wt). However, the levels of mercury in the olfactory bulbs could not be correlated to the number of amalgam fillings. Mercury is known to alter olfaction and olfaction-related behavior in fish (Hara et al., 1976; Baatrup et al., 1990). To our knowledge the potential toxicity of mercury to the olfactory sense has not been examined in mammals. In view of the observation that Hg 2+ appears to be taken up in the olfactory neurons from the circulation and with regard to the possible oxidation of Hg° in these cells, this is an issue which needs to be explored. The ability of mercury to be taken up from the blood into the primary olfactory neurons may be a part of a more general capacity of mercury to be taken up in neurons with access to the systemic circulation and then reach the CNS via transport in these neurons. In the study in rats given 203 HgCl2 i.p. (Henriksson and Tjalve, 1998) we observed a labeling of the superior and inferior colliculi which exceeded the background labeling of the brain. We also found a labeling of the optic nerves. The superior colliculi are terminals of the axons of the retinal ganglion cells. It is possible that mercury is taken up in these cells from the circulation and then is transported in the optic nerves to the superior colliculi. The inferior colliculi are terminals of the central auditory tract. Conceivably mercury, in a similar manner as in the optic nerves, is taken up from the circulation into ganglion cells in the inner ear and then is transported to the inferior colliculi. Studies by Arvidson (1992) in rats have indicated that mercury is taken up in motoneurons from the circulation. Thus, following parenteral injection of mercuric chloride mercury deposits were observed in motoneurons of the brainstem and of the anterior horns of the spinal cord. Unilateral ligation of the hypoglossal nerve prior to the injection of the mercuric chloride prevented accumulation of mercury deposits in the ipsilateral hypoglossal nucleus. It was assumed that the mercury via the circulation reaches the neuromuscular junctions (which are situated outside the blood-brain barrier) and that the metal then, following uptake into the nerve terminals, is transported to the motoneurons in the brainstem and the spinal cord (Arvidson, 1992). Studies at our department in trout exposed to inorganic mercury via aquarial water indicate that the metal reaches the CNS via axonal transport in various water exposed sensory neurons (Rouleau et al., 1999). This applies to the olfactory neurons and in addition to neurons innervating mechanoreceptors of the lateral line organs,

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neurons innervating cutaneous sensory cells, such as tactile, pressure and nociceptive sensory cells, and neurons innervating receptor cells of taste buds in the oral mucosa. Transport along these nerves allows waterborne mercury to circumvent the blood-brain barrier and reach the central nervous system.

7. ABUNDANCE OF METALS IN THE OLFACTORY BULB In addition to mercury, there is a relative abundance of some other metals in the olfactory bulb, compared to other regions of the brain. Thus, it has been shown that the concentration of cadmium in the olfactory bulb is much higher than in the remaining brain in untreated rats (Suzuki and Arito, 1975; Clark et al., 1985). Bonilla et al. (1982) reported higher levels of manganese in the olfactory bulbs than in other brain regions in human brains sampled at autopsy. In untreated control rats Fechter et al. (2002) reported higher manganese levels in the olfactory bulbs than in other brain regions. In two strains of untreated mice Ono and Cherian (1999) found higher levels of zinc and copper in the olfactory bulbs than in several other parts of the brain. Domingo et al. (1996) reported much higher levels of aluminum in the olfactory bulbs than in other areas of the brain in untreated control rats. It is possible that the high levels of metals in the olfactory bulbs are related to metal transport in the primary olfactory neurons following uptake from the circulation. The metals may reach the unmyelinated olfactory neurons by diffusion through the walls of the fenestrated capillaries underlying the olfactory epithelium. Thus, this may be a route which can be used by several metals to circumvent the blood-brain barrier and gain access to the CNS.

8. METAL TRANSPORT INTO THE CEREBROSPINAL FLUID VIA THE OLFACTORY PATHWAY It has been shown that some drugs and other xenobiotics applied on the olfactory mucosa will penetrate into the cerebrospinal fluid (Jackson et al., 1979; Mathison et al., 1998; Ilium, 2000). Two possible routes for this passage have been considered: One is an intraaxonal transport along the olfactory neurons to the olfactory bulb followed by a diffusion from the bulb into the cerebrospinal fluid (designated "the

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olfactory nerve pathway"); the other is a movement of molecules along the perineural space that is continuous with the subarachnoid space (designated "the olfactory epithelial pathway"). The latter pathway could explain the very rapid movement of molecules into the cerebrospinal fluid reported by some authors. The transfer of metals into the cerebrospinal fluid via the olfactory pathway is less well documented. However this phenomenon has been observed for manganese. Thus, studies in pike showed that manganese which moved along the olfactory nerve to the olfactory bulb penetrated into the cerebrospinal fluid of the surrounding meningeal sac (Tjalve et al., 1995). It was proposed that manganese diffuses into this fluid from the synaptic nerve clefts through the extracellular space of the bulb. Manganese moves at a constant rate by fast axonal transport in the long olfactory neurons to the olfactory bulbs, in which high levels of the metal are obtained. It is unlikely that manganese will reach the meningeal fluid via diffusion in the perineural space. As described above other metals such as mercury, nickel, zinc and cobalt, have been shown to accumulate in the terminal parts of the olfactory nerves in the glomeruli of the bulb and low levels will also leave the synapses in the glomeruli. It is possible that the metals in this way may diffuse in the extracellular space of the bulbs and then reach the cerebrospinal fluid, but this possibility has not been tested experimentally. Czerniawska (1970) injected colloidal gold (198Au) under the mucous membrane of the nasal olfactory region in rabbits and found a penetration of the metal into the cerebrospinal fluid surrounding the olfactory bulb. The author concluded that there is a connection between the mucous membrane of the olfactory region of the nose and the cerebrospinal fluid. However, it is not known whether the gold will be transferred via "the olfactory nerve pathway" or "the olfactory epithelial pathway" or via both these pathways. It should be noted that in the olfactory mucosa the receptor cells and the sustentacular cells form tight junctions with one another (Farbman, 1992). Therefore transfer of metals and hydrophilic or large-molecular-weight organic xenobiotics from the surface of the olfactory epithelium to the extracellular compartment of the olfactory mucosa probably occurs to a very limited extend. The transfer of such materials from the surface of the olfactory mucosa to the cerebrospinal fluid can therefore be assumed to occur mainly via uptake in the primary olfactory neurons and a transfer via "the olfactory nerve pathway".

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9. CONCLUSIONS In this article we have reviewed literature demonstrating that the olfactory epithelium can serve as a direct pathway to the CNS, bypassing the systemic circulation and therefore also the blood-brain barrier. An important question that remains unresolved is the relative importance of the olfactory transport route to the brain in humans. In this connection models which allow extrapolations between species is of great importance (Kimbell et al., 2001). Even if the significance of the entry of toxic metals into the CNS via the olfactory pathway in humans is unknown the role of this route must be taken into account in future risk assessments of occupational metal exposure via inhalation. It is known that severe involvement of the olfactory system occurs early and frequently in patients with neurodegenerative diseases such as Alzheimer's and Parkinson's disease (Herzog and Kemper, 1980; Pearson et al., 1985; Doty, 1991; Doty et al, 1995). It is not yet known whether the preferential olfactory system pathology represents the initiation of the diseases or whether it reflects a part of the general neurodegenerative processes. However, it has been proposed that the entry of toxic metals or other neurotoxins into the CNS via the olfactory route may in part contribute to the etiology of these disorders (Roberts, 1986; Ferrera-Moyano and Barragan, 1989; Perl and Good, 1991; Zatta, 2000).

ACKNOWLEDGMENTS Our studies have been supported by the Swedish Council for Working Life and Social Research and the Foundation for Strategic Environmental Research (MISTRA).

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Rouleau C, Brog-Neczak K, Gottofrey J, Tjalve H. Accumulation of waterbome mercury(II) in specific areas offish brain. Environ Sci Technol 1999; 33:3384-3389. Scott JW. The olfactory bulb and central pathways. Experientia 1986; 42:223-232. Shipley MT. Transport of molecules from nose to brain: Transneuronal anterograde and retrograde labeling in the rat olfactory system by wheat germ agglutinin-horseradish peroxidase applied to the nasal epithelium. Brain Res Bull 1985; 15:129-142. Sichak SP, Mavis RD, Finkelstein JN, Clarkson TW. An examination of the oxidation of mercury vapor by rat brain homogenate. J Biochem Toxicol 1986; 1:53-68. Sloot WN, Gramsbergen JBR Axonal transport of manganese and its relevance to selective neurotoxicity in rat basal ganglia. Brain Res 1994; 657:124-132. Spranger M, Schwab S, Desiderato S, Bonmann E, Krieger D, Fandrey J. Manganese augments nitric oxide synthesis in murine astrocytes: A new pathogenetic mechanism in manganism? Exp Neurol 1998; 149:277-283. Stewart WB. Labeling of olfactory bulb glomeruli following horseradish peroxidase lavage of the nasal cavity. Brain Res 1985; 347:200-203. Stromberg PC, Ferrante JG, Carter S. Pathology of lethal and sublethal exposure of fathead minnows, Pimephales promelas, to cadmium: A model for aquatic toxicity assessment. J Toxicol Environ Health 1983; 11:247-259. Sun TJ, Miller ML, Hastings L. Effects of inhalation of cadmium on the rat olfactory system: Behavior and morphology. Neurotoxical Teratol 1996; 18:89-98. Suzuki Y, Arito H. Cadmium content of the olfactory bulb of Cd-administered rats for a long term. Ind Health 1975; 13:77-79. Takeda A, Ohnuma M, Sawashita J, Okada S. Zinc transport in the rat olfactory system. NeurosciLett 1997; 225:69-71. Takeda A, Kodama Y. Ohnuma M, Okada S. Zinc transport from the striatum and substantia nigra. Brain Res Bull 1998; 47:103-106. Tallkvist J, Henriksson J, d'Argy R, Tjalve H. Transport and subcellular distribution of nickel in the olfactory system of pikes and rats. Toxicol Sci 1998; 43:196-203. Tallkvist J, Persson E, Henriksson J, Tjalve H. Cadmium-metallothionein interactions in the olfactory pathways of rats and pikes. Toxicol Sci 2002; 67:108-113. Tatarskaya AA. Occupational diseases of upper respiratory tract in persons employed in electrolytic nickel refining departments. Gig Trud ProfZabol 1960; 6:35-38. Thorne RG, Emory CR, Ala TA, Frey WH II. Quantitative analysis of the olfactory pathway for drug delivery to the brain. Brain Res 1995; 692:278-282. Tjalve H, Gottofrey J, Bjorklund I. Tissue disposition of 109Cd2+ in the brown trout (Salmo trutta) studied by autoradiography and impulse counting. Toxicol Environ Chem 1986; 12:31-^5. Tjalve H, Mejare C, Borg-Neczak K. Uptake and transport of manganese in primary and secondary olfactory neurones in pike. Pharmacol Toxicol 1995; 77:23-31. Tjalve H, Henriksson J, Tallkvist J, Larsson BS, Lindquist NG. Uptake of manganese and cadmium from the nasal mucosa into the central nervous system via olfactory pathways in rats. Phamacol Toxicol 1996; 79:347-356. Tjalve H, Henriksson. Uptake of metals in the brain via olfactory pathways. Neuwtoxicology 1999; 20:181-196.

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Vallee RB, Bloom GS. Mechanisms of fast and slow axonal transport. Annu Rev Neurosci 1991;14:59-92. Vitarella D, Wong BA, Moss OR, Dorman DC. Pharmacokinetics of inhaled manganese phosphate in male Sparague-Dawley rats following subacute (14 d) exposure. Toxicol Appl Pharmacol 2000; 163:279-285. Walsh CT, Sandstead HH, Prasad AS, Newberne PM, Fraker PJ. Zinc: Health effects and research priorities for the 1990s. Environ Health Perspect 1994; 102 (suppl):5^16. Zatta P, Favarato M, Nicolini M. Deposition of aluminum in the brain tissues of rats exposed to inhalation of aluminum acetylacetonate. Neuro Report 1993; 4:1119-1122. Zatta P. Aluminum neurotoxicity: implications in neurodegenerative diseases. In: Centeno JA, Collery P, Vernet G, Finkelman RB, Gibb H, Etienne JC, editors. Metal Ions in Biology and Medicine, Vol. 6. Paris: John Libbey Eurotext, 2000: 443^146.

CHAPTER 4

Aluminum in Neurological Disorders and Systemic Chelation Therapy Theo P Kruck, Walter J Lukiw

ABSTRACT Human neurological disorders that display complex pathogeneses involve multiple etiologic factors. Aging remains the most significant risk factor for the development of any neurological disorder and this in itself suggests collective and cumulative effects over decades of life. Alzheimer's disease (AD), a common, progressive degeneration of the human brain characterized by memory loss and the deterioration of higher cognitive function, is the leading cause of acquired neurological dysfunction in our aging population. A highly intricate maze of factors appears to contribute to the development and progression of AD. Mutations and/or susceptibility polymorphisms in gene coding or control regions, traumatic head injury, previous viral infection of the brain, cerebral vascular disease, ischemia and hypoxia are factors that appear to contribute substantially to the development and/or progression of AD. In addition, numerous epidemiological studies suggest that relatively high concentrations of neurotoxic metal ions, such as copper, iron, zinc, and in particular aluminum, are important environmental factors contributing to the development and/or progression of AD neuropathology. Systemic chelation therapies dealing with the removal of these environmental metallic toxins from living brain tissues therefore represent one reasonable and attractive strategy to alleviate the development and/or progression of neurological disorders which are linked to metal ion intoxication. The following chapter will briefly review the postulated role of aluminum in neurological disease, focusing on AD, and the potential application of systemic aluminum chelators such as desferrioxamine (DFO) and Feralex-G in the treatment and amelioration of this devastating disorder of the human mind. 99

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Kruck TP & Lukiw WJ Keywords: Aluminum; Alzheimer's disease; amyloid-(3; chelation therapy; desferoxamine; Feralex-G; molecular shuttle chelation.

1. INTRODUCTION Human neurological disorders display complex pathogeneses involving multiple etiologic factors. Alzheimer's disease (AD), a common, progressive degeneration of the human brain characterized by memory loss and the deterioration of higher cognitive function, is the leading cause of acquired neurological dysfunction in our aging population. Aging remains the most significant risk factor for the development of neurological dysfunction and this, in itself, suggests collective and cumulative effects over decades of life. A highly intricate maze of factors appears to contribute to the development and progression of neurological dysfunction and neural degeneration in AD. Mutations and/or susceptibility polymorphisms in gene coding or control regions, traumatic head injury, cerebrovascular disease, ischemia, and hypoxia are factors that may contribute substantially to the development and/or progression of AD (Terry and Katzman, 1983; Smith et al., 2000; Bazan and Lukiw, 2002). In addition, numerous epidemiological studies suggest that di- and tri-valent neurotoxic metal ions, such as copper, iron, zinc, and in particular aluminum, are important environmental factors contributing to the development and/or progression of AD neuropathology (Crapper McLachlan et al, 1991; Hollosi et al, 1994; Cuajungco et al., 2000; Cherny et al., 2000; Exley and Korchazhkina, 2001; Lukiw, 2001; Carpenter, 2001). Systemic chelation therapies dealing with the removal of these environmental toxins from living brain tissues, therefore, represent a reasonable strategy to alleviate the development and/or progression of neurological disorders such as AD. This chapter will briefly review the postulated role of aluminum in neurological disease, focusing on AD, and the potential application of systemic aluminum chelators, such as desferoxamine (DFO) and Feralex-G, in the treatment and/or amelioration of this devastating disorder of the human brain.

2. METAL NEUROTOXICITY AND ALZHEIMER'S DISEASE Unique to the more evolutionarily recent structures of the human neocortex, AD represents a progressive degeneration of the brain's synaptic circuitry culminating in cognitive dysfunction. Classically, two argyrophilic

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brain lesions, neurofibrillary tangles (NFTs) and amyloid-beta (A3) peptides have been correlated with AD development, progression, and severity (Terry and Katzman, 1983; Uylings and de Brabander, 2002). NFTs are composed of hyperphosphorylated bundles of microtubule-associated tau protein; A(3 peptides are derived from nonhomeostatic beta- and gammaO-7) secretase cleavage of the membrane spanning gylcoprotein amyloid-beta protein precursor (ApJPP). Importantly, the integral membrane proteins presenilin-1 and -2 (PS1 and PS2) appear to have a determining role in the generation of A3 peptides from A(3PP. Neocortical densities and the degree of aggregation of NFTs, in particular A(3 peptides, are correlated with neurotoxic mechanisms that culminate in brain cell dysfunction and death. Normal neural cell functions are also directly compromised via A3 peptide-mediated microglial activation. Release from activated microglia of toxic cytokines, such as interleukin-l(3, and microglia-associated elevations of reactive oxygen species (ROS), such as OH-, 0 2 -, and H 2 0 2 , and other oxidative disturbances appear to be central to the development and propagation of AD etiopathology (Joshi, 1991; Vyas and Duffy, 1995; Lukiw and McLachlan, 1995; Smith et al, 2000; Lukiw and Bazan, 2000; Lukiw, 2001; McGeer and McGeer, 2001). In particular, highly condensed A3 peptides, which form the senile plaque, are thought to lie at the core of an accelerated brain-specific inflammatory response in the AD brain (Nilsson et al., 1998; Kurihara and Pardridge, 2000; Lukiw and Bazan, 2000; Kontush, 2001; McGeer and McGeer, 2001). NFT formation and, to an even greater extent, amyloidosis, A3 processing, crosslinking, aggregation, and stabilization have been associated with elevated brain copper (Atwood et al., 1998; Multhaup et al., 1998; Miura et al., 2000; Zou and Sugimoto, 2000; Regland et al., 2001; Kontush, 2001), iron (Bodovitz et al., 1995; Cuajungco et al., 2000; Rottkamp et al., 2001), zinc (Huang et al., 1997, 2000; Miura et al., 2000; Yang et al, 2000; Regland et al., 2001), and in particular aluminum (Crapper et al., 1980; Lukiw et al., 1987, 1989, 1989a, 1992; Vyas and Duffy, 1995; Murayama et al., 1999; Lukiw, 2001; Hamai et al., 2001; Szutowicz, 2001; Carpenter, 2001; Strong, 2001; Perl et al., 2001; Rondeau and Commenges, 2001; Savory et al., 2001; Kiss and Hollosi, 2001; Shin, 2001; Exley and Korchazhkina, 2001; Campbell et al., 2002; Pratico et al., 2002). Notably, the neurotoxicity of aggregated NFTs and A3 peptides may be modulated by the nonphysiological actions of these metals through elevations in the intercellular concentrations of ROS which, in turn, drive pathological

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responses at the plasma and nuclear membrane, as well as at the extracellular, intracellular, and genetic levels. Apart from the role of PS1 and PS2 in facilitating the processing of AfSPP into A|3, their roles in moderating metal-mediated neurotoxicity in neurodegenerative disease are not known.

3. ALUMINUM AND ALZHEIMER'S DISEASE Aluminum has been repeatedly associated with the etiopathology of several neurological disorders, including AD (reviewed by Crapper et al., 1980; Kruck and McLachlan, 1989; Joshi, 1991; Lukiw, 2001; Hamai et al., 2001; Carpenter, 2001; Szutowicz, 2001; Rondeau and Commenges, 2001; Pratico et al., 2002; Kruck et al., 2002, 2002a). Once aluminum has bypassed important epithelial, gastrointestinal, physiological, and blood-brain barriers, this environmentally abundant trivalent neurotoxin has been shown to adversely affect the physiological, intracellular, neurochemical, and genetic integrity of mammalian nervous tissue. Compartmentalization of aluminum or localized accumulation of a critical mass of metabolic errors resulting from this neurotoxin, rather than a single intoxicating event, superimposed over a time course of aging (typically for many decades) may be essential to collectively precipitate neurological diseases, such as AD (Joshi, 1991; Lukiw and McLachlan, 1995; Lukiw, 2001; Kruck et al., 2002). Apart from its strong effect on the processing, crosslinking, aggregation, and stabilization of NFT and A(3, aluminum may have a particular and focused attraction for the genetic material of brain cells; in fact, one recent review summarizes the deleterious effects of aluminum on the structure and function of neural genetic material from 27 independent international laboratories using 45 different bioanalytical and biophysical methods (Lukiw, 2001).

4. SYSTEMIC CHELATION THERAPY FOR PHYSIOLOGICALLY BOUND NEUROTOXIC METALS Recent clinical trials in patients showing neurological symptoms, animal and neurochemical models involving chelation therapy for aluminum, copper, iron, and zinc overload cases using l-n-butyl-2-methyl-3hydroxypyridin-4-one (HNBP; Bondy et al., 1998), the alpha-ketohydroxypyridine chelator l,2-dimethyl-3-hydroxypyrid-4-one (HDDP; L1JNN/

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OH O

O

OH O

O

OH O

NH2(CH2)5N-C(CH2)2CNH(CH2)5N-C(CH2)2CNH(CH2)5N-C-CH3 Fig. 1. Desferoxamine.

BAN: deferiprone; Kontoghiorghes, 1995; Blanusa et al., 2000; Yokel, 2002), penicillamine (McDonald and Lake, 1995; Cherny et al., 2000), clioquinol (Regland et al., 2001; Gouras and Beal, 2001), trientine, bathophenanthroline and bathocuproine (Cherny et al., 2000), various pyridyl donors (Caravan et al., 1997), DFO (Fig. 1; Crapper McLachlan et al., 1991; Ye and Connor, 1999; Cherny et al., 2000), and [1,2-dimethyl3-hydroxypyrid-4-one + DFO together] (Blanusa et al., 2000) have demonstrated varying degrees of pharmacological success (Gouras and Beal, 2001; Yokel, 2002). Those published studies involving copper, iron, and zinc chelation will not be considered further here.

5. POTENTIAL OF SYSTEMIC ALUMINUM CHELATION THERAPY In one 256-day distribution and retention study in rats receiving intravenous infusions of aluminum-transferrin or aluminum-citrate containing 26-aluminum at concentrations which approximated physiological conditions, approximately 0.005% of the 26-aluminum entered the brain and repeated DFO treatments were shown to accelerate the reduction of brain aluminum (Yokel et al., 2001). In one 24-month clinical trial study wherein the aluminum chelating agent DFO was administered to 25 patients with advanced AD and compared to a 23-member control group, a statistically significant difference in the rate of mental deterioration between the DFO-treated and untreated groups was found. Based upon assessment of activities of daily living, and employing a battery of standardized clinical tests and videotaped measures of activities to assess the effects of DFO, the average rate of mental decline was half as rapid for the DFO-treated group as for the control group that received oral lecithin as placebo or no drug treatment (Crapper McLachlan et al., 1991; Kruck et al., 2002). In another study, post-mortem brain aluminum measurements also showed that, in 21 neocortical regions of three controls and three

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DFO-treated, age-matched AD patients, whole tissue aluminum content was reduced from mean values of 4.09 (xg/gm to 2.69 |Jig/gm dry weight of cerebral gray matter, respectively (Kruck et al., 2002, 2002a). In addition, analysis of small cortical blood vessels excised from areas affected by AD pathology showed a significant reduction in aluminum content between the control and DFO-treated group, from a mean value of 10.5 |JLg/gm to 5 |xg/gm, respectively. These preliminary data, therefore, suggest that DFO treatment in AD reduces total neocortical aluminum concentrations to near control values. This reduction appears to be associated with a slowing down of the clinical progression of AD and an improvement in the quality of life for the AD patient (Crapper McLachlan et al., 1991; McLachlan et al., 1993; Kruck et al, 2002a). These preliminary studies indicate that aluminum chelation therapy may be a rational route for the treatment of AD.

6. EXPERIMENTAL REMOVAL OF ALUMINUM FROM NUCLEI TREATED WITH ALUMINUM LACTATE: CONCEPT OF MOLECULAR SHUTTLE CHELATION The hypothesis that known aluminum chelators in tandem combination might be more efficient than singly administered chelators in removing aluminum from physiological systems has been tested in human brain nuclei. Such chelation of aluminum for potential neurobiological applications has been investigated using a newly developed technique, which we have called "molecular shuttle chelation" (Kruck et al., 1990, 2002, 2002a). In this in vitro technique, chelating agents with a high affinity for aluminum were employed in tandem combination with DFO for the removal of aluminum from normal human neocortical nuclei that were preincubated with aluminum lactate (Table 1). These experiments were carried out to examine the reversibility of aluminum binding to human neocortical nuclei in vitro, and to more closely examine the high affinity of the aluminum cation for DNA containing structures of human neocortical nuclei (Lukiw et al., 1989, 1989a; Hamai et al., 2001). Briefly, aluminum, in the form of a freshly prepared aluminum lactate solution, was added to a suspension of nuclei and 1,895 ng of aluminum cation was added for each 150 |xg of Deoxyribonucleic acid (DNA), a concentration which approximates nuclear heterochromatin or Brodman

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Table 1. Molecular shuttle chelation.

Chelation Method

Al

Li

CIT

ASC

3

— 3 30 3 30 -

— 3 30 3 30

30 3 30 -

DFO

A

B

1

99

1 10 10 1 1 10 10 1 1 10 10

99 86 89 87 88 82 86 79 80 87 83

— 84 85 79 80 75 75 85 81

Al = aluminum, ASC = ascorbate, CIT = citrate, DFO = desferrioxamine, Li = lithium.

A22 dinucleosome aluminum levels in the AD brain (Lukiw et al., 1987, 1992; Lukiw and McLachlan, 1995; Lukiw, 2001). Several chelators were employed, either by themselves or in tandem combination, in an attempt to remove aluminum which had bound to the nuclear compartment. In each case, DFO was used as the ultimate aluminum acceptor. After preincubation with aluminum, two methods were used to treat nuclei with chelating agents: (A) Nuclei with aluminum were incubated for 18 hours at zero to 4°C followed by incubation with prechelators for 18 hours at zero to 4 °C. This was followed by application of DFO for three hours at zero to 4°C followed by centrifugation at 16,000 G av for 30 minutes, and by electrothermal atomic absorption spectroscopic (EAAS) analysis of high-speed supernatants and pellets. (B) Nuclei with aluminum were incubated for 18 hours at zero to 4°C followed by incubation with prechelators and DFO for 18 hours at zero to 4°C. This was followed by centrifugation at 16,000 G av for 30 minutes followed by EAAS analysis of high-speed supernatants and pellets.

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A summary of some preliminary experiments using the above methods is shown in Table 1 (Kruck et al., 2002). The numbers under columns A or B represent the mean of three to five separate experiments. Note that the vast majority of the added aluminum (75% to 99%) remains bound to the nuclear compartment and is inaccessible to the chelating agents used in this in vitro test system. This emphasizes the concept that once aluminum has accessed brain-specific compartments containing electron-rich structures, such as the genetic material, it tends to remain there and is highly refractory to removal by systemic chelation (Lukiw et al., 1992; Lukiw, 2001; Yokel et al., 2001). Also, note that method B (prechelators and DFO incubated together) had a slightly higher chelation efficiency than was observed with method A. It is also apparent in this in vitro system that combinations of ascorbate (ASC) and DFO combinations using method B showed the most promise for therapeutic removal of aluminum bound to nuclear ligands. Using molar ratios of ASC to DFO of three to one, for example, 25% of nuclear bound aluminum could be removed in vitro using molecular shuttle chelation. The enhanced chelation efficiency using the combination of ASC with DFO is especially interesting, since co-administration of ASC with DFO greatly enhances the excretion of iron complexes in vivo in patients with chronic iron storage disease (Kruck et al., 2002, unpublished observations). In the latter case, ASC may reduce biologically bound iron (Fe3+) to Fe 2+ , thereby changing the affinity for its biological ligands and allowing its dissociation followed by DFO chelation. However, since aluminum cannot be reduced by physiological oxido-reductive processes, the enhancement of aluminum removal in the simultaneous presence of ASC with DFO may follow an alternate mechanism and is probably due to the greater accessibility to nuclear bound aluminum by ASC (MW= 176.12) when compared to DFO (MW = 560.71). It is suggested that the nuclear bound aluminum is initially chelated by ASC at its binding site and subsequently transported, or "shuttled" to an extra nuclear matrix compartment where DFO can competitively remove the aluminum, thereby liberating free ASC that can re-enter the nuclear matrix and start the cycle again. Hence, the concept of molecular shuttle chelation. Even though nuclear bound aluminum is particularly refractory to removal, the application of this novel chelation technique might be useful in the treatment of human diseases, such as AD, where aluminum

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accumulates on DNA containing structures of the nucleus (reviewed by Lukiw, 2001; Kruck et al., 2002, 2002a).

7. EXPERIMENTAL REMOVAL OF ALUMINUM FROM HYPERPHOSPHORYLATED PAIRED HELICAL FILAMENT-TAU PROTEIN Paired helical filaments (PHFs), forming in part the NFT, form an essential part of the AD pathology. These structures are formed from hyperphosphorylated PHF-tau protein. Utilizing a specific antibody against hyperphosphorylated PHF-tau and DFO to chelate aluminum in hippocampal AD brain sections, it was shown that aluminum removal released antigenic sites revealed by specific antibody staining of hyperphosphorylated PHFtau. It was further demonstrated that aluminum binding was phosphorylation state-dependent and its action was cumulative. It should be noted that in order for DFO to react with physiologically bound aluminum, the preparations had to be autoclave-treated. Furthermore, PHF-tau aggregation by A1C13 has been shown in vitro (Murayama et al., 1999). Phosphorylation state-dependent direct aluminum PHF-tau interaction provides strong evidence implicating aluminum ions in the stabilization or generation of the PHF part of the NFTs. Together, the co-localization of aluminum in NFTs and in the nuclear compartment, both constituting critical sites of toxic challenge to cell survival, supports the idea that aluminum removal from these sites might mitigate or even revert some of the toxic effects. At the same time, it points out the difficulty in finding a chelator and a chelation treatment regimen that removes aluminum from both specific and critical subcellular sites.

8. SYNTHESIS OF FERALEX-G: A NOVEL ALUMINUM/IRON CHELATING COMPOUND Recognizing the need for having available for use as a medicine an in vivo applicable Al 3 + /Fe 3 + chelation compound for treatment of toxic aluminum overload conditions, a novel chelation compound, 2-deoxy-2(N-carbamoylmethyl-[N'-2'-methyl-3'- hydroxypyrid-4'-one] )-D-glucopyranose (Feralex-G), was designed and subsequently synthesized from

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readily available and naturally occurring nontoxic products, namely, glucosamine, glycine, and maltol. First, maltol was chemically linked to glycine via a highly efficient aminolysis reaction resulting in the intermediate product l-carboxymethyl-3-hydroxy-2-methylpyrid-4-one, also named "TPAK-G". TPAK-G was then subsequently joined to glucosamine via a di-cyclo-hexyl-carbodiimide-promoted peptide coupling method to produce Feralex-G, which is the desired end product. This product was subjected to physical and chemical analysis; nuclear magnetic resonance (NMR) analysis permitted assignment of resonant frequencies for all covalently attached hydrogen atoms, which are consistent with the proposed molecular structure. Analysis by electron spray ionization mass spectroscopy yielded the expected molecular mass of 344 (Kruck and Burrow, 2002; Kruck et al., 2002). The stoichiometric formula is given in Fig. 2.

9. FERALEX-G IN VITRO CHELATION STUDIES To test Fe 3+ and Al 3+ binding by Feralex-G, proton displacement analysis was performed. This analytical pH titration technique is based on earlier work by Kruck and Sarkar (1973). Briefly, the maltol moiety of the

OH

Fig. 2. Feralex-G.

OH

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Feralex-G molecule (Fig. 2) carries a high pH dissociable proton, part of the 3'-hydroxy group in maltol, which can be used to study metal complex formation. Al 3+ and Fe 3+ both bind to the maltol group by coordination with the oxygen of the 4' keto group and the oxygen of the 3' hydroxy group, thus displacing the proton from the 3' hydroxy group as a result of bond formation. This proton displacement can be measured by pH base titration analysis and is related to co-ordination bond formation. Applying this proton displacement analysis yielded three Feralex-G molecules bound to one central Al 3+ or Fe 3+ ion over the measurable pH range of three to 10.5. Since the Fe 3+ maltol complexes are colored (red to purple) the observation range can be extended beyond the pH range. On lowering the pH, colored Fe 3+ maltol complexes persist down to pH = 1 and below (fading of color). Since Al 3+ maltol complexes do not show light absorption in the visible range, no conclusion can be inferred beyond the lower measurable, instrumental pH limit (Kruck and Burrow, 2002).

10. ALUMINUM REMOVAL FROM HYPERPHOSPHORYLATED PHF-TAU IN HUMAN HIPPOCAMPAL BRAIN SECTIONS The experimental system to show direct Al3+-PHF-tau interaction, as developed by Murayama et al. (1999), was used to compare the efficiency of Feralex-G in removing hippocampal PHF-tau bound Al 3+ , which is considered to be involved in the pathogenic process. The experiment using DFO to remove PHF-tau bound Al 3+ was repeated with comparable molar concentrations of Feralex-G. It became evident that Feralex-G was a stronger chelator than DFO, since it removed Al 3+ at 37 °C compared to DFO, which required autoclave heating to achieve Al 3+ removal (Shin and Kruck, submitted, 2002). This tight Al 3+ binding to PHF-tau, which is considered a prime determinant of the AD pathology and the relatively low efficiency of DFO to effect Al 3+ removal in the in vitro study, might explain why, in the human clinical trial subjecting AD patients to DFO chelation therapy, two years are needed for treatment to become effective (Crapper McLachlan et al., 1991). Therefore, Feralex-G might prove, in the future, to be more effective than DFO for systemic aluminum chelation therapy in the treatment of AD patients, and shows promise as a component of the molecular shuttle chelation strategy.

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11. COMPLICATIONS The development of novel, systemic, and orally effective chelators with increased Al selectivity is a highly desired goal of pharmacology. However, the design of aluminum-selective chelators is a difficult challenge due to the affinity of these compounds for iron, copper, magnesium, zinc, and other essential trace metals. Replacement therapy with these latter essential metal ion co-factors may be a necessary adjunct to any current systemic aluminum chelation therapy. For example, the bidentate 3-hydroxypyridine-4-ones have been shown to increase urinary excretion of aluminum in rabbits and rats, but have toxicities comparable to or greater than DFO (Yokel, 1994; Yokel et al., 1996). This suggests that they also have an effect in chelating other essential trace metals. Alternatively, these compounds may have differential access to compartmentally restricted toxic Al 3+ or Fe 3+ deposits. Molecular shuttle chelation strategies may provide another reasonable approach using recently designed aluminum chelators, such as Feralex-G. This approach requires further study. To prevent the development of anemic conditions through chelation-induced iron elimination in the clinical DFO treatment trial involving AD patients (Crapper McLachlan et al, 1991), treatment protocols were designed to allow intermittent treatment and/or no treatment periods (three days of DFO followed by one day without drug, then by two days of DFO followed by one day without drug). In addition, dietary iron was supplemented to replenish the body's physiological iron requirements. In this study, over the two-year treatment period, no iron deficiency was detected. Iron supplementation on no-drug days was considered; however, in this case, no additional iron supplement was required. In conclusion, differential chelation combined with molecular shuttle chelation and biologically essential trace metal replacement (such as iron replacement) are components to be considered in designing chelation compounds for metal toxicity treatments. Further studies will show whether Feralex-G will be useful as a primary chelator in conjunction with, or without, molecular shuttle chelators.

12. SUMMARY Neurological disorders represent graded disturbances in rational thought, cognition, emotion and creativity. Due partly to the demographics of our

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aging population, the number of cases of human neurological dysfunction is rising rapidly and, without treatment, will overwhelm our healthcare system and ability to adequately care for afflicted people. There is currently no cure for any human neurological disorder, including Alzheimer's disease (AD), which represents the single most prevalent form of senile dementia. Human neurological disorders involve highly varied etiopathologies. Besides aging, an intricate maze of factors appears to contribute to the development and progression of neurological dysfunctions, such as AD, and molecular genetic evidence indicates considerable etiopathologic heterogeneity in the predisposition to AD. While specific genotypes are associated with different relative risks and age of onset distributions for AD etiopathology, genetic variation also influences several aspects of clinical AD phenotype, such as severity, symptomatology, and duration. Environmental metal toxins are strongly implicated in the onset of the most common form of AD, which is idiopathic and of unknown origin. The use of metal-specific chelators to reverse metalinduced neurological deficits has been described; compounds such as DFO and Feralex-G hold promise in those instances where aluminumtriggered neurological dysfunctions are indicated.

ACKNOWLEDGMENTS The authors thank Dr. Hilary Thompson, Department of Ophthalmology, for statistical analysis and Vittorio Colangelo, Carla Cline, Carole Lachney, and Aileen Pogue for expert technical assistance; Dr. Moire Percy for helpful suggestions and critically editing the manuscript. This work was supported, in part, by the United States Public Health Service grant AG 18031 (WJL) from the National Institute on Aging, National Institutes of Health, Bethesda, Maryland, and by a grant from the Ear, Eye, Nose and Throat Foundation of the State of Louisiana.

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Rottkamp CA, Raina AK, Zhu X, Gaier E, Bush AI, Atwood CS, Chevion M, Perry G, Smith MA. Redox-active iron mediates amyloid-beta toxicity. Free Radicals Biol Med 2001; 30:447^150. Savory J, Ghribi O, Forbes MS, Herman MM. The rabbit model system for studies of aluminum-induced neurofibrillary degeneration: Relevance to human neurodegenerative disorders. In: Exley C, editor. Aluminum and Alzheimer's Disease: the Science that Sescribes the Link. New York: Elsevier Press, 2001: 203-219. Shin R.-W. Aluminum, tau and neurofibrillary degeneration. In: Exley C, editor. Aluminum and Alzheimer's Sisease: The Science that Describes the Link. New York: Elsevier Press, 2001: 411—420. Smith MA, Nunomura A, Zhu X, Takeda A, Perry G. Metabolic, metallic, and mitotic sources of oxidative stress in Alzheimer's disease. Antioxidants and Redox Signaling 2000; 2:413-420. Strong MJ. Aluminum as an experimental neurointoxicant: The neuropathology and neurochemistry. In: Exley C, editor. Aluminum and Alzheimer's Disease: The Science that Describes the Link. New York: Elsevier Press, 2001: 189-202. Szutowicz A. Aluminum, NO, and nerve growth factor neurotoxicity in cholinergic neurons. J Neuroscience Res 2001; 66:1009-1018. Terry RD, Katzman R. Senile dementia of the Alzheimer type. Ann Neurol 1983; 14:497-506. Uylings HB, de Brabander JM. Neuronal changes in normal human aging and Alzheimer's disease. Brain Cognition 2002; 49:268-276. Vyas SB, Duffy LK. Stabilization of secondary structure of Alzheimer beta-protein by aluminum(III) ions and D-Asp substitutions. Biochem Biophys Res Comm 1995; 206:718-723. Yang DS, McLaurin J, Qin K, Westaway D, Fraser PE. Examining the zinc binding site of the amyloid-beta peptide. Eur J Biochem 2000; 267:6692-6698. Ye Z, Connor JR. Screening of transcriptionally regulated genes following iron chelation in human astrocytoma cells. Biochem Biophys Res Comm 1999; 264:709-713. Yokel RA. Aluminum chelation principles and recent advances. In: Zatta P, editor. Coordination Chemistry Reviews. Recent Topics in Aluminium Chemistry 2002; 228:97-113. Yokel RA, Rhineheimer SS, Sharma P, Elmore D, McNamara PJ. Entry, half-life, and desferrioxamine-accelerated clearance of brain aluminum after a single (26) Al exposure. Toxicol Sci 2001; 64:77-82. Yokel RA, Ackrill P, Burgess E, Day JP, Domingo JL, Flaten TP, Savory J. Prevention and treatment of aluminum toxicity including chelation therapy: Status and research needs. J Toxicol Environ Health 1996; 48:667-683. Yokel RA. Aluminum chelation: Chemistry, clinical, and experimental studies and the search for alternatives to desferrioxamine. J Toxicol Environ Health 1994; 41:131-174. Zou J, Sugimoto N. Complexation of peptide with Cu2+ responsible for inducing and enhancing the formation of alpha-helix conformation. Biometals 2000; 13:349-359.

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CHAPTER 5

Aluminum and Central Nervous System Morphology in Hemodialysis Erich Reusche

ABSTRACT Aluminum is a well-known neurotoxicant that causes encephalopathies in long-term hemodialysis and discussed in Alzheimer's disease for decades. Using silver staining variants, we were able to demonstrate the morphology of Alzheimer's disease and, for the first time, the characteristic morphological changes in hemodialysis patients. This so-called dialysis-associated encephalopathy is, nowadays, apparently caused by long-term ingestion of aluminum-containing drugs (up to 2.5 kg of "pure" aluminum). It comprises pathognomonic aluminum-induced argyrophilic, proteinaceous, lysosomalderived inclusions in the cytoplasm of choroid plexus, gray matter glia, and neurons of the cortex and brainstem. Statistically, there was no increase in the morphology of Alzheimer's disease in 50 hemodialysis patients, but in patients over 60 years normally age related AD changes (pA4 p< 0.001, tangles p< 0.001). However, there was a correlation between the degree of dialysisassociated encephalopathy and ingested amounts of aluminum-containing drugs (p< 0.001). Laser microprobe revealed significant aluminum signals in the neuronal cytoplasm of dialysis-associated encephalopathy and, to a lesser degree, in the nuclei of neurons and glia in dialysis-associated encephalopathy and controls. In conclusion, our results suggest that aluminum does not cause an increase in the morphology of Alzheimer's disease, at least not in terms of bioavailable aluminum in drugs or hemodialysis. Keywords: Aluminum; atomic absorption spectrometry; dialysis; encephalopathy; laser microprobe; morphology.

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1. ALUMINUM AND THE ENVIRONMENT Aluminum (Al) is, after oxygen and silicon, the third most abundant element in the earth's crust, of which it comprises 8%. It occurs only in combined form: as an oxide in bauxit, and in complex aluminosilicates such as micas and feldspars. In contrast to its abundance in the earth's crust, its concentration in the ocean is below 1 |xg/L; natural water contains an insignificant amount of Al. With the advent of acid rain, metal ions such as Al, mercury, and lead escape from mineral deposits and appear in fresh waters (Martin, 1991). In food supply, Al originates from natural sources, including water, food additives, and contamination by Al utensils and containers. Most adults consume 1 mg to 10 mg of Al daily. Intake from Al food additives varies from zero to 95 mg daily, with a median intake of about 24 mg daily (Greger et al., 1985; Greger, 1992). In Al-rich foods, such as processed American cheese, home-made cornbread, and yellow cake with icing, food additives were the major source of Al (Pennington and Jones, 1989). Plants, such as tea, accumulate A13 + ; older leaves contain, for example, up to 3% of A13 + , while younger ones contain only 0.01%, a three hundred fold increase. Generally, the intake of Al from foods is less than 1% that of pharmaceuticals consumed, such as antacids, buffered analgesics, antidiarrheal agents, and certain ulcer drugs (Lione, 1983, 1985). For instance, 840 mg and 5,000 mg are possible daily doses in buffered analgesics and antacids, respectively (Lione, 1985). Citrate is the low molecular weight, and transferrin the main high molecular weight, carrier of A13+ in rat serum (Van Ginkel et al., 1990). Most brain Al entry is probably through the blood-brain barrier by transferrin receptor-mediated endocytosis (Roskams and Connor, 1990; Zatta et al. 1991). There appears to be a transporter-mediated brain Al efflux, probably as Al citrate (Yokel, 2001); a proton-dependent monocarboxylate transporter is at least partially mediating the efflux of Al from the brain's extracellular fluid (Ackley and Yokel, 1998). Removal of Al is effective by chelation with desferrioxamine (DFO) and oral administration of hydroxypyridones in rats (Florence et al., 1995; Gomez et al., 1998, 1999).

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2. ALUMINUM NEUROTOXICITY IN EXPERIMENTS AND INDUSTRY Experimentally, the neurotoxicity of Al was first demonstrated in animals by Dollken (1897). Subcutaneous inoculation of Al solutions resulted in bulbar paralysis, tremor, spasticity, weight loss, and spinal cord changes. Kopeloff et al. (1942) induced recurrent epileptic seizures by direct application of Al paste to the cortex of monkeys. Al was shown to be a potent experimental neurotoxicant. It can induce metabolic disturbances by modifications of cytoskeletal protein gene expression, such as the inhibition of DNA repair mechanisms, reduced rate of DNA synthesis, increased DNA replication errors, inhibition of chromatindependent RNA synthesis, or suppression of cytoskeletal mRNA steadystate levels. On the other hand, there are modifications of cytoskeletal proteins, such as increased microtubule-associated protein phosphorylation. Furthermore, there are alterations in membrane stability (with increased membrane rigidity, oxidative injury, and cholinergic effects) and signal transduction, including alterations in calcium homeostasis and activation of pro-apoptotic pathways (see references in Strong, 2001). Industrially, in 1921, a 46-year-old man who had been dipping red-hot metal articles into concentrated nitric acid using an Al holder was reported to have developed "loss of memory, tremor, jerking movements, and impaired co-ordination" (Spofforth, 1921). In 1962, a 49 year-old Al worker was reported to have inhaled Al dust and rapidly developed progressive encephalopathy with epileptiform seizures (McLaughlin et al., 1962). Years before his death, he suffered from short-term memory loss and speech impairment followed by increased muscle twitching. The electroencephalogram was abnormal, showing generalized slowing and spike activity; his cerebral state deteriorated irresistibly. After his death from bronchopneumonia, high levels of Al were found in all tissues examined, including the brain (480 |xg/g dry weight, normal range 0.6 (xg/g dry weight). In 1985, three patients who had worked for more than 12 years in the same Al smelting plant were reported to be suffering from neurological disorders (Longstreth et al., 1985). In 1990, this series was expanded to include 25 symptomatic workers from the same Al melting plant. The results of this study supported the existence of a syndrome characterized

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by poor co-ordination and memory, impairment in abstract reasoning, and depression. Al exposure in the potroom seemed to be the most likely cause (White et al., 1990). The Mclntyre powder, a mixture of Al and Al oxide, was used as a prophylactic agent against silicotic lungs between 1944 and 1979 in the mines of northern Ontario in Canada. Neurotoxic effects were studied in a morbidity prevalence study between 1988 and 1989. Exposed miners performed less well than unexposed miners on cognitive state examinations; the proportion of men with scores in the impaired range was greater in the exposed than in the nonexposed group. The likelihood of scores in the impaired range increased with duration of exposure, findings which were consistent with the putative neurotoxicity of chronic Al exposure (Rifat et al., 1990). The Camelford incident had exposed Al neurotixicity by water contamination. In 1988, 20 tonnes of Al sulphate were accidentally emptied into the treated water reservoir at Lowermoor Water Treatment Works, which supplied water to 20,000 people in the Camelford area of Cornwall. Within a few days, local residents and holiday-makers reported rashes, gastrointestinal disturbances, and mouth ulcers. The water was heavily contaminated with Al (up to 620 |xg/L; the European guideline for tap water is less than 0.2 |xg/L). In the weeks and months following the incident, exposed individuals complained of joint and muscle pains, malaise, fatigue, and impairment of concentration and memory (Clayton, 1991). A number of veterinary reports emerged, most notably that of fishes dying from Al poisoning in the local rivers following their contamination (Allen and Samson, 1989). Two years later, approximately 400 people were suffering from symptoms attributed to the incident. The standardized hospital discharge ratios in the five years following the incident were far greater than other areas of Cornwall, although no single diagnosis prevailed (Owen and Miles, 1995). Almost three years after the Camelford incident, 55 people were studied who had problems with poor short-term memory and concentration following exposure to the contaminated water (Altmann et al., 1989, 1999; Altmann, 2001).

3. DIALYSIS ENCEPHALOPATHY The introduction of long-term hemodialysis (HD) in chronic renal failure caused a new endemic picture of lethal encephalopathies, of unknown

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origin, characterized by EEG abnormalities, dementia, myoclonic jerks, and seizures (Alfrey et al., 1972). After the introduction of flameless atomic absorption spectrometry (AAS), Alfrey et al. (1976) discovered that Al-contaminated dialysis fluids of tap water was the obvious cause of this fatal "dialysis encephalopathy" (DE). They found gray matter Al values of 25 ppm in a group of uremic patients on dialysis who died from DE compared to 2.2 ppm in a control group. Both gross and microscopical neuropathologic abnormalities were minimal. (Because of clinical similarities to the Creutzfeldt-Jacob syndrome, one patient's brain was injected into primates, which was lacking in result after 3.5 years). Sabouraud et al. (1978) reported 16 patients with progressive myoclonic encephalopathy, which appeared after a mean duration of 32 months of HD, leading to death in 4.5 months and characterized by myoclonus, speech disorders, epileptic seizures, and changes in the mental status. DE-like symptoms have also been reported in undialyzed patients. Bakir et al. (1986) described four patients — two were undialyzed, one with HD for one month and one with peritoneal dialysis — who developed acute hyperaluminemic encephalopathy with myoclonus, refractory convulsions, coma, and rapid fatal course within one month. All had been taking Al hydroxide as a phosphate binder and Shol's solution, a buffer of citric acid and sodium citrate as an alkalinizing agent. They had clearly died from excessive Al absorption from the bowel. Two patients were diabetic, a state believed to increase Al absorption. On the other hand, the concomitant ingestion of Al hydroxide and large doses of Shol's solution might have facilitated Al absorption because citric acid in the solution is a strong chelator of Al. Another 12 undialyzed patients with DE were cited, with resolution of the symptoms upon cessation of Al intake. Russo et al. (1992) reported two undialyzed patients with DE. The first had used large amounts of Al-containing phosphate binders for two years; her serum Al level was 25.34 (xmol/L. Despite chelating therapy with DFO and HD, she died. An autopsy of the cerebral cortex revealed elevated Al levels of 19 mcg/gm. The second patient had never taken phosphate binders, but ingested several grams of citrate for at least six months. The discontinuation of citrate and additional HD resulted in a resolution of symptoms and the return of the Al level to normal. Bolla et al. (1992) studied neurocognitive effects of Al in 35 HD patients. As Al levels increased, patients with lower vocabulary scores

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showed a decline in attention/concentration and frontal lobe functions, while patients with higher vocabulary scores revealed no Al-related decline. These results suggested that patients with lower verbal intelligence may possess less well-developed compensatory strategies to overcome the toxic neurocognitive effects associated with Al. Morphologically, only unspecific cerebral changes have been found in DE, including lipofuscinosis, loss of neurons, and neurofibrillary degeneration, especially in the motor cortex, red nucleus, and dentate-olivary system. The intracellular binding of Al was shown by a histochemical fluorescent Morin stain (Sabouraud et al., 1978). Burks et al. (1976) reported shrinkage of neurons, increased paired astrocytes, microglial reactions, and spongiform changes in the second and third cortical layers, changes which were no different from those found in the brains of dialysis patients dying from other causes. In the latest report, the neuropathologic aspects of DE "have remained obscure. ... Spongy changes of the cerebral cortex consisted of vacuoles in the neuropil and inside nerve cell bodies and astrocytic processes. Thus, DE is a disease of neurons and astrocytes ..." (Winkelman and Ricanati, 1986).

4. DIALYSIS-ASSOCIATED ENCEPHALOPATHY In 1991, we reported new effective methods for the demonstration of Alzheimer morphology by applying new silver staining variants of the Ag NOR method (Howell and Black, 1980; Reusche, 1991; Reusche, 1997). Paraffin sections were silver stained as follows: a colloidal developer of 2% gelatine — the use of type 4078 or 4070 (Merck, Darmstadt, Germany) is recommended to obtain optimal results — was dissolved in 1 % formic acid and left stable for several weeks in the dark. A 50% solution of silver nitrate — at room temperature "maturing" for two weeks or at 60°C overnight (Quinn and Conklin, 1996) and left stable for more than one year in the dark — was prepared. Developer and silver were mixed at a ratio of one to two immediately before the staining procedure. Sections of 10 |jim thick were incubated with the mixed solution in a horizontal position at room temperature for 30 to 45 minutes in the dark and covered with a coverslip. For optional lightening of the background, the sections were dipped in 0.1% goldchloride for two minutes. This was followed by repeated rinsing with distiled water and a two-minute incubation in

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a 3% sodium thiosulfate solution. After rinsing in tap water for 30 minutes, the sections were dehydrated and mounted in a synthetic resin. In 1993, 30 years after the introduction of HD, we demonstrated — for the first time — characteristic changes in long-term hemodialyzed patients by applying our new silver staining variants (Reusche and Seydel, 1993). Unlike the clinical lethal dialysis of dementia in the 1960s and 1970s this so-called "dialysis-associated encephalopathy" (DAE) comprises a morphological term. Nowadays — using modern techniques of water purification deionized fluids are applied in HD — DAE is caused mainly by long-term ingestion of Al-containing drugs to control hyperphosphatemia, up to 2.5 kg of "pure" Al (Reusche et al., 1996). Morphological changes in a post-mortem study of 10 patients who had undergone long-term HD have demonstrated pathognomonic argyrophilic inclusions. These Al-induced degradation products were silver stained in the cytoplasm (but not in lysosome-free nuclei) of choroid plexus epithelia ("kidney of the brain"), cortical/subcortical glia, and numerous neurons of the cortex and brainstem. In addition, a manifold increase in Al was demonstrated in the subcellular structures of neurons and glia by laser microprobe mass analysis (LAMMA).

4.1. Correlation of DAE Morphology, Al Intake, and Long-Term HD The CNS tissue and peripheral organs of 50 patients with chronic renal failure and HD were evaluated for Al-containing inclusions. Morphological alterations were correlated with the duration of HD and the amount of ingested Al-containing drugs. Significant correlations were found between the degree of morphological changes and Al intake of up to 2.5 kg "pure" Al (p = 0.0003), as well as morphology and duration of long-term HD of up to 178 months (p = 0.001). Beside well-known deposits in the choroid plexus, glia, and neurons of the CNS, typical argyrophilic Al-containing inclusions were found in the neurons of autonomic ganglia, heart muscle, ovary, testis, parathyroid, and adrenal and pituitary glands. Deposition of degradation products seemed to be almost irreversible. Even after renal transplantation with termination of drug-related Al intake and normalized renal Al excretion, proteinaceous inclusions remained in the cellular cytoplasm in an unchanged fashion for up to 10 years (Reusche et al., 1996).

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5. ALUMINUM, HEMODIALYSIS, AND ALZHEIMER'S DISEASE In 1964, the same year that HD was introduced in chronic renal failure, neurofibrillary changes of neurons were induced experimentally in rabbit nerve cells with the injection of Al salts for the first time. Since then, the technique has been reproduced repeatedly in different applications and species (Klatzo et al., 1965; Terry and Pena 1965; Wisniewski and Wen, 1992; Savory et al., 2001). A comparison of Al-induced neurofibrils with neurofibrillary tangles (NFTs) of the Alzheimer type revealed differences in peptide composition, protofilament composition, immunoreactivity, fluorescence after thioflavin S stain, and birefringence after congo red stain. Moreover, NFTs of the Alzheimer type are also found in amyotrophic lateral sclerosis, Down's syndrome, Picks's disease, Hallervorden-Spatz disease, dementia pugilistica and head trauma, and Guam Parkinson dementia (Wisniewski and Wen, 1992). Nevertheless, both events — experimentally Al-induced neuronal changes and Al-induced dialysis dementia — have given rise to more than 30 years of speculation as to whether morphological alterations associated with Alzheimer's disease (AD) may, in fact, be linked to Al metabolism. The subject remains controversial, having both its supporters and its opponents. Two short review articles, published in the same issue of Archives of Neurology, demonstrate the controversy of opinions associated with this problem and subject: Forbes and Hill (1998) claim that exposure to aluminum is indeed a risk factor for the development of Alzheimer's disease' while Munoz (1998) is of the view that this is not true. In particular, controversial epidemiological results were obtained by a number of studies, which addressed the problem of Al in drinking water that was used as dialysate in the 1960s and 1970s, in connection with the incidence of AD. Both positive results (Flaten, 1990; McLachlan et al., 1996) and a lack of correlation (Broe et al., 1990; Forster et al., 1995) were found. Interestingly, Martyn et al. (1989) reported a correlation between the incidence of AD and drinking water. In a subsequent paper, they concluded that "there was little association between AD and higher aluminum or lower silicon concentrations in drinking water when cases were compared with any of the control groups" (1997). These two articles, albeit from the same group, are a telling example that serve to highlight the controversy of opinion associated with this problem and subject.

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The possibility to visualize AD and DAE morphology simultaneously in silver stained paraffin sections provides a new tool to collect new information about the role of Al in the pathogenesis of AD.

5.1. Differences in Morphology of AD and DAE The morphologies of AD and DAE are completely different. On the one hand, silver stained AD morphology (Reusche, 1991) is characterized by the well-known amyloid-beta 4 (A£4) and NFTs consisting of paired helical filaments (Reusche and Seydel, 1993). On the other hand, DAE presents pathognomonic argyrophilic, Al-containing, lysosomal-derived inclusions in the cytoplasm of the choroid plexus epithelia (Fig. 1), gray

Fig. 1. Choroid plexus. (A) Laser microprobe mass analysis (LAMMA) spectrum with prominent mass signal at m/z 27 as evidence for marked Al storage ("kidney of the brain"). Note the lack of silver at 107/109 in nonsilver stained specimen (arrow). Inset: laser perforations. (B) Silver stained paraffin section with numerous (Al-containing) argyrophilic cytoplasmic inclusions. (C) Electron micrograph with mostly electron dense (arrow) Al=aluminum.

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Fig. 2. Gray matter glia. (A) Paraffin section with silver stained delicate astrocytic processes (arrows) (magnification X200). (B) Electron micrograph with irregular astrocytic, cytoplasmic electron dense inclusion (magnification X7,200).

matter glia (Fig. 2), and neurons in the cortex and brainstem (Fig. 3) (Reusche and Seydel, 1993).

5.2. Epidemiology of AD and DAE The investigation of nearly 1,000 cases of suspected AD never presented characteristic argyrophilic inclusions of the DAE type. On the other hand, microscopic evaluation of 127 long-term dialysis patients revealed only one single 72-year-old patient, which demonstrated light and electron microscopically argyrophilic neuronal and glial deposits of the DAE type, as well as and distinct amounts of age-related neuropil threads and NFT of the Alzheimer type. Both morphological changes were intertwined with each other, but can be distinguished easily and unequivocally (Reusche, 1997). In addition, one single case with severe neurological symptoms also presented distinct DAE changes and age-related Ap4 of the AD type. (Reusche et al., 1999).

5.3. Statistical Study of 50 Long-Term Hemodialyzed Patients A study has examined the role of Al in the etiology of AD morphology. To this end, modified silver stain enabled the morphological demonstration and clear, simultaneous discrimination of AD changes from that of DAE on paraffin sections of the hippocampal and frontal regions and subcortical

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Fig. 3. Hypoglossal neuron. (A) Paraffin section with numerous fine granular, deeply black argyrophilic exclusively cytoplasmic inclusions (silver stain, magnification X920). (B) Electron micrograph with irregular, partly electron-lucent (asterisk), partly electrondense cytoplasmic inclusion. Note the lack of inclusions in the neuronal nucleus and nucleolus (arrow).

gray matter. Statistically, we studied 50 brains from patients with a long history of HD. The cumulative amounts of "pure" Al ingested in Alcontaining drugs (prescribed for the control of hyperphosphatemia and gastric complaints) were recorded retrospectively. Data were collected from 60 hospitals and dialysis units in Hamburg and Northern Germany.

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The accurate Al content of each drug, recorded in the prescriptions, was calculated in detail. The morphological criteria of CERAD were used to quantify A04 (Mirra et al., 1991) and NFT (Mirra et al., 1993) of the Alzheimer type on a three-point scale. DAE changes were assessed semi-quantitatively in a similar manner, using a three-point scale (Reusche et al., 2001a), thus enabling some comparison with the morphological changes of AD. No apparent microscopic increase in AD morphology was found. No AD changes were seen in patients below the age of 60, despite a long history of HD with ingestion of "pure" Al of up to 2.5 kg. Patients above 60 years of age occasionally presented with sparse deposits of A(34 and/or a low incidence of NFT of the Alzheimer type. In accordance with CERAD criteria, AD changes were identified as normal, age-related phenomena (p < 0.001 for A(34 and p < 0.001 for NFT) (Figs. 4 and 5).

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Fig. 5. Correlation of amounts of neurofibrillary tangles with age of 50 patients with long-term hemodialysis or dialysis-associated encephalopathy. Degree of deposition was determined by CERAD criteria (Mirra et al., 1993). Reprinted from Reusche et al., 2001a, courtesy of the publisher.

However, a significant statistical difference was found between the amounts of drug-related Al ingested and the degree of DAE-related morphological changes (p< 0.001) (Table 1).

5.4. LAMMA Investigation of Al Content in DAE and AD Finally, subcellular Al content was evaluated by LAMMA in the hippocampal neurons, glial nuclei, and neuropil of seven age-matched controls and six patients with DAE. The data were averaged by cells to make cell-to-cell comparisons possible. In addition, a relation between the aluminum-related peak at m/z 27 and all other peaks was made to correct interferences of differences due to sample thickness and background. In each specimen, the neuronal cytoplasm, nucleus, glial nuclei, and neuropil were investigated.

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Table 1. Statistical analysis of 50 patients with a long history of HD. Variable HD by DAE Al in drugs by DAE Age by AD-amyloid Age by AD-NFT DAE by AD-amyloid DAE by AD-NFT Al in drugs by AD-amyloid Al in drugs by AD-NFT HD by AD-amyloid HD by AD-NFT DAE by age DAE by gender

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Aluminum and CNS Morphology in Hemodialysis

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The Al content in the cytoplasm of DAE neurons was statistically significantly different compared with the controls. Apparently, and due to long-term failing autolysosomal processes, Al is enriched in characteristic proteinaceous, therefore argyrophilic inclusions. (Lysosome-free) nuclei of the DAE neurons and glial nuclei presented Al signals within the range of neurons and glial nuclei of the control group (see box-plot diagram in Fig. 7).

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6. SUBACUTE FATAL AL ENCEPHALOPATHY AFTER RECONSTRUCTIVE OTONEUROSURGERY A 52-year-old female was reported who underwent otoneurosurgery in order to resect acoustic neurinoma (Reusche et al., 2001b). Bone reconstruction was performed with an Al-containing cement. Six weeks later, the patient suffered loss of conciousness, myoclonic jerks, and persistent grand mal seizures, clinical symptoms that resembled those of lethal DE of the 1960s and 1970s. She died six months later from septic complications. Light and electron microscopic investigation of CNS revealed Al-containing intracytoplasmic, argyrophilic inclusions in the choroid plexus epithelia, neurons, and cortical glia. AAS revealed an increase in mean bulk Al concentration of the cortex and subcortex of up to 9.3 (xg/g (normal range, <2|jLg/g; Table 2a). LAMM A demonstrated an increase in Al in the subcellular structures (Table 2b). This unique case again demonstrates the extraordinary neurotoxicity of Al. The Al encephalopathy described here presented similar clinical signs as four patients from France (Renard et al., 1994) and Belgium (Hantson et al., 1994), which were reported in Lancet, comprising limited clinical and laboratory information, but no morphological results or details. All five patients underwent translabyrinthine approach for otoneurosurgery followed by bone reconstruction with "Ionocem" cement. All of them presented retroauricular CSF collections, obviously due to an incomplete closure of dural defects. Regular dural closure is of paramount importance to ensure the avoidance of any direct contact of brain tissue with Al ions/ compounds. Al had apparently been released from "Ionocem" directly into the subarachnoid space by CSF leakages, which subsequently infiltrate the brain parenchyma. Elevated Al levels in CSF had been documented in all five patients. In our patient, a small amount of approximately 30 mg Al might have initiated the fatal clinical course with refractory grand mal seizures (1.5 g "Ionocem" cement contained approximately 20% Al; a loss of about 10%, observed in surgical repair, amounts to 30 mg Al). After the presentation of our results, Ionos Company became bankrupt. In conclusion, our morphological, epidemiological, and statistical results show a pathognomonic morphology in long-term HD, due apparently to the ingestion of large amounts of Al-containing drugs. There is a significant statistical correlation between DAE morphology and HD/A1containing drugs. On the other hand, the results suggest that Al does not

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ACKNOWLEDGMENT Support for this work was provided by Deutsche Forschungsgemeinschaft Grant RE 1301 1/2.

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Campbell A. The potential role of aluminum in Alzheimer's Disease. Nephrol Dial Transplant 2002; 17:17-20. Clayton B. Water pollution of Lowermoor, North Cornwall (second report). London: Department of Health, 1991. Dollken V. Uber die Wirkung des Aluminiums mit besonderer der durch das Aluminium verursachten Lasionen im Centralnervensystem. Arch Exp Pathol Pharmacol 1897; 40:98-120. Flaten TP. Geographical associations between aluminum in drinking water and death rates with dementia (including Alzheimer's disease), Parkinson's disease and amyotrophic lateral sclerosis in Norway. Environ Geochem Health 1990; 12:152-167. Florence AL, Gauthier A, Ward RJ, Crichton RR. Influence of hydroxypyridones and desferrioxamine on the mobilization of aluminum from tissues of aluminum-loaded rats. Neurodegeneration 1995; 4:449-455. Forbes WF, Hill GB. Is exposure to aluminum a risk factor for the development of Alzheimer's disease? Yes. Arch Neurol 1998; 55:740-741. Forster DP, Newens M, Kay DW, Edwardson JA. Risk factors in clinically diagnosed presenile dementia of the Alzheimer type: A case control study in northern England. J Epidemiol Community Health 1995; 49:253-258. Gomez M, Esparza JL, Domongo JL, Singh PK, Jones MM. Comparative aluminum mobilizing actions of desferoxamine and four 3-hydroxypyrid-4-ones in aluminum-loaded rats. Toxicology 1998; 130: 175-181. Gomez M, Esparza JL, Domongo JL, Singh PK, Jones MM. Chelation therapy in aluminum-loaded rats: Influence of age. Toxicology 1999; 137:161-168. Gorsky JE, Dietz AA, Spencer H, Osis D. Metabolic balance of aluminum studied in six men. Clin Chem 1976; 25:1739-1743. Graves AB, White E, Koepsell TD, Reifler BV, van Belle G, Larson EB, Raskind M. A case control study of Alzheimer's disease. Ann Neurol 1990; 28:744-766. Greger JL. Dietary and other sources of aluminum intake. In: Proceedings of CIBA Foundation Symposium an Aluminum in Biology and Medicine. New York: John Wiley & Sons, 1992: 26^19. Greger JL, Goetz W, Sullivan D. Aluminum levels in foods cooked and stored in aluminum pans, trays and foil. J Food Prot 1985; 48:772-777. Hantson P, Mahieu P, Gersdorff M, Sindic CJ, Lauwerys R. Encephalopathy with seizures after use of aluminum-containing cement. Lancet 1994; 344:1647. Heyman A, Wilkinson WE, Stafford JA, Helms MG, Sigmon AH, Weinberg T. Alzheimer's disease: A case study of epidemiological aspects. Ann Neurol 1984; 15:335-341. Howell WM, Black DA. Controlled silver staining of nucleolus organizer regions with a protective colloidal developer: A one-step method. Experientia 1980; 36:1014—1015. Klatzo I, Wisniewski H, Streicher E. Experimental production of neurofibrillary degeneration. JNeuropathol Exp Neurol 1965; 24:187-199. Kopeloff LM, Barrera SE, Kopeloof N. Recurrent convulsive seizures in animals produced by immunologic and chemical means. Am J Psych 1942; 98:881-902.

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Lione A. The prophylactic reduction of aluminum intake. Food Chem Toxicol 1983; 21:103-109. Lione A. Aluminum intake from nonprescription drugs and sucralfate. Gen Pharmacol 1985; 16:223-228. Longstreth WT, Rosenstock L, Heyer NJ. Potroom pals? Neurologic disorders in aluminum smelting workers. Ann Int Med 1985; 145:1972-1975. Martin RB. Aluminum in biological system. In: Nicolini M, Zatta PF, Corain B, editors. Aluminum in Chemistry, Biology and Medicine. New York: Raven Press, 1991: 3-20. Martyn CN, Osmond C, Edwardson JA, Barker DJP, Lacey RE Geographical relation between Alzheimer's disease and aluminum in drinking water. Lancet 1989; 1:59-62. Martyn CN, Coggon DN, Inskip H, Lacey RF, Young WF. Aluminum concentrations in drinking water and risk of Alzheimer's disease. Epidemiology 1997; 8:281-286. McDermott JR, Smith IA, Ward MK, Parkinson IS, Kerr DNS. Brain aluminum concentration in dialysis-encephalopafhy. Lancet 1979; 1:901-904. McLachlan DR, Bergeron C, Smith JE, Boomer D, Rifat SL. Risk for neuropathologically confirmed Alzheimer's disease and residual aluminum in municipal drinking water employing weighted residential histories. Neurology 1996; 46:401^105. McLaughlin AIG, Kazantzis G, King E, Teare D, Porter RJ, Owen R. Pulmonary fibrosis and encephalopathy associated with the inhalation of aluminum dust. Brit J Indust Med 1962; 19:253-263. Mirra SS, Heyman A, McKeel D, Sumi SM, Crain BJ, Brownlee LM, Vogel FS, Hughes MS, Van Belle G, Berg L. The consortium to establish a registry for Alzheimer's disease (CERAD). Part II. Standardization of the neuropathologic assessment of Alzheimer's disease. Neurology 1991; 41:479-486. Mirra SS, Hart MD, Terry RD. Making the diagnosis of Alzheimer's disease. Arch Pathol Lab Med 1993; 117:132-144. Munoz DG. Is exposure to aluminum a risk factor for the development of Alzheimer's disease? No. Arch Neurol 1998; 55:737-739. Owen PJ and Miles DP. A review of hospital discharge rates in a population around Camelford in North Cornwall up to the fifth anniversary of an episode of aluminum sulphate absorption. J Public Health Medicine 1995; 17:200-204. Pennington J AT, Jones JW. Dietary intake of aluminum. In: Gitelman HJ, editor. Aluminum and health: A Critical Review. New York: Marcel Dekker, 1989: 67-100. Quinn B, Conklin L. Alzheimer impregnation: studies with the Reusche method. Lab Invest \996;1A:U2A. Renard JL, Felten D, Bequet D. Post-otoneurosurgery aluminum encephalopathy. Lancet 1994; 344:63-64. Reusche E. Silver staining of senile plaques and neurofibrillary tangles in paraffin sections: A simple and effective method. Pathol Res Pract 1991; 187:1045-1048. Reusche E. Argyrophilic inclusions distinct from Alzheimer neurofibrillary changes in one case of dialysis-associated encephalopathy. Acta Neuropathol 1997; 94: 612-616.

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Reusche E, Seydel U. Dialysis-associated encephalopathy: Light and electron microscopic morphology and topography with evidence of aluminum by laser microprobe mass analysis. Acta Neuropathol 1993; 86:249-258. Reusche E, Ogomori K, Diebold J, Johannisson R. Electron microscopic study of paired helical filaments and cerebral amyloid using a novel en bloc silver staining method. VirchArchivA 1992;420:519-525. Reusche E, Koch V, Friedrich HJ, Niinninghoff D, Stein P, Rob PM. Correlation of drug-related aluminum intake and dialysis treatment with deposition of argyrophilic aluminum-containing inclusions in CNS and organ systems of patients with dialysisassociated encephalopathy. Clin Neuropath 1996; 15:342-347. Reusche E, Gerke P, Kriiger S, Rohwer J, Lindner B, Rob PM. Long-term organic brain syndrome and stroke-like brainstem symptoms in undiagnosed dialysis-associated encephalopathy. Dtsch Med Wschr 1999; 124:176-181. Reusche E, Koch V, Friedrich HJ, Harrison AP Alzheimer morphology is not increased in hemodialysis and dialysis-associated encephalopathy. Acta Neuropathol 2001a; 101:211-216. Reusche E, Pilz P, Oberascher E, Gloeckner K, Trinka K, Iglseder E, Lindner B, Ladurner P. Subacute fatal aluminum-encephalopafhy after otoneurosurgery. Hum Pathol 2001b; 32:1136-1140. Rifat SL, Eastwood MR, McLachlan DR, Corey PN. Effect of exposure of miners to aluminum powder. Lancet 1990; 336:1162. Roskams AJ and Connor JR. Aluminum access to the brain: A possible role for transferrin and its receptor. Proc Natl Acad Sci USA 1990; 87:9024-9027. Russo LS, Beale G, Sandroni S, Ballinger WE. Aluminum intoxication in undialysed adults with chronic renal failure. J Neurol Neurosurg Psych 1992; 55:697-700. Sabouraud O, Chatel M, Menault F, Dien Peron J, Carder F, Garre M, Gary J, Pecker S. Progressive myoclonic encephalopathy in dialysis patients. Clinical, electroencephalographic and neuropathological study. Pathogenetic discussion. Rev Neurol (Paris) 1978; 134:575-600. Savory J. et al. The rabit model system for studies of aluminum-induced neurofibrillary degeneration: Relevance to human neurodegenerative disorders. In: Exley C, editor. Aluminum and Alzheimer's Disease. Amsterdam: Elsevier, 2001: 203-220. Spofforth J. Case of aluminum poisoning. Lancet 1921; 1:1301. Strong MJ. Aluminum as an experimental neurotoxicant: The neuropathology and neurochemistry. In: Exley C, editor. Aluminum and Alzheimer's Disease. Amsterdam: Elsevier, 2001: 189-202. Terry RD, Pena C. Experimental production of neurofibrillary degeneration. / Neuropathol Exp Neurol 1965; 24:200-210. Van Ginkel WF, van der Voet GB, van Eijk HG, de Wold FA. Aluminum binding to serum constituents: A role for transferrin and its receptors. J Clin Chem Clin Biochem 1990; 28:459-463.

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White DM, Longstreth, WJ, Rosenstock L, Claypoole KH, Brodkin CA, Townes BD. Neurologic syndrome in 25 workers from an aluminum smelting plant. Arch Int Med 1990; 152:1443-1448. Winkelman MD, Ricanati ES. Dialysis encephalopathy: Neuropathologic aspects. Hum Pathol 1986; 17:823-833. Wisniewski HM, Wen GY. Aluminum and Alzheimer's disease. CIBA Foundation Symposium 1992; 169:142-154. Yokel RA. Aluminum and the blood-brain barrier. In: Exley C, editor. Aluminum and Alzheimer's Disease. Amsterdam: Elsevier, 2001: 233-260. Zatta PF, Nicolini M, Corain B. Aluminum (III) toxicity and blood-brain barrier. In: Nicolini M, Zatta PF, Corain B, editors. Aluminum in Chemistry, Biology and Medicine. New York: Raven Press, 1991: 96-112. Zatta PF, Suwalsky M. Aluminum, membranes and Alzheimer's disease. In: Exley C, editor. Aluminum and Alzheimer's Disease. Amsterdam: Elsevier, 2001: 279-292.

CHAPTER

6

Zinc, Brain, and Aging Eugenio Mocchegiani, Mario Muzzioli, Robertina Giacconi, Tiziana Casoli, Giuseppina DiStefano, Patrizia Fattoretti

ABSTRACT Zinc is relevant to the maintenance of brain functions. It is involved in glutaminergic transmission in the gene expression of transcriptional factors and in nerve growth factor activity. Zinc turnover in the brain is mediated by metallothionein (MT) and the zinc traffic into the brain is due to ZnTl-4 transporter proteins. Alterations in zinc turnover lead to brain dysfunction. During aging, zinc turnover is altered, coupled with decreased brain functions and impaired cognitive performances. This decrease may lead to neurodegeneration. One of the causes of altered zinc turnover in aging may be due to altered zinc-bound MT homeostasis, which transforms from being a protective shield against stress into a harmful factor in aging because of the exclusive sequestration of zinc and lack of subsequent zinc release by MT for brain functions. The beneficial effect of zinc supplementation is discussed in aging and neurodegeneration. Keywords: Zinc; brain; behavior; zinc supplementation; aging; neurodegeneration.

1. INTRODUCTION Zinc (Zn) is a relevant trace element in the body. The discovery of its essentiality in 1930 was followed by extensive studies on its physiological role, which has extraordinarily enlarged the impact of Zn in various cell and body functions (Mills, 1989). Some of the most relevant findings include the role of Zn as a catalytic component of more than 200 enzymes, its requirement as a structural constituent of many proteins, and, most likely, its function in preventing free radical formation (McCall et al.,

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2000). The ensuing relevance to cell division and differentiation, as well as for programmed cell death, gene transcription, biomembrane functioning, and many other enzymatic activities, have led to the consideration of Zn as a leading element in ensuring the correct functioning of various tissues, organs, and systems. Of great interest is the importance of Zn in neurobiology, which has been acknowledged and reviewed by several authors (Sandstead, 1985; Smart et al., 1994; Takeda, 2000a). Pioneering studies on the fundamental role of Zn in brain functions were carried out by Hurley and Swenerton (1966), Agpar (1968), and Caldwell et al. (1970) in Zn-deficient rat embryos displaying abnormal development of the central nervous system (CNS) with subsequent altered behavior. Further studies have shown that the involvement of Zn in the CNS occurs throughout ontogenesis. In rodents, early prenatal Zn impoverishment seriously impairs embryonic neurulation and development of the primitive neural tube (Hurley and Shrader, 1972), and results in a high incidence of exencephalus and spina bifida, which together represent the most severe type of Zn-related neural defects (Dreosti, 1983). More recent studies have focused Zn on in various parts of the brain, particularly on the hippocampus where it is found to accumulate in the mossy fiber pathway, neurons, and synaptic vesicles (Weiss et al., 2000). High concentrations in the synaptic vesicles of the special "Zn-containing" neurons in the forebrain (Frederickson et al., 2000), together with its function in biochemical myelination and release of neurotransmitters such as 7-aminobutyric acid (GABA) (Ben-Ari and Cherubini, 1991) and glutamate, indicate that Zn is a key modulator of neuronal excitability. There is also evidence that Zn deficiency results in lowered levels of long co-3 and co-6 chains, thereby causing impaired fatty acid metabolism in the neurons (Wauben et al., 1999). Moreover, Zn is important for neurogenesis, neuronal migration, and synaptogenesis; its deficiency interferes with neurotransmission and subsequent neurophysiological development (Colvin et al., 2000; Frederickson et al., 2000). Zn is also important in p-adrenoreceptor density in the brain cortex (Viticchi et al., 1999), as well as in the release of hormonal factors from the hypothalamus (Fabris et al., 1997b). Of these, thyrotropin-releasing hormone (TRH) and somatostatin are of interest because both affect the turnover of thyroid hormones which, in turn, influences the CNS (Morley et al.,

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1980). Moreover, Zn is also required for "Zn-fingers" proteins involved in the developing brain (LIMK-1, LIMK-2, Krox-20, and zif268) (Mori et al., 1997) and in maintaining circadian rhythms from brain genes clock (c-fos, egr-3, and nur77) in the suprachiasmatic nucleus (Morris et al., 1998); the maintenance of circadian rhythms is indispensable in conferring plasticity on the many homeostatic mechanisms of the human body, which ensures good health (Mocchegiani et al., 2002b). From these considerations, it can be seen that Zn is pivotal for brain functions (development and maintenance). The mechanisms by which Zn is taken up in the brain, as well as transport into the brain, are still undefined. The brain barrier system (Takeda, 2000a), proteins (metallothioneins [MTs]) (Aschner, 1997), and ZnTl-T4 transporter peptides family (McMahon and Cousins, 1998) have been suggested in the traffic and uptake of Zn in the brain. During aging, Zn ion bioavailability is low in both the brain and at the periphery (Mocchegiani et al., 2001). This fact leads to an impairment in the functionality of many organs and systems, including brain functions (Fabris, 1997a). Many authors reported that Zn deficiency in aging alters some brain functions (synaptic transmission, GABA and N-methylD-aspartate (NMDA) receptors' activity, and neuronal excitability) and impairs the release of the most relevant factor of growth of neurons, such as nerve growth factor (NGF) (Rylett and Williams, 1994). Such biological alterations are coupled with impairments in cognitive functions in animals put on a Zn-deprived diet, old rats, and the elderly (Mills, 1989; Bhatnagar and Taneja, 2001). It is also intriguing and, at the same time, of interest that some age-related neurodegenerative diseases— amyotrophic lateral sclerosis (ALS), brain ischemia, Parkinson's disease (PD), and Alzheimer's disease (AD)—display low Zn bioavailability with impairments in brain functions (Fabris and Mocchegiani, 1995). In this chapter, we analyze the role of Zn in brain functions and cognitive performance under normal conditions, during aging, and in neurodegeneration. We also discuss the possible mechanisms involved in Zn alteration in the brain. The intervention or prevention, by means of Zn supplementation or other antioxidants, in brain dysfunctions is also discussed in the elderly and in some age-related neurodegenerative diseases, such as PD and AD.

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2. BIOLOGY OF ZINC IN THE BRAIN AND AGING 2.1. Zinc Release, Uptake, Receptors' Interaction, and Aging Zinc concentration in the brain increases with growth after birth and remains constant in the adult brain (Markesbery et al., 1984). The intracellular Zn concentration in the brain was estimated to be approximately 150 |jLmol/L, judging from the average total brain Zn concentration (Takeda, 2000a). Serum Zn concentration is approximately 15 (xmol/L. On the other hand, the concentration of free Zn ions in mammalian serum and in some organelles has been estimated to be as low as 10~9 to 10~10M (Magneson et al., 1987). Therefore, the brain has the same high Zn content as all organs. In particular, neurons and glia cells display a higher Zn content. However, the distribution of Zn ions is not uniform, with higher concentrations found in gray than white matter, and the highest concentrations are present in certain forebrain regions, including the hippocampus, amygdala, and cortex (Frederickson, 1989). During aging, the distribution of Zn in the brain changes in relation to the area involved. Decrements are observed in the cerebellum and cerebral cortex, while increments occur in the hippocampus and dentate gyrus of old rats (Sawashita et al., 1997). Using the model of senescenceaccelerated mouse prone 10 (SAMP-10), which displays brain atrophy and defects in learning and memory, the Zn content is low in eight regions of the brain, including the hippocampus and cerebral cortex, with a strong imbalance in other metals, such as calcium (Ca) (Saito et al., 2000). Such an imbalance also occurs in the human brain, indicating that the balance between Zn and Ca is fundamental to the maintenance of brain functions during aging (a schematic representation is reported in Fig. 1). The uptake of Zn in neurons and its transport into the brain occur with various mechanisms. One mechanism of Zn transport into the brain is via the brain barrier system. It has been reported that the transport of Zn into the brain may occur by means of binding it to L-histidine (His) using bloodbrain barrier. Zn-His transfers Zn ions to the plasma membrane protein DCT1, which is a divalent metal transporter (Gunshin et al., 1997). DCT1 mediates active transport, that is, proton-coupled and strictly dependent on the membrane potential of the cells (Gunshin et al., 1997). Alternatively, Zn is transported into the brain via the blood-CSF barrier. This is shown

Zinc, Brain, and Aging

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T

Y

Krox-20, NF-kB, Sp1 c-fos, c-jun n

NO

No protein Altered intercellular phosphorylation signaling

Altered circadian rhythms

Impaired neuronal functions

Fig. 1. Schematic representation of the role of Zn starting from the extracellular compartment until its entry into neurons in aging, and the possible effect on glutaminergic transmission and MT homeostasis leading to dysfunctions in the brain's biochemical pathways (PARP-1 activity, NO production, PKC activity, membrane depolarization, and brain genes clock functions related to circadian rhythms). (A) The reduced number of zn ions present in the extracellular compartment during aging are transported into neurons preferentially by ZnTl-T4 transporter proteins. Low Zn ion bioavailability in the extracellular compartment may provoke low Ca + + regulation, leading to dysfunctions in the glutaminergic system. (B) When Zn ions enter the neurons, they are captured by MT. This leads to the limited release of Zn by MT due to altered glutathione activity, which is caused by persistent stress in aging. This condition provokes a series of altered Zn-dependent biochemical pathways, finally resulting in impaired neuronal functions. AMPA=alpha-amino-3-hydroxy5-methyl-4-isoxazolepropionic acid, DNA=deoxyribonucleic acid, GABA=7-aminobutyric acid, GSH=glutathione, GSSG=glutathione desulfide, MT=metallothionein, NMDA=N-methyl-D-aspartate, NO= nitric oxide, PARP=poly (ADP-ribose) polymerase, PKC=protein kinase C, Zn=zinc.

in the increased amount of Zn in the choroid plexus after one hour of 65 Zn injection. On the third day after injection, relatively high levels of 65Zn were seen in the dentate gyrus and CA3 region of the hippocampus and cerebral cortex (Takeda et al., 1994). After Zn is transported into the brain

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via the blood-brain barrier, the uptake in the brain parenchyma occurs by means of the Zn transporter proteins family (ZnTl-4), which acts as a pump in order to maintain a balance between intra- and extracellular Zn concentration (McMahon and Cousins, 1998). As such, Zn is taken up by the neurons and glia cells, and is then incorporated into Zn-binding proteins, mainly MT-III (McMahon and Cousins, 1998). Concomitantly, the Zn-T3 protein in Zn glutaminergic neurons transports Zn ions in presynaptic vesicles and are then utilized as neuromodulators (Frederickson, 1989). Zn-Tl, Zn-T2, and Zn-T4 transport the excess Zn ions out of the cells across the plasma membrane (McMahon and Cousins, 1998). MT-III is also a key protein in intracellular Zn traffic. It is expressed only in the brain and is preferentially located in regions that have a high density of Zn-containing neurons, such as the hippocampus (Masters et al., 1994). Mice lacking the MT-III gene display normal Zn in their Zncontaining boutons (Erickson et al., 1997), indicating that MT-III is not an obligatory carrier in the process of getting Zn into neurosecretory vesicles. Other MT isoforms (I+11) are also involved in trafficking Zn into the brain (Mocchegiani et al., 2001). The main task of MT-III is to sequester and release Zn under transient stress, such as in freeze lesioned rat brain (Carrasco et al., 1999), into brain Zn-dependent enzymes in order to prevent oxidative damage. The absence of MT-III has been suggested in the development of AD (Uchida et al., 1991; Uchida, 1994). However, high concentrations of MT (I+11) are present in the brains of AD patients (Zambenedetti et al., 1999) and old rats (Mocchegiani et al., 2001), coupled with Zn deficiency in both conditions (Fabris and Mocchegiani, 1995). These last findings suggest that Zn-bound MT (all isoforms) plays a pivotal role in brain Zn homeostasis during development and aging (see next section). In contrast, Zn-T3 is more involved in trafficking and concentrating Zn within the secretory vesicles destined for the boutons. Knockout mice lacking the Zn-T3 gene display no histochemically reactive Zn in their vesicles (Federickson et al., 2000) and they are especially seizure-prone (Cole et al., 2000). It has also been reported that the gene expression of Zn-T3 is markedly reduced in the hippocampal region of a young SAMP-10 mouse (Saito et al., 2000). In this context, no data are available in the old brain. In any case, findings in the SAMP-10 mouse suggest that Zn transport by Zn-T3 is impaired during aging, with subsequent

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deficiencies in the synaptic vesicles of the mossy fiber and low glutaminergic excitotoxicity in the hippocampal neurons (Saito et al., 2000). On the other hand, reduced Zn in the mossy fibers (Mocchegiani et al., 2001) and decreased synaptic number are also reported in old rats with impaired synaptic plasticity (Bertoni-Freddari et al., 1996). The uptake of Zn in the cytosol for transport into the vesicles by Zn-T3 may also occur via the voltage-gated calcium channel, NMDA receptor, and calcium permeable alpha-amino-3-hydroxy-5-methyl-4isoxazolepropionic acid (AMPA)/kainate receptor (Weiss et al., 2000). From these findings, the relevance of Zn in the synaptic vesicles is evident, particularly in the hippocampal mossy fiber system. From the presynaptic vesicles, Zn is released into the post-synaptic vesicles via the NMDA receptor, assuming thus the role of a neuromodulator. However, the physiological role of presynaptic Zn release is relatively little understood. In any case, it is clear that Zn is involved in the function of glutaminergic synapses. It has been reported that Zn is co-released with glutamate from the presynaptic vesicles into the post-synaptic vesicles via the NMDA and AMPA receptors present in the post-synaptic vesicles (Smart et al., 1994). Electrophysiological studies on cultured neurons found that Zn weakens the NMDA receptor-mediated response through both voltage-dependent and voltage-independent mechanisms, and potentates the AMPA-receptor-mediated response (Smart et al., 1994; Dingledine et al., 1999). Thus, the co-release of Zn with glutamate might have the net effect of shifting the excitation from the NMDA receptors to favor the net activation of AMPA and/or kainate receptors. Indeed, recent electrophysiological studies have shown that Zn release from the mossy fibers provides tonic inhibition of the NMDA receptors on CA3 pyramidal neurons (Vogt et al., 2000). Ca + + ions use independent NMDA or AMPA receptors for synaptic transmission (Dingledine et al.,1999). Thus, Zn is a regulator of Ca + + channels in synaptic transmission. However, excess Zn may also translocate in post-synaptic vesicles, thereby inducing neurotoixicity (Weiss et al., 2000), which is due to the interaction of Zn with both ionotropic and metabotropic glutamate receptors (Bresink et al., 1996). But, at the same time, the remaining Zn ions are taken up by glia cells for Zn translocation to the presynaptic vesicles in the newly regulated NMDA and AMPA receptors for Ca + + transmission (Takeda, 2000a). Thus, if the second uptake of Zn ions is low, a defective circle appears with subsequent

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impaired glutaminergic transmission. Therefore, correct Zn ion homoestasis may be relevant to normal synaptic transmission. Although this assumption requires further study, neurodegenarative diseases, such as PD, display Zn deficiency (Forsleff et al., 1999), dysfunction in the NMDA receptors (Le and Lipton, 2001), and altered glutaminergic transmission (Eisen and Calne, 1992). Studies in aging revealed that the brain glutaminergic system is weakened, particularly in the frontal cortex, hippocampus, and striatum (for a review, see Segovia et al., 2001). Although Palmer (2000) reports that NMDA receptors are unaffected by aging, data abounds which show defects in the NMDA receptors during aging in both animals and human. The hippocampus and neocortex area are the most involved, leading to impaired learning and memory (Clayton et al., 2002; Magnusson, 2000a; Magnusson et al., 2000b, 2002; Villares and Stavale, 2001; Cady et al, 2001; Kuehl-Kovarik et al, 2000; Migani et al., 2000; Newman et al., 2002). Moreover, AMPA receptors decrease in certain areas of the old brain (Magnusson and Cotman, 1993), while kainate receptors are unaffected by age (Nicolle et al., 1996). Caloric and dietary restriction, but not Zn-restricted diet, restores NMDA and AMPA receptors' activity in old rats (Eckles-Smith K et al., 2000; Magnusson, 2001). As described above, low Zn concentration is present in these areas of the old brain. Therefore, Zn ion bioavailability is fundamental to glutaminergic neurotransmission and subsequent synaptic plasticity during development and aging. Besides these effects on glutamate receptors, Zn binds to the GABA receptors and weakens the GABA-mediated response, particularly on immature neurons that possess receptors which incorporate the 7-subunit (Smart et al., 1994). Conversely, in the adult brain, Zn enhances synchronized GABA release (Smart et al., 1994). Because Zn is released at glutaminergic terminals, the direct GABA receptor-mediated effect is uncertain. Interactions between glutaminergic and GABA receptors may then take place. Indeed, neuronal death in cerebral ischemia is partly due to the ratios of glutamate, glycin, and GABA released extracellularly (excitotoxic index) (Globus et al., 1991). Interaction of Zn with these receptors could, therefore, modify the excitotoxic index and be regionspecific due to the localization of neurons containing vesicular Zn. Indirect evidence of the interactions between Zn and these receptors include the collapse of GABA receptors by high Zn release in kindling

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models of epilepsy displaying aberrant mossy fiber sprouting (Coulter, 2000). Zn modulates both the glutamate and GABA responses of neurons that contain only specific splice-variant receptor subunits of NMDA and GABA, respectively (White and Gurley, 1995). The dissociation constants for Zn antagonism of NMDA and GABA responses are approximately 13 (jumol/L and 11 |xmol/L, respectively (Mayer and Vyklicky, 1989). These dissociation constants are quite similar and are well within the concentrations released during synaptic activity. This implies a delicate balance between the NMDA and GABA responses via Zn, with subsequent Zn involvement in the regulation of neurotransmission. On aging, there is a paucity of data on GABA receptors and Zn. GABA receptors and their response, as well as interactions with the glutaminergic system, seem diminished in the nucleus accumbes, but remain unaffected by age in the striatum (Segovia et al., 1999). Other authors have reported an enhancement of GABA in the forebrain neurons of aged rats (Griffith and Murchison, 1995). A decrease in binding site affinity, rather than in receptor density, has been reported in the brains of old rats (Turgeon and Albin, 1994). Further studies are needed to clarify this point.

2.2. Zinc, Enzymes, Neurotransmitters, Hormones, Cytokines, and Aging 2.2.1. Zinc and brain enzymes Several Zn-related enzymes that are relevant to brain development and function are listed in Table 1. The relevance of these enzymes has been studied in the brain during Zn deficiency. Low Zn ion bioavailability is often the cause of diminished activity of Zn-dependent antioxidant enzymes in brain injury (Lewen et al., 2000) and aging, in which oxidative stress and the appearance of free radicals are persistent (Mocchegiani et al., 2000a, 2000b). On the other hand, the relevance of Zn-dependent brain enzymes against persistent oxidative stress has also been observed in accelerated aging in humans (Down's syndrome), displaying Zn deficiency and altered brain functions (Fabris et al., 1993). Observations of the decreased activities of the myelin marker enzyme 2',3'-cyclic nucleotide phosphohydrolase and brain alkaline phosphatase, as a result of Zn deficiency, are important as these enzymes are involved in myelination and brain maturation (Dreosti, 1984). With advancing age,

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Table 1. Effect of Zn status on the activity of enzymes, neurotransmitters, neurohormones, cytokines, transcriptional factors, and brain genes clock in murine brain*.

Variable 2'-3'-cyclic nucleotide 3' -phosphohy drolase Alkaline phosphatase Glutamate dehydrogenase, in vivo in vitro Dopamine-(3-hydroxylase Phenylethanolamine-N-methyltransferase Glutamate decarboxylase Intracerebral Zn Thymidine kinase Superoxide dismutase

Zn status

+ +

Activity

Brain region Ce Ce, Hi Fetal brain Ce, Hi Hi, Nc Co, Ce, Hi Co, Ce, Hi A, CN, Ht Hi Fetal brain Nc

Neurotransmitters Norepinephrine Dopamime Opioid system Enkephaline

Pvn Pvn Hi Hi

Neurohormones Thyrotropin-releasing hormone Somatostatin Gonadotrophin-releasing hormone Melatonin

Ht Ht Ht SCh

Cytokines Interleukin-1 Interleukin-6 Tumor necrosis factor-a 7S-nerve growth factor

Ps Ps Ps Nerve

Transcriptional factors NF-kB, TFIIIA, SP1 LIMK-1, LIMK-2, Krox-20, Zif-268 (zinc fingers) c-fos, c-jun, egrl, egr3, nur77 (brain genes clock)

Neurons Neurons SCh

*The same alterations also occur in the old brain (see text). For references, see text. — = diminished, + = increased, 0 = unaffected. A = amygdala, Ce = cerebellum, CN = caudate nucleus, Co = cortex, Hi = hippocampus, Ht = Hypothalamus, Nc = neocortex, Pvn = paraventricular nucleus, Ps = paravesicular spaces, SCh = suprachiasmatic nucleus, Zn = zinc.

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the activity of alkaline phosphatase decreases, leading to diminished permeability of the cerebral microvessels and, subsequently, impairment of the blood-brain barrier (Agrawal et al., 1996). Alkaline phosphatase, via adenylate cyclase, is also involved in p-adrenergic receptor activity (Yamashita et al., 1990), which in turn is reduced in the brain cortex (Viticchi et al., 1999) and cerebral microvessels (Mooradian and Scarpace, 1991) of old rats. Another enzyme, superoxide dismutase (SOD), which protects against damage by superoxide radicals, is important as it increases dramatically in the first two months, postnatally in rat brain, and declines thereafter with low activity in aging (Gupta et al., 1991). Such a decline is often associated with brain membrane injury (Prasad, 1993) and, in some cases, with neuronal cell death during oxidative stress, such as in aging (Cookson and Shaw, 1999). SOD protects catecholamines from being oxidated by superoxide. Indeed, during development, its activity is much higher in synaptosomes and catecholaminerich area of the brain (Ledig et al., 1982), and catecholamines decrease in aging (Rehman and Masson, 2001). Changes in the activity of glutamate dehydrogenase, as a result of Zn deficiency, has an important effect on the metabolism of neurotransmitter glutamic acid and glutaminergic neural system (Dreosti, 1984). Thymidine kinase activity, which is related to cell proliferation, is strongly reduced (53%) in brains of Zn-deficient fetuses (Record and Dreosti, 1979). The activity of these last two enzymes declines during aging in the synapses, mitochondria, and cerebellum (Caron and Unsworth, 1978), thereby impairing neurotransmission and spatial memory. Glutamate decarboxylase, an enzyme involved in the synthesis of the inhibitory neurotransmitter GAB A, has been studied in relation to excess cerebral Zn rather than Zn deficiency. Intraventricular injection of Zn into the brains may be epileptogenic (Ebadi and Pfeiffer, 1984). At physiological concentrations, Zn stimulates the enzyme pyridoxal kinase to form the co-factor pyridoxal phosphate, thereby enhancing the activity of glutamate decarboxylase. At supraphysiological levels, Zn inhibits glutamate decarboxylase directly and reduces the binding capacity of GABA receptor sites (Mills, 1989). Epilepsy in humans, as well as epilectic fits in rats, by commissural kindling display indeed increased the level of hippocampal Zn (Mills, 1989). Recently, the activity of glutamate decarboxylase was studied in old mice and rats and, therefore, under the

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condition of Zn deficiency. Its activity is gender-dependent and decreases in the neocortex, hippocampus, and striatum (brain area is poor in Zn in aging) in old male mice (Frick et al., 2002). In old rats, glutamate decarboxylase activity is higher in the vestibular nuclei after vestibular lesion, suggesting a recall of Zn for the normal GAB A system (Giardino et al., 2002). The last finding is in line with that of Mills (1989) and reflects the distribution of Zn in various brain areas. Moreover, dopamine-(3-hydroxylase and phenylethanolamineN-methyltransferase enzymes, which convert dopamine to norepinephrine and norepinephrine to epinephrine, respectively, are involved in neurophysicological symptoms. It has been reported that the levels of norepinephrine and dopamine are higher in the brain of Zn-deficient weaning rats with a role in catecholamine alterations (Wallwork et al., 1982). Others have reported that these enzymes are involved in dopamine activity and are decreased in certain regions of Zn-deficient rat brain (Wenk and Stemmer, 1982). However, decrements in norephinephrine and dopamine, especially during the dark period in the paraventricular nucleus from Zn-deprived rats (Huntington et al., 2002) and in aging (Segovia et al., 1999), occur. Therefore, the neurotransmitters norepinephrine and dopamine are altered by Zn deficiency and in aging.

2.2.2. Zinc and brain

neurotransmitters

Of interest is the interaction between zinc and opioid peptides in hippocampal mossy fibers (Table 1). Early findings have shown that Zn ions block [3H]-D-Ala2-Met5-enkephelamide binding to rat membrane in the hippocampus. Thus, Zn plays a role in regulating opioid peptides in the brain regions where they are co-localized (Stengaard-Pedersen et al., 1981). Thiol reducing agents restore the binding capacity of ZnCl2-treated membranes, suggesting that oxidation of opioid receptor SH groups by Zn ions is the mechanism through which Zn blocks opioid binding (Stengaard-Pedersen et al., 1981). The following evidence suggest interactions between Zn and opioid peptides in the hippocampus: Zn and enkephaline-containing opioid peptides are selectively localized in the hippocampal mossy fibers; Zn blocks opioid binding; Zn complexes with enkephalines in vitro; and Zn and opioid peptides, when administered intraventricularly, induce limbic seizures (for a review see Mills, 1989). These findings suggest that opioid peptides and Zn in the mossy fibers interact in the physiological regulation

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of hippocampal excitability. These neurotransmitters (opioids) act synergistically with an excitatory amino acid during hippocampal synaptic transmission. Together, they could effectively regulate the normal excitability and seizure threshold in limbic circuits, possibly by altering the degree of GABAergic inhibition in the hippocampus. It is not a simple coincidence that anorexia displays reduced appetite, impaired synaptic transmission, a strongly altered opioid system, and abnormalities in limbic functions (behavior and cognitive functioning); anorexia is the major symptom of Zn deficiency in the brain (Takeda, 2000a; Takeda et al., 2000b). In this context, it parallels aging, taking into account that Zn deficiency and an altered opioid system followed by abnormalities in the limbic system are also observed in aging (Rehman and Masson, 2001).

2.2.3. Zinc and brain hormones Zinc is essential to a variety of neurohormones (hormone-releasing factors) secreted by the hypothalamus and hormones which secondarily affect brain functions (Table 1). In the first group, TRH and somatostatin are of interest because both affect the pituitary-thyroid axis which, in turn, affects brain functions (Morley et al., 1980). Moreover, gonadotrophin releasing hormone (GnRH) affects the sexual glands. Lack of maturation of the sexual glands provokes hypogonadism, which is characterized by Zn deficiency and delay in brain development (Prasad, 1993). In the second group, melatonin production from the pineal gland in the suprachiasmatic nucleus is peculiar because it affects circadian rhythms, including those related to Zn turnover and brain functionality, such as glucocorticoids (Mocchegiani et al, 1998b). Somatostatin release from the hypothalamus is lower in Zn-deficient rats and in aging (Rains and Shay, 2001), leading to an impairment in limbic functions (Takeda et al., 2000b). In contrast, increased somatostatin, as it occurs in hypothermia, enhances Zn in the mossy fibers by protecting neuronal cells through cell death (Johansen et al., 1993). This supports the notion that Zn in the hypothalamus helps to maintain the brainneuroendocrine network during aging (Fabris, 1997a; Fabris et al., 1997b). Moreover, Zn potentates NMD A receptor activity via somatostatin (Tapia-Arancibia, 1990). Taking into account the fact that somatostatin affects growth hormone/IGF-1 axis, which is in turn Zn-dependent and decreases in aging (Fabris et al., 1997b), the recent discovery of

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IGF-1 in restoring NMDA receptors in aging (Sonntag et al., 2000) is further evidence of the role of Zn in neurohormones' release and activity. TRH decreases Zn deficiency (Morley et al., 1980). This affects TSH production and turnover of peripheral thyroid hormones, leading to hypothyroidism (Beck-Peccoz and Persani, 1994) with the subsequent appearance of impaired cognitive functions, which occurs in Down's syndrome subjects, the elderly, and experimental hypothyroidism (propyltiouracil treated mice) (Mocchegiani et al., 2002a). Indeed, Zn deficiency and thyroid hormone shortage which occur in both cretinism and myxedema, have similar symptoms, that is, delayed growth, reduced appetite, and activity (Hartoma et al., 1979). Although no correlation exists between Zn and GnRH in the hypothalamus, indirect evidence shows that GnRH release and production is controlled by the Zn-finger protein DAX-1 (Kottler et al., 1999). Decrements in GnRH affect sexual maturation with the appearance of hypogonadism, which displays Zn deficiency and impaired cognitive functions (Ozata et al., 1999). In aging, hypogonadism does not exist, but decrements in GnRH and the production of sexual hormones by peripheral glands do occur (Khoury and Sowers, 1988). As for melatonin production by the pineal gland in the suprachiasmatic nucleus, Zn plays a pivotal role as the nocturnal peak of melatonin decreases in Zn deficiency and in aging (Mocchegiani et al., 1998b). The decrement in melatonin production in aging affects circadian rhythms in adrenocorticotropic release, with increments of glucocorticoids in the nocturnal period (Mocchegiani et al., 1998b) leading to cell death, including neurons (Nichols et al., 2001). A correlation has been shown between decrement in nocturnal melatonin production and affective disorders in multiple sclerosis patients (Sandyk and Awerbuch, 1993), who also display Zn deficiency (Fabris and Mocchegiani, 1995). Although no conclusive data is available on the precise role of Zn in hormone-releasing factors from the hypothalamus, these findings pinpoint on the fact that Zn deficiency in the brain and aging may also affect the neuroendocrine network. This induces an impaired response from the peripheral endocrine glands, which in turn affect brain functions by means of specific receptors in the brain with resultant negative consequences on behavior and cognitive functioning (Morley et al., 1980; Fabris et al., 1997b; Mocchegiani et al., 2002a, 2000b).

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2.2.4. Zinc and brain cytokines Cytokines are various polypeptides that mediate cellular response in immunity and inflammation in the peripheral nervous system and CNS (Rothwell and Hopinks, 1995). An inflammatory condition is known to deplete Zn ion bioavailability (Taubeneck et al., 1995) and inflammation is known to persist in aging (Mocchegiani et al., 2000a, 2000b). A mechanism by which Zn is depleted is through enhanced activities of Zn requiring the enzymes (involved in DNA/nucleic acid synthesis) of mitogen or cytokine-responsive cells (Odeh, 1992). Some cytokines, especially those related to inflammatory status, such as TNF-a, IL-1, and IL-6, have been implicated in agerelated neurological dysfunctions and neurodegenerative conditions (McGeer and McGeer, 1995). These cytokines are Zn-dependent (Fabris et al., 1997b) and, at the same time, affect Zn turnover (Mocchegiani et al., 1998a). TNF-a causes in vivo alterations in Zn metabolism, leading to Zn depletion known as hypozincemia (Taubeneck et al., 1995). Increments of TNF-a induce cell death, via the NF-kB transcriptional factor (Beg and Baltimore, 1996), and are an index of poor prognosis in constant and severe inflammations (Rodriguez-Gaspar et al., 2001). IL-1 (Goldblum et al., 1987) and IL-6 (Mocchegiani et al., 1998a) also induce hypozincemia in inflammation (in aging). However, they are strictly correlated with depressed brain functions, such as synapses number (Bertoni-Freddari et al., 1996), (3-adrenoreceptor density (Viticchi et al., 1999), and altered behavior and cognitive functioning (Reichenberg et al., 2001). Zinc has also been histochemically identified in the oligomer 7S-NGF, a complex with three distinct subunits: a, (3, and 7 (Pattison and Dunn, 1975). 7S-NGF has high affinity for Zn (Dunn et al., 1980). Two molecules of Zn are present in the complex and Zn participates by holding the structure together. It has been observed, in vitro, that chelation of Zn from 7S-NGF using 2,2',2"-terpyridine or 8-hydroxyquinoline-5-sulfonic acid activates the -y-esteropetidase enzyme. This leads to the liberation of a, p, and 7 subunits from the oligomer and inactivation of NGF (Dunn et al., 1980). NGF is required for the survival and development of sympathetic and sensory neurons, as well as for a wide range of other cells. NGF is present on the nucleus and at the synaptic ending (Dunn et al., 1980). NGF also plays a crucial role in cytokine/neurotrophin interaction, particularly in the signaling pathways for neurogenesis (MacDonald et al., 2000).

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NGF is an antioxidant and interacts with glucocorticoid receptors to prevent apoptosis (McLay et al., 1997). In aging, NGF production decreases. This has been suggested as a cause for the appearance of classical age-related neurodegenerative disorders, such as dementia (Rylett and Williams, 1994). Indeed, treatments with NGF have been suggested to prevent neurodegeneration (Williams, 1995) and to restore the activity of Zn-dependent antioxidant enzymes (Nistico et al., 1992), as well as to improve cognitive functions (Kiprianova et al., 1999).

2.2.5. Zinc, brain metallothionein,

and aging

MT is crucial to Zn homeostasis (Vallee and Falchuk, 1993). It is a low molecular weight protein with 61 to 68 amino acids, 20 of which are represented by cysteine residues in conserved positions. Currently, only the functional mammalian isoforms (MT-I, MT-II, MT-III, and MT-IV) have been identified (Palmiter, 1998). It is able to bind up to seven atom Zn/ molecule of the thionein protein (for a review see Vallee, 1995). MT is ubiquitous in most organs and can be induced, via activation of the factor MTF-1, as a response to stress (Jacob et al., 1999) by different endogenous or exogenous stimuli, including heavy metals, glucocorticoids, proinflammatory cytokines (IL-1, IL-6, and TNF-a) (Kagi and Schaffer, 1988; Vallee, 1995; Mocchegiani et al., 2000b), and Zn (Thakran et al., 1989). MT modulates the transfer and homeostasis of metal ions, of which Zn is pivotal (Kagi and Scahffer, 1988). MT prevents Zn deficiency and toxicity in vivo (Kelly et al., 1996); it acts as a detoxifying agent of reactive metals and free radicals (Sato and Bremner, 1993). In the cultured Ehrlich cell, the cellular concentration of Zn-bound MT is approximately 13 |xmol/L (Krezoski et al., 1988). MT, however, is not a long-term storage for Zn as it has a short biological half-life in these cells (Krezoski et al., 1988). In vesicles, Zn is bound to MT (Palmiter, 1994) and provides a crucial role for Zn homeostasis in the brain. The expression of MT isoforms is tissuespecific. MT-I and MT-II are expressed in most mammalian organs, including the brain, while the MT-III isoform is specific to the brain and MT-IV is present in the squamosa epithelia (Palmiter, 1998). MT-I and MT-II are localized in the astrocytes, perivascular spaces, and pia mater (Nishimura et al., 1992; Penkowa et al., 1999), while MT-III is abundant

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in neurons and hippocampal mossy fibers (Masters et al., 1994). MT-III may be expressed independently by MT-I and MT-II, as evidenced by the increased intensity of Timm's stain in the mossy fibers (Masters et al., 1994). MT-III sequesters Zn, (Mocchegiani et al., 2001). The role of MT isoforms (I and II) is to sequester and release Zn, particularly under stresslike conditions for Zn-dependent antioxidant enzyme activities (SOD and GSH) and other Zn-dependent enzymes involved in base excision DNA repair (PARP-1), cellular proliferation (PKC), and prevention of apoptosis (endonuclease) (Mocchegiani et al., 2000b) (see Fig. 1). Even if the role of MT-III is mainly to sequester excess Zn, which may lead to neurotoxicosis (Palmiter, 1998; Masters et al., 1994), MT-III is also involved in Zn release during stress (Mocchegiani et al., 2001). Therefore, all MT isoforms are involved in Zn homeostasis in the brain. This role of MT is peculiar to transient stress-like conditions, as it may occur in the young adult. In aging, stress-like conditions persist throughout the whole circadian cycle (Mocchegiani et al., 2002b). This provokes continuous sequestration of Zn by MT with no Zn release, leading to low Zn ion bioavailability for many biological functions, including the brain functions (Mocchegiani et al., 2001) described here. Therefore, increments of MT isoforms may transform from being useful under transient stress to being harmful under constant stress, such as aging (Mocchegiani et al., 1998a; Mocchegiani et al., 2000b). Increments of MT-I and MT-II have been found in the astrocytes of old rats (Mocchegiani et al., 2001) and AD patients (Zambenedetti et al., 1998). MT-III gene expression is also enhanced in the hippocampus of old mice (Giacconi et al., 2002). Conversely, MT-III is poorly expressed in the brains of AD patients, thereby implicating it in the development of AD (Uchida et al., 1991; Uchida, 1994). However, the presence of high MT I+II isoforms in the brains of old mice and AD patients leads one to interpret the increments of MT isoforms as being harmful to brain functions in aging and dementia. The existence of low Zn ion bioavailability and impaired brain functions in aging and dementia (Fabris and Mocchegiani, 1995) is in line with this interpretation. However, the presence of compensatory phenomena among the various isoforms of MT (I, II, and III) in the brain (Penkowa and Hidalgo, 2000) gives rise to intriguing aspects of the role of MT isoforms in the brain in aging. This represents an interesting topic for future research in the aged brain.

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2.2.6. Zinc and gene expression Zinc also plays a key role in DNA "Zn finger" binding motifs. As such, it is involved in the gene expression of many proteins, transcriptional factors, and receptors, which are important to the functioning of body homeostatic mechanisms. Zn fingers are crucial for brain development and maintenance. The removal of Zn provokes an incorrect production of Zn finger proteins and subsequent damage to organs or tissues, including the brain (Ebadi et al, 1990; Vallee and Falchuk, 1993). LIMK-1 and LIMK-2 are two Zn finger motifs expressed preferentially in the CNS during mid-to-late gestation. They are mainly present in the deep nuclei of the cerebellum. In adult life, their expression decreases and are mainly expressed in the cerebral cortex (Mori et al., 1997). This last fact also leads to low gene expressions of these Zn-finger proteins in aging, even if no data are still available. The Zn finger Krox-20 is expressed in the hindbrain and cranial nerves during embryogenesis governing the process of segmentation (Schneider-Maunoury et al., 1993). No data exist in the literature on the role of Krox-20 in aging. However, knockout Krox-20 mice display delayed or absent myelination (Mirsky and Jessen, 1999), which is important in nerve regeneration in CNS injury (Levine et al., 2001) and in aging (Lalonde and Badescu, 1995). The Zn finger Zif268 is involved in synaptic plasticity. It is expressed in hippocampal granule cells and induced by NMDA receptors (Worley et al., 1993). However, its expression is strictly related to other important gene expressions, such as c-fos, c-jun, and egrl, which are also Zn finger proteins (Morris et al., 1998). The lack of expression in c-fos, c-jun, and egrl provokes an impaired synaptic plasticity despite the fact that Zif268 is normally expressed (Worley et al., 1993). This was recently shown in egrl knockout mice (Mataga et al., 2001). Therefore, c-fos, c-jun, and egr-1 gene expressions are crucial to synaptic transmission. The relevance of these proteins is still more intriguing because c-fos, together with egr-1, egr-3, and nur77, is the brain gene clock in the suprachiasmatic nucleus driving circadian rhythms (Morris et al., 1998). Also, egr-3 and nur77 are codified by Zn finger motifs. Taking into account the relevance of circadian rhythms in the maintenance of body homeostatic mechanisms (Touitou et al., 1997), the bioavailability of Zn ions is also crucial to brain circadian rhythms.

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Indeed, preliminary data from our laboratory show that the gene expressions of c-fos and c-jun are low in old mice, coupled with the absence of Zn circadian rhythms, compared to young mice (Mocchegiani, unpublished observation). Zn is also vital for the gene expression of many transcriptional factors and enzymes, which are also relevant to other cell functions. Among them, TFIIIA, Spl, and NF-kB are the most well-known Zn-dependent transcriptional factors involved in brain functions (Cuajungco and Lees, 1997). The removal of Zn by chelation disables TFIIIA, SP1, or NF-kB binding activity to DNA (Yang et al., 1995). The gene expression of the estrogen receptor (EsR), erythroid receptors (GATA-1, NF-E1, and GF-1), and glucocorticoid receptors is also affected by Zn ion in the brain. It is also interesting to note that Zn-dependent enzymes (PKC and PARP-1), Zn-dependent antioxidant enzymes (SOD and GSH), and endonuclease enzymes, which are involved in the prevention of apoptosis, are also affected by Zn in the brain (for a review see Cuajungco and Lees, 1997). During aging, PKC activity is downregulated in the brain, leading to apoptosis (Jin and Saitoh, 1995; Pascale et al., 1998). Poly(ADP-ribose) polymerase-1 (PARP-1) is activated by DNA strand breakage, which induces DNA repair under transient stress and cell death under persistent stress (Szabo and Dawson, 1998). In aging, low Zn ion bioavailability induces PARP-1 towards cell death because of diminished NAD+, which is due to too much energy consumption (Mocchegiani et al., 2000b, 2002c). The activity of endonuclease enzyme, together with low Zn ion bioavailability, is impaired in aging with limited prevention of apoptosis (Barbieri et al., 1992). A plethora of data report that SOD and GSH activities decrease in the old brain. Subsequently, there is no protection against oxidative damage by free radicals (for a review see Melov, 2002). EsRs in the hippocampus are gender-dependent: they decreased in the old male and are maintained in the old female (McEwen et al., 1997). This fact may justify more efficient cognitive functions in the old female than in the old male (Clausen et al., 1989). Erythroid receptors decrease in aging, leading to impaired transferrin transport (Adrian et al., 1996). As for glucocorticoid receptors, they increase in the hippocampus during aging (Carroll, 2002). Knowing the effect of glucocorticoids in inducing apoptosis in aging (Fabris et al., 1997b), the increase in glucocorticoid receptors in the old brain is a sign of cellular brain aging. However, recent

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data show a decrease in glucocorticoid receptors in the hippocampus during aging, attenuating to the beneficial effect of glucocorticoids on hippocampal memory (Murphy et al., 2002). While this last finding may be in line with the beneficial effect of moderate stress (van der Klink et al., 2001), it contradicts the fact that prolonged stress induces cell death, as in aging (Fabris et al., 1997b). Therefore, more studies are needed to clarify this point.

3. ZINC, BEHAVIOR, COGNITIVE FUNCTIONING, AND AGING The role of Zn in behavior and cognitive functioning has been studied in relation to Zn deficiency during development and in adulthood. There is a paucity of data on this subject in aging. The common symptoms of Zn deficiency are anorexia and growth retardation. Bulimia is also associated with Zn deficiency and cognitive neurological dysfunctions (McClain et al., 1992). Changes in behavior, especially reduced activity and responsiveness, may be one of the components of the regulatory system of Zn that provides a sensitive index of inadequate dietary supply (Golub et al., 1995). Periods of rapid growth, such as pregnancy and infancy, are most susceptible to dietary Zn deficiency (Favier, 1992). Zn deficiency during prenatal development is teratogenic in rats (Hurley and Swenerton, 1966). Zn deprivation in rapid brain growth has effects similar to those of protein malnourishment (Halsted et al., 1972). When behavior was tested during a period of Zn deprivation in immature animals (rat and monkey), lethargy and impaired learning and memory were observed (Halas and Sandstead, 1975; Golub et al., 1994). After a limited period of Zn deprivation during development, the impairment in behavior, learning, and memory persists in to maturity (Halas and Sandstead, 1980). Impairments of cognitive performance, such as visual attention and short-term-memory, by moderate Zn deprivation were observed in young mature monkeys (Golub et al., 1994) and rats weeks of Zn-deficient diet, suggesting that long-term memory is not affected by Zn deprivation (Takeda et al., 2000b). In humans, the earliest observations of an impairment in cognitive functioning were made by Prasad et al. (1961) in poor developing countries (Iran and Egypt), where the diet is low in Zn. Children in these countries had severe growth retardation, hypogonadism, and mental lethargy. Similar observations in other developing countries (Guatemala, India, and

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Bazil), where the diet is low is Zn or contains other substances (phytates and fiber) that induce Zn loss in feces, have confirmed the earlier observations in Iran and Egypt. A paradox seen in humans relates to anorexia and bulimia, where the dopaminergic and serotinergic systems in the hypothalamus are impaired in both conditions (Patrick, 2002). These brain defects lead to depression, anxiety, and somatization disorders in anorexia and bulimia (low in anorexia and high in bulimia). Both conditions are characterized by Zn malabsorption with subsequent Zn deficiency (Patrick, 2002). In aging, a paucity of data exist on cognitive functions related to Zn. Using the Morris water maze, it has been shown that aged rats display an increase in escape latency and low Zn in synaptic vesicles compared to young rats (Guidolin et al., 1992) (Table 2). This finding suggests that spatial memory is strongly reduced in aged rats and is strictly related to Zn content in the hippocampal mossy fibers. This assumption has recently been confirmed by Keller et al. (2001), who showed that old rats have deficits in short-term memory. In the elderly, Clausen et al. (1989) demonstrated impairment in visual and cognitive functions related to low Zn bioavailability. A more precise study has been performed in a human model of accelerated aging (Down's syndrome), which displayed a marked level of low Zn ion bioavailability (Fabris and Mocchegiani, 1995). Impairments in co-ordination and attention (Das et al., 1995),

Table 2. Escape latency and Zn staining in the hippocampus of rats*. Variable

Young rat

Adult rat

Old rat

Escape latency (sec) Zn staining (optical density)

8.5 ± 1.3 0.3 ± 0 . 5

11.4 ± 1.7 0.24 ± 0 . 3

62.5 ± 7.4+ 0.15±0.4t

*Spatial memory was assessed using the Morris water maze task. Each rat received two blocks of four trials per day for five days during the screening week. Escape latency to reach the hidden platform was automatically recorded. A series of l-u,mol/L semi-thin sections of the dorsal hippocampus was obtained. Sections of the CA3 region were stained in Timm's reagents to visualize Zn and be used for light microscopy. Zn granules in the vesicular region were analyzed by a computer-assisted image analysis system (IBAS; Kontron, Germany) and measured as optical density. tp<0.001 when compared to young and adult rats (ANOVA test). Zn = zinc. Redrawn, from Guidolin et al. (1992) with permission.

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memory (Roeden and Zitman, 1995), and comprehension (Licastro et al., 2001) are observed. These deficits in cognitive functioning are often present in the elderly (Capurso et al., 2000). More studies are, however, required in aging.

4. MECHANISMS WHICH LINK ZINC TO BRAIN AGING AND NEURODEGENERATIVE DISEASES Of all the mechanisms linking Zn to brain aging, oxidative stress and apoptosis play a pivotal role. Both conditions increase in aging and, coupled with a persistent inflammatory status (Mocchegiani et al., 2000a, 2000b), lead to neurodegeneration and correlated diseases, of which PD and Alzheimer-type dementia are the most common. In this context, Zn may be crucial in that it is involved in preventing oxidative damage via Zn-dependent antioxidant enzymes and proteins and, in preventing apoptosis, via endonuclease enzymes or PARP-1 activity. However, Zn may also be toxic in brain functions because, as discussed above, it may accelerate excitotoxicity in the synaptic vesicles with subsequent damage in synaptic transmission (Weiss et al., 2000). Here we discuss oxidative stress and the neurotoxicity of Zn in the brain. For apoptosis, the reader is referred to the excellent review by Troy and Salvesen (2002).

4.1. Oxidative Stress It has been proposed that oxidative stress due to diminished antioxidant defenses and/or increased production of reactive oxygen species (ROS) and free radicals could initiate a cascade of events that lead to neurodegeneration (Olanow, 1993). However, the possibility that the production of free radicals is a consequence of underlying degenerative processes, rather than a major cause of degeneration, requires more corroboration (for a review see Coyle and Puttfarcken, 1993). Recent reports suggest Zn involvement in the regulation of oxidative stress and, in particular, an association with the glutathione (GSH) antioxidative system (Ryu et al., 2002). "Energy release" involves aerobic and anerobic mechanisms. In the aerobic process, the reduction in the flow of molecular oxygen (0 2 ) to water (H 2 0) leads to the formation of ROS (Olanow, 1993). ROS and free radical species, such as superoxide radical (0 2 ), hydrogen peroxide (H 2 0 2 ), peroxynitrite (ONOO ), nitric oxide (NO), hydroxyl radical (OH"), and

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hypochlorous acid (HOC1), can cause oxidative stress and cell death when their rate of production exceeds the cellular capacity to metabolize them (Halliwell and Gutteridge, 1984). The glutathione peroxidase (GPO) family is an important cellular antioxidant defense mechanism (Jenner, 1993). Peroxidases catalyze the reduction of H 2 0 2 to H 2 0 and 0 2 . Oxidation of GSH, catalyzed by GPO, results in the formation of oxidized glutathione (GSSG; glutathione disulfide). Glutathione reductase (GR) catalyzes the reduction of GSSG back to GSH. In vivo depletion of GSH shows a marked correlation with a decrease in GR activity in rat brains, thus reducing the capacity to lower GSSG and increasing the vulnerability to oxidative stress (Barker et al., 1996). GSH may also be converted into mercapturate products catalyzed by glutathione S-transferase (GST) or degraded back to cysteinylglycine and -/-glutamyl-transpeptidase (Jenner, 1993). GSH and GSH-related enzymes are differentially distributed in the CNS (Huang and Philbert, 1995). Disulfide products are abundant in cellular systems and were recently found to interact with MT by facilitating the release of Zn from its clusters (Maret, 1995). Therefore, alterations in the GSH/GSSG ratio that result in increased concentrations of GSSG and other biological disulfides could release and elevate cytoplasmic Zn levels. As a homeostatic response, increased production of MT and/or efflux of Zn may occur (see Fig. 1). Both Zn (Bray and Bettger, 1990) and MT (Sato and Bremner, 1993) have been proposed as antioxidants against ROS and free radicals. However, as discussed in the section on Zn and brain MTs, under constant stress in aging MT isoforms sequester Zn, but are not able to release Zn (Mocchegiani et al., 1998a, 2000b, 2001). While increments in Zn-bound MT may be useful against oxidative stress, low Zn ion bioavailability appears to expedite the efficiency of other brain functions, including synaptic transmission. Hence, an imbalance arises between MT and the GSH oxidative system. This is because the latter needs Zn for its activity (Ryu et al., 2002). Therefore, a correct Zn turnover, via MT and the GSH system, must exist to maintain brain efficiency. The correct balance between Zn turnover and MT is also needed for NO production in the maintenance of the immune response in aging (Mocchegiani et al., 2000b). This is because NO is also involved in Zn release from MT (Zangger et al., 2001). Thus, Zn-bound MT homeostasis, via GSH or NO, is crucial to the maintenance of a correct Zn turnover. Indeed, both low (as discussed above)

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and high Zn are toxic in many apparatus and systems, including the brain. The interplay among Zn, MT, and GSH or the NO antioxidant system is an important event linking their functions to cellular senescence and death. In the next section, we report evidence of Zn toxicity in the brain.

4.2. Neurotoxicity of Zinc Zn is rather nontoxic compared to other transition metals, such as manganese and iron. However, it has been demonstrated that excess Zn in the extracellular fluid is neurotoxic in the brain (Choi and Koh, 1998). Zn promotes in vitro aggregation of amyloid-(3 protein, the main component of the senile plaques typically observed in the AD brain (Bush et al., 1994). A high concentration of extracellular Zn triggers off the formation of amyloid plaques in the brain (Huang et al., 2000). Synaptically released Zn enters post-synaptic neurons in toxic excess during seizures, resulting in Zn loss in the post-synaptic vesicles. The excessive influx of Zn into post-synaptic neurons leads to neurodegeneration (Choi and Koh, 1998). The translocation of Zn from presynaptic neuron terminals into postsynaptic neurons also occurs after cerebral ischemia (Park and Koh, 1999) and traumatic injury (Suh et al., 2000). After transient forebrain ischemia, Zn released into the synaptic clefts accumulates in neuronal cell bodies followed by degeneration of the neurons (Koh et al., 1996). The prevention of Zn translocation using a Zn chelator has been shown to be neuroprotective in both seizures and ischemia (Koh et al., 1996). Preferential Zn influx through the calcium permeable AMPA/kainate receptor after exposure to NMDA, kainate, or high K + in the presence of 300 |jLmol/L Zn + + causes prolonged mitochondrial superoxide production (Weiss et al., 2000). Zn-dependent production of ROS may be associated with the degeneration of post-synaptic neurons. In the case of exposure of cortical cultures to Zn + + , synaptically released Zn-induced neuronal death exhibits features of both apoptosis and necrosis, and it is mediated by free radical injury (Kim et al., 1999). Recently it has been shown that inZn-T3 null mice, which lack the Zn transporter into the brain, the accumulation of Zn in the synaptic vesicles leads to a degeneration of neurons in the CA1 and CA3 areas of the hippocampus (Lee et al., 2000). In aging, an accumulation of Zn in the synaptic vesicles is not reported. The available data in adult rats (12 months of age) show the highest

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concentration of Zn in CA3 and dentate gyrus, and a decrease in Zn in the cerebellum and cerebral cortex (Sawashita et al, 1997). However, data from our laboratory show a decrease in Zn content in the hippocampus of old rats (28 months of age) (Mocchegiani et al., 2001). This finding suggests that the possible accumulation of Zn in the hippocampus may not occur in aging with no subsequent toxic effect. Thus, it is clear that more studies are necessary to better understand the real reasons why neurodegenerative diseases, such as AD, may appear in aging, since AD has a characteristic accumulation of Zn in the brain as a toxic metal (Bush et al., 1994).

5. ZINC AND AGE-RELATED NEURODEGENERATIVE DISORDERS Zn turnover is different in neurodegenerative disorders. It is diminished in some, but increased in others (see Table 3). In any case, age-related neurodegenerative disorders (AD, PD, and dementia) display diminished Zn levels. Table 3. Plasma Zn status in neurological disorders in humans*. Disorder

Zn status

Alzheimer's disease Dementia Depression Epilepsy Fifth-day fits Mental retardation Pick's disease Schizophrenia Parkinson's disease Multiple sclerosis Amyotrophic lateral sclerosis Brain ischemia Autism Comatose status (brain injury) Anorexia Bulimia

Diminished Diminished Diminished Increased Diminished Diminished Increased Diminished Diminished Diminished Diminished Diminished Diminished Diminished Diminished Diminished

*For references, see Dreosti, 1984; Mills, 1989; McClain et al., 1992; Prasad, 1993; Fabris and Mocchegiani, 1995. Zn=zinc.

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5.1. Parkinson's Disease Oxidative stress may be an etiologic factor in PD (Jenner et al., 1992). According to Dexter et al. (1989a), post-mortem studies have revealed that the polyunsaturated fatty acid levels in the substantia nigra (SN) of patients with PD were reduced compared to those in other brain regions and in control (normal) tissue. Moreover, malondialdehyde levels were elevated in the SN, suggesting lipid peroxidation (Dexter et al., 1989a). Mitochondrial SOD activities in the SN of patients with PD were also markedly increased (Dexter et al., 1989b; Dexter et al., 1991; Dexter et al, 1992; Jenner et al., 1992), while GSH levels were reduced in both earlyand late-stage PD (Jenner et al., 1992; Sofic et al., 1992). In contrast, there were no significant alterations in the activity of H 2 0 2 degrading enzymes (catalase and GPO) or in the concentrations of free radical scavengers (ascorbic acid and a-tocopherol) (Jenner et al., 1992) and GSSG (Sofic et al., 1992). Recently, it has been shown that Zn deficiency is present in PD (Forsleff et al., 1999). Such a deficiency is correlated with vision problems, olfactory loss, and loss of taste (Forsleff et al., 1999). Conversely, increments in MT have been found in PD (Ebadi et al., 1991). Based on the concepts described above an abnormal increments in Zn-bound MT, which are harmful in aging as no Zn is released due to persistent oxidative stress, it is tempting to ascribe a harmful role to Zn-bound MT in PD by inducing low zinc ion bioavailability. Hence, many brain functions are altered in PD. An intriguing point to support this assumption is the strong increase in NO in PD, with an exacerbation of the disease (Hirsch and Hunot, 2000). An abnormal increase in NO induces limited release of Zn by MT, with subsequent PARP-1 activity towards cell death rather than base excision DNA repair (Mocchegiani et al., 2000b). This condition occurs in damaged immune response in age-related diseases, such as severe infections, and displays high oxidative stress and a persistent inflammatory status (Mocchegiani et al., 2002c). The same mechanism might also occur in the PD brain because it resembles the oxidative and inflammatory status in severe infections in aging.

5.2. Alzheimer's Disease Due to the bulk of data on the biological and biochemical aspects of the AD brain, (see the excellent review by Christen, 2000), we will only

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report on some aspects of the brain in relation to Zn in AD. Zn induces amyloid-fJ aggregation in senile plaque (Bush et al., 1994). As such, the progress of AD may be accelerated because amyloid-(3 aggregation correlates with the expression of the e4 allele of apolipoprotein E, which is a characteristic in late-onset AD (Huang et al., 2000). Zn amyloid-p precipitates at low concentration < 1 0 - 6 M (Bush et al., 1994). However, other studies (Clements et al., 1996; Esler et al., 1996) do not support the report of Bush et al. (1994). Hence, the subject is rather controversial. It is interesting to note that Zn-mediated amyloid- p ^ o precipitation "per se" is not sufficient for neurodegeneration as the co-addition, specifically, of Zn in rat hippocampal primary cultures in a dose-dependent manner reduced amyloid-(31^0 neurotoxicity (Fuson et al., 1996). Post-mortem studies report that Zn is significantly reduced in the temporal lobes, including the hippocampus, and interior parietal and primary visual cortices of AD (Andrasi et al., 1995). Other investigators report no change or an increase in a specific brain area (Deibel et al., 1996). Thus, the specificity and location in brain regions/neurons of any changes in Zn require more study. It is not known if Zn depletion is simply due to a reduction in the number of Zn-containing neurons or the cause of neuronal death in specific areas of AD brains. Decrease in brain Zn concentration contrasts with the role of Zn in amyloid-|3 aggregation. This paradox may be resolved or better understood by taking into account the role of MT in the brain. The absence of MT-III has been reported to be the cause of AD as there is no prevention against aberrant neuronal sprouting and neurofibrillary tangles in AD (Uchida et al., 1991; Palmiter, 1996). Conversely, increments in MT-I+II have been found in AD brains (Zambenedetti et al., 1998). Decrements in MTIII and increments in MT-I+II may be due to compensatory phenomena among MT isoforms, as previously suggested (Penkowa and Hidalgo, 2000). Taking into account the harmful role of MT-I+II because of its exclusive sequestration of Zn with no subsequent Zn release in permanent stress and inflammation, which occurs in aging (Mocchegiani et al., 1998a), it is tempting to interpret MT-I+II as the cause of brain dysfunctions in AD, since it leads to low Zn ion bioavailability. Hence, neuronal cell death occurs with subsequent neurodegeneration. The same phenomena occur in damaged immune response in severe infections in old patients (Mocchegiani et al., 2002c). On the other hand, oxidative stress and

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persistent inflammatory status are also present in AD (Christen, 2000). Thus, while Zn may be harmful to amyloid-P aggregation, it prevents neuronal death in AD. In this context, Zn-bound MT-I+II homeostasis plays a key role. Therefore, it is necessary to examine, in more detail, this aspect of AD by studying the MT isoforms as a collective group, and not individually as has been done to date. This suggestion is supported by the fact that Down's syndrome subjects who develop AD in the long run display high MT-I+II (Mocchegiani et al., 2002b). Stressed trisomy 16 mouse (experimental model of human Down's syndrome) displays no protective role confered by MT-I+II in the brain (Scortegagna et al., 1998).

6. EFFECT OF ZINC SUPPLEMENTATION IN THE AGING BRAIN Based on the findings and concepts presented in this chapter, it is evident that Zn supplementation may be useful in the aged brain in preventing age-related neurodegenerative disorders. Physiological supplementation with Zn improves and restores the deranged immune-endocrine functions in aging, prolongs the life of old mice (Mocchegiani et al., 2000c), and enhances the resistance to infections in HIV patients (Mocchegiani et al., 1999) and in the elderly (Mocchegiani et al., 2000b). The beneficial effect of Zn supplementation in brain function is controversial in Zn deficiency, aging, and age-related neurodegenerative diseases. Some authors believe that Zn supplementation improves myelination (Dreosti, 1984), while others believe this has deleterious effects (such as cell death or necrosis) since Zn can easily enter the neurons via the Ca ++ -A/K channels, leading to its accumulation in the post-synaptic vesicles and culminating in neuronal toxicity (Weiss et al., 2000). High Zn ion concentrations are certainly toxic due to competition between Zn and other metals, including Ca + + , with the same valence shell electronic structure (Mocchegiani et al., 1998a). Moreover, a high Zn concentration is mitogenic and, as such, might lead to the proliferation of mutated DNA cells, as it does for tumoral cell growth via DNA/RNA polymerase enzyme activity (Mocchegiani et al., 1998a). Therefore, limited bioavailability exists for each metal, being narrow for some and broad for others. The beneficial effect of Zn is strictly dependent on the dose and length of treatment. Physiological doses (two to three times the RDA/day) administered over

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short periods (one to two months) and in periodical cycles are beneficial (Mocchegiani et al., 1998a). Indeed, many authors have reported on the beneficial effect of Zn supplementation (RDA dose of 15 mg Zn ++ /day over a short period of one to seven months) on cognitive functions in children from poor developing countries (for a review see Bhatnagar and Taneja, 2001). The same treatment regimen was found to improve the cognitive function of Down's syndrome subjects (Fabris et al., 1993), the elderly (Clausen et al., 1989), patients with severe head injury (Young et al., 1996), anorexics, and bulimics (Patrick, 2002). Zn supplementation also restores the low activity of (3-adrenoreceptors in the cerebral cortex of old mice (Viticchi et al., 1999). However, caution is necessary in Zn supplementation, since Zn may accelerate amyloid- (34 aggregation in senile plaque with the risk of AD developing (Bush et al., 1994). Therefore, despite the benefits that physiological Zn supplementation confers on the cognitive functions of the elderly and Down's syndrame subjects, caution is required in Zn protocols: one should bear in mind that the former may develop AD with advancing age (Leverenz and Raskind, 1998). Zn supplementation in aging does not lead to further significant increments in the already high MT protein. This suggests that MT is fully saturated by pre-existing Zn ions (Mocchegiani et al., 2002a). Moreover, Zn supplementation provokes faster degradation of MT in lysosomes and favors the complete release of Zn (Klaassen et al., 1994). Therefore, Zn supplementation plays two important roles in aging. First, it gives rise to huge Zn ion bioavailability through faster MT degradation. Second, it avoids continuous sequestration of Zn by MT. Hence, a plateau in the MT protein is maintained with a protective role for MT (Mocchegiani et al., 2002a). Zn supplementation also restores thyroid hormones turnover in aging and Down's syndrome (Fabris et al., 1993), which represents one of the mechanisms involved in improving cognitive functions in both conditions after Zn supply (Licastro et al., 2001). Therefore, good Zn ion bioavailability, via correct Zn-bound MT homeostasis, is crucial to brain function. The satisfactory nutritional profile, good antioxidant activity, normal thyroid hormones turnover, low MT expression, moderate stress, and good cognitive performances in healthy centenarians (Mocchegiani et al., 2002b, 2002c) strongly support this assumption. Altered thyroid status (Thomas et al., 1987), high MT-I+II (Zambenedetti et al, 1998), and low Zn concentration (Andrasi et al., 1995)

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also appear in AD Even if Zn supplementation is performed as cautioned above, it may benefit the cognitive functions in AD. Alternatively, other antioxidants may be used, especially those related to the Zn pool. Arginine may be useful because its action, other than as a substrate for NO production, is exerted by means of the Zn pool (Fabris and Mocchegiani et al., 1992). In AD, arginine may have two roles. First, it establishes a balance between iNOS and cNOS, which is necessary for NO to go from being neurotoxic to becoming neuroprotective in AD (Law et al., 2001). Second, it leads to endogenous Zn ion bioavailability that avoids the addition of exogenous Zn, which can be harmful. This speculation is supported by the fact that arginine supplementation leads to decreased tumor growth in cancer patients (Fabris and Mocchegiani et al., 1992). Otherwise, they may present increased tumor growth after Zn supplementation due to the mitogenic role of high Zn concentrations (both exogenous and endogenous) (Mills, 1989). It is important to remember that Zn supplementation is always useful in prevention (Mocchegiani et al., 1998a). Therefore, it is recommended in periods of relatively good health. In diseases, it may have contraindications as described in this chapter.

7. CONCLUSIONS AND FUTURE DIRECTIONS It is clear that Zn ion bioavailability plays a crucial role in brain functions during development and aging. It affects synaptic transmission, hormonereleasing factors by the hypothalamus, brain enzymes, gene expression of transcriptional factors, and genes clock (c-fos and egr-1) with reflexes on behavior and cognitive functioning. This complex brain machinery may be regulated by Zn-bound MT homeostasis. Changes in this homeostasis provoke different Zn ion bioavailabilities. Hence, an altered physiological cascade occurs that results in brain dysfunctions. During aging, this physiological cascade is impaired because of the presence of low Zn ion bioavailability, which is due to the continuous sequestration of Zn by MT with no subsequent Zn release. This may lead to reduced uptake of Zn by NMDA and AMPA/kainate receptors, resulting in altered gluatminergic transmission. The exacerbation of this defect may lead to the appearance of agerelated neurodegenerative diseases, particularly PD because Zn is required for correct myelination (Dreosti, 1984). As for AD, the role of Zn is still

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controversial as it can favor amyloid-P aggregation in senile plaque, which may lead to the progression of the dementia. In any case, Zn supplementation in aging is beneficial as it improves cognitive performance and ameliorates the health status. However, the precise role of Zn-bound MT isoforms (I, II, and III) in the aged brain is still not clear due to the presence of compensatory phenomena among the MT isoforms. Hence, the data in the literature are controversial due to the fact that MT-III and MT-I+II have been studied separately. One direction for future research is, thus, to study MT isoforms collectively in an attempt to gain insight into the role of Zn metabolism in the brain during aging and in age-related neurodegenerative diseases. Moreover, no data have been reported on Zn supplementation in PD. An interesting direction for future research is to carry out Zn supplementation in this pathology or, alternatively, to use arginine supplementation in both AD and PD. This is because arginine may act on the endogenous Zn pool and avoid the harmful effects of exogenous Zn in neurodegeneration. However, it is always important to remember that Zn is useful in prevention. Therefore, physiological supplementation of Zn in the healthy elderly is recommended for good health and longevity. Conversely, Zn supplementation must be carried out with caution in the presence of clear neurodegeneration.

ACKNOWLEDGMENTS This paper was supported by the Italian National Research Centers on Aging (INRCA), Italian Health Ministry (RF 99/107 to EM), and CEE (ImAginE project: no. QLK6-CT-1999-02031).

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Sonntag WE, Lynch C, Thornton P, Khan A, Bennett S, Ingram R. The effects of growth hormone and IGF-1 deficiency on cerebrovascular and brain aging. J Anat 2000; 197:575-585. Stengaard-Pedersen K, Fredens K, Larsson LI. Enkephalin and zinc in the hippocampal mossy fiber system. Brain Res 1981; 212:230-233. Suh SW, Chen JW, Motamedi M, Bell B, Listiak K, Pons NF, Danscher G, Frederickson CJ. Evidence that synaptically-released zinc contributes to neuronal injury after traumatic brain injury. Brain Res 2000; 852:268-273. Szabo C, Dawson VL. Role of poly(ADP-ribose) synthetase in inflammation and ischaemia-reperfusion. Trends Pharmacol Sci 1998; 19:287-298. Takeda A. Movement of zinc and its functional significance in the brain. Brain Res Brain Res Rev 2000a; 34:137-148. Takeda A, Akiyama T, Sawashita J, Okada S. Brain uptake of trace metals, zinc and manganese in rats. Brain Res 1994; 640:341-344. Takeda A, Takefuta S, Okada S, Oku N. Relationship between brain zinc and transient learning impairment of adult rats fed zinc-deficient diet. Brain Res 2000b; 859:352-357. Tapia-Arancibia L, Astier H. Pharmacological properties of the NMDA receptor involved in somatostatin release from cortical neurons. Eur J Pharmacol 1990; 186:319-322. Taubeneck MW, Daston GP, Rogers JM, Gershwin ME, Ansari A, Keen CL. Tumor necrosis factor-alpha alters maternal and embryonic zinc metabolism and is developmentally toxic in mice. J Nutr 1995; 125:908-919. Thakran P, Leuschen MP, Ebadi M. Metallothionein induction in rat hippocampal neurons in primary culture. In Vivo 1989; 3:191-197. Thomas DR, Hailwood R, Harris B, Williams PA, Scanlon MF, John R. Thyroid status in senile dementia of the Alzheimer type (SDAT). Acta Psychiatr Scand 1987; 76:158-163. Touitou Y, Bogdan A, Haus E, Touitou C. Modifications of circadian and circannual rhythms with aging. Exp Gerontol 1997; 32:603-614. Troy CM, Salvesen GS. Caspases on the brain. J Neurosci Res 2002; 69:145-150. Turgeon SM, Albin RL. GABAB binding sites in early adult and aging rat brain. Neurobiol Aging 1994; 15:705-711. Uchida Y, Takio K, Titani K, Ihara Y, Tomonaga M, The growth inhibitory factor that is deficient in the Alzheimer's disease brain is a 68-amino acid metallothionein-like protein. Neuron 1991; 7:337-347. Uchida Y. Growth-inhibitory factor, metallothionein-like protein, and neurodegenerative diseases. Biol Signals 1994; 3:211-215. Vallee BL. The function of metallothionein. Neurochem Int 1995; 27:23-33. Vallee BL, Falchuk KH. The biochemical basis of zinc physiology. Physiol Rev 1993; 73:79-118. Van der Klink JJ, Blonk RW, Schene AH, van Dijk FJ. The benefits of interventions for work-related stress. Am J Public Health 2001; 91:270-276. Villares JC, Stavale JN. Age-related changes in the N-methyl-D-aspartate receptor binding sites within the human basal ganglia. Exp Neurol 2001; 171:391-404.

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Viticchi C, Moresi R, Piantanelli L. Modulation of mouse brain cortex adrenoceptor in old mice by supplementation of zinc and thymomodulin. Gerontology 1999; 45:265-268. Vogt K, Mellor J, Tong G, Nicoll R. The actions of synaptically released zinc at hippocampal mossy fiber synapses. Neuron 2000; 26:187-196. Wallwork JC, Botnen JH, Sandstead HH. Influence of dietary zinc on rat brain catecholamines. J Nutr 1982; 112:514-519. Wauben IP, Xing HC, Wainwright PE. Neonatal dietary zinc deficiency in artificially reared rat pups retards behavioral development and interacts with essential fatty acid deficiency to alter liver and brain fatty acid composition. J Nutr 1999; 129:1773-1781. Weiss JH, Sensi SL, Koh JY. Zn(2+): A novel ionic mediator of neural injury in brain disease. Trends Pharmacol Sci 2000; 21:395-401. Wenk GL, Stemmer KL. Activity of the enzymes dopamine-beta-hydroxylase and phenylethanolamine-N-methyltransferase in discrete brain regions of the copper-zinc deficient rat following aluminium ingestion. Neurotoxicology 1982; 3:93-99. White G, Gurley DA. Alpha subunits influence Zn block of gamma-2 containing GABAA receptor currents. Neuroreport 1995; 6:461^-64. Williams LR. Oxidative stress, age-related neurodegeneration, and the potential for neurotrophic treatment. Cerebrovasc Brain Metab Rev 1995; 7:55-73. Worley PF, Bhat RV, Baraban JM, Erickson CA, McNaughton BL, Barnes CA. Thresholds for synaptic activation of transcription factors in hippocampus: Correlation with longterm enhancement. J Neuwsci 1993; 13:4776^1786. Yamashita A, Kurokawa T, Fujii Y, Yasuda H, Ishibashi S. Difference in sensitivity to alkaline phosphatase treatment between rat reticulocyte membranes in which beta-adrenoceptor desensitization was induced by isoproterenol, dibutyryl cAMP and phorbol ester. Eur J Pharmacol 1990; 188:229-234. Yang JP, Merin JP, Nakano T, Kato T, Kitade Y, Okamoto T. Inhibition of the DNA-binding activity of NF-kappa p by gold compounds in vitro. FEBS Lett 1995; 361:89-96. Young B, Runge JW, Waxman KS, Harrington T, Wilberger J, Muizelaar JP, Boddy A, Kupiec JW. Effects of pegorgotein on neurologic outcome of patients with severe head injury. A multicenter, randomized controlled trial. JAMA 1996; 276:538-543. Zambenedetti P, Giordano R, Zatta P. Metallothioneins are highly expressed in astrocytes and microcapillaries in Alzheimer's disease. J Chem Neuroanat 1998; 15:21-26. Zangger K, Oz G, Haslinger E, Kunert O, Armitage IM. Nitric oxide selectively releases metals from the amino-terminal domain of metallothioneins: Potential role at inflammatory sites. FASEB J 2001; 15:1303-1305.

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CHAPTER 7

Transition Metals, Oxidation, Lipoproteins, and Amyloid-p: Major Players in Alzheimer's Disease Anatol Kontush ABSTRACT Since transition metals are known to accumulate in Alzheimer's disease lesions, increased oxidative stress represents a well-established characteristic of Alzheimer's disease, extracellular fluids of the brain contain lipoproteins that easily carry oxidizable lipids and can be oxidatively modified, and amyloid-p, a peptide containing 39 to 42 amino acids and a major component of amyloid plaques, is associated with lipoproteins and possesses a structure that implies a strong influence on oxidative processes, it is proposed that the complex interplay of transition metals, increased oxidative stress, lipoproteins and amyloid-p plays an underlying role in the pathogenesis of Alzheimer's disease. Increased production of amyloid-p in a form of lipoprotein antioxidant under the action of increased oxidative stress in aging with subsequent chelation of transition metal ions (first of all, copper), accumulation of amyloid-p metal lipoprotein complexes, their maturation into amyloid plaques, production of reactive oxygen species, and neurotoxicity are reviewed and postulated to form the temporal sequence of events in the development of Alzheimer's disease. Chelation of copper by amyloid-P is proposed to be the most important component of this amyloid-P-copper model. Keywords: Alzheimer's disease; amyloid-p; transition metals; oxidation; lipoproteins; antioxidants.

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1. INTRODUCTION Deposition of amyloid-|3 (ApS) is thought to play a central role in the development of Alzheimer's disease (AD). Accumulation of ApJ in the form of senile plaques is a pathological hallmark of the disease. According to the widely accepted amyloid cascade hypothesis, increased production of A(3, especially of its longer and more amyloidogenic form A(342, leads to the formation of amyloid plaques (Hardy, 1997). The extracellularly located plaques subsequently cause the formation of intracellularly located neurofibrillary tangles (NFTs), another essential feature of the AD brain, and neuronal death. There is good experimental evidence to support the primary role of A (3 in this temporal sequence. Mutations which have been linked genetically to AD, that is, those in the genes coding for amyloid-(3 protein precursor (ApPP), presenilin-1 (PS-1), and presenilin-2 (PS-2), bring about alteration in Ap metabolism, resulting in the elevation in the brain of either total Ap or Ap42 (Selkoe, 1998). However, 5% of all cases of AD are associated with such mutations and are classified as familial AD (FAD) (Smith and Perry, 1998), whereas an overwhelming majority of AD cases are sporadic. It remains unexplained how amyloid plaques can be formed in the absence of any genetically determined increase in A|3 synthesis. Remarkably, the amyloid cascade hypothesis does not contain any explanation for the basal ApS production that is known to occur in neurons and many other cells (Haas et al., 1992). It is, therefore, no wonder that a physiologic role of AfJ continues to be a mystery. In contrast to FAD mutations, normal aging represents a factor that is a clear contributor to all AD cases (Smith et al., 2000c), including those of FAD (which do not develop until the brain has aged to a required stage, despite increased levels of A(3 production). It is unclear which factors are responsible for Ap deposition in the aging brain. One of the earliest pathological events in AD is oxidative damage to vulnerable neurons (Smith et al., 2000c). The damage is not limited to AD lesions, but instead involves the neuronal cytoplasm and precedes lesion formation. Increased intracellular production of reactive oxygen species (ROS) by abnormal mitochondria caused by damage to mitochondrial enzymes has been proposed to be responsible for the observed effects (Bonilla et al., 1999). It is surely not a coincidence that qualitatively similar changes are found in normal aging (Shigenaga et al., 1994). Importantly,

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the cytoplasm of vulnerable neurons shows a striking increase in transition metals, which are potent catalysts of ROS production (Perry and Smith, 1998). Imbalance in the metabolism of transition metals and their extra- and intracellular accumulation in the AD brain have been repeatedly demonstrated (Bush, 2000). Thus, it is feasible that accumulation of ApS in AD is related to increased oxidative stress and imbalanced metabolism of transition metals experienced by the brain in aging. It is important to emphasize that neuronal cells, first of all astrocytes, are the major source of A(3 in brain (Haas et al., 1992; Busciglio et al., 1993). Secreted A(3 is associated with lipoprotein particles (Koudinov and Koudinova, 1997). Thus, ApS is an apolipoprotein. This can be expected, due to the fact that C-terminus of Ap is highly hydrophobic (Brasseur et al., 1997). Taking into account the extracellular location of amyloid plaques, one can assume that A|3 plaque originates from brain lipoproteins. The lipoprotein origin of A(3 in plaques is supported by a close correspondence between the deposition of apolipoprotein E (apoE), the major brain apolipoprotein (Koch et al., 2001), and A(3 in AD brains (Han et al., 1994), as well as by the presence of apoE in amyloid plaques (Wisniewski and Frangione, 1992).

2. TRANSITION METALS Metabolism of transition metals is heavily impaired in the AD brain (Bush, 2000). The brain is a specialized organ that concentrates transition metals. For example, the cerebral cortex contains the highest concentrations of zinc (Zn) in the body (Bush et al., 1994). Therefore, the brain must have efficient mechanisms to prevent abnormal distribution of metals. Transition metals are known to accumulate in two types of AD lesions: amyloid plaques and NFTs. A comparison of neuropil obtained from AD patients and control subjects reveals elevation of Zn, iron (Fe), and copper (Cu) in the former. The highest concentration of transition metals are measured in amyloid plaques (Lovell et al., 1998). Significant elevation of Fe and Zn was observed in multiple regions of the AD brain compared to the controls (Cornett et al., 1998). A significant increase in Zn and Fe was found in the AD hippocampus and amygdala, areas showing severe histopathologic alterations in AD. None of these elements were significantly imbalanced in the cerebellum, which is minimally affected

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(Deibel et al., 1996). Accordingly, Cu, Fe, and Zn are increased in the cerebrospinal fluid (CSF) of AD patients (Hershey et al., 1983; Basun et al., 1991). Brain homeostasis of Fe is disrupted in AD. In AD brain tissue, Fe is present in the NFTs and amyloid plaques. Both amyloid plaques and NFTs contain redox-active Fe, which is not bound to normal Fe-binding proteins and can catalyze oxidation in situ (Smith et al., 1997). Transferrin, an important Fe-binding protein, is homogenously distributed around amyloid plaques and is apparently extracellular. In addition, transferrin is decreased in AD brain tissue, particularly in the cortical regions (Connor et al., 1992b). Ferritin, another Fe-binding protein, is abundant in amyloid plaques and many blood vessels in AD brains (Connor et al., 1992a). Brain homeostasis of Cu is also severely affected. Ceruloplasmin (CP) is increased by more than 60% in the AD brain compared to elderly controls, in all brain regions (Loeffler et al., 1996). In the cortex, 22% of amyloid plaques contain CP, which is present intracellularly in neurons and astrocytes and extracellularly in neuropil. In AD, CP is also increased in CSF (Loeffler et al., 1994). In addition, metallothionein (MT) III, a strong chelator for Cu and Zn, is reduced in the AD cortex (Yu et al., 2001).

3. OXIDATION Transition metal ions, such as Cu(II) and Fe(III), are capable of promoting oxidative stress by the production of highly reactive hydroxyl radicals via the Fenton reaction. Usually, transition metal ions are tightly bound in a redox-inactive state to their transport or storage proteins. Under some conditions, transition metals may be pathologically released and/or reduced to their highly active low valency form. This makes them potent oxidants. For example, human CP, which contains six to seven Cu atoms per molecule of protein, can efficiently promote oxidation in the presence of superoxide-generating systems that reduce CP Cu (Fox et al., 2000). There has been increasing evidence that metal-catalyzed oxidation is particularly important for AD as pathologically deposited metal ions represent a feature of this disorder (Bush, 2000). There are various studies demonstrating elevated products of lipid peroxidation, including thiobarbituric acid-reactive substances (Lovell

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et al., 1995) and 4-hydroxy-2-nonenal (HNE) (Markesbery and Lovell, 1998), in the brain tissue of AD patients compared to the controls. These studies are supported by more recent publications which observe increased F2-isoprostanes, stable markers of peroxidation of arachidonic acid, in the brains of AD patients compared with age-matched controls (Pratico et al., 1998). Interestingly, the differences in F2-isoprostanes are highest in the temporal and frontal cortices, brain regions which are particularly affected in AD. Acrolein, another endproduct of lipid peroxidation which is more toxic than HNE and is produced to a much higher extent, has also been found to be increased in AD brains (Lovell et al., 2001). It is important to mention that increased accumulation of oxidation products in AD is not confined to lipids, but is also relevant to other biomolecules such as proteins and DNA (Smith et al., 2000c). In addition, antioxidant enzymes are frequently elevated in the brains of AD patients, probably in response to increased oxidative stress (Markesbery, 1997). The discovery of increased levels of oxidation in the brains of AD patients led to the measurement of lipid peroxidation in body fluids, especially in CSF. Case-control studies in AD patients reveal a set of alterations in lipid peroxidation in CSF that correspond to the observations made in the brain. Antioxidative vitamins C and E, as well as easily oxidizable polyunsaturated fatty acids (PUFAs), are decreased in the CSF of AD patients (Schippling et al., 2000). In addition, CSF F2-isoprostanes are increased in AD (Pratico et al., 1998; Montine et al., 1999). Elevated levels of oxidation in AD are not restricted to the brain and CSF compartments, but to some extent are also observed as systemic oxidative stress in plasma (Schippling et al., 2000) and urine (Tuppo et al., 2001), body fluids that are more easily available for possible monitoring of antioxidant pharmacotherapy.

4. LIPOPROTEINS Human CSF contains primarily spherical lipoproteins of approximately 13nm to 22 nm in diameter with a density of approximately 1.09 g/mL to 1.15 g/mL, which corresponds to the density of high and very high density lipoproteins (HDL and VHDL) of human plasma (Pitas et al., 1987, Borghini et al., 1995). ApoE and apolipoprotein A-I (apoA-I) are the

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major CSF apolipoproteins and are contained in separate populations of lipoproteins. The major lipoprotein fraction consists of particles of 13 nm to 20 nm containing apoE and apoA-I, as well as apoA-IV, apoD, apoH, and apoJ (Koch et al., 2001). In the second particle class (13 nm to 18 nm), mainly apoA-I and apoA-II, but no apoE, are detected. In the third class, there is a small number of large particles (18 nm to 22 nm) containing no apoA-I, but apoE, associated with apoA-IV, apoD, and apoJ. CSF also contains low levels of small particles (10 nm to 12nm) with low lipid content containing apoA-IV, apoD, apoH, and apoJ. It is essential to emphasize that lipoproteins with similar properties are expected to be present in extracellular fluids of the brain. Similar to plasma lipoproteins, CSF lipoproteins carry a variety of easily oxidizable lipids and can be oxidatively modified in vitro (Arlt et al., 2000). Interestingly, the CSF lipoproteins of AD patients are more sensitive to in vitro oxidation than those of controls. This was demonstrated in a post-mortem study (Bassett et al., 1999) and antemortem study (Bassett et al., 1999; Schippling et al., 2000). Oxidation of CSF lipoproteins can have similar pathophysiological consequences as oxidation of brain lipids. Oxidized lipids have a plethora of toxic effects towards neuronal cells (Keller et al., 1999). It has been demonstrated that oxidized lipoproteins of human CSF are neurotoxic by disrupting neuronal microtubule organization in neuronal cell culture (Bassett et al., 1999).

5. A3 Soluble A3, a peptide containing 39 to 42 amino acids, is associated with CSF lipoprotein particles of 17 nm and approximately 200 kDa of relative molecular mass (Koudinov et al., 1996). The main ApS species associated with CSF-HDL is A^l-40. Similarly, soluble A|3 is associated with lipoprotein particles in normal human plasma, in particular with the HDL and VHDL fractions where it is complexed to apoJ and, to a lesser extent, to apoA-I (Koudinov et al., 1994). Human hepatoma HepG2 cells secrete soluble A(3 as an apolipoprotein in the culture media. Soluble A(3 in the cell supernatant is detected in 200- to 300-kDa lipoprotein complexes in association with apoA-I, apoJ, phospholipids, triglycerides, and free and esterified cholesterol (Koudinov and Koudinova, 1997). When added to plasma HDL, exogenous Api-40 is bound to many HDL apolipoproteins, mainly to apoA-I, apoA-II, apoE, and apoJ (Koudinov et al., 1998).

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Reconstituted, protein-free HDL lipid particles also bind Ap and inhibit its aggregation. ApS is one of several known oblique-oriented peptides that have a hydrophobicity gradient and are partially inserted into lipids by their more hydrophobic C-terminal tail (Brasseur et al., 1997). Ap binding to lipids may play an important role in maintaining the peptide in solution. Ap possesses a structure that implies a strong influence on oxidative processes. A(3 has two major sites that are potentially important for its activity towards oxidation. The first site is located in the hydrophilic Nterminal part of the peptide and consists of three histidine (at positions 6, 13, and 14) and one tyrosine (at position 10) residues, all of which are known to efficiently chelate transition metal ions (Lovstad, 1987). Chelation of transition metals in a redox-inactive form at this site may theoretically serve to inhibit metal-catalyzed oxidation of biomolecules. The second site is found in the lipophilic C-terminal part of A(3 and consists of single methionine residue at position 35. The free sulfhydryl group of the methionine can both scavenge free radicals (Soriani et al., 1994) and reduce transition metals to their high-active low valency form (Lynch and Frei, 1997), thereby possessing both anti- and pro-oxidative properties. Recently, Ap has been shown to be a very efficient chelator for transition metal ions (Atwood et al., 2000). A particularly strong binding is observed for Cu, which is a more efficient catalyst of oxidation than other transition metals. A(31-42 has a higher affinity to Cu(II) than A(3140, with an apparent stability constant of Ap-Cu complexes reaching 2 • 1017 NT 1 and 1.6 • 1010 M _ 1 , respectively (Atwood et al., 2000). This is comparable to the affinity of the best metal chelators ever known, such as ethyleneaminetetraacetic acids. Compared to Cu, Fe is a less suitable ligand for Ap. Ap appears to possess two binding sites for Cu (located between residues 6 to 14), which differ in their affinity. Cu presumably binds to the nitrogen atoms of all three histidine residues of Ap (Miura et al., 2000), as well as to amide groups of the N-terminus (Atwood et al., 2000). Compared to chelation (which occurs instantly), reduction of transition metals by A p is slow. Its rate constant can be estimated at about l O ' l V P ^ 1 (Huang et al., 1999a). It is also only efficient at high (micromolar) concentrations of Ap and presumably occurs through metal interaction with Met35. Reduced metal ions are highly active oxidants and can catalyze further oxidation of biomolecules. It is worth mentioning that

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A(342 is a more effective reductant than AfS40 (Huang et al., 1999a), which can be related to its higher efficiency as a metal chelator (Atwood et al., 2000). Thus, the efficiency of metal reduction by A(3 can be influenced by the efficiency of metal binding to the peptide. Concurring with this observation, Ap reduces Cu(II) more efficiently than Fe(III).

6. INTERPLAY BETWEEN TRANSITION METALS, LIPOPROTEINS, OXIDATION, AND A0 IN ALZHEIMER'S DISEASE Increased oxidative stress is related to the development of AD (Smith et al., 2000c) and A(3 is considered to be an important pro-oxidant in this process (Markesbery, 1997; Varadarajan et al., 2000). The pro-oxidative properties of A(3 have been known for about a decade. In various biochemical systems, Ap efficiently initiates oxidation of different biomolecules. It induces peroxidation of membrane lipids and lipoproteins, generates H 2 0 2 and HNE in neurons, damages DNA, and inactivates transport enzymes. Importantly, to induce oxidation, Ap must be present at high concentrations, typically in a micromolar range. In addition, Ap preparations must be "aged" (incubated over a relatively long period of time at room temperature) to become aggregated and fibrillated. Fibrillated Ap is able to initiate free radical processes that result in protein oxidation, lipid peroxidation, ROS formation, and cellular dysfunction, leading to calcium ion accumulation and subsequent neuronal death. Chain-breaking antioxidants, such as vitamins E or C, can abrogate these processes (Markesbery, 1997; Varadarajan et al., 2000). The production of H 2 0 2 is thought to be central to Ap toxicity (Behl et al., 1994). To be toxic, Ap must be fibrillated (Iversen et al., 1995). It has been shown that Ap aggregation to fibrils, which are essential to its toxicity and pro-oxidative activity, is caused by traces of transition metals inevitably present in laboratory buffers (Moir et al., 1999). In vitro, Ap is readily aggregated by transition metal ions, such as Cu(II), Fe(III), Zn(II), and Al(III) (Atwood et al., 1998). Transition metals are such efficient precipitation agents that binding of even one atom of Cu or Zn leads to Ap precipitation (Atwood et al., 2000). In contrast, in the absence of metals Ap is monomeric, has a-helix conformation, and does not form

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aggregates (Liu et al., 1999). ApJl-42 is more prone to aggregation, probably due to its higher metal affinity, compared to A31-40 (Atwood etal., 1998). The molecular mechanism of A@ aggregation by Zn or Cu includes the formation of intermolecular crosslinks between (3-sheets of A3 by the atoms of metal. The crosslinks are formed between the nitrogen atoms of all three histidine residues in A(3 (Miura et al., 2000). Histidine-13 seems to be essential to A3 aggregation (Liu et al., 1999). As a result, the ApJl40 aggregates formed have about three to four metal atoms per molecule of A3 (Atwood et al., 1998). Of particular importance is the fact that the presence of transition metals is not only required for A3 aggregation, but also for its pro-oxidative activity. Fe is required for the toxicity and pro-oxidative activity of aged preparations of ApS25-35 and A3142 to neuronal cells, whereas Fe chelators protect the cells from A3 (Schubert and Chevion, 1995; Rottkamp et al., 2001). Cu potentiates the neurotoxicity of human Ap (Huang et al., 1999b). Incubation of A|3140 and A|3142 with transition metals leads to the generation of H 2 0 2 (Huang et al., 1999a). Therefore, A3 toxicity is likely to be mediated by a direct interaction between A(3 and transition metals, with the subsequent generation of ROS (Huang et al., 1999b; Rottkamp et al., 2001). Another factor essential to the pro-oxidative activity of A3 seems to be the presence of Met35 (Walter et al., 1997). The triple requirements of fibrillation, transition metals, and presence of Met35 for the pro-oxidative activity of A3 can be understood by taking into account its redox properties. In order to function as a pro-oxidant, A3 must first bind metals to its metal-binding site(s) and then reduce them in its metal-reducing site to produce ROS (such as hydroxyl radicals from H 2 0 2 ). However, metals are bound to the N-terminal hydrophilic part of A3, whereas metal reduction occurs at its C-terminal part. Since metals must be placed in the vicinity of the reductant to be reduced, fibrillation is likely to fulfil this task by forming complexes where metal atoms bound to the N-terminal part of one molecule of A3 are simultaneously available for the reductive Met35 residues belonging to other A3 molecules. The reduced transition metal ions formed can participate in further redox reactions by generating various free radical species. Due to the relatively slow reduction of metals by A3 (Huang et al., 1999a), this mechanism is only operative at high (micromolar) concentrations of the peptide.

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Thus, to accelerate oxidation, A(3 must be present in concentrations greatly exceeding those usually measured in biological fluids, that is, micromolar versus nanomolar (Kontush, 2001; Kontush et al., 2001a; Markesbery, 1997). In addition, A(5 must be aggregated to fibrils by transition metals. Fibrillated A(3 is highly toxic to neurons and other cells (Iversen et al., 1995). In contrast, at low nanomolar concentrations (that is, those circulating in CSF and plasma), A(3 is monomeric and functions as an antioxidant (Kontush, 2001; Kontush et al., 2001a). At these low concentrations, ApS is nontoxic and even has beneficial effects on neuron survival, axonal length, and neurite outgrowth (Whitson et al., 1990; Yankner et al., 1990; Koo et al., 1993). These activities may be related to the antioxidative properties of the peptide. Moreover, if A(3 is kept monomeric, it functions as an antioxidant even at micromolar concentrations by protecting neurons from transition metals (Zou et al., 2002). Unlike pro-oxidative properties, the antioxidative activity of A@ peptides is hardly studied. Recently, exogenously added A(3 has been demonstrated to inhibit metal-catalyzed oxidation of lipoproteins from human CSF and plasma (Kontush, 2001; Kontush et al., 2001a). More importantly, the effect is observed at the peptide concentration measured in these biological fluids (0.1 nM to 1 nM), whereas at higher A(3 concentrations its antioxidant action is abolished. Both ApS140 and A(3142 are efficient antioxidants, whereas the A(325-35 fragment is much less effective. In contrast, all Afi peptides are unable to considerably influence metal-independent lipoprotein oxidation, suggesting that the antioxidative activity of A(3 is mainly mediated by chelating transition metal ions via its hydrophilic moiety rather than by free radical scavenging through Met35. Nevertheless, the latter mechanism contributes to the antioxidative activity of A(3, since A(325-35 is able to inhibit lipid peroxidation (at low, compared to lipids, concentrations of the peptide) and since replacement of Met35 by Leu considerably weakens the effect (Walter et al., 1997). Endogenous A(3 present in the CSF can also act as an antioxidant. This is suggested by the positive correlation between CSF resistance to oxidation and its levels of A|3 (Kontush et al., 2001b). The level of A£42 correlates better with CSF oxidative resistance than that of A@40 (Kontush et al., 2001b), which accords with stronger metal binding to A(3142 than Apl40 (Atwood et al., 2000). CSF from AD patients has lower oxidative resistance than CSF from control subjects (Schippling

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et al., 2000), in accordance with increased oxidative stress known to occur in AD (Smith et al., 2000c). Since Ap has antioxidative properties at its CSF concentrations, this agrees with its lower CSF levels typically measured in AD patients as compared to control subjects (Pirttila et al., 1994; Kontush et al., 2001b). An antioxidant role for A(3 in vivo is in agreement with recent data on the distribution of oxidative damage to AD neurons. 8-Hydroxyguanosine (80HG), a major product of nucleic acid oxidation, markedly accumulates in the cytoplasm of cerebral neurons in AD. Unexpectedly, an increase in A3 deposition in the AD cortex is associated with a decrease in the neuronal level of 80HG, that is, with decreased oxidative damage (Nunomura et al., 1999). A similar negative correlation between A3 deposition and oxidative damage is found in patients with Down's syndrome (DS) (Nunomura et al., 2000). These findings indicate that in brains of patients with AD and DS, A(3 deposition is related to decreased oxidative damage. Thus, the formation of amyloid plaques may be considered as a compensatory response that reduces oxidative stress (Smith et al., 2000c). In lipoproteins, a more hydrophilic metal-binding region of A(3 extends into the aqueous phase where it can bind transition metals (Brasseur et al., 1997). Neuronal cell cultures secrete a high molecular weight product, presumably a lipoprotein complex, that possesses an antioxidative activity (Berndt et al., 1998). Taken together with the antioxidative properties of A(3, these data suggest that A3 is an antioxidant secreted as a part of lipoprotein complexes. A3 can function as a preventive lipoprotein-associated antioxidant that binds transition metal ions in inactive form and prevents them from catalyzing lipoprotein oxidation. Protection from hydroxyl radicals that can be generated by transition metal ions must occur in the nearest vicinity of the protected molecules. This implies that lipoproteins must be protected in their nearest vicinity, a task that is easily performed by a lipoprotein-associated chelator such as A3- The amphiphilic properties of A(3 may allow extracellular chelation of metal ions, which have avoided binding by hydrophilic chelators and reached the lipoproteins. The physiologic relevance of the antioxidative properties of A (3 is determined by its physiologic concentration and level of oxidative stress in vivo. The level of A3 in CSF, that is, 0.1 nM to 1 nM, (Pirttila et al., 1994; Kontush et al., 2001b) corresponds to about one molecule of

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A(3 per 100 lipoprotein particles. Exogenous A(3 at 0.1 nM, that is, at about one molecule of A(3 per 100 lipoprotein particles, delays autoxidation of tenfold diluted CSF in vitro (Kontush et al., 2001a). This implies that the endogenous level is sufficient to considerably delay autoxidation. Mild oxidation of lipoproteins in vitro (such as autoxidation) should better reflect their hypothetical in vivo oxidation than strong oxidation, such as that induced by micromolar amounts of Cu(II) (see Neuzil et al., 1997). Taken together, these data suggest that A(3 may well function as an antioxidant for CSF lipoproteins under normal physiologic conditions. Altogether, data on the activities of Ap to oxidation imply that it may change from being an antioxidant into a pro-oxidant if its concentration increases enough to induce its substantial fibrillation, and if transition metal ions are available to catalyze this process. Various stress conditions are known to increase A(3 production. More importantly, A@ production increases under oxidative stress induced in different ways. Both H 2 0 2 and ultraviolet irradiation elevate the production of A(3 peptides in monkey eye lenses (Frederikse et al., 1996) and neuroblastoma cells (Zhang et al., 1997; Olivieri et al., 2001). H 2 0 2 upregulates both the secretion of ApJ in the cell medium (Olivieri et al., 2001) and levels of A|3 in the cell (Misonou et al., 2000). The antioxidants Trolox and dimethyl sulfoxide are able to block this effect (Misonou et al., 2000). Increased production of A(3 in the presence of H 2 0 2 is not related to increased synthesis of A(3PP, but rather to increased generation of A(3 from A(3PP (Misonou et al., 2000). The upregulation of ApJ production from ApSPP is likely to occur through the activation of A(3-l transcription factor (Frederikse et al., 1996). Other sources of oxidative stress, though less common than H 2 0 2 , similarly lead to increased A(3 production in cell culture. Inorganic mercury decreases cellular glutathione and increases the release of Af5 from neuroblastoma cells (Olivieri et al., 2000). Paired helical filaments from AD patients generate superoxide radicals and increase the release of A(3 from neurons (Yan et al., 1995). Interestingly, secretion of A|3 is also increased when oxidative stress is induced by micromolar concentrations of ApJ itself (Olivieri et al., 2001). This provides a feedback loop mechanism that allows A(3 to increase its own production, which can be considered a vicious circle (Zhang et al., 1997). A(3 generation can also be increased when cells are subjected to a more general metabolic stress. For example, serum deprivation increases

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A(3 production by human neurons (LeBlanc, 1995) and inhibition of energy metabolism results in increased amyloidogenic ApiPP processing by (3-secretase (Gabuzda et al., 1994). Finally, AfS production increases in vivo after brain injury. In patients with head injury, both A(340 and, especially, A(342 increase in CSF during the first week following the trauma (Raby et al., 1998). Fatal head injury results in the formation of diffuse parenchymal deposits of A(3 in the brain, all of which contain A(342 as a major component (Gentleman et al., 1997). Notably, the post-traumatic deposits of A(3 do not arise as a result of passive leakage from damaged cerebral blood vessels, but are similar to the early AJ342 deposits observed in AD and DS. In addition, Ap accumulates in the brain as a response to ischemic/hypoxic injury localized to the cerebral cortex (Jendroska et al., 1995). Taken together, these data strongly suggest that A(3 behaves as a stressrelated protein whose synthesis is increased under stress conditions. The antioxidant metal-chelating properties of A(3 may form a rationale for this phenomenon. Indeed, an increase in A(3 production may be aimed at chelating potentially harmful transition metal ions which can be released, such as from metal-binding proteins, during abnormal cellular metabolism and otherwise catalyze adverse oxidation of biomolecules. This mechanism was proposed recently (Berthon, 2000). It is supported by the fact that increased levels of oxidative damage (measured as neuronal 80HG immunoreactivity) occur prior to the onset of A(3 deposition in the brains of patients with DS (Nunomura et al., 1999). The increase in A3 production may be a regulatory response which helps cells to cope with abnormal metabolism of transition metals (Smith et al., 2000b). Since A3 binds Cu stronger than Fe and other transition metals (Atwood et al, 2000), and Cu is a more efficient catalyst of oxidation than other transition metals (Sayre et al., 2000), chelation of Cu by Afi can be viewed as the most important part of this ApJ-Cu (A(3C) model. Processing of A(3PP to A3 has been suggested more than a decade ago to represent the release of an active peptide ligand and to constitute a part of the reactive plasticity response to neuronal loss (Whitson et al., 1989). Now, when A3 production is known to occur by a fundamental mechanism of regulated intramembrane proteolysis (Brown et al., 2000), its obligatory physiologic significance is even more prominent. Assuming that oxidative stress causes the increase in A(3 generation in AD, its source becomes central in explaining this pathology. Oxidative damage to neurons

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is one of the earliest pathological events in AD (Smith et al., 2000c). Mitochondria have long been known to be the major source of ROS in actively metabolic cells (Cadenas and Davies, 2000). This is especially true of neurons, since the brain accounts for 20% to 25% of the total body oxygen consumption, but for less than 2% of the total body weight. Superoxide is a major ROS produced by the mitochondrial respiratory chain as a result of direct "electron leakage" to oxygen. Superoxide can be further metabolized to H 2 0 2 by catalase. Steady-state concentrations of superoxide and H 2 0 2 in the mitochondrial matrix are about 10~10M and 10~8M, respectively (Cadenas and Davies, 2000). H 2 0 2 may diffuse from the mitochondrial matrix into the cytosol and extracellular space, where it can react with redox-active transition metal ions to produce highly reactive hydroxyl radicals. More importantly, superoxide leakage from the mitochondrial respiratory chain increases sharply in aging due to accumulated damage to the mitochondrial DNA and respiratory chain enzymes (Shigenaga et al., 1994). It is worth mentioning that AD most strongly affects brain regions with the highest metabolic rate and expression of mitochondrial enzymes. Thus, mitochondria may represent an important source of ROS in aging and AD. Increased ROS production may lead to increased generation of A(3 as a compensatory response, similar to increased synthesis of antioxidant enzymes well-documented in AD (Markesbery, 1997). This mechanism implies that transition metal ions become abnormally sequestered and need to be chelated in a redox-inactive form by A(3. It is noteworthy that Ap aggregation by transition metals is accelerated at acidic pH (Atwood et al., 1998). Since it is well-known that inflammation decreases pH in the vicinity of the inflammation site, it can facilitate the formation of amyloid plaques. In addition, inflammation leads to increased secretion of CR a Cu-transporting protein, from the liver and accelerated transfer of Cu to the inflammation site (Berthon, 1993). This cascade may represent an important source for redox-active Cu in AD, since microglial activation and inflammation typically accompany this disease (McRae et al., 1997) and CP is increased in the AD brain (Loeffler et al., 1996). While free Cu does not exist in the cytosol (Rae et al., 1999), extracellular Cu may be more readily exchangeable and increased levels of CP-bound Cu may facilitate Cu interaction with A(3. In addition, transition metals released into extracellular space from synapses

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upon depolarization (Kardos et al., 1989), as well as Fe produced from mitochondrial heme upon its degradation in the cells (Smith et al., 2000b), may serve as other potential sources for metals in A(3-metal complexes. The A(3-metal complexes formed must be efficiently removed, which may occur through lipoprotein receptors known to be abundant in the CNS (Danik et al., 1999). The balance between synthesis and degradation is very fine, since in most early-onset FAD cases A|3 accumulation is caused by only about a 50% increase in A3 anabolism (Iwata et al., 2000). Degradation is likely to be effective in young age and, in the absence of genetically linked increase in A(3 production is observed in FAD. In contrast, increased oxidative stress in the aged brain can lead to increased production of redox-active transition metals which can, in turn, lead to increased production of A^-metal complexes. At one stage, the efficient removal of A(3-metal complexes can be overtaken by their disproportionately high generation. This can result in their accumulation in a form of A(3 oligomers and/or early (diffuse) senile plaques. Transition metal ions are indeed highly enriched in the plaques (Lovell et al., 1998). Diffuse plaques do not contain fibrillated A(B and are not toxic (Yankner, 1996). Their accumulation can, thus, be considered as a final nonpathological stage of this protective pathway and should, inversely, correlate with oxidative damage, as has recently been reported (Nunomura et al., 2001). In contrast, A(3 oligomers have a fibrillated structure, are highly toxic, and may represent the toxin responsible for neurodegeneration in AD (Klein et al., 2001). It is interesting to speculate that diffuse plaques may represent a form of detoxification of ApJ oligomers, such as deactivation by Zn, as has recently been suggested (Cuajungco et al., 2000). Late (compact) amyloid plaques can be another toxic form of aggregated A(3. They contain fibrillated A(3 and can lead to the formation of pathological structures, such as NFTs, in neurons (Mann and Esiri, 1989). Remarkably, compact plaques contain redox-active transition metal ions that can catalyze H202-dependent oxidation in vitro, and may thereby exert pro-oxidant activity in vivo (Sayre et al., 2000). This finding suggests that compact amyloid plaques may represent a pathological stage of the Ap production pathway, when metal sequestration in the redox-inactive form becomes ineffective. However, a negative correlation between the accumulation of compact plaques and 80HG (Nunomura et al., 2000) contradicts this assumption.

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Whatever the nature of the toxic form of Af3, the antioxidant activity of A(3 evolves into pro-oxidant activity, representing a typical gain-offunction transformation which can further stimulate A(3 production and prove a feedback loop mechanism to accelerate plaque growth via a "seeding" mechanism (Irizarry et al., 1997). This can further worsen the situation. Massive accumulation of A(3 in the brain of AD patients might be accordingly considered as a hyper-response to increased oxidative stress in aging. This model is in accordance with the recently proposed three-stage mechanism of neurodegeneration in AD comprising protein aggregation in neural tissue, oxidation of neural tissue mediated by redoxactive metal ion interaction with a target protein, and functional demise (Bush, 2000). It also agrees with the accumulation of early amyloid plaques in cognitively normal individuals, which can be considered as a successful compensation for aging. In contrast, an unsuccessful compensation, that is, when "the primary pathogenic force" (Smith et al., 2000a) is too strong, is accompanied by an uncontrollable growth of plaques and represents AD. Finally, this mechanism allows an explanation of a well-known epidemiological association between aluminum exposure and incidence of AD (Wisniewski and Wen, 1992) by accelerated formation of A(3 aggregates in the presence of aluminum ions, which are well-established initiators of A(3 aggregation (Mantyh et al., 1993). Similarly, increased incidence of AD in subjects having the apoE4 allele (Smith, 2000) can be related to its decreased, compared to apoE3, ability to bind transition metals (Miyata and Smith, 1996), thereby preventing their binding to A|3.

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Wisniewski HM, Wen GY. Aluminum and Alzheimer's disease. Ciba Found Symp 1992; 169:142-164. Wisniewski T, Frangione B. Apolipoprotein E: A pathological chaperone protein in patients with cerebral and systemic amyloid. Neurosci Lett 1992; 135:235-238. Yan SD, Yan SF, Chen X, Fu J, Chen M, Kuppusamy P, Smith MA, Perry G, Godman GC, Nawroth P, al e. Nonenzymatically glycated tau in Alzheimer's disease induces neuronal oxidant stress resulting in cytokine gene expression and release of amyloid betapeptide. Nat Med 1995; 1:693-699. Yankner BA. Mechanisms of neuronal degeneration in Alzheimer's disease. Neuron 1996; 16:921-932. Yankner BA, Duffy LK, Kirschner DA. Neurotrophic and neurotoxic effects of amyloid beta protein: Aeversal by tachykinin neuropeptides. Science 1990; 250:279-282. Yu WH, Lukiw WJ, Bergeron C, Niznik HB, Fraser PE. Metallothionein III is reduced in Alzheimer's disease. Brain Res 2001; 894:37^5. Zhang L, Zhao B, Yew DT, Kusiak JW, Roth GS. Processing of Alzheimer's amyloid precursor protein during H 2 0 2 - induced apoptosis in human neuronal cells. Biochem Biophys Res Commun 1997; 235:845-848. Zou K, Gong JS, Yanagisawa K, Michikawa M. A novel function of monomelic amyloid beta-protein serving as an antioxidant molecule against metal-induced oxidative damage. J Neurosci 2002; 22:4833-4841.

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CHAPTER 8

Molecular Basis of Copper Transport: Cellular and Physiological Functions of Menkes and Wilson Disease Proteins (ATP7A and ATP7B) David R Kramer, Roxana M Llanos, Julian FB Mercer

ABSTRACT Eukaryotic cells prevent copper-induced, free radical damage to cell components by employing copper-binding proteins and transporters that minimize the likelihood of free copper ions existing in the cell. In the cell, copper is actively transported from the cytoplasm during the biosynthesis of secreted coppercontaining proteins and, as a protective measure, when there is an excess of copper. In humans, this is accomplished by two related copper-transporting ATPases (ATP7A and ATP7B), which are the affected genes in two distinct human genetic disorders of copper transport, Menkes disease (copper deficiency) and Wilson disease (copper toxicosis). The study of these ATPases has revealed their molecular mechanisms of copper transport and their roles in physiological copper homeostasis. Both ATP7A and ATP7B are expressed in specific brain regions and neurological abnormalities are important clinical features in Menkes and Wilson disease. Keywords: Copper; Menkes disease; Wilson disease; ATPase.

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1. INTRODUCTION The ability to accept and donate electrons makes copper (Cu) a useful co-factor for oxido-reductase enzymes and oxidative respiration. However, by exploiting this property of Cu, cells expose themselves to a metal ion that is a potential catalyst of reactive oxygen radicals which can damage cell membranes, proteins, and nucleic acids (Kumar et al., 1978). Eukaryotic cells from yeast to humans express a highly conserved set of Cu-binding proteins and transporters which ensure that the uptake and distribution of Cu is tightly regulated, thereby minimizing the likelihood of free Cu ions existing in the cell (Kumar et al., 1978). Saccharomyces cerevisiae has been the basic model system in which many of the genes regulating Cu uptake and distribution have been discovered. In such unicellular organisms, the metabolic demand of continual cycles of cell division and growth requires a constant supply of Cu. Saccharomyces cerevisiae accomplishes this by transcriptional induction and post-translational negative regulation of the major Cu import protein Ctrl (Dancis et al., 1994; Ooi et al., 1996), ensuring that sufficient, but not excessive, uptake of environmental Cu occurs. In contrast, human cells control intracellular Cu levels by the efflux of excess Cu from the cell. This activity is largely dependant on the activity of two related Cu-transporting ATPases (ATP7A and ATP7B) (Chelly et al., 1993; Mercer et al., 1993; Vulpe et al., 1993; Bull et al., Tanzi et al., 1993; Yamaguchi et al., 1993), which are orthologues of the yeast Cu pump, Ccc2 (Yuan et al., 1995). Like Ccc2, ATP7A and ATP7B transport Cu into the lumen of the transGolgi network (TGN) for incorporation into secreted cuproenzymes (such as lysyl oxidase and ceruloplasmin (CP), respectively). However, what makes ATP7A and ATP7B proteins so important to human Cu homeostasis is their remarkable ability to rapidly shuttle from the TGN to the cell periphery to catalyze the removal of excess intracellular Cu (Petris et al., 1996). Besides protecting cells from potential oxidative damage, the latter function is central to physiological Cu homeostasis, as it is the likely mechanism by which Cu is transferred by ATP7A across the cellular boundaries of the intestine and blood-brain barrier (Qian et al., 1998). It also appears to be the molecular mechanism by which ATP7B orchestrates the excretion of Cu into the bile (Roelofsen et al., 200). Our understanding of these respective physiological roles of ATP7A and ATP7B has been gained from the study of two human genetic

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disorders of Cu transport, Menkes and Wilson diseases (MD and WD), in which the activity of these pumps is lost. The association of Cu (and other metals) with several neurodegenerative diseases has increased the interest in molecular mechanisms of cellular Cu transport and Cu-binding proteins. Alterations in specific Cu-binding proteins have been suggested to promote some of the pathologies seen in neurodegenerative diseases, such as Alzheimer's disease (AD), Creutzfeld-Jakob disease, and other prion diseases, while mutation in Cu/zinc (Zn) superoxide dismutase (SOD) is linked to familial amyotrophic lateral sclerosis (FALS) (Campbell et al., 2001). Profoud neurological disturbances occur in MD and WD patients, with the former demonstrating the essentiality of Cu for normal brain development and function and, for the latter, the potential for dysregulation of Cu to cause neurological damage (Strausak et al., 2001). While both ATP7A and ATP7B are expressed in the brain, the relative importance of functional ATP7A and ATP7B in brain Cu homeostasis and in neurodegenerative processes remains unresolved.

2. MOLECULAR FEATURES OF ATP7A AND ATP7B The Menkes gene, ATP7A, is located within a 150 kb region of the X chromosome. Its 23 exons encode an mRNA of approximately 8.5 kb comprising a single open reading frame, with the ATG codon in exon 2 and the TAA stop codon in exon 23, followed by a 3.8-kb untranslated region (Dierick et al., 1995; Turner et al., 1995). The sequence of ATP7A predicts a 1,500-amino acid protein with eight transmembrane domains (Vulpe et al., 1993). There is limited information on the promoter region of ATP7A (Levinson et al., 1996), although it is unlikely to contain a metal response element as the expression of the mouse orthologue, Atp7a, appears to be unchanged by Cu exposure (Paynter et al„ 1994). The Wilson gene, ATP7B, is contained within human chromosome 13 (Bull et al., 1993) and has 21 exons that encode an mRNA of 7.5 kb with a 162base pair 5' untranslated region (Petrukhin et al., 1994). Four copies of a metal response element have been discovered in the 1.3 kb promoter region of ATP7B, (Xie et al., 1998; Oh et al., 1999) and the expression of ATP7B has been shown to possibly involve interaction between these elements and the Ku-80 transcription factor (Oh et al., 2002). ATP7B is

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predicted to be 1,411 amino acids in length and is very similar to ATP7A with an amino acid identity (Bull et al., 1993) of 57%. The structural features of ATP7A and ATP7B indicate that they belong to a subclass of cation P-type ATPases called CpX-ATPases (Solioz and Vulpe, 1996; Lutsenko and Kaplan, 1995). A composite diagram of ATP7A and ATP7B highlighting the conserved and functional domains of these P-type ATPases is presented in Fig. 1. Confirmation that ATP7A functions as a Cu-specific P-type ATPase was obtained using a vesicle assay for Cu transport (Voskoboinik et al., 1998), which showed that Cu transport was inhibited by orthovanadate, a specific inhibitor of P-type ATPases.

Golgi lumen

Fig. 1. Hypothetical representation of ATP7A and ATP7B. Each possesses six metal binding sites (MBSs) containing the GMTCxxC motif. The eight transmembrane domains (depicted as cylinders traversing the transGolgi membrane) are thought to form a pore through which Cu(I) ion is transported. One of these expresses a CPC motif, thought to be crucial for Cu binding during transit. Also represented within the Golgi lumen are clusters of methionine and histidine residues (M, H), which are speculated to be potential low affinity Cu-binding sites. The reaction cycle in P-type ATPases requires the phosphorylation of an invariant aspartic acid residue (D). Also shown are the ATP binding domain, an endogenous phosphatase domain (P-ase), and a C-terminal di-leucine motif thought to be an endocytic signal in ATP7A.

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Common to all P-type ATPases are an ATP-binding site and an invariant aspartic acid residue (D) which becomes phosphorylated and dephosphorylated during the reaction cycle (Lutsenko and Kaplan, 1995). Heavy metal ATPases also contain a conserved CPC motif within the channel formed by the transmembrane domains (Forbes and Cox, 1998). The cysteines in the motif are thought to co-ordinate Cu and are essential to enzyme activity (Forbes and Cox, 1998), while the conserved proline is thought to be required for suspected conformational changes that facilitate cation transport (Jencks, 1992). The cytoplasmic amino terminal region of ATP7A and ATP7B possesses six metal-binding sites (MBSs) (Jensen et al., 1999a, 1999b; Lutsenko et al., 1997) and the solution, a three-dimensional structure of MBS4 of ATP7A, has been determined (Gitschier et al, 1998). Each MBS possesses a canonical sequence, GMTCxxC, that is thought to co-ordinate Cu via the cysteine residues (Jensen et al., 1999b; Cobine et al., 2000). Alternative forms of both ATP7 A and ATP7B have been reported, but their functional significance is not understood. An alternative form of ATP7A mRNA lacking exon 10 is present in most tissues of normal individuals (Diereick et al., 1995), but the truncated product lacks two of the transmembrane domains and mislocalizes to the endoplasmic reticulum. Hence, it is presumed to be nonfunctional (Francis et al., 1998). Spliced forms of ATP7B mRNA have also been found. Interestingly, two variants of ATP7B may be produced in the brain by alternative processing of mRNA. One variant cloned from a human brain cDNA library was found to lack exons 6, 7, and 8, which encode the first two transmembrane domains, as well as exon 12, which encodes a portion of cytoplasmic loop between the fourth and fifth transmembrane domains (Yang et al., 1997). The product of this cDNA was found to localize to the cytoplasm of cultured cells. Another curious variant whose expression appears to be regulated by light occurs in the pineal gland and retina. It is known to be active in Cu transport despite a lack of MBS (Borjigin et al., 1999).

2.1. Cellular Uptake and Transfer of Copper to ATP7A and ATP7B The delivery of Cu to ATP7A and ATP7B is thought to require a minimum of two steps: uptake of Cu across the plasma membrane followed by the delivery of Cu to the MBS of ATP7A and ATP7B. The uptake of extracellular Cu is a facilitated process thought to involve the recently

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described high-affinity Cu import protein hCTRl (Zhou and Gitschier, 1997; Moller et al., 2000; Lee et al., 2000). Disruption of the murine orthologue mCtrl was found by two groups to result in severe developmental abnormalities and fetal death, suggesting an essential role for CTR1 in Cu uptake by mammalian cells (Kuo et al., 2001; Lee et al., 2001). Consistent with this role is the ubiquitous expression of the mRNA (Zhou and Gitschier, 1997) and the apparent lack of human disorders due to mutation in this gene. The genetic sequence of hCTRl predicts an integral membrane protein with three transmembrane domains and an extracellular N-terminus containing two methionein-rich MxxMxM motifs (Moller et al., 2000). A recent biochemical and genetic study has identified key residues in the MxxMxM motif adjacent to the membrane, as well as two conserved methionines within the second transmembrane domain that are critical to Cu transport (Puig et al., 2002). Plasma membrane expression of hCTRl has been shown in some cell lines (Lee et al., 2002; Klomp et al., 2002), while others have shown predominantly vesicular localization (Klomp et al., 2002). Klomp et al. (2002) have suggested that this pattern of localization may represent a pool that circulates between the plasma membrane and vesicles and, thus, may represent a form of regulated Cu uptake. Lee et al. (2002) showed that incubation of hCTRl expressing HEK239 cells with a crosslinking agent generated higher molecular weight forms of hCTRl, suggesting that hCTRl may be a component of an oligomeric complex at the plasma membrane. The distribution of Cu within the cell is accomplished by an evolutionary conserved set of Cu chaperones that bind the ion and, subsequently, deliver it to specific target proteins. This ensures that adequate Cu is supplied for Cu-dependant processes and, at the same time, limits the ability of Cu(I) ion to oxidize cellular components. Three Cu chaperones have been described in human cells: COX 17 is the chaperone that transports Cu to the mitochondria for incorporation into cytochrome c-oxidase (Amaravadi et al., 1997); CCS is the chaperone that delivers Cu into Cu/ZnSOD in the cytosol (Wong et al., 2000); and ATOX1 is the chaperone that delivers Cu to ATP7A and ATP7B (Komp et al., 1997). The structural and functional similarities to their yeast counterparts and to the Cu-binding domains of the target protein(s), with which they interact, have been reviewed (Pena et al., 1999; Harrison et al., 2000; Huffman and O'Halloran, 2001). The specificity and mechanism of Cu donation to a

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given target is thought to involve complementary interfaces between the chaperone's and target protein's Cu-binding sites (Huffman and O'Halloran, 2001). The structure of ATOX1 is homologous to a single Cu-binding domain of the Cu ATPases (Huffman and O'Halloran, 2001; Pufahl et al., 1997). Based on studies of the yeast orthologues, it is believed that ATOX1 accepts Cu from hCTRl (Xiao and Wedd, 2002) and transports the ion through the cytoplasm to the MBS of ATP7A and ATP7B (Pufahl et al., 1997) which, under normal conditions, are localized to the TGN (Petris et al., 1996). Other investigators have suggested that Cu may also be delivered to ATP7A by glutathione and/or metallothionein (MT) complexes (Jensen et al., 1999a).

2.2. Mechanism of Copper Transport by ATP7A and ATP7B P-Type ATPases P-type ATPases are termed as such due to the formation of a high-energy phosphoryl-enzyme intermediate during the reaction cycle (Lutsenko and A

T2*1

El

transition

E2

Fig. 2. Model of Cu binding and transport through ATP7A and ATP7B. The reaction cycle in ATP7A and ATP7B is thought to involve sequential conformational changes. The enzyme is thought to exist in an initial state, El, that is capable of binding ATP and to receive Cu from the ATOX1 chaperone at the N-terminal metal-binding sites (MBSs). Cu binding to N-terminal MBS is thought to induce conformational changes that facilitate the transfer of Cu to the CPC site and the phosphorylation of the aspartic acid (D). The phosphorylated state, E2, is thought to adopt a conformation that permits the transfer of the ion across the membrane. The aspartic acid is then dephosphorylated and the enzyme returns to the original El state.

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Kaplan, 1995; Kuhlbrandt et al., 1998) (Fig. 2). The reaction cycle is thought to involve a minimum of two conformational states and is modeled on the calcium (Ca) P-ATPase (Jencks, 1992). In the El state, the high-affinity CPC Cu-binding site in the channel is accessible to Cu on the cytoplasmic side of the membrane (Jencks, 1992). The binding of Cu to the cytoplasmic MBSs causes significant conformational changes that are presumably related to the Cu transport cycle (Cobine et al., 2000; DiDonato et al., 2000). In P-type ATPases, the binding of a metal ion to the CPC allows the phosphorylation of the aspartic acid to form the E2 state, and causes further conformational changes which then facilitate the transfer of the ion across the membrane to the lower-affinity binding site (MacLenna et al., 1992). For ATP7A and ATP7B, this low affinity site is not known, but is likely to involve one or more clusters of methionines and histidines on the luminal/extracellular side of the channel. An endogenous phosphatase domain, found in the cytoplasmic loop between transmembrane domains four and five of ATP7A (Fig. 1), completes the cycle by catalyzing the E2 to El transition (Jencks, 1992). In addition to their presumed role in the delivery of Cu to the CPC binding site within the membrane channel, the presence of six MBSs may also serve to defect (2000) the Cu concentrations in the cell (Vulpe et al., 1993; Petris et al., 1996). DiDonato et al. (2000) showed that a 70-kd fragment containing the six Cu-binding domains of ATP7B undergoes both secondary and tertiary structural changes upon Cu binding. Such Cudependent, conformational changes may facilitate intramolecular protein interactions, such as that demonstrated between the N-terminal and the ATP binding regions of ATP7B (Tsivkovskii et al., 2001). This interaction was proposed to be part of the overall conformational changes in the protein that are involved in Cu transport. Functional analyses of the individual MBS through in vitro mutagenesis studies, truncations, and domain swapping experiments support the notion that the specific roles of individual MBS may differ between the two homologues. Altering the MBS to the non-Cu binding GMTSxxS in MBS 1-3 of ATP7A abrogated the ability of the mutated ATP7A to complement a Cu transport defect in yeast (Payne and Gitlin, 1998). In contrast, using truncated proteins and domain swapping, Forbes et al. (1999) found that only MBS6 of ATP7B was essential to Cu transport activity in yeast. Interestingly, a mutated version of ATP7A, in which the Cu binding ability of all six MBSs was

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abrogated, retained the ability to catalyze the delivery of Cu into a vesicle derived from mammalian cells, although this mutant had lost the ability to traffick in response to elevated Cu (Voskobionik et al., 1999). Further evidence for specialization of the MBS relates to the ability of the MBS to interact with the Cu chaperone ATOX1, which is thought to deliver Cu to the MBSs of ATP7A and ATP7B. ATOX1 has been shown to co-immunoprecipitate with ATP7B. (Hamza et al., 1999) This interaction has also been shown to occur at the first four MBSs of ATP7B (Larin et al., 1999). A model has been proposed involving the stepwise transfer of Cu from ATOX1 to the MBSs, from which Cu is subsequently donated to the CPC site in the channel (Forbes et al., 1999) (dotted lines in Fig. 2). The observation that ATP7A, without functional MBSs, retains Cu transport activity into mammalian vesicles (Voskobionik et al., 1999) suggests that there are other ways for Cu to access the channel. Such mechanisms could include donation of Cu from glutathione or MT complexes that may occur during times of elevated intracellular Cu (Freedman et al., 1989; Hidalgo et al., 2001). Glutathione-Cu complexes are also likely to be increased during times of Cu exposures. However, a direct role for glutathione-mediated delivery of Cu to ATP7B is not likely, given the lack of an effect of glutathione inhibitors on the rate of Cu efflux into the bile (Nederbrag, 1989).

3. BIOLOGICAL FUNCTIONS OF COPPER ATPASES ARE GOVERNED BY CELLULAR LOCATION AND TISSUE EXPRESSION Most mature tissues express predominately one form of Cu ATPase. ATP7A mRNA is normally found in the kidneys, lung, heart, brain, testis, gut mucosa, placenta, skeletal muscle, fibroblasts, lymphoblasts, and mammary gland carcinoma cells (Chelly et al., 1993; Vulpe et al., 1993; Payner et al, 1994; Ackland et al., 1997). Very low levels of ATP7A mRNA have been detected in mature liver, although their expression appears to be higher in the developing liver (Paynter et al., 1994; Kuo et al., 1997). The range of tissues expressing ATP7B is more restricted than those expressing ATP7A. ATP7B expression is highest in the liver and, to a lesser extent, in the kidneys, brain, hypothalamus, placenta, mammary gland and pancreas, intestine, and ovary (Bull et al., 1993; Tanzi et al., 1993; Michalczyk et al., 2000; Lockhart et al., 2000). There

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are some differences in the above patterns of expression of ATP7A and ATP7B during the developmental stages of an animal and increased expression in the lactating breast and in the placenta, which are suggestive of some specific functions in the delivery of Cu to the fetus and infant (Kuo et al., 1997). ATP7A and ATP7B are required for the biosynthesis of secreted Cu-containing proteins and this is accomplished by the ATPdependent pumping of cytosolic Cu into the lumen of the TGN (Petris et al., 1996; Yamaguchi et al., 1996; Dierick et al., 1997; Schaefer et al., 1999). ATP7A is required for the biosynthesis of the Cu-dependent enzymes expressed in extra hepatic tissues, while ATP7B is required for the synthesis of secreted Cu-dependent enzymes in the liver, including CP, which catalyzes the conversion of the ferrous ion to the ferric form required for transferrin-mediated iron (Fe) uptake. While ATP7A and ATP7B do not directly take part in the biosynthetic pathways of cytoplasmic or mitochondrial cuproenzymes, such as Cu/ZnSOD and cytochrome c-oxidase, their role in physiological Cu homeostasis enables an adequate Cu supply required for the normal activities of these enzymes.

3.1. Copper-Induced Trafficking of ATP7A and ATP7B Facilitates Copper Export Changes in the levels of Cu alter the steady-state location of the Cu ATPases within the cell and determine whether the pumping of Cu by the ATP7A or ATP7B is a part of a biosynthetic or protective function. The pool of ATP7A molecules within the cell is continuously being recycled, via the vesicles, between the TGN and plasma membrane. During normal (safe) Cu exposures, the rate of retrieval of ATP7A from the plasma membrane and its return to the TGN (Petris et al., 1996) is greater than the movement of TGN to the plasma membrane. Cu exposure alters the dynamics of this process (Petris and Mercer, 1999) (Fig. 3). When Cu levels exceed a putative threshold (shown in vitro by increasing the Cu content in the extracellular culture fluid), the rates of ATP7A trafficking change, favoring a plasma membrane localization of the protein (Petris et al., 1996) where ATP7A functions to efflux excess Cu from the cell. Consistent with this regulation, ATP7A contains protein motifs that are important for determining the cellular location of the protein. A 38-amino acid sequence in the third transmembrane domain of ATP7A was identified

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High copper

L o w copper

Fig. 3. Steady-state localization of ATP7A. ATP7A continuously recycles, via the vesicles, between the TGN and plasma membrane. Under normal physiological Cu exposures, however, the majority of the ATP7A pool is retained at the TGN, where the protein functions to deliver Cu for the biosynthesis of the Cu containing enzymes in the endoplasmic reticulum. However, when intracellular Cu levels are raised, there is a shift in steady-state localization such that more ATP7A is associated with the plasma membrane, thereby allowing efflux of the ion. The precise mechanism of the Cu-induced trafficking of ATP7 A is not fully understood, but it is thought to involve an increase in the rate of transport from the Golgi, and is possibly due to a loss of a retention signal.

as a Golgi localization signal, and may be involved in the retention of the protein in the TGN during basal or safe Cu levels (Francis et al., 1998). Furthermore, Petris et al. (1998) and Francis et al. (1999) have shown that a di-leucine motif in the C-terminal region of ATP7A is required for retrieval of the protein from the plasma membrane. Mutation of the dileucine to di-alanine resulted in a molecule that was constitutively located on the plasma membrane. ATP7B also undergoes Cu-induced trafficking, but the distribution pattern after Cu exposure differs from ATP7A. In nonpolarized cells, such as Chinese hamster ovary cells, Cu induces ATP7B to traffick to a vesicle compartment that resembles late endosomes (La Fontaine et al., 2001). However, Roelofsen et al. (2000) have provided convincing data that, in polarized cultured hepatocytes, ATP7B trafficks to the apical membrane (which in vivo corresponds to the canalicular membrane) via an intermediary pool of subapical, vesicular structure. Although there have been no reports of specific domains of ATP7B that determine protein localization, a tri-leucine motif is found in the

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cytoplasmic region of ATP7B, which may be required for retrieval of ATP7B from the cell periphery (J Mercer, unpublished). The conformational changes that the N-terminal region undergoes upon Cu binding have been suggested to be part of the signaling mechanism that stimulates the Cu-induced trafficking of ATP7A and ATP7B (Cobine et al., 2000; DiDonato et al., 2000; Forbes et al., 1999). However, as was discovered for the catalytic function of the protein, not all the MBSs of the proteins may be essential to the response. Data from Strausak et al. (1999) revealed that ATP7A with intact MBS1-3 and mutated MBS4-6 was incapable of relocalizing in high Cu, while no effect was found when only the first three MBSs were mutated. Furthermore, a truncated version of ATP7A, in which the first four MBSs were deleted and only one functional MBS (that is, MBS5 or MBS6) was present, retained the ability to traffick in response to Cu. Thus, either MBS5 or MBS6 of ATP7A is sufficient for Cu-induced trafficking (Strausak et al., 1999). In contrast, Goodyer et al. (1999) showed that an ATP7A molecule with any one of the six MBSs intact could still undergo Cu-induced trafficking. The reason for this discrepancy is not clear.

3.2. Sequential Roles of ATP7A and ATP7B in Physiological Copper Homeostasis The regulation of the overall Cu status of the body occurs by balancing the amount of dietary Cu absorbed with the amount excreted in the bile. The key roles of ATP7A and ATP7B, respectively, in these processes are evident from the pattern of Cu accumulation in MD and WD patients. Studies in human volunteers fed isotopic Cu under controlled conditions revealed that there is little net absorption of Cu over a range of exposures, suggesting that regulatory mechanisms operative within the intestinal epithelium affect the efficiency of Cu absorption (Turnlund et al., 1989). MD patients accumulate Cu in the absorptive epithelial cells of the small intestine due to a blockade in the transfer of absorbed dietary Cu to the portal venous system (Danks, 1995). The known Cu-induced trafficking of ATP7A to the plasma membrane (Petris et al, 1996) suggests that ATP7A might localize to the basolateral membrane after Cu ingestion to deliver Cu to the portal blood (Fig. 4). The liver is the first organ to receive absorbed nutrients and toxicants, and much of the absorbed

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dietary Cu is taken up by the liver, thereby buffering the exposure of vital organs to large fluxes in the concentration of absorbed Cu. This is an example of the "first-pass effect" (Fig. 4). The liver is the primary regulator of Cu distribution and elimination, and this is achieved by modulation of the rate of biliary excretion mediated by ATP7B versus incorporation of Cu into CP (Harris, 2000). The majority of post-hepatic circulating Cu is contained within CP, but this pool is not thought to be immediately accessible to tissues (Harris, 2000). The role of CP as a Cu carrier has been cast in doubt as patients with aceruloplasminemia have defects in Fe homeostasis without appreciable changes in Cu status or other Cu-dependent processes (Gitlin, 1998). Excess absorbed Cu is apparently defected within the hepatocyte and, in response, ATP7B migrates from the TGN towards the cell periphery (La Fontaine et al., 2001). ATP7B is thought to catalyze the efflux of Cu from the cell by pumping it into vesicles, which may either fuse with lysosomes (Gross et al., 1989) or the canalicular membrane and release the Cu directly into the bile (Ballatori, 1991). These conclusions, based on studies in cultured cells, are supported by the distribution of ATP7B in rat liver. Schaefer et al. (1999) found that in Cudeficient rats, ATP7B was localized in the TGN of hepatocytes. However, when the animals were Cu loaded, the protein was distributed in multiple vesicular structures and The authors did not find any ATP7B on the canalicular membrane. ATP7A and ATP7B have additional roles in physiological Cu homeostasis which are depicted in Fig. 5. For instance, ATP7A is required for the delivery of Cu across the blood-brain barrier and for its reabsorption in the kidney. Adequate Cu is essential to normal human growth and development, and dysregulation of Cu homeostasis has profund effects on development. ATP7A and ATP7B both appear to be important in the delivery of Cu to the fetus and newborn, although the precise roles of the homologues in these processes have not been fully resolved. A recent model of placental Cu transport suggests that it involves the sequential activities of ATP7B and ATP7A (Harris et al, 1998). ATP7B expression was localized to the maternal side of the placenta in Long Evans Cinnamon (LEC) rats, a murine model of WD which accumulates Cu in the placenta (Muramatsu et al., 1998). Affected fetuses in mouse models of MD also exhibit placental accumulation of Cu, implicating a role for the mouse orthologue, Atp7a, in placental Cu transport. Also, expression

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Fig. 5. The transport of Cu across cellular boundaries requires Cu ATPase activity. Studies on Cu transport defects in MD patients and in mouse models of MD suggest that ATP7A is required for the uptake of dietary Cu across the intestinal epithelium (1), the reabsorption of Cu by the renal tubules (2), the transport of Cu into the brain (3), and the efficient uptake of Cu across the fetal side of the placenta (4). Studies of WD and related animal models suggest primary roles for ATP7B in the transfer of Cu across the maternal side of the placenta (4), the biosynthesis of CP (5), biliary excretion of Cu (6), and the delivery of Cu to the milk (7). Note that the lack of systemic Cu deficiency in WD suggests that some Cu is able to bypass the liver or is released by the liver as Cu complexes, such as Cu-histidine (Cu-His), via mechanisms that may not require a functional ATP7B.

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of ATP7A by the human placental cell line, BeWo, was found to correlate with the ability of the cells to efflux Cu. These data support the theory that ATP7A functions to transport Cu absorbed by the fetal-derived cells of the placenta to the fetal blood (Harris et al., 1998). A potential role for ATP7B in the delivery of Cu to the milk was suspected from the findings that milk produced by the "toxic milk" mouse mutant (a mouse model of WD) was Cu-deficient (Rauch, 1983). This is supported by the accumulation of Cu in the mammary gland of Atp7b knockout mice (Buiakova et al., 1999). Both ATP7A and ATP7B are expressed in mammary tissue and are located in the TGN of nonlactating breast tissue, but are redistributed to vesicular-like structures in the lactating tissue (Michalczyk et al., 2000; Ackland et al., 1999). Although the physiological stimulus for the change in the intracellular location of the proteins in the lactating gland has not been determined, these studies suggest that the trafficking of ATP7B (and perhaps ATP7A) is an important part of the delivery of Cu into breast milk.

4. GENETIC DISORDERS OF COPPER TRANSPORT 4.1. Menkes Disease and Animal Models MD is one of three X-linked, human Cu deficiency disorders caused by mutations within the ATP7A gene. The others, occipital horn syndrome (OHS) and mild MD, represent subsets of the phenotypes seen in classical MD (Danks, 1995). MD is a rare disorder with an estimated frequency of between one per 100,000 and 300,000 live births (Tonnesen et al., 1991) and likely often arises within the affected families from spontaneous mutations. Classical MD always results when mutations in ATP7A prevent the formation of any functional protein. Partial or complete deletions of ATP7A are found in about 15% of MD patients (Turner et al., 1999). Many missense mutations causing classical MD and the milder variants have also been identified, (Turner et al., 1997) some of which result in aberrant RNA splicing (Das et al., 1994). Some MD patients and patients with the less severe form of OHS appear to retain some residual activity of the mutant ATP7A, or continue to produce small amounts of normal protein (Moller et al., 2000). The occurrence of variant clinical phenotypes and the variable success rate of Cu therapy in MD patients have been suggested to result from the different effects of particular mutations on the protein (Kaler,

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1996; Mercer, 2001), such as reduction in Cu transport activity, altered intracellular location, and defective trafficking in response to Cu. Classical MD is a fatal disorder due to severe Cu deficiency that results from the combined effects of an inability to transfer Cu across cell boundaries of the intestine, brain endothelium, and kidney tubules (Danks, 1995). Transfer of Cu to the affected fetus is also diminished (Xu et al., 1994). The brains of patients with MD are severely deficient in Cu and the effects on neurological function in these patients are probably responsible for the fatal outcome. In MD , the affected brain has only 2% of total body Cu, compared with a normal brain which contains 35% of total body Cu (Horn et al., 1978). Brain Cu deficiency results in myelin abnormalities and atrophy of the cerebrum and cerebellum, and is associated with an array of major neurological symptoms including mental retardation, seizures, and hypothermia (Strausak et al., 2001). While patients with OHS generally show only minor neurological impairment (Strausak et al., 2001), there has been at least one report of Menkes-like neuropathology in a mentally retarded and dysmorphic 26-year-old patient with OHS (Palmer and Percy, 2001). A recent hypothesis has linked cerebral Cu imbalances, particularly Cu accumulation in the lentiform nuclei, in some patients with idiopathic adult-onset dystonia (Becker et al., 2001). A reduction in the expression of ATP7A has been reported in both the

Table 1. Reduced activity of Cu-dependent enzymes and resultant phenotypes in MD. Phenotype

Defect

Affected enzyme

Connective tissue defects: abnormalities of bone and weak vascular walls

Impaired crosslinking of collagen and elastin

Lysyl oxidase

Impaired melanin biosynthesis

Tyrosinase

Impaired catecholamine biosynthesis and neuropetide processing Electron transport

Dopamine-p-hydroxylase Peptidylglycine-aamidating mono-oxygenase Cytochrome c-oxidase

Sensitivity to oxidative stress

Free radical defense

Cu/ZnSOD

Weakness, hypothermia

Electron transport

Cytochrome c-oxidase

Hypopigmentation Neurological abnormalities

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lentiform nuclei and in leukocytes in a significant number of patients with cervical dystonia (Berg et al., 2000; Kruse et al., 2001). Cu deficiency in MD profoundly affects the formation of connective tissues, as well as the formation and function of the brain, due to the low activity of specific cuproenzymes (see Table 1). ATP7A is required for the biosynthesis of the Cu-dependent enzymes tyrosinase, involved in the melanin biosynthesis pathway (Petris et al., 2000), and lysyl oxidase, required for the crosslinking of elastin and collagen within the extracellular matrix (Kosonen et al., 1997). Hence, MD patients, as indicated in Fig. 6 Kinky hair Defective copper transport

Neurological defects metal retardation hypothermia feeding difficulties

Vascular defects Muscular weakness

Defective copper absorption * Hypopigmentation Fig. 6. Diagram illustrating the defects in Cu transport (left side) and some of the clinical outcomes of the resultant Cu defciency (right side) in affected boys with classical Menkes syndrome. Due to a loss of catayltic activity of ATP7A, the intestinal epithelial cells of Menkes children are unable to transfer absorbed Cu to the blood. The small amount of Cu that is absorbed by ATP7A-independent mechanisms (or by a partially active mutant ATP7A) subsequently becomes trapped within tissues, including the kidney tubules that normally reabsorb Cu from the urine. Cu is also trapped in the endothelial cells of the brain. MD patients have a plethora of clinical symptoms that result from the reduced activities of Cu dependent enzymes and processes (right side of diagram) (see Table 1). Some of the neurological deficits are thought to be related to impaired generation of ATP due to reduced electron transport, while other neurological features may result from imbalances in neurotransmitter biosynthesis and death of neurons in specific brain regions.

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and Table 1, suffer from hypopigmentation, flaccid muscles, and weakened endothelial walls due to the loss of the activities of tyrosinase and lysyl oxidase (Danks, 1995; Kuivaniemi et al., 1985). (These connective tissue defects found in MD patients become predominant features in OHS (Danks, 1995).) The neurological defects are possibly caused by the low activity of cytochrome c-oxidase, a Cu-dependent enzyme in the electron transport chain (Danks, 1995). The low activity of other Cu-dependent enzymes in the brain may also contribute to brain abnormalities (Kaler, 1994) and to the observed abnormal plasma and cerebrospinal fluid (CSF) neurochemical patterns in MD patients (Kaler et al., 1993). These enzymes include Cu/ZnSOD, peptidylglycine-a- amidating mono-oxygenase, and dopamine- ^-hydroxylase. Interestingly, brains from MD patients may compensate for reduced levels of Cu/ZnSOD with increased expression of MnSOD (Shibata et al., 1995). The animal models closest to MD are the mottled mice: a collection of mutant strains with reduced or null activity of Atp7a, the murine orthologue of Atp7A (Mercer et al., 1999). Of these, the macular and brindled strains are considered to be the most accurate representations of classical MD in humans (Mercer, 1999). The mutation in brindled Atp7a has been shown to be an in-frame deletion of six nucleotides (Grimes et al., 1997). In macular mice, probes to Atp7a hybridize to the hippocampus, denate gyrus, olfactory bulb nuclei, cerebral granule layer, choroid plexus, ependyma, and cerebral Purkinje cells (Iwase et al., 1996). Strong expression of Atp7a in the choroid plexus has also been reported in the developing mottled mouse (Kuo et al., 1997). This pattern of expression of the mutant Atp7a is in overall agreement with the findings from histochemical staining of Cu deposits in the macular mouse brain, demonstrating that Cu accumulates in those regions that express the mutant gene (Kuo et al., 1997; Michalczyk et al., 2000; Iwase et al., 1996). Furthermore, the effects of Cu deficiency may occur during both prenatal and postnatal brain development in mice (Kuo et al., 1997). Brindled mice can be rescued from death by injections of Cu salts, provided the injection regimen is commenced prior to 10 days after birth. This suggests that a critical period of Cu-sensitive brain development takes place around this time in the mouse (Fujii et al., 1990). The precise nature of the critical period is unresolved, although there have been suggestions that it may coincide with the expansion of astrocytes in the developing brain (Kuo et al., 1997). In humans,

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Cu-histidine (Kaler, 1994; Sarkar et al., 1993) therapy results in clinical improvements and can prolong survival into adolescence, but treatment must be commenced as soon as possible after birth and appears to be successful in only a subset of patients (Turner et al., 1996; Christodoulou et al., 1998). In responding patients, dopamine and norepinephrine levels can normalize after Cu-histidine therapy (Kaler et al., 1993). A recent report of the post-mortem examination of a Cu-histidine treated 12-year-old MD patient revealed minimal pathology in the central nervous system (CNS) (George and Casey, 2001). Despite the success in alleviating some of the neurological features of MD, the connective tissue abnormalities are not corrected and the Cutreated MD patients display many of the clinical features and pathology normally associated with OHS (Turner et al., 1996; Christodoulou et al., 1998; Geroge and Casey, 2001). In the mouse models of MD, treatment with Cu chloride restored brain cytochrome ooxidase and Cu/ZnSOD activity (Yoshimura et al., 1993). However, despite these improvements, some brain abnormalities persist after therapy, including abnormal innervations, heterotypic sprouting of serotonergic neurons (Martin et al., 1994), and reduced number of Purkinje cells (Kuo et al., 1997; Yamano et al., 1988). Neuronal cell death in macular and brindled mice has been reported to involve both necrosis and, more recently, apoptosis. Apoptosis is particularly evident in the cerebral cortex, thalamus, and pyramidal layer of the hippocampus. It begins around day 10 when the affected mice begin to show overt symptoms of neurodegeneration (Rossi et al., 2001; Ohno et al., 2002). Although the timing of the onset of apoptosis is variable in the mice, the time frame is consistent with the therapeutic window of initiation of Cu therapy in affected male mice. The role of Cu deficiency in triggering off apoptosis is supported by the observation that Cu treatment reduces the level of apoptosis (Danks, 1995). The brain regions undergoing apoptosis were found to be those that normally express low levels of SOD, suggesting that the trigger may not be directly related to oxidative damage associated with reduced SOD activity (Ohno et al., 2002). Apoptosis in the cerebral cortex and hippocampus was recently found to be associated with a dramatic decrease in the anti-apoptotic protein Bcl-2 (Rossi et al., 2001). The authors of this study also found that the brains of the brindled mice had significantly increased levels of cytosolic cytochrome C, a 50% reduction in ATP and a 30% reduction in cytochrome

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c-oxidase activity. They suggest that damage to the mitochondria, due to a Cu-deficient state, may trigger off apoptosis in the affected neurons (Rossi et al., 2001).

4.2. Wilson Disease and Animal Models WD is an autosomal recessive Cu toxicity disorder affecting both the liver and CNS. In WD, mutations of ATP7B result in reduced biliary excretion of Cu and low incorporation of Cu into CP in the liver (Danks, 1995). However, not all patients have low CP levels. In the liver of WD patients, much of the excess metal becomes associated with MTs (Elmes et al., 1989) and accumulates in lysosomes (Evering et al., 1991). The accumulation of Cu eventually causes severe liver damage, liver failure, and death (Danks, 1995). The progressive damage to hepatocytes is thought to result in release of Cu, which is subsequently taken up by other liver cells and the brain (Fig. 7). Cu also deposits in the cornea, resulting in the formation of Kayser-Fleischer rings, which are a valuable diagnostic indicator of the

Fig. 7. Progression of Cu accumulation and toxicosis in WD. In WD, the impaired biliary excretion results in a net uptake of Cu and its accumulation in hepatocytes (steps 1 and 2). The continual accumulation of Cu in the liver of WD patients is thought to eventually overwhelm the capacity of the hepatocytes to store the metal and to repair associated oxidative damage. This results in the ongoing death of hepatocytes and release of the metal (step 3). Cycles of Cu release and uptake by hepatocytes (step 4) exacerbate the damage to the liver and can progress to fulminant hepatitis. The brain also accumulates Cu (step 5), primarily in the basal ganglia with the onset of neurological symptoms often, but not always, secondary to significant liver damage.

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disorder (Danks, 1995; Liu et al., 2002). Cu accumulation in the brain is associated with neurological abnormalities, including Parkinsonian symptoms, dystonia, rigidity, and psychological manifestations ranging from depression to psychotic symptoms that can be misdiagnosed as schizophrenia. (Strausak et al., 2001; Rathbun, 1996). The neuropathology appears to be directly related to Cu accumulation as the neurological symptoms are associated with increased Cu levels in the CSF of WD patients (Kodama et al., 1998). Cu accumulates predominantly in the basal ganglia of WD brains and degeneration in the basal ganglia is a common finding in magnetic resonance images (van Wassenaer-van Hall, 1996). The presence of periodic acid-Schiff positive glial cells are also characteristic of the brain changes in WD (Strausak et al., 2001). Demyelination within the cerebrum occurs late in the disease (van Wassenaer-van Hall, 1996). WD is commonly treated with the Cu chelators penicillamine (Walsh, 1973) or ammonium tetrathiomolybdate (Brewer et al., 1994), which aim to mobilize the accumulated Cu (Brewer et al., 1987b) or to reduce the uptake of Cu in the small intestine with large doses of oral Zn, which is thought to lower Cu absorption through induction of MTs (Brewer et al., 1987b). The successful treatment of WD appears to be related to the stage of the disease (Brewer et al., 1987b). Treatment has demonstrated that some of the neurological symptoms are reversible. However, patients with advanced disease frequently experience an initial worsening of their neurological symptoms (Brewer et al., 1987a). One recent study has suggested that disruptions to the blood-brain barrier due to the therapy may account for this outcome (Stuerenburg, 2000). Liver transplantation is employed to treat WD patients with fulminant hepatitis or cirrhosis, and this has been found to improve the neurological features of WD (Wu et al., 2000; Stracciari et al., 2000). LEC rats are an animal model of WD (Li et al., 1991; Wu et al., 1994). In the parental Long Evans rat, Atp7b was localized by immunochemistry to the neuronal cells of the hippocampus, olfactory bulb, cerebellar Purkinje cells, cerebral cortex, and brainstem (Saito et al., 1999). The distribution in the brain was found to correlate to both the distribution of Cu and the normal pattern of dopamine-(^-hydroxylase in the LEC rat (Saito et al., 1999). Saito et al. (1999) also noted the overlap of Atp7b expression in the rat with the published localization of nitric oxide synthase. The brains of

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LEC rats also show evidence of oxygen radial damage to DNA (Yamamoto et al., 1993). Cu accumulates predominantly in the basal ganglia, subthalamic nuclei, and gray and white matter of LEC rats (Sato et al., 1994). The effects of altered brain Cu metabolism on nerve fiber densities in the brains of LEC rats have been investigated by Kawano et al. (2001) who found that, despite no differences in Cu levels between control and LEC rat brains at 10 weeks of age, there are likely to be significant reductions in norepinephrine producing neurons based on the reduction in tyrosine hydroxylase (an enzyme in the biosynthetic pathway of norepinephrine) positive neurons. An increase in 5-hydroxytrypamine positive neurons in the cortex, hippocampus, and cerebellum of four- and 10-week-old rats was also found (Kawano et al., 2001). Diagnosis of WD is often difficult due to variations in the age of presentation (from two to more than 40 years) and clinical features at diagnosis. Such variations are thought to arise from the mutations causing different degrees of loss of function in the mutant ATP7B proteins. Over 200 mutations have been described and are catalogued on the Internet (http://www.medgen.med.ualberta.ca/database). This work has revealed a diverse array of mutations, although some, such as the Hisl069Gln mutation, appear more frequently in specific populations. Interestingly, large deletions of ATP7B have not been found in WD patients (Thomas et al., 1995). Similar to MD variants, mutations that cause mislocalization of the protein or changes in Cu-induced trafficking may result in distinct phenotypes, such as WD patients with normal CP (Forbes and Cox, 2000). Patients who are homozygous for severe mutations have an earlier onset of disease and are sometimes not recognized as WD because of this fact (Thomas et al., 1995). Milder mutations may present with later-onset neurological disease (Thomas et al., 1995). However, many WD patients are compound heterozygotes (that is, they carry different ATP7B mutations on each of the two chromosome 13s (Thomas et al., 1995) which complicates the genotype/phenotype correlation). The clinical severity of WD might also be influenced by environmental factors, such as the amount of dietary Cu ingested, or allelic variants of modifying genes, such as MTs. Such factors could explain why the common Hisl069Gln mutations are associated with a range of clinical presentations (Due et al., 1998).

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Fig. 8. Proposed role of ATP7A in the stepwise transfer of Cu across the blood-brain barrier. Astrocytes adjacent to the blood vessels are in a position to partially buffer the exposure of neurons to Cu. It is not known if Cu ions are preferentially taken up at the astrocytic endfeet (1), or if the pool of interstitial Cu ions are equally acccessible to both astrocytes and neurons (2). Whether or not astrocytes distribute metal ions to neurons via—controling the rates of storage of metal ions, and subsequent release such as in MTs, is speculative. Nevertheless, the release of Cu from astrocytes is presumed to require ATP7A (3).

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5. ROLES OF ATP7A AND ATP7B IN COPPER HOMEOSTASIS IN THE CNS 5.1. A Specific Role of ATP7A in Copper Transport in the CNS For Cu to be delivered to the CNS, it must be transported across either the blood-brain barrier or the CSF barrier. Endothelial cells in the mature brain display a low rate of endocytosis and are joined by specialised "high electrical resistance" tight junctions that, together, form the blood-brain barrier (Rubin and Staddon, 1999). Astroglial cells are adjacent to the endothelial cells and make contact with the vessels by membranous processes called "endfeet" (Fig. 8). Cu accumulates within brain capillaries of macular and brindled mice (Yoshimura et al., 1995; Yoshimura, 1994; Kodama et al., 1993) and within cultured astrocytes from macular mice (Kodama et al., 1991). In agreement with this pattern of Cu accumulation, Atp7a mRNA has been detected in primary mouse cell lines from cultured brain endothelial explants (Qian et al., 1998) and is expressed in the rat glial cell line, C6 (Qian et al., 1995, 1997). These observations have been incorporated into a model in which ATP7A catalyzes the stepwise transport of Cu across brain endothelial cells and astrocytes to supply neurons with a regulated supply of Cu (Qian et al., 1998; Tiffany-Castiglioni and Qian, 2001). A corollary of this model suggests that the severe Cu deficiency of neurons in MD patients is due to the combined effects of reduced endothelium to brain Cu transport and the accumulation of the already reduced brain Cu supply within astrocytes (Tiffany-Catiglioni and Qian, 2001). This proposed mechanism of brain uptake of Cu follows the mechanisms proposed for brain Fe metabolism (Bradbury, 1997). Fe uptake occurs across the cerebral vascular endothelium, after which if becomes associated with transferrin and is then taken up by glial cells and neurons. An unresolved issue concerns the role of the choroid plexus in brain Cu transport. Unlike most other brain regions, the choroid plexus is supplied by fenestrated capillaries and venule sinusoids that permit the rapid exchange of water and solutes between the plasma and the extracellular fluid (Segal, 2001). Integrity of the brain-CSF boundary results from the expression of tight junctions in the choroidal epithelium (Segal, 2001). The epithelium of the choroid plexus is the major site of production of CSF and delivers micronutrients to the CSF, as well as remove potentially

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toxic xenobiotics and organic anions from the CSF (Segal, 2001). Cu accumulates in the choroid plexus of macular mice and Atp7a is highly expressed in the choroid plexus in both developing mottled embryos and adult mice. Mouse choroid plexus also expresses the Cu transporter mCtrl and Atoxl chaperone (Nishihara et al., 1998). A second model of Cu transport to the brain proposed that the choroid plexus may function as an adjunct to Cu transport mediated by brain endothelium and astroglial cells (Kuo et al., 1997). The choroid plexus expresses CP (Aldred et al., 1987) and probably secretes it into the CSF. ATP7B has not been localized to the choroid plexus It is possible that ATP7A in the choroid plexus delivers Cu to CP, which is then secreted into the CSF. (ATP7A may also be important in delivering Cu to CP in astroglial cells, which express a glycosylphosphatidalinositol-anchored form of CP (Hellman and Gitlin, 2002) and have also not been reported to express ATP7B.) Alternatively, ATP7A may catalyze the apical transport of Cu across the epithelium of the choroid plexus. Both proposed functions are analogous to the role of ATP7B in the liver and, indeed, anatomical and physiological comparisons between hepatocytes and the choroid plexus epithelium have been discussed (Segal, 2001). An alternative hypothesis that the choroid plexus may reabsorb Cu from the CSF, which occurs in some metabolic waste products, cannot be ruled out at this time (Segal, 2001).

5.2. Potential Role of ATP7B in the Brain The development of neurological symptoms and pathology in the majority of WD patients is progressive and can often be prevented by early intervention. Moreover, many established neurological features improve with Cu chelation therapy or after liver transplantation. These data suggest that the neurological features of WD are likely to be linked to persistent exposure to elevated Cu and its accumulation in the brain. The degeneration observed in the basal ganglia in many patients with advanced WD likely results from Cu accumulation in these cells. Neurons within the basal ganglia are presumed to express ATP7B (based on information from rat brains), and the loss of ATP7B activity in these neurons may restrict their ability to efflux Cu. Other neurological features may result from alterations in brain chemistry, rather than permanent neuronal loss. One likely role of ATP7B in the CNS is to deliver Cu into the TGN

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for incorporation into Cu-dependent enzymes that are involved in neurotransmitter biosynthesis, such as dopamine-(^-hydroxylase and peptidylglycine-a-amidating mono-oxygenase. Studies in the rat have colocalized ATP7B expression in neurons that also express dopamine-phydroxylase (Saito et al, 1999). Reduced activity of ATP7B in these neurons may cause reduced neurotransmitter biosynthesis and affect the observed changes in the frequencies of specific types of nerve cells, such as the changes observed in the monoaminergic neurons in the rat.

6. CONCLUSIONS There have been rapid advancements in the understanding of the molecular mechanisms of Cu transport in recent years. Central roles for the Cu-induced trafficking of ATP7A and ATP7B in regulating cellular Cu levels and Cu status in the body have been recognized. These processes are necessarily dependent on controlling the delivery of Cu to ATP7A and ATP7B by the ATOX1 chaperone, but the mechanisms that regulate the distribution of Cu among the chaperones are not understood. Work on hCTRl suggests that its cellular location may differ within cell types, suggesting that Cu uptake may also be a regulated process in some tissues (Klomp et al., 2002). The role of Cu in a number of neurodegenerative diseases has become apparent (Campbell et al., 2001). Still, our understanding of the molecular mechanisms of Cu transport in the brain is largely modeled on the cellular and physiological control of Cu homeostasis outside the CNS. For instance, the normal cellular location of ATP7A and ATP7B in the neurons in which they are expressed, and the ability of the proteins to undergo Cuinduced trafficking in neurons, has yet to be demonstrated. There is also evidence that some components of cellular Cu homeostasis may have brainspecific functions, including the aforementioned variants of ATP7B. In addition, although the expression of ATOX1 in the brain appears to match the distribution of ATP7A (Naeve et al., 1999), ATOX1 has also been suggested to be important in protecting neurons from oxidative stress (Kelner et al., 2000). Protection from oxidative stress may also be a function of the prion protein which is known to bind, Cu (Brown et al, 1997) and which apparently recycles between the plasma membrane and endosomes (Pauly and Harris, 1998). Where the latter observation fits into the cellular Cu homeostasis story and the potential roles of ATP7A and ATP7B in this

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process remain a mystery. Lastly, the mechanisms of Cu elimination from the brain have not been addressed. Based on the Fe model,(Bradbury, 1997) it is likely that excess Cu is removed from the CNS via the CSF, perhaps bound to an unknown brain-specific Cu carrier, and returned to the circulation at the arachnoid villi. Whether or not a disruption to the process of Cu efflux from the brain contributes to the accumulation of Cu in WD or other neurodegenerative diseases is not known. It is anticipated that further research into the mechanisms of brain Cu homeostasis will reveal the links between Cu transporters and neurodegenerative diseases associated with Cu.

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CHAPTER 9

Importance of Copper and Zinc in Alzheimer's Disease and the Biology of Amyloid-p Protein and Amyloid-p Protein Precursor Avi L Friedlich, Xudong Huang, Seiichi Nagano, Jack T Rogers, Lee E Goldstein, Ashley I Bush, Gerd Multhaup, Konrad Beyreuther, Wolfgang Stremmel, Thomas Bayer

ABSTRACT Alzheimer's disease is an age-dependent neurodegenerative disorder associated with parenchymal and cerebrovascular deposition of fibrillized amyloid-P protein. Strong genetic evidence implicates the amyloid-p protein and its precursor, the amyloid-p protein precursor in the pathogenesis of Alzheimer's disease. Amyloid-p and amyloid-p protein precursor are both metalloproteins, and significant progress has been made toward understanding their biologic functions. A growing body of evidence links pathophysiologic copper and zinc metabolism to Alzheimer's disease. The role of copper and zinc in Alzheimer's disease, as well as possible physiological and pathophysiological interactions between these metals and amyloid-p and amyloid-P protein precursor, is reviewed here and therapeutic implications are discussed. 245

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1. INTRODUCTION The ~4kDa amyloid-f} (A(3) protein deposits as amyloid in the neuropil and cerebrovasculature in Alzheimer's disease (AD). Strong genetic evidence links A(3 and its precursor, the amyloid- (3 protein precursor (A(3PP), to AD pathogenesis. Although much progress has been made over the past few years toward understanding the biology and pathobiology of A(3 and A(BPP, consensus on the biologic and pathogenic functions of each protein has not been reached. The proteolytic cleavage of A(3PP is a necessary event in pathogenesis. The generation of A|3 occurs in a regulated cascade of cleavage events in A(3PP. The production of Ap is initiated by the transmembrane aspartyl protease f3-site amyloid precursor protein cleaving enzyme (BACE) that cleaves A(3PP at the N-terminus of A(3. Presenilin-1 is essential to gammasecretase cleavage and is part of the catalytic complex by which the Cterminal end of A(3 is liberated. The AfSPP copper (Cu)- binding domain (CuBD) in the amino terminus of A(3PP plays important roles both in modulating Cu homeostasis and in A(3 production (Borchardt et al., 1999), as described below. Substantial evidence links metal ion homeostasis to AD pathophysiology. This evidence derives mainly from in vitro, ex vivo, and in vivo studies of A(3 and A(3PP, as well as biochemical, pathologic, and therapeutic studies in AD. We review the growing body of evidence linking Cu and zinc (Zn) to AD and to Ap/A^PP function and pathogenicity, and discuss the therapeutic implications for Alzheimer patients.

2. METABOLISM OF COPPER AND ZINC IS ALTERED IN ALZHEIMER'S DISEASE Substantial evidence suggests that microscopic Cu and Zn distribution is altered in AD brain. First, accumulation of these metals in AD amyloid deposits has been demonstrated by microparticle X-ray emission spectroscopy (PIXE; Lovell et al., 1998). Cu was localized predominantly to the senile plaque rim, while Zn was elevated in the senile plaque rim and core. In addition, Zn was elevated in the AD neuropil, compared to

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age-matched control. Another PIXE study demonstrated increased Zn levels in the Alzheimer hippocampus and amygdala in unstained cryostat sections, though no correlation with plaques was made (Danscher et al., 1997). Synchrotron radiation-induced X-ray fluorescence has been employed to identify Cu and Zn in association with diffuse and mature plaques in untreated, fresh frozen cryostat sections of AD brain (Friedlich et al., manuscript in preparation). Histochemical methods for Cu and Zn detection have also been utilized to demonstrate abnormal distribution of these metals in AD. Rubeanic acid and rhodanine have been used to detect Cu in mature plaques, neurofibrillary tangles, and amyloid angiopathy (Friedlich et al., manuscript in preparation). Histochemically reactive Zn (N-(6-methoxy8-quindinyl-4-methylbenzene sulfonamide) (TSQ) fluorescence) has been localized to amyloid plaques and amyloid angiopathy in AD tissue (Suh et al., 2000) and in APPP2576 transgenic mouse brain (Lee et al., 1999). Gross, abnormalities in cerebral Cu and Zn metabolism in AD are less clear. Numerous studies have quantified metal levels in gross brain specimens or homogenates using instrumental neutron activation analysis (INAA) and inductively coupled plasma mass spectroscopy (ICPMS), with conflicting results. ICPMS studies have demonstrated decreased Zn in the AD hippocampus, thalamus, and gyri (Panayi et al., 2002), as well as in the frontal, occipital, and temporal cortices (Corrigan et al., 1993). An INAA study found decreased Cu and increased Zn in the AD hippocampus and amygdala (Deibel et al., 1996). An INAA subcellular fractionation study found decreased nuclear Zn in the temporal lobe (Wenstrup et al., 1990). Some evidence suggests that systemic Cu or Zn metabolism may be altered in AD. In the nun study, Tully et al. (1995) reported that fasting serum Zn about one year prior to death negatively correlated with plaque density in seven brain regions. A consensus is emerging that serum Cu is elevated in Alzheimer patients compared to age-matched control subjects (Kapaki et al., 1993; Squitti et al., 2002), and elevated serum Cu and Zn in AD has been reported to be associated with the apolipoprotein E-4 (apoE4) allele (Gonzalez et al., 1999). In AD cerebrospinal fluid (CSF), one study has reported an increase in Cu (Basun et al., 1991), two studies have reported an increase in Zn (Hershey et al., 1983; Rulon et al., 2000), and two studies have reported a decrease in Zn (Kapaki et al., 1993;

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Molina et al., 1998). Other reports have found no differences between AD and control CSF, with respect to Cu and Zn (Hershey et al., 1983; Sahu et al., 1988). Cerebral expression and distribution patterns of Cu and Zn transport and sequestration proteins provide additional evidence for disrupted metabolism of these cations in AD. Expression of ceruloplasmin (Castellani et al., 1999), metallothioneins I, II, and III (MT-I, -II, and -III; Yu et al., 2001), matrix metalloproteinases (MMP-1; Leake et al., 2000), MMP-2 (Backstrom et al., 1992), and MMP-9 (Asahina et al., 2001) have all been reported to be altered in the Alzheimer brain. MT-III is deficient in AD brain tissue (Uchida et al., 1988, 1991), which may impair the buffering capacity of the cortical tissue and contribute to the extracellular pooling of Zn and Cu that occurs in AD. Whereas abnormal Cu elevation may drive the toxicity of A@ (Huang et al., 1999b), interstitial Zn elevation may reflect a homeostatic antioxidant response. Mechanistically, this could be due to Zn 2+ release from the MT pool upon glial activation (Penkowa et al., 1999) or MT thiols being oxidized by hydrogen peroxide (H202) (Maret & Vallee, 1998), which could be elevated in AD tissue due to production from Ap (Huang et al., 1999a, 1999b). The hypothesis that Zn2+ elevation forms amyloid is supported by the distribution of chelatable (loosely-bound) Zn 2+ in the brain, which is most highly concentrated in the corticofugal system (Frederickson, 1989) and, therefore, parallels the anatomical sites most prone to amyloid deposition. Other Ap-associated proteins may also modulate the precipitation of A(3 in the presence of Zn 2+ , and so play a role in amyloid formation. The Zn-binding properties of alpha-2-macroglobulin, a genetic risk factor for AD (Blacker et al, 1998), modulate its binding to A(3 (Du et al, 1997). Also, apoE preserves A(3 solubility in the presence of Zn 2+ and the apoE4 isoform, a risk factor for amyloid deposition and AD, is the poorest solubility chaperone under these conditions (Moir et al., 1999). Therefore, in apoE4 carriers, A(3 is more likely to be precipitated by Zn.

3. COPPER AND ZINC BIND AMYLOID PRECURSOR PROTEIN Several fatal neurodegenerative disorders, such as AD, familial amyotrophic lateral sclerosis (FALS), and prion-related diseases (also known

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as the transmissible spongiform encephalopathies), are associated with the misfolding of a Cu-binding protein that is central to the specific disease (Strausak et al., 2001). We have investigated different aspects of the structure-function relationships of the underlying Cu-binding proteins A(BPP and prion protein (PrP), and have studied their cell biology, physiologic role in Cu transport, and role in human neurodegenerative diseases. The latter aspects are greatly facilitated by a number of transgenic and knockout mouse models.

3.1. Copper Binding Modulates A(3PP Homodimerization and Proteolysis Cu binding and oligomerization of A[3PP are important characteristics with implications for A(3PP function and amyloidogenesis. It has been shown that, in brain, a distinct percentage of A(3PP is present on the cell surface as a membrane protein of type I. This cell surface A^PP may mediate the transduction of extracellular signals into the cell via its C-terminal tail. Involvement of AfSPP in neuronal development, synaptogenesis, and synaptic plasticity indicated that the observed function is not restricted to secreted ApPP, raising the possibility that some aspects of synaptic plasticity are mediated by cell-associated ApPP A(3PP belongs to a multigene family of homologous proteins with different amyloid precursor-like protein lineages, including the ancestral drosophila [3-amyloid precursor like (APPL) and caenorhabditis elegans (3-amyloid precursor like-1 (APL-1) (Bayer et al., 1999). There is increasing evidence that all members of the superfamily have similar functional properties in cell-cell, cell-substrate adhesion, and Cu homeostasis. AfJPP has a CuBD located in the N-terminal cysteine-rich region, which can strongly bind Cu 2+ and reduce it to Cu + in vitro (Hesse et al., 1994; Multhaup et al., 1996). The CuBD sequence is similar among the A(3PPfamily paralogs, amyloid precursor-like proteins (APLP1 and APLP2), and its orthologs (including drosophila melanogaster, xenopus laevis, and caenorhabditis elegans), suggesting an overall conservation in its function or activity (Simons et al., 2002; White et al., 2002). The ApPP CuBD is involved in modulating Cu homeostasis and A(3 production. By three lines of evidence (crosslinking experiments, size exclusion chromatography, and mutational analysis), we could demonstrate that,

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under native conditions, cellular Af3PP and recombinant A(3PP from pichia pastoris are capable of forming noncovalent homodimers and tetramers (Scheuermann et al., 2001). AfJPP dimers appear to assemble in the endoplasmic reticulum shortly after the synthesis, suggesting that the homodimeric state could even be an essential prerequisite for its sorting in the transGolgi network and to secretory vesicles. The N-terminal domain is both necessary and sufficient for homodimerization, since the assembly of oligomers only depends on the N-terminal domain and does not require the C-terminal domain. This interaction does not require additional factors, but may be regulated by A(3PP-specific ligands, such as heparin, Cu 2+ , or Zn 2+ . Two highly conserved regions of AfSPP, the N-terminal AfJPP residues 91-111 and the C-terminal A(3PP residues 448-465 representing the collagen-binding site, are of critical importance for the regulation of homo-oligomerization of full-length A(3PP AfSPP dimerization occurs in a zipper-like mechanism. The interaction mediated between the N- and C-terminal domains of A(3PP behaves in a co-operative manner, with the N-terminal one being a prerequisite for efficient interaction and the C-terminal domain linking two pre-existing N-terminally-bound AfJPP molecules to one another. In accordance with this model, a Cys-mutant K624C of A|3PP695 formed disulfide bridges which rearranged spontaneously, possibly in an extended formation of the proposed linear zipper-like array with a third contact between juxtamembrane and intramembrane a-helices enabling a spatial proximity of the A|3 regions of two A|3PP molecules. A(3PP homodimers are a favored substrate of the A|3PP cleaving enzymes beta- and gamma- secretases, which are involved in the processing pathway that generates Ap. Mutant A(3PP with a single cysteine in the ectodomain juxtamembrane position generates stabilized dimers and produces six- to eightfold more amyloids in neuroblastoma cells than normal ApPP. These constitutively formed ApJPP oligomers, which also play a crucial role in AfJPP function. Such oligomers accumulate at the cell surface in populations of intact cells and enhance cell adhesion functions of A(3PP (Multhaup et al., unpublished observations). Taken together, our current efforts to investigate AfSPP dimerization will reveal its function for cell-cell and cell-matrix interactions of A|3PP and if it is the basic event in amyloid formation in AD. A remaining question is whether other APLPs dimerize like A(3PP The conservation of the cysteine-rich N-terminal domain is consistent

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with the possibility that all APLPs can dimerize and the variable residues in this region could provide the necessary dimerization specificity. To identify novel risk factors for AD and possible inhibitory compounds, it will obviously be important to determine whether ApJPP and APLPs can heterodimerize, and how A|3PP dimerization and increased A(S production are linked to the signaling function of A(3PP A(3PP also binds Zn 2+ through a binding site in its cysteine-rich amino terminus (residues 181 to 189, Kd»750nM), which is homologous in all members of the ApSPP, APLP1, and APLP2 superfamily, and preserved in caenorhabditis elegans and drosophila (Bush et al., 1993, 1994a). Zn 2+ binding at this site promotes ApSPP binding to heparin, which may be important for the interaction of A(3PP with the extracellular matrix and matrix metalloproteinases. Zn 2+ also increases the ability of the Kunitzprotease inhibitory isoform of A(3PP (AfSPP-KPI) to inhibit coagulation factor XIa (Van Nostrand, 1995), which may play a role in blood coagulation since Zn 2+ and A(3PP are releases during platelet activation (Bush et al., 1990). Zn 2+ also inhibits the oxidative fragmentation of A(3PP by Cu 2+ , indicating that Zn 2+ may serve a protective, antioxidant function in binding ApPP (Multhaup et al., 1998).

3.2. Importance of A|JPP for Copper Homeostasis ApPP expression modulates Cu homeostasis since ApPP / _ mice have elevated Cu levels in the liver and cerebral cortex compared to APPP + / + mice (White et al., 1999). In addition, elevated Cu concentrations decrease A(3 production and increase secretion of A(3PP in a cell line transfected with human A(3PP cDNA. This effect could be influenced by Zn or with Zn and Cu chelators (Borchardt et al., 2000). These studies provide strong evidence that AfSPP has an important role in modulating cellular Cu metabolism in certain tissues, including the brain. In general, alterations to A(3PP and/or Cu metabolism, as found in AD, could potentially result in increased A(3PP-Cu+-mediated oxidative stress and altered A(3PP processing to A(3 (Multhaup et al., 1998). In our cell culture studies, ApPP-Cu toxicity was specifically mediated by the Cu-binding ectodomain between residues 135 and 166 of human A(3PP. Mutagenesis of the ApPP CuBD revealed that ApPP-mediated Cu toxicity was dependent on the central histidine residues H147, H149, and H151. Importantly, A(3PP orthologs with different amino acid residues at the his-

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tidine positions have dramatically altered phenotypes. The caenorhabditis elegans APL-1 peptide (APL-lCuBD), which has tyrosine 147 and lysine 151, is strongly protected against Cu toxicity. The reason is that higher species CuBDs have a decreased affinity for Cu 2+ and high Cu2+-reducing activities; ancestral CuBDs form very tight binding sites for Cu 2+ ions and have low Cu2+-reducing activities. Thus, the ApPP lineage displays a gain in activity towards promoting Cu 2+ reduction and Cu + release. These findings identify a significant evolutionary change in the function of the CuBD. The data also highlight the important role of A[3PP CuBD in A(3PP metabolism. Thus, as AD is a typically common multifactorial disease in that environmental and genetic factors interact, a perturbed Cu homeostasis might result in a disease state and vice versa. Our recent data obtained with a mouse model system for AD indicate that Cu has a positive impact on A(3PP metabolism, and that bioavailable Cu cannot be regarded as a general risk factor in the pathogenesis of AD. Biochemical studies are in progress to verify the data on the molecular level. In a pilot study, we have treated three groups of aged AfSPP transgenic mice (A(3PP23 mice expressing human A(3PP751 with the Swedish mutation) and littermate controls with + sucrose, two with Cu-specific chelators DTPA and BC, and three with Cu sulfate. Whereas AfSPP overexpressing mice have a mean probable duration of life that is shorter than that of the littermate controls, a statistical analysis of our pilot study revealed that this effect is rescued in the group of animals that have been treated with Cu sulfate. The transgenic ApPP23 mice of the Cu group show an extended life duration compared with chelator-treated animals. Only the Cu-treated animals reach the ideal value for life duration of the control group, indicating that supplemented Cu rescued the toxic effect caused by the overexpression of human A(3PP, which is known to induce Cu depletion (Multhaup et al., unpublished observations).

4. COPPER AND ZINC BIND THE BETA AMYLOID PROTEIN AfJ possesses high and low affinity binding sites for Cu and Zn (Bush et al., 1994b; Atwood et al., 1998; Miura et al., 2000). The affinity of the Zn 2+ binding sites on AfJl-40 are 100nM and 5 |xM (Table 1), indicating that they might be occupied under physiological conditions (Atwood

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Importance of Copper and Zinc in Alzheimer's Disease Table 1. Cu and Zn binding affinities of the residue A(340 and -42 proteins. Data are from Atwood et al. (2000). Kd (M)

High affinity

Low affinity

Apl-40/Zn

IX10" 7

1.3 X10" 6

Apl-42/Zn A(31-40/Cu Api-42/Cu

7

1.3 X 10~6 1.3 X10" 8 5X10" 9

IX 10~ 4.6 X 1 0 _ u 7X10~ 18

et al., 1998, 2000). The highest affinity Cu 2+ binding site on Apl-42 has a measured K a « 10" 18 M, which is much greater than the highest affinity Cu 2+ binding site on Apl-40 (Ka% 10"11 M) (Atwood et al., 1998, 2000). This is important since Api-42 is enriched in cerebral amyloid pathology and its overexpression is linked to familial AD.

4.1. Copper and Zinc Binding to A(3 Mediates H 2 0 2 Production The differential affinity of A(342 and A(340 for Cu 2+ may account for the differential redox behavior and toxicity of these two species (Atwood et al., 2000). The Cu2+-A|31-42 complex has a strong reduction potential (+550mM vs Ag/AgCl) compared to the blue Cu proteins. Ap$ binds Cu 2+ and Zn 2+ to form a superoxide dismutase (SOD)- like structure (Curtain et al., 2001). Hence, the oxidative damage induced by A(3 may be mechanistically related to the oxidative stress induced by mutant SOD1 (Atwood et al., 1998, 2000). H 2 0 2 , implicated in AD pathogenesis, is produced by Cu A0 (Huang et al, 1999a). Among A(3 species, Cu A£42 produces H 2 0 2 at a faster rate than Cu A(340, while mouse Cu Ap produces much less H 2 0 2 (Huang et al., 1999a). The redox activities of these Cu A(3 species correlate with their respective neurotoxicity in culture, which is largely mediated by H 2 0 2 formed by Cu Ap (Huang et al., 1999a). H 2 0 2 formation by Cu A(3 and CuZn AfS can also be inhibited by chelation (Huang et al., 1999a), and the H202-mediated toxicity of A(3 in culture can be exaggerated by Cu 2+ and ameliorated by excess Zn 2+ (Cuajungco et al., 2000). Recently, we have determined that the production of H 2 0 2 by the A|3-Cu complex obeys a catalytic relationship (Km^i9 |xM and Vmax*30 nM for dopamine), where biological

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reducing agents are consumed as the reservoir of electrons for the reduction of dioxygen as the co-substrate (Opazo et al., 2002). Various biological reducing agents could drive the catalytic formation of H2Oz, such as dopamine and cholesterol. The neurotoxicity of AfS (in nanomolar concentrations) in cell culture was shown to be mediated by H 2 0 2 and dependent upon the presence of Cu 2+ and biological reducing agents.

4.2. Copper and Zinc Binding to AfJ Alters Protein Conformation It is now well-established that Af3 is rapidly precipitated by physiological concentrations of Zn 2+ , a physiologically inert cation. Under mildly acidic conditions (pH 6.8 to 7), the redox-active metals Cu 2+ and Fe 3+ induce greater A(3 aggregation than does Zn (Atwood et al., 1998). Significantly, the turbidity of rodent Apl-40 (with substitutions of Arg —> Gly, Tyr —> Phe, and His —> Arg at positions 5, 10, and 13, respectively) is unaffected by Zn 2+ or Cu 2+ at low micromolar concentrations (Atwood et al., 1998), perhaps explaining why rodents do not deposit cerebral amyloid. The molecular risk factor, apoE, modulates the precipitation of A|3 by Cu 2+ and Zn 2+ (Moir et al., 1999). We found that that the apoE4 isoform, which is an associated risk factor for the development of AD, is the poorest solubility chaperone for A(3 when compared to the other forms, apoE2 and apoE3. This finding holds regardless of whether the precipitating stress is Cu 2+ or Zn 2+ . CuZn selective chelators markedly enhance the resolubilization of Afi deposits from post-mortem AD brain samples (Cherny et al., 1999), supporting the possibility that Cu and Zn ions play a significant role in assembling these deposits. The metallochemistry of A(3 links oxidative damage and amyloidosis in AD. In vitro precipitation of Ap by Cu 2+ or Zn 2+ can be completely reversed by chelation (Huang et al., 1997). In the post-mortem AD brain, selective Cu or Zn chelators can induce the resolubilization of A$ from plaques (Cherny et al., 1999). Oxidative damage may be the earliest pathologic event in AD (Nunomura et al., 2001) and a negative correlation exists between amyloid burden and levels of 8-hydroxyguanosine, a marker of hydroxyl radical activity (Cuajungco et al., 2000). This is possibly because Zn suppresses H 2 0 2 formation from Ap. Thus, the mature plaque in AD may form through a compensatory

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mechanism where Zn 2+ is sequestered into accumulations of A(3. However, the quenching of H 2 0 2 formation by Zn 2+ is inefficient and, therefore, if the mass burden of plaque amyloid becomes sufficient, oxidative damage predominates (Opazo et al., 2002).

5. COPPER AND ZINC METABOLISM IS A PROMISING THERAPEUTIC TARGET IN ALZHEIMER'S DISEASE Preclinical and clinical data now support Cu and Zn metabolism as a valid therapeutic target in AD. Clioquinol (CQ), a retired antibiotic with a 0.5 billion patient-day history, was evaluated by Cherny et al. (2001) for efficacy in 21-month-old A(3PP2576 transgenic mice. Administration by lavage of 30 mg/kg for nine weeks resulted in a 49% decrease in brain A(3 load and a decrease in serum A(3, with no evidence of drug-induced toxicity or systemic loss of metals. General health and body weight parameters were significantly improved in the treated animals after only 16 days of treatment. Treatment of A(3PP2576 mice with CQ induced a «15% increase in brain Cu and Zn, indicating that the therapeutic mechanism of CQ is not simply chelation and clearance. A phase II double-blinded clinical trial of CQ in Alzheimer patients has recently been completed. CQ decreased plasma Ap levels and, in more advanced AD patients, slowed the rate of cognitive decline (Masters et al., personal communication). Continued investigation into Cu and Zn metabolism, the metallobiology of A(3 and A|3PP, and the mechanism of action of CQ seems likely to increase the understanding of AD pathogenesis and lead to new treatments for the millions of patients suffering from the disorder.

REFERENCES Asahina M, Yoshiyama Y, Hattori T. Expression of matrix metalloproteinase-9 and urinarytype plasminogen activator in Alzheimer's disease brain. Clin Neuropathol 2001; 20:60-63. Assaf SY, Chung S-H. Release of endogenous Zn 2+ from brain tissue during activity. Nature 1984; 308:734-736. Atwood CS, Moir RD, Huang X, Bacarra NME, Scarpa RC, Romano DM, Hartshorn MA, Tanzi RE, Bush Al. Dramatic aggregation of Alzheimer Ap by Cu(II) is induced by conditions representing physiological acidosis. J Biol Chem 1998; 273:12817-12826.

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Atwood CS, Huang X, Moir RD, Tanzi RE, Bush AI. Role of free radicals and metal ions in the pathogenesis of Alzheimer's disease. Met Ions Biol Syst 1999; 36:309-364. Atwood CS, Scarpa, RC, Huang X, Moir RD, Jones WD, Fairlie DP, Tanzi RE, Bush AI. Characterization of copper interactions with Alzheimer AB peptides—identification of an attomolar affinity copper binding site on AB1-42. JNeurochem 2000; 75:1219-1233. Backstrom JR, Miller CA, Tokes ZA. Characterization of neutral proteinases from Alzheimer-affected and control brain specimens: Identification of calcium-dependent metalloproteinases from the hippocampus. J Neurochem 1992; 58:983-992. Basun H, Forssell LG, Wetterberg L, Winblad B. Metals and trace elements in plasma and cerebrospinal fluid in normal aging and Alzheimer's disease. J Neural Transm Park Dis Dement Sect 1991; 3:231-258. Bayer TA, Cappai R, Masters CL, Beyreuther K, Multhaup G. It all sticks together—the ABPP-related family of proteins and Alzheimer's disease. Mol Psychiatry 1999; 4:524-528. Blacker D, Wilcox MA, Laird NM, Rodes L, Horvath SM, Go RC, Perry R, Watson B Jr, Bassett SS, Mclnnis MG, Albert MS, Hyman BT, Tanzi RE. Alpha-2 macroglobulin is genetically associated with Alzheimer' disease. Nat Genet 1998; 19:357-360. Borchardt T, Camakaris J, Cappai R, Masters CL, Beyreuther K, Multhaup G. Copper inhibits B-amyloid production and stimulates the non-amyloidogenic pathway of amyloid precursor protein (ABPP) secretion. Biochem J 1999; 344:461-467. Borchardt T, Schmidt C, Camakaris J, Cappai R, Masters CL, Beyreuther K, Multhaup G. Differential effects of zinc on amyloid precursor protein (ABPP) processing in copperresistant variants of cultured Chinese hamster ovary cells. Cell Mol Biol 2000; 46:785-795. Bush AI, Martins RN, Rumble B, Moir R, Fuller S, Milward E, Currie J, Ames D, Weidemann A, Fischer P. The amyloid precursor protein of Alzheimer's disease is released by human platelets. J Biol Chem 1990; 265:15977-15983. Bush AI, Multhaup G, Moir RD, Williamson TG, Small DH, Rumble B, Pollwein P, Beyreuther K, Masters CL. A novel zinc(II) binding site modulates the function of the AB4 amyloid protein precursor of Alzheimer's disease. J Biol Chem 1993; 268:16109-16112. Bush AI, Pettingell WH, de Paradis M, Tanzi RE, Wasco W. The amyloid-B protein precursor and its mammalian homologues. Evidence for a zinc-modulated heparin-binding superfamily. J Biol Chem 1994a; 269:26618-26621. Bush AI, Pettingell WH, Multhaup G, Paradis MD, Vonsattel JFG, Beyreuther K, Masters CL, Tanzi RE. Rapid induction of Alzheimer AB amyloid formation by zinc. Science 1994b; 265:1464-1467. Bush AI. Metals and neuroscience. Curr Opin Chem Biol 2000; 4:184-191. Castellani RJ, Smith MA, Nunomura A, Harris PL, Perry G. Is increased redox-active iron in Alzheimer's disease a failure of the copper-binding protein ceruloplasmin? Free Radic Biol Med 1999; 26:1508-1512. Chelly J, Turner Z, Tonnesen T, Petterson A, Ishikawa-Brush Y, Tommerup N, Horn N, Monaco AP. Isolation of a candidate gene for Menkes disease that encodes a potential heavy metal binding protein. Nat Genet 1993; 3:14-19.

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Cherny RA, Legg JT, McLean CA, Fairlie D, Huang X, Atwood CS, Beyreuther K, Tanzi RE, Masters CL, Bush AI. Aqueous dissolution of Alzheimer's disease A(3 amyloid deposits by biometal depletion. J Biol Chem 1999; 274:23223-23228. 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. Corrigan FM, Reynolds GP, Ward NI. Hippocampal tin, aluminum and zinc in Alzheimer's disease. Biometals 1993; 6:149-154. Cuajungco MP, Goldstein LE, Nunomura A, Smith MA, Lim JT, Atwood CS, Huang X, Farrag YW, Perry G, Bush AI. Evidence that the beta-amyloid plaques of Alzheimer's disease represent the redox-silencing and entombment of abeta by zinc. J Biol Chem 2000;275:19439-19442. Curtain CC, Ali F, Volitakis I, Cherny RA, Norton RS, Beyreuther K, Barrow CJ, Masters CL, Bush AI, Barnham KJ. Alzheimer's disease amyloid binds Cu and Zn to generate an allosterically-ordered membrane-penetrating structure containing SOD-like subunits. J Biol Chem 2001; 276:20466-20473. Danscher G, Jensen KB, Frederickson CJ, Kemp K, Andreasen A, Juhl S, Stoltenberg M, Ravid R. Increased amount of zinc in the hippocampus and amygdala of Alzheimer's diseased brains: A proton-induced X-ray emission spectroscopic analysis of cryostat sections from autopsy material. J Neurosci Methods 1997; 76:53-59. Deibel MA, Ehmann WD, Markesbery WR. Copper, iron, and zinc imbalances in severely degenerated brain regions in Alzheimer's disease: Possible relation to oxidative stress. J Neurol Sci 1996; 143:137-142. Du Y, Ni B, Glinn M, Dodel RC, Bales KR, Zhang Z, Hyslop PA, Paul SM. Alpha2macroglobulin as AP-amyloid peptide-binding plasma protein. J Neurochem 1997; 69:299-305. Frederickson CJ. Neurobiology of zinc and zinc-containing neurons. Int Rev Neurobiol 1989;31:145-328. Frederickson CJ, Suh SW, Silva D, Thompson RB. Importance of zinc in the central nervous system: The zinc-containing neuron. J Nutr 2000; 130:S1471-S1483. Glenner GG, Wong CW. Alzheimer's disease: Initial report of the purification and characterization of a novel cerebrovascular amyloid protein. Biochem Biophys Res Commun 1984; 120:885-890. Gonzalez C, Martin T, Cacho J, Brenas MT, Arroyo T, Garcia Berrocal B, Navajo JA, Gonzales Buitrago JM. Serum zinc, copper, insulin and lipids in Alzheimer's disease epsilon 4 apolipoprotein E allele carriers. Eur J Clin Inv 1999; 29:637-642. Hambidge M, Krebs NF. Interrelationship of key variables of human zinc homeostasis: Relevance to dietary zinc requirements. Ann Rev Nutr 2001; 21:429^152. Hartter DE, Barnea A. Brain tissue accumulates 67copper by two ligand-dependent saturable processes. J Biol Chem 1988a; 263:799-805.

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Hartter DE, Bamea A. Evidence for release of copper in the brain: Depolarization-induced release of newly taken-up 67copper. Synapse 1988b; 2:412-415. Hershey CO, Hershey LA, Varnes A, Vibhakar SD, Lavin P, Strain WH. Cerebrospinal fluid trace element content in dementia: Clinical, radiologic, and pathologic correlations. Neurology 1983; 33:1350-1353. Hesse L, Beher D, Masters CL, Multhaup G. The beta A4 amyloid precursor protein binding to copper. FEBS Lett 1994; 349:109-116. Howell GA, Welch MG, Frederickson CJ. Stimulation-induced uptake and release of zinc in hippocampal slices. Nature 1984; 308:736-738. Huang X, Atwood CS, Moir RD, Hartshorn MA, Vonsattel J-P, Tanzi RE, Bush AL Zincinduced Alzheimer's AfU-40 aggregation is mediated by conformational factors. J Biol Chem 1997; 272:26464-26470. Huang X, Atwood CS, Hartshorn MA, Multhaup G, Goldstein LE, Scarpa RC, Cuajungco MP, Gray DN, Lim J, Moir RD, Tanzi RE, Bush, AL The AB peptide of Alzheimer's disease directly produces hydrogen peroxide through metal ion reduction. Biochemistry 1999a; 38:7609-7616. Huang X, Cuajungco MP, Atwood CS, Hartshorn MA, Tyndall J, Hanson GR, Stokes KC, Leopold M, Multhaup G, Goldstein LE, Scarpa RC, Saunders AJ, Lim J, Moir RD, Glabe C, Bowden EF, Masters CL, Fairlie DP, Tanzi RE, Bush AL Cu(II) potentiation of Alzheimer AB neurotoxicity: Correlation with cell-free hydrogen peroxide production and metal reduction. / Biol Chem 1999b; 274:37111-37116. Jacob C, Maret W, Vallee BL. Control of zinc transfer between thionein, metallothionein, and zinc proteins. Proc Natl Acad Sci USA 1998; 95:3489-3494. 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. Kapaki EN, Zournas CP, Segdistsa IT, Xenos DS, Papageorgiou CT. Cerebrospinal fluid aluminum levels in Alzheimer's disease. Biol Psychiatry 1993; 33:679-681. King JC, Shames DM, Woodhouse LR. Zinc homeostasis in humans. J Nutr 2000; 130:S1360-S1366. Krebs NF. Overview of zinc absorption and excretion in the human gastrointestinal tract. J Nutr 2000; 130:S1374-S1377. Leake A, Morris CM, Whateley J. Brain matrix metalloproteinase 1 levels are elevated in Alzheimer's disease. Neurosci Lett 2000; 291:201-203. Lee DY, Prasad AS, Hydrick-Adair C, Brewer G, Johnson PE. Homeostasis of zinc in marginal human zinc deficiency: Role of absorption and endogenous excretion of zinc. J Lab Clin Med 1993; 122:549-556. Lee J-Y, Mook-Jung I, Koh J-Y. Histochemically reactive zinc in plaques of the Swedish mutant beta-amyloid precursor protein transgenic mice. J Neurosci 1999; 19:1-5. Lovell MA, Robertson JD, Teesdale WJ, Campbell JL, Markesbery WR. Copper, iron and zinc in Alzheimer's disease senile plaques. J Neurol Sci 1998; 158:47-52. Maret W, Vallee BL. Thiolate ligands in metallothionein confer redox activity on zinc clusters. Proc Natl Acad Sci USA 1998; 95:3478-3482.

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Penkowa M, Giralt M, Moos T, Thomsen PS, Hernandez J, Hidalgo J. Impaired inflammatory response to glial cell death in genetically metallothionein-I- and -II-deficient mice. Exp Neurol 1999; 156:149-164. Pullen RG, Franklin, PA, Hall GH. 65Zinc uptake from blood into brain and other tissues in the rat. Neurochem Res 1990; 15:1003-1008. Rulon LL, Robertson JD, Lovell MA, Deibel MA, Ehmann WD, Markesbery WR. Serum zinc levels and Alzheimer's disease. Biol Trace Elem Res 2000; 75:79-85. Sahu RN, Pandey RS, Subhash MN, Arya BY, Padmashree TS, Srinivas KN. CSF zinc in Alzheimer's type dementia. Biol Psychiatry 1988; 24:480-482. Sandstead HH. Causes of iron and zinc deficiencies and their effects on brain. J Nutr 2000; 130:S347-S349. Scheuermann S, Hambsch B, Hesse L, Stumm J, Schmidt C, Beher D, Bayer TA, Beyreuther K, Multhaup G. Homodimerization of A(3PP and its implication in the amyloidogenic pathway of Alzheimer's disease. J Biol Chem 2001; 276:33923-33929. Simons A, Ruppert T, Schmidt C, Schlicksupp A, Pipkorn R, Reed J, Masters CL, White AR, Cappai R, Beyreuther K, Bayer TA, Multhaup G. Evidence for a copper-binding superfamily of the amyloid precursor protein. Biochemistry 2002; 41:9310-9320. Squitti R, Rossini PM, Cassetta E, Moffa F, Pasqualetti P, Cortesi M, Colloca A, Rossi L, Finazzi-Agro A. d-penicillamine reduces serum oxidative stress in Alzheimer's disease patients. Eur J Clin Invest 2002; 2:51-59. Strausak D, Mercer JF, Dieter HH, Stremmel W, Multhaup G. Copper in disorders with neurological symptoms: Alzheimer's, Menkes, and Wilson diseases. Brain Res Bull 2001;55:175-185 Suh SW, Jensen KB, Jensen MS, Silva DS, Kesslak PJ, Danscher G, Frederickson CJ. Histochemically-reactive zinc in amyloid plaques, angiopathy, and degenerating neurons of Alzheimer's diseased brains. Brain Res 2000; 852:274-278. Szerdahelyi P, Kasa P. Histochemistry of zinc and copper. Int Rev Cytol 1984; 89:1-29. Tanzi RE, Petrukhin K, Chernov I, Pellequer JL, Wasco W, Ross B, Romano DM, Parano E, Pavone L, Brzustowicz LM, Devoto M, Peppercorn J, Bush AI, Sternlieb I, Pirastu M, Gusella JF, Evgrafov O, Penchaszadeh GK, Honig B, Edelman IS, Soares MB, Scheinberg IH, Gilliam TC. Identification of the Wilson's disease gene; a copper transporting ATPase with homology to the Menkes' disease gene. Nat Genet 1993; 3:344-350. Tully CL, Snowdon DA, Markesbery WR. Serum Zinc, senile plaques, and neurofibrillary tangles: Findings from the Nun study. Neuroreport 1995; 6:2105-2108. Uchida Y, Ihara Y, Tomonaga M. Alzheimer's disease brain extract stimulates the survival of cerebral cortical neurons from neonatal rats. Biochem Biophys Res Commun 1988; 150:1263-1267. Uchida Y, Takio K, Titani K, Ihara Y, Tomonaga M. The growth-inhibitory factor that is deficient in the Alzheimer's disease brain is a 68-amino acid metallothionein-like protein. Neuron 1991; 7:337-347. Vallee BL. The function of metallothionein. Neurochem Int 1995; 27:23-33.

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Van Nostrand WE. Zinc(II) selectively enhances the inhibition of coagulation factor XIa by protease nexin-2/amyloid-beta-protein precursor. Thromb Res 1995; 78:43-53. Wenstrup D, Ehmann WD, Markesbery WR. Trace element imbalances in isolated subcellular fractions of Alzheimer's disease brains. Brain Res 1990; 533:125-131. White AR, Multhaup G, Galatis D, McKinstry WJ, Parker MW, Pipkorn R, Beyreuther K, Masters CL, Cappai R. Contrasting, species-dependent modulation of copper-mediated neurotoxicity by the Alzheimer's disease amyloid precursor protein. J Neurosci 2002; 22:365-376. White AR, Reyes R, Mercer JFB, Camakaris J, Zheng H, Bush AI, Multhaup G, Beyreuther K, Masters CL, Cappai R. Copper levels are increased in the cerebral cortex and liver of ApPP and APLP2 knockout mice. Brain Res 1999; 842:439-444. Wong PC, Waggoner D, Subramaniam JR, Tessarollo L, Bartnikas TB, Culotta VC, Price DL, Rothstein J, Gitlin, JD. Copper chaperone for superoxide dismutase is essential to activate mammalian Cu/Zn superoxide dismutase. Proc Natl Acad Sci USA 2000; 97:2886-2891. Yu WH, Lukiw WJ, Bergeron C, Niznik HB, Fraser PE. Metallothionein III is reduced in Alzheimer's disease. Brain Res 2001; 894:37-45.

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CHAPTER 10

Copper-Zinc Superoxide Dismutase and Familial Amyotrophic Lateral Sclerosis Moon B Yim, P Boon Chock, Earl R Stadtman

ABSTRACT More than 90 individual mutations in copper-zinc superoxide dismutase are associated with familial cases of amyotrophic lateral sclerosis and they are mostly fatal within one to five years of the onset of the disease. Results obtained from studies with transgenic mice, transfected cells, and patients suggest that familial amyotrophic lateral sclerosis copper-zinc superoxide dismutase mutants have gains or enhancements in one or more cytotoxic properties, which are not related to the superoxide dismutation activity. The proposed nature of the cytotoxic gain-of-function of the familial amyotrophic lateral sclerosis copperzinc superoxide dismutase mutants under debate includes enhanced free radicalgenerating (peroxidative) activity, enhanced activity of peroxynitrite-mediated tyrosine nitration, and familial amyotrophic lateral sclerosis mutant-associated aggregate formation due to failure of protein folding. We review these proposals and summarize our findings from the studies with purified familial amyotrophic lateral sclerosis copper-zinc superoxide dismutase mutants. Keywords: Copper-zinc superoxide dismutase; familial amyotrophic lateral sclerosis; free radical; protein oxidation; protein aggregation.

1. INTRODUCTION Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease, is an age-dependent, fatal degenerative disorder of motor neurons in the spinal cord, motor cortex, and brainstem. Up to 10% of all ALS patients 263

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are familial ALS (FALS) and about 20% of FALS are associated with dominantly inherited missense mutations in the coding regions of superoxide dismutase 1 (SOD1), the gene for copper-zinc SOD (Cu,ZnSOD) (Rosen et al., 1993; Deng et al., 1993), which catalyzes the dismutation of superoxide radical anions (0 2 ") to hydrogen peroxides (H 2 0 2 ) and oxygen molecules (0 2 ). Over 90 individual point mutations in Cu,ZnSOD are known to cause FALS, which is usually fatal within one to five years of onset of the disease, generally at the age of 45 years and older (Gaudette et al., 2000; Cleveland and Rothstein, 2001), when the antioxidant defenses start to deteriorate (Moskovitz et al., 2002). The mutation sites are scattered throughout the structure of the wild-type Cu,ZnSOD protein. Several mutated residues are positioned within the electrostatic active site channel and in the active site (H46R, H48Q, or H80R). Another group of mutations causes truncation of the final 20 to 33 amino acids. The most abundant sites of mutation are located in conserved interaction regions critical to the subunit fold and dimer contact. Initial studies suggested that erythrocytes and brain tissues of FALS patients exhibited a marked reduction in Cu,ZnSOD activity compared to that of normal individuals (Deng et al., 1993). This reduction in SOD activity may induce oxidative damage and cause FALS symptoms. However, several studies with transgenic mice (Gurney et al., 1994; Ripps et al., 1995; Bruijn et al., 1997; Wong et al., 1995), transfected cells (Borchelt et al., 1995; Rabizadeh et al., 1995), and lymphoblasts of patients (Deng et al., 1993; Borchelt et al., 1994) revealed that levels of total Cu,ZnSOD activities remain unchanged or higher than normal. These results imply that the FALS mutants have gained or enhanced one or more cytotoxic properties, which are not related to superoxide dismutation (SOD) activity. Although the nature of the cytotoxic gain-of-function caused by Cu,ZnSOD FALS mutants is debatable, several mechanisms have been proposed. They include enhanced free radical-generating (or peroxidative) activity with H 2 0 2 and small anions as substrates (Yim et al., 1996, 1997; Wiedau-Pazos et al., 1996), enhanced activity of peroxynitritemediated tyrosine nitration, especially with Zn-depleted enzymes (Crow et al., 1997a, 1997b; Estevez et al., 1999), and FALS mutant-associated aggregate formation due to failure of protein folding (Bruijn et al., 1998; Chou et al., 1998). For the first two mechanisms, oxidation reactions catalyzed by Cu ions at the active sites or other bound sites are required. In

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contrast, the aggregate formation for the last mechanism may not require Cu incorporation into Cu,ZnSOD, even though enhanced free radicalgenerating activity would lead to aggregation of macromolecules. We review these proposals and summarize our findings obtained from the studies with purified FALS Cu,ZnSOD mutants.

2. STRUCTURE OF Cu,ZnSOD Cu,ZnSOD is a homodimer with each subunit containing 153 amino acids and an active site that contains one Cu ion at the catalytic site (ligated by the four histidines (His) of 46, 48, 63, and 120) and one Zn ion at the structural site (ligated by the three His of 63, 71, and 80 and one Asp, 83) (Tainer et al., 1982; Getzoff et al., 1983). The Cu and Zn ions are bridged by His-63. The catalytic site can be reached by the substrate through a positively charged channel shaped like a funnel, which leads to the catalytically essential Arg 143 and a narrow hydrophobic access (< 4 A) to the Cu ion (see Fig. 1). One oxygen molecule of the superoxide anion binds to the Cu ion at the active site, while the other oxygen molecule forms a hydrogen bond with the positively charged guanidinum nitrogen on Arg 143, which is located 5 A away from the Cu ion. This positively charged channel, which consists the basic residues Arg 143, Lys 122, and Lys 136, is responsible for the favorable electrostatic guidance of the anionic substrate 0^" to the active site to yield an unusually rapid catalytic rate constant (2 X 109 M _ 1 s _1 ) for superoxide dismutation (Klug et al., 1977; Tainer et al., 1982; Getzoff et al., 1983). Other small anions, such as azide, cyanide, halides, and phosphate, are also known to have easy access to the channel and bind to either the active site or the positively charged guanidinum nitrogen on Arg 143 (Rigo et al., 1977; Mata de ( ^ ^

24A

> J

*Thr5§\

5A |

5A

("

L

10A

Argl43"-\

Glu 133

>J (Thrl37

WA^ Cu

Fig. 1. Shape and dimension of the active site channel. The Lys 136 and Lys 122 located front and back of the paper, respectively, at the top of the channel.

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Freitas and Valentine, 1984). Thus, these anions, as well as many other small anions, behave as inhibitors in the superoxide dismutation reaction and exert profund effects on the general reactions involving Cu,ZnSOD.

3. PRO-OXIDANT, FREE RADICAL-GENERATING (PEROXIDATIVE) ACTIVITY OF Cu,ZnSOD Besides its superoxide dismutation activity, Cu,ZnSOD also reacts with its own reaction product, H 2 0 2 , which leads to its inactivation (Hodgson and Fridovich, 1975a; Fuchs and Borders, 1983). The inactivation appears to be caused by the oxidation of one or more amino acid residues of the enzyme. This includes His, which is ligated to the Cu ion at the active site of the enzyme. A reaction mechanism has been proposed for the inactivation reaction (Hodgson and Fridovich, 1975a): Enz-Cu(II) + H 2 0 2 -> Enz-Cu(I) + 0 2 * + 2H + , Enz-Cu(I) + H 2 0 2 -> Enz-CuQIKOH + OH", Enz-Cu(II)-"OH + ImH -> Enz-Cu(II) + Im" + H 2 0.

(1) (2) (3)

The intermediate product, Enz-Cu(II)-"OH, is a highly oxidizing species, such as free, caged, or Cu-bound "OH radical. The reactive species can react with the enzyme itself, resulting in damage to amino acid residues, such as the imidazole moiety of a His residue at the active site. Analysis of the stable products of the inactivated SOD revealed that 2-oxohistidine (His-118 for bovine SOD) was produced during the inactivation reaction by H 2 0 2 (Uchida and Kawakishi, 1994). Recent spin-trapping electron paramagnetic resonance (EPR) spectroscopic experiments have shown that 2-oxohistidine was formed by the reaction of 0 2 with the 2-histidinyl radical intermediate. This was confirmed by comparing the EPR spectrum of the authentic histidinyl radical generated by the reaction of the free "OH radical with His (Gunther et al., 2002). Some small anions, such as azide and formate, which have easy access to the active channel, protect the enzyme against inactivation caused by H 2 0 2 by scavenging free or metal-bound "OH radicals generated in reaction 3 (Hodgson and Fridovich, 1975b). During this reaction, the scavenger anions were oxidized. Hence, Cu,ZnSOD exhibits peroxidative activity (Hodgson and Fridovich, 1975b). The scavengers that can be oxidized are not limited to small anions that protect the enzyme

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against inactivation, but also include proteins and relatively large molecules, such as ferrocytochrome-c and bilirubin, or neutral molecules, such as alcohols. These molecules or proteins do not protect the Cu,ZnSOD against inactivation by H 2 0 2 . These results may suggest that reactive species were released from the Cu ion to oxidize larger molecules or proteins. In the initial spin-trapping EPR experiments (Yim et al., 1990, 1993), it was demonstrated that during these reactions "OH radicals and other free radical species originating from scavengers are generated and some stable free radicals may diffuse away from the active site to exert oxidative damage on other macromolecules in the cell. Thus, Cu,Zn-SOD can function as a pro-oxidant to generate free radicals that may initiate harmful chain reactions in vivo (Yim et al., 1990, 1993, 1999).

4. FREE RADICAL SPECIES GENERATED BY Cu,ZnSOD In initial studies (Yim et al., 1990, 1993), two spin traps, 5,5'-dimethyl-1pyrroline-N-oxide (DMPO) and N-tert-butyl-a-phenylnitrone (PBN), were used to convert transient free radicals to stable free radical adducts according to the following reaction: DMPO (or PBN) + R' -» DMPO-R* (or PBN-R').

(4)

The nature of the trapped free radical was identified by EPR spectroscopy. When H 2 0 2 was added to the solution containing active Cu,Zn-SOD and DMPO, EPR resonance lines of the hydroxyl radical adduct of DMPO (DMPO-'OH) were observed (Yim et al., 1990). The formation of this radical adduct was greatly affected by various buffer systems: the signal amplitude of DMPO-'OH obtained in phosphate (25 mM, pH 7.3) or borate (25 mM, pH 8.1) buffers was increased dramatically in the NaHC0 3 (23.5 mM)/C0 2 buffer at pH 7.6 (Yim et al, 1990). A recent study (Sankarapandi and Zweier, 1999a) suggested that bicarbonate may bind to the Arg 141 of bovine Cu,ZnSOD and anchors H 2 0 2 at the active site Cu for easier redox cleavage. In the presence of "OH radical scavengers with anionic characteristics, such as HCO^, N J , or glutathione, DMPO adducts of the scavenger-derived radicals, DMPO-C0 2 \ DMPO-Nj, or DMPO-GS' were observed besides DMPO-'OH (Yim et al., 1990; Kwak et al., 1995). Surprisingly, with neutral 'OH radical scavengers, such as ethanol, DMPO-hydroxyethyl radical adducts were

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formed, but in very small quantities compared to those of anionic scavengers. However, with a different spin trap, PBN, in place of DMPO, PBN-hydroxyethyl radical adducts were formed in a quantity for a competition reaction for free *OH radicals. These seemingly contradictory results can be explained by differences in the affinities of the spin traps used to bind to the positively charged active channel of Cu,ZnSOD, where the "OH radicals are generated (Yim et al., 1990, 1993). For this purpose, we measured binding constants of the spin traps to the active site/channel using a negatively charged chromogen, 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS) (Yim et al., 1993). The ABTS is known to react with the "OH radical at a diffusion-controled rate (reaction 5) to produce a stable oxidized free radical, ABTS+", which has strong absorption at 415 nm (E415 - 3.6 X 104 JVT' cm - 1 ): ABTS + "OH -> ABTS + " + OH~

k = 1.2 X 1010 M _ 1 s"1.

(5)

Addition of active Cu,ZnSOD also produced ABTS+" in the presence of H 2 0 2 and ABTS. The formation of ABTS+* followed first-order kinetics with respect to Cu,ZnSOD and H 2 0 2 . However, it showed a binding isotherm with ABTS that yielded a dissociation constant for ABTSCu,ZnSOD of Kd = 7.1 ± 0.5. The formation of ABTS+" was inhibited by the presence of DMPO or PBN in the reaction mixture. By performing a competition study, Kd values for DMPO and PBN were obtained as 0.63 mM and 11 mM, respectively. A radical scavenger, formate anion, also inhibits the formation of ABTS+", whereas ethanol does not. These results indicate that DMPO and anionic scavengers have much more accessibility to the cavity of the active channel than PBN and neutral ethanol. Consequently, DMPO or anionic scavengers are in a position to intercept the newly formed "OH radicals and drastically reduce the probability for the reaction of "OH radicals with ethanol. Therefore, the concentrations of DMPO-hydroxyethyl radical adducts observed are much lower than that expected under equally competitive conditions. PBN and ethanol, however, are able to compete in the bulk solution for free "OH radicals released from the active site of Cu,ZnSOD. Thus, both PBN-hydroxyethyl radical and PBN-OH can be detected. Conversely, these results indicate that free 'OH radicals and scavenger-derived radicals can reach the bulk solution and may cause oxidative damage to the biological environments.

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Considering the short half-life of "OH radicals, the majority of the radicals released to biological environments will be secondary cascading radicals formed by scavenging the initial 'OH radicals within a short distance of their formation (Yim et al, 1990, 1993, 1999). The term "bulk solution" should not be misunderstood. It is defined as a few bonding distances away (5 A) from the active Cu ion site due to the unique topography of the channel. As shown in Fig. 1, the Cu ion is located about 5 A inside the narrow channel (<4 A width), such that it can be reached by Br~ (diameter of 3.9 A), but not I" (diameter of 4.32 A) (Rigo et al., 1997). Thus, it is quite possible that, in the absence of the small scavenging anion accessible to the Cu ion, some fraction of the newly synthesized 'OH radicals can be released and trapped by DMPO (or oxidized by other molecules) located about 5 A away, but DMPO (or other oxidizing molecules) cannot protect against self-inactivation of Cu,ZnSOD caused by the damage to the Cu ligands: A—

DMPO

-^ Enz-Cu(II) + 'A > DMPO-'A, (6) Enz-Cu(II)-"OH —> 'Enz-Cu(II) —> Inactivation, oxidized protein, (7) -> Enz-Cu(II) + 'OH > DMPO-'OH, B'. (8) DMPO orBH

Several groups (Sankarapandi and Zweier, 1999b; Singh et al, 1998; Goss et al., 1999; Zhang et al., 2000, 2002) carried out similar experiments and concluded against the release of free "OH radicals from metal ions for various reasons. Singh et al. (1998) and Goss et al. (1999) carried out DMPO spin-trapping experiments using O-17-enriched H 2 17 0 2 and H 2 17 0. Their experimental spectra clearly showed that a significant fraction (—65%) of DMPO-'OH (and also with a different spin trap, DEPMPO-'OH) formed during SOD reaction was derived from oxygen of H 2 0 2 , but DMPO cannot protect the enzyme against inactivation. This result supports the above mechanism, although they reached a different conclusion. A recent report (Zhang et al., 2000) by the same group, however, claimed that 100% of oxygen in DMPO-'17OH originated from H 2 17 0, and not from H 2 17 0 2 . They suggested that the DMPO-'OH adducts observed originated entirely from the hydrolysis of DMPO-carbonate radical adducts, which were formed by

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the scavenging reaction that occurred in the bicarbonate buffer. However, DMPO-'OH adducts can also be observed in phosphate and borate buffers (Yim et al., 1990). In free radical chemistry, propagating radicals and chain carriers frequently govern the final products, rather than initiating species of free radical reactions. Thus, a variety of secondary free radicals can be produced by the reaction involving Cu,Zn-SOD and H 2 0 2 in vivo, irregardless of the identity of the initiating species, be it free, caged, or metal-bound "OH. These secondary free radical species, including radicals originating from glutathione (Kwak, et al., 1995), formate (Yim et al., 1990, 1993), bicarbonate (Goss et al., 1999), neurotansmitters (Yim et al., 1993), and many other radicals that have yet to be identified, may diffuse away from the active channel. These diffused radicals could initiate damaging reactions to proteins and other macromolecules and/or behave as second messengers to induce apoptosis (Guegan et al., 2001).

5. ENHANCEMENT OF FREE RADICAL GENERATION BY FALS Cu,ZnSOD MUTANTS Yim et al. (1996, 1997) and Wiedau-Pazos et al. (1996) have investigated whether there are any differences in the free radical-generating activity between the wild-type and FALS mutant Cu,ZnSOD. For this purpose, we cloned the wild-type and FALS G93A and A4V cDNA of human Cu,ZnSOD, overexpressed them in insect cells (Sf9), purified proteins, and measured their enzymatic activities. Our results revealed that the G93A, A4V, and wild-type Cu,ZnSOD have similar dismutation activities. However, the free radical-generating function of the G93A and A4V mutants measured by the spin-trapping method is enhanced relative to that of the wild-type enzyme (wild-typeG93A (25 mM)>A4V (13 mM)]. Thus, A4V and G93A have higher affinities for H 2 0 2 than the wild type. This increased affinity for H 2 0 2 could favor its conversion to "OH radicals. This enhancement of free radical-generating activity may provide, in part, an explanation for the association of the FALS Cu,ZnSOD mutants to ALS. We have also investigated whether FALS mutants have enhanced capacity for tyrosine nitration mediated by peroxynitrite (unpublished

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results) by using two substrates, free tyrosine and a synthetic pentadecameric peptide, which correspond to the tyrosine phosphorylation site of p34cdc2 kinase used in our previous work (Kong et al., 1996). The results showed that the nitration activity of the FALS mutants is virtually identical to that of the wild-type enzyme. Several reports, however, suggested that FALS mutants have a lower affinity to Zn ion (Lyons et al., 1996; Crow et al., 1997a) and Zn-deficient enzyme, either wild-type or mutant, shows enhanced capacity for nitration involving peroxynitrite (Estevez et al., 1999). Considerable experimental results support the involvement of free radicals in FALS. Hall et al. (1998) and Andrus et al. (1998) demonstrated that lipid peroxidation and protein carbonyl levels, a marker of protein oxidative damage, in G93A transgenic mice were elevated in comparison to nontransgenic mice, respectively. Liu et al. (1998) carried out spin trapping by using azulenyl nitrone, which produces azulenyl aldehyde through reaction with free radicals. The results obtained from G93A transgenic mice showed significant enhancement of free radical generation in the spinal cord prior to motor neuron degeneration compared to wild-type transgenic control mice. Bogdanov et al. (1998) used a microdialysis method to measure oxidation of 4-hydroxybenzoic acid to 3,4-dihydroxybenzoic acid. They observed elevated hydroxyl radical generation in vivo in G93A transgenic mice, but not in wild-type transgenic mice. Aguirre et al. (1998) demonstrated that the fibroblasts obtained from FALS patients showed an increased sensitivity to H 2 0 2 stress, which was explained by the increased affinity of the mutants to H 2 0 2 . Liu et al. (2002) showed that infection of mouse NSC-34 motor neuron-like cells with the G93Acontaining vector increased cellular oxidative stress, indicating mitochondrial dysfunction. Pretreatment of cells containing the mutant with highly oxidizable polyunsaturated fatty acid elevated lipid peroxidation and enhanced cell death, but not with cells containing wild-type Cu,ZnSOD. Pretreatment with DMPO prevents mutant-mediated mitochondrial dysfunction and cell death. Roe et al. (2002) trapped free radicals generated in the FALS mutants (G93A, G93C, L38V, and A4V) expressed in yeast cells using the a-(pyridyl-4-N-oxide)-N-tert-butylnitrone (POBN) spin trap. They found that mutant Cu,ZnSOD produced a greater concentration of trapped free radical adducts. Zhang et al. (2002) used the spinal cords of homogenized mice to detect the oxidation of dichlorodihydrofluoroscein

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(DCHF) to dichlorofluoroscein. Their results showed a significant increase in the oxidation of DCHF in spinal cord extracts from G93A transgenic mice (90 days' old) relative to that of wild type. This observation strongly suggests that G93A has enhanced free radical-generating activity, unlike their previous assertion that their EPR data obtained with G93A and wild-type Cu,Zn-SOD showed no difference in free radicalgenerating activity (Singh et al, 1998).

6. AGGREGATION INDUCED BY FALS MUTANTS Bruijn et al. (1998) suggested that poorly or unstably folded mutants mitigate Cu,ZnSOD-containing aggregates that are toxic to motor neurons. They suggested that this process is induced by an as yet unidentified chemical reaction, rather than the involvement of free radicals. Subramaniam et al. (2002) reported results recently obtained from FALS transgenic mice (G93A, G37R, or G85R), in which the gene encoding the Cu chaperone for SOD1 (CCS) has been disrupted. They found that the incorporation of Cu ion into mutants was very much diminished in the absence of CCS. However, it did not modify at all (neither accelerate nor delay) the onset and progression of motor neuron disease in transgenic mice, relative to the mice with intact CCS. They concluded that the CCS-dependent Cu loading to mutant Cu,ZnSOD plays no role in the pathogenesis of disease in these mouse models (Subramaniam et al., 2002). The results, however, are not easily understood by invoking the aggregation hypothesis alone. In general, the apo-forms of metalloproteins are less stable and aggregate more easily than their holo forms (Rodriguez et al., 2002). Thus, if mutant-mediated aggregation is the cause of Cu,ZnSOD-associated FALS disease, the onset and progression of the disease should be accelerated in the CCS-disrupted FALS mouse model. Protein aggregates containing Cu,ZnSOD are commonly observed in FALS patients and transgenic mice expressing human Cu,ZnSOD (Kaytor and Warren, 1999; Julien, 2001; Watanabe et al., 2001). Johnston et al. (2000) have studied the formation of FALS Cu,ZnSOD-associated aggregates and insoluble protein complexes (IPCs) in G93A and G85R transfected human embryonic kidney and G93A transgenic mice. They found that SOD IPCs were present in the spinal cords of transgenic mice expressing mutant, but not wild-type SOD, at least three months before either SOD inclusion bodies or motor neuron dysfunction were first

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manifested. They proposed that the accumulation of SOD IPC could cause neuronal death by disrupting the capacity of the ubiquitin-proteasome pathway (Johnston et al., 2000). Several groups have reported the involvement of glycation in the formation of the aggregates. These include the observation of advanced glycation endproducts in neurofilament-associated conglomeration of motor neurons (Chou et al., 1998), detection of glycated SODl-positive inclusions (Kato et al., 2000), and observation of protein glycoxidation in the spinal cords of sporadic ALS patients (Shibata et al., 2001). Advanced glycation endproducts are heterogeneous products that may include various crosslinked proteins. It has been shown that oxidative conditions enhance glycation reactions. In addition, we have shown previously that protein glycation creates catalytic sites for free radical generation in protein-crosslinked products (Yim et al., 1995, 1998; Lee et al., 1998). Thus, the aggregation hypothesis may not exclude the possibility that the aggregation is mediated by free radicals initially, and these crosslinked proteins will then function as free radical generators to create autocatalytic phenomena for the progression of the disease (Yim et al., 1999).

7. CONCLUSIONS It is clear that the mechanism(s) by which FALS Cu,ZnSOD mutants induce ALS is far from resolved. The available data suggest that the processes caused by FALS mutants will eventually lead to the formation of Cu,ZnSOD-containing aggregates, which are commonly found in FALS patients and are believed to be toxic to motor neurons. The formation of aggregates can be facilitated by free radical reactions, protein misfolding and demetallation, and/or protein glycation. All these processes may be mechanistically linked as causes (enhanced free radical-generating activity and/or protein glycation) and consequences (protein glycation and aggregates). Thus, effects of wild-type and mutant Cu,ZnSOD on the formation of aggregates in vitro and characterization of their associated proteins found in the aggregates of FALS patients deserve further investigation.

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Sankarapandi S, Zweier JL. Bicarbonate is required for the peroxidase function of Cu,Znsuperoxide dismutase at physiological pH. J Biol Chem 1999a; 274:1226-1232. Sankarapandi S, Zweier JL. Evidence against the generation of free hydroxyl radicals from the interaction of copper,zinc-superoxide dismutase and hydrogen peroxide. J Biol Chem 1999b; 27:34576-34583. Shibata N, Nagai R, Uchida H, Horiuchi S, Yamada S, Hirano A, Kawaguchi M, Yamamoto T, Sasaki S, Kobayashi M. Morphological evidence for lipid peroxidation and protein glycoxidation in spinal cords from sporadic amyotrophic lateral sclerosis patients. Brain Res 2001; 917:97-104. Singh RJ, Karoui H, Gunther MR, Beckman JS, Mason RP, Kalyanaraman B. Re-examination of the mechanism of hydroxyl radical adducts formed from the reaction between familial amyotrophic lateral sclerosis-associated Cu,Zn superoxide dismutase mutants and H 2 0 2 . Proc Natl Acad Sci USA 1998; 95:6675-6680. Subramaniam JR, Lyons WE, Liu J, Bartnikas TB, Rothstein J, Price DL, Cleveland DW, Gitlin JD, Wong PC. Mutant SOD1 causes motor neuron disease independent of copper chaperone-mediated copper loading. Nature Neuroscience 2002; 5:301-307. Tainer JA, Getzoff ED, Beem KM, Richardson JS, Richardson DC. Determination and analysis of the 2A structure of bovine copper, zinc superoxide dismutase. J Mol Biol 1982;160:181-217. Uchida K, Kawakishi S. Identification of oxidized histidine generated at the active site of Cu,Zn-superoxide dismutase exposed to H 2 0 2 . Selective generation of 2-oxohistidine at the histidine 118. J Biol Chem 1994; 269:2405-2410. Watanabe M, Dykes-Hoberg M, Culotta VC, Price DL, Wong PC, Rothstein JD. Histological evidence of protein aggregation in mutant SOD1 transgenic mice and in amyotrophic lateral sclerosis neural tissues. Neurobiol Dis 2001; 8:933-941. Wiedau-Pazos M, Goto JJ, Rabizadeh S, Gralla EB, Roe JA, Lee MK, Valentine JS, Bredesen DE. Altered reactivity of superoxide dismutase in familial amyotrophic lateral sclerosis. Science 1996; 271:515-518. Wong PC, Pardo CA, Borchelt DR, Lee MK, Copeland NG, Jenkins NA, Sisodia SS, Cleveland DW, Price DL. An adverse property of a familial ALS-linked SOD1 mutation causes motor neuron disease characterized by vacuolar degeneration of mitochondria. Neuron 1995; 14:1105-1116. Yim, H-S, Kang J-H, Chock PB, Stadtman ER, Yim MB. A familial amyotrophic lateral sclerosis-associated A4V Cu.Zn-superoxide dismutase mutant has a lower Km for hydrogen peroxide. Correlation between clinical severity and Km value. J Biol Chem 1997; 272:8861-8863. Yim H-S, Kang S-O, Hah Y-C, Chock PB, Yim MB. Free radicals generated during the glycation reaction of amino acids by methylglyoxal—a model study of proteincrosslinked free radicals. J Biol Chem 1995; 270:28228-28233. Yim MB, Chock PB, Stadtman ER. Copper, zinc superoxide dismutase catalyzes hydroxyl radical production from hydrogen peroxide. Proc Natl Acad Sci USA 1990; 87:5006-5010. Yim MB, Chock PB, Stadtman ER. Enzyme function of copper, zinc superoxide dismutase as a free radical generator. J Biol Chem 1993; 268:4099^1105.

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CHAPTER 1 1

Copper and Prion Disease Judyth Sassoon, David R Brown

ABSTRACT The prion protein, PrP c , is a neuronal cell surface glycoprotein. In an abnormal isoform, termed PrPSc, it is associated with the family of neurodegenerative conditions called prion diseases. PrP c was recently shown to bind copper and there is strong evidence that it has a role in the regulation of brain copper metabolism. Its expression alters copper uptake into cells and enhances copper incorporation into superoxide dismutase enzymes. Also, PrP c has a superoxide dismutase-like function and may, therefore, protect neurons from the onslaught of reactive oxygen species. Furthermore, several lines of evidence have suggested that copper ions play a role in the biology of both PrP c and PrPSc and may influence the conversion of PrP c to PrPSc. This chapter provides an overview of the latest findings in this field and discusses structural and functional aspects of the prion protein. Keywords: Antioxidant; copper; neurodegeneration; prion; PrP.

1. INTRODUCTION Prion diseases are a group of fatal neurodegenerative conditions, such as Creutzfeldt-Jakob disease (CJD) in humans and bovine spongiform encephalopathy and scrapie in ruminant animals (Collinge, 2001). The infectious factors associated with these conditions are proteins, composed of PrPSc a post-translationally modified form of a normal cellular protein, designated PrP c (Prusiner, 1998) (Fig. 1). Spectroscopic studies have revealed that PrP c has a high a-helical content, whereas PrPSc is rich in P-sheets (Caughey et al., 1991; Pan et al., 1993; Liu et al., 1999; Zahn et al., 2000). These two isoforms differ biochemically, with PrPSc displaying reduced solubility in nondenaturing detergents and partial resistance to 279

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#

*

*

%

B Fig. 1. Detection of PrPSc in the brains of mice with experimental mouse scrapie. The histoblot technique was used to examine brain sections of mouse brain infected with the ME7 strain of scrapie at (A) 30 days and (B) 120 days post-inoculation. The sections were treated with proteinase K to destroy protease-sensitive PrR An antibody to PrP then allowed detection of PrPSc. This is detected heavily in the 120-day brain.

protease digestion (Oesch et al, 1985; Meyer et al, 1986). The "proteinonly hypothesis" proposed by Prusiner (Prusiner, 1982, 1991) implies that prpSc a i o n e c a n a c t a s t k e infectious agent. However, experiments with recombinant prion protein converted to the PrPSc-like form failed to induce disease in mice (Hill et al., 1999). It is, therefore, possible that some other element is necessary for infectivity or that PrP interacts with some other factor which catalyzes the conversion of PrP c to PrPSc. Although there is still no evidence for a virus or DNA element in prion diseases, a modified version of the protein-only hypothesis is now clearly required to explain the mechanism of infection. Although much data is available about PrPSc in relation to prion diseases, the physiological function of the normal cellular PrP c is still a matter of contention. There is clear evidence that the expression of PrP c and its conversion to PrPSc is connected in some way with disease-associated

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neurodegeneration. It has been shown that mice lacking expression of PrP c cannot be infected with the disease and suffer no neurodegeneration, or any of the symptoms associated with prion diseases (Biieler et al., 1992, 1993). Furthermore, cultured neurons lacking PrP c expression are resistant to the neurotoxicity of PrPSc or peptide homologs, both in vivo and in vitro (Brown et al., 1994; Brandner et al., 1996). In the course of research directed towards understanding the function of PrP c , several lines of evidence suggested that copper (Cu) might play a key role in prion biology (Brown et al., 1997a). Most importantly, through studies on synthetic peptides and recombinant protein, several laboratories have shown that Cu ions bind to the octapeptide repeat region in the N-terminal portion of the mammalian PrP c (Hornshaw et al., 1995a; Brown et al., 1997a; Stockel et al., 1998; Miura et al., 1999; Viles et al., 1999; Whittal et al., 2000). Binding of Cu is pH-dependent and apparently induces a conformational change in the normally unstructured N-terminus of the molecule. In addition, it was shown that Cu rapidly and reversibly stimulated endocytosis of PrP c from the surface, raising the possibility that PrP c may serve as a receptor for cellular uptake and efflux of Cu (Pauly and Harris, 1998; Brown et al., 1999a). An enzymatic function for PrP c has also been claimed based on the observation that Cu binding confers superoxide dismutase (SOD) activity on the protein (Brown et al., 1999b). Other connections between PrP c and Cu have been proposed, but have remained controversial. PrP c was postulated to be a major Cubinding protein in brain based on the observation that the content of Cu is 5% to 50% of normal in membrane fractions derived from the brains of mice which carry a disrupted PrP gene (Brown et al., 1997; Herms et al., 1999). The activity of SOD-1 was also reported to be 50% of normal in the brains of these mice, and neurons cultured from the animals were found to be more susceptible to oxidative stress, further implying a role for PrP c in protection from oxidative damage (Brown and Besinger, 1998; Brown et al., 1999b). There are also several other results suggesting interactions between Cu and the abnormal prion, PrPSc. A role for Cu in mediating the disease process has been proposed for the following reasons. First, Cu facilitated the restoration of protease resistance and infectivity during refolding of guanidine-denatured PrP Sc (McKenzie et al., 1998). Secondly, the protease cleavage pattern of PrPSc derived from the brains

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of patients with CJD was altered by addition or chelation of Cu and another trace metal zinc (Zn), suggesting a role for metal occupancy in determining prion strain properties (Wadsworth et al., 1999). In addition, Cu added at high concentration to the prion protein causes it to refold into a protease-resistant form. (Quaglio et al., 2001). Finally, it was reported almost 25 years ago that administration of the Cu- chelating agent cuprizone to mice caused spongiform degeneration of the brain similar to scrapie (Pattison and Jebbett, 1971a). In view of all this, much current research is now being directed towards defining and characterizing the function of Cu within the prion protein.

2. PRION PROTEIN AND COPPER BINDING A link between prion disease and Cu metabolism was first proposed in the early 1970s. Pattison and Jebbett (1971a) noticed that the histopathology of mouse scrapie resembled that induced by the chemical, cuprizone. Cuprizone (bis-cyclohexanone oxaldihydrazone) is a Cu chelator which interferes with oxidative phosphorylation and induces spongiform encephalopathy and gliosis following chronic exposure. Interestingly, this resembles the well-known phenomenon in Wilson's disease, a human disease related to the loss of activity of a Cu-transporting ATPase, which also demonstrates spongiform degeneration (Tanzi et al., 1993). When cuprizone is injected into animals, it depletes the brain of Cu and modifies the activity levels of Cu-dependent enzymes (Venturini, 1973). This effect is not observed if the chelator is first charged with Cu before injection. Many of the symptoms observed in cuprizone-treated hamsters are similar to the enzymatic changes seen in scrapie-infected hamsters (Kimberlin et al., 1974; Kimberlin et al., 1976). Further studies were made in which scrapie-infected mice were treated with cuprizone to see if this would alter incubation times. Although a number of effects were observed, no clear picture emerged (Kimberlin and Millson, 1976). Research on cuprizone, in relation to prion diseases, was not pursued with any great enthusiasm following the publication of studies on PrPSc in the early 1980s (Prusiner, 1982) and the proposal of the "protein-only hypothesis". At that time, the emphasis turned towards determining the proposed function of PrPSc in infectivity rather than to look at the range of other possible factors involved in the disease.

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2.1. PrPc Structure There is now a considerable amount of biochemical information on the structure and amino acid sequence of PrP c . Translation of the original DNA sequences of the mouse and hamster prion genes revealed a 254amino acid protein (253 in humans) encoded by a single copy gene, Prnp (Locht et al., 1986) (see Fig. 2), The coding region of the prion protein is preceded by a 22-amino acid signal peptide at the N-terminus and followed by a 23-amino acid region at the C-terminus (Easier et al., 1986). Under normal physiological conditions, these N- and C-terminal signal peptides are proteolytically cleaved, leaving residues 23 to 231 in the mature mammalian protein. A glycosyl phosphatidylinositol (GPI) anchor is attached at serine 231. This marks the protein for expression at the cell surface and makes it conceivable that PrP c may be similar to other GPI-anchored proteins that are commonly associated with cell to cell signaling, adhesion, or cellular defense (Sendo et al., 1998). The C-terminal region of PrP c forms a globular structure comprising three a-helices and two short (3-strands Octamerie repeats (5) N-terminal cleavage

Sugar chain attachment

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Signal peptide Hydrophobic core

O 23

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51

90

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II ,!

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231

135

Main Cu binding site (four sites)

MS*. 252

Alternative fifth site Histidineat 186

Proposed fifth site Histidine at 111 Fig. 2. Schematic representation of the human prion protein primary sequence showing the main features. Indicated are the main Cu binding sites and two of the possible sites at which a fifth atom of Cu may bind.

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(Riek et al., 1996, 1997; Donne et al., 1997) and, during the process of prion infection, can refold into protease-resistant (3-sheet enriched aggregates (McKinley et al, 1983; Oesch et al, 1985; DeArmond et al., 1985; Caughey et al., 1991; Pan et al., 1993). In contrast, the N-terminal domain is highly flexible and typically includes at least four tandem copies of a conserved octapeptide repeat motif (Riek et al., 1997; Donne et al., 1997; Oesch et al., 1985). Evidence suggests that Cu binding occurs predominantly within this region. The N-terminal domain of human PrP c contains these "octarepeat" sequences (PHGGGWGQ: within residues 60 to 91) and one homologous sequence lacking the histidine residue (PQGGGWGQ: residues 51 to 59) (Kretzschmar et al., 1986) Residues 51 to 91 of mouse PrP c consists of four octarepeat sequences (PHGGSWGQ: residues 60-89) and one homologous sequence lacking histidine (PQGGTWGQ: residues 51 to 59) (Westaway, 1987).

2.2. Copper Binding in the Octameric Repeat Region Although the octarepeat motifs within PrP c have no sequence homology to classical Cu-binding proteins (Bazan et al., 1987), evidence suggests that these motifs bind Cu with a remarkable degree of selectivity; as such, they may comprise the prototype of a new class of Cu binding motif. Recombinant PrP c (rPrPc) can be expressed and purified in large amounts from inclusion bodies in an E.coli expression system (Wong et al., 1999). The protein is denatured in 8 M urea and subsequently refolded by gradual removal of urea. rPrP c can be refolded in the presence of Cu, which binds specifically in the octameric repeat region and was also demonstrated to have increased solubility (Brown et al., 1999a). Cu binding to the N-terminal octarepeat region of human recombinant PrP23-98 has now been demonstrated using equilibrium dialysis (Brown et al., 1997a) and in synthesized peptides using mass spectrometry (Hornshaw et al., 1995a; Whittal et al., 2000), fluorescence Spectroscopy (Whittal et al., 2000), Raman spectroscopy (Miura et al., 1999), circular dichroism, proton nuclear magnetic resonance (NMR) spectroscopy (Viles et al., 1999), electron paramagnetic resonance (EPR), and electron spin-echo envelope modulation spectroscopy (Aronoff-Spencer et al., 2000). Cu binding has also been reported for nearly full-length forms of Syrian hamster (residues 29 to 231) (Stockel et al., 1998) and human PrP

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(residues 91 to 231) (Jackson et al., 2001) and full-length mouse PrP (residues 23 to 231) (Kramer et al., 2001). Affinity chromatography using immobilized Cu ions has been used to purify mature, glycosylated PrP c isolated from hamster brain (Pan et al., 1993). With regard to stoichiometry, the number of Cu(II) binding sites in the N-terminal region of PrP c has been variously reported as between two and 5.6 and pH-dependent (Brown et al., 1997a; Whittal et al., 2000; Viles et al., 1999; AronoffSpencer et al., 2000; Stockel et al., 1998; Hornshaw et al., 1995b). At neutral pH, Cu binding to the N-terminal domain occured in the micromolar range with positive co-operativity. There was a remarkably close correlation of Hill coefficients calculated by different laboratories: 3.4 (PrP23 to 98) (Brown et al., 1997a), 3.3 (PrP58 to 91) (Viles et al., 1999), and 3.6 (PrP23 to 98) (Kramer et al., 2001). More recently, the suggestion was made that there are two independent high affinity Cu binding sites of 10" 14 M and 4 X 10 _14 M deduced from the analysis of PrP58 to 98 and PrP91 to 231, respectively (Jackson et al, 2001). Other authors provided different data regarding the binding of Cu at the octameric repeat region, using a mutant protein lacking this region, PrPA59-91. They found that four Cu atoms were missing from each molecule of the mutant protein compared to full-length rPrP c . This corresponded well with evidence showing that the histidines in the octameric repeat region co-ordinate with the four Cu atoms (Brown, 1999a). Other mutants lacking one, two, or three octarepeats were also studied and showed that the amount of Cu binding is directly proportional to the number of repeats, although affinity values were not calculated (Brown et al., 2000). Several other authors also confirmed that Cu can bind to PrP c along the more structured C-terminal domain of the protein (Cereghetti et al., 2001; van Doorslaer et al., 2001). Continuous wave EPR studies demonstrated that, on binding, Cu first fills the C-terminal binding sites before occupying the octarepeats at the N-terminus (Cereghetti et al., 2001).

2.3. Copper Co-Ordination in PrPc Cu in the form of Cu(II) can adopt a range of co-ordination geometries in proteins and peptides and can interact with nitrogen, oxygen, and sulfur atoms. Electron spin resonance spectra have shown that, in PrP peptides, the complexes co-ordinate with tetragonal symmetry (Aronoff-Spencer

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et al., 2000). The first study to determine the co-ordination of Cu in the octameric repeat region of PrP c was performed by Viles et al. (1999). Their model proposed that each Cu atom was co-ordinated by nitrogens of two histidine imidazole rings and a nitrogen from a proline residue, as well as with the oxygen of a water molecule. Aronoff-Spencer et al. (2000), however, disputed this model. Their EPR studies suggested that their spectra are composed of two components. The first apparently arose from three nitrogen atoms, and one oxygen atom, while the second consisted of two nitrogens and two oxygens. The first component came from studies of peptides that are equal to or longer than a single octarepeat, and the sequence HGGGW gave a pure spectrum of this kind. Thus, the fragment probably bound Cu(II) in a way that is nearly equivalent to that of peptides containing multiple repeats and, therefore, comprises the fundamental binding unit. Thus, each of the four metal ions bound to the octameric repeat region probably binds to a single HGGGW region. In the three nitrogen co-ordination component, Aronoff-Spencer et al. (2000) proposed that the first nitrogen is contributed by a histidine imidazole ring and the other two originate from deprotonated backbone amide groups. The idea that each Cu is bound by residues primarily within the octarepeat region is further supported by titration experiments on one, two, and four octarepeat-containing peptides (Aronoff-Spencer et al., 2000; Burns et al., 2002). Thus, the ratio of Cu bound within an octarepeat region is one to one and this conflicts with the findings of Viles et al. (1999), who proposed that the Cu interacted with two imidazole rings. An imidazole bridge between a single pair of Cu(II) ions leads to exchange interactions and the resulting EPR transitions take place solely among the two-spin triplet levels. If, in addition, the exchange interaction is large compared to the kT, the Boltzmann factor will favor the singlet ground state, thereby decreasing the integrated EPR absorption signal relative to that expected from uncoupled spins. The work of Aronoff-Spencer et al. (2000) found no evidence for this, suggesting that the exchange coupling does not occur in the binding of Cu to multirepeat peptides. The finding that one repeat binds one Cu ion also agrees with the findings of Miura et al. (1999), whose work was based on Raman spectroscopy studies. They suggested that HGGG is the fundamental binding unit. For a fully Cu(II) loaded protein, the model of Aronoff-Spencer et al. (2000) implies that the metal ion binding sites in the N-terminal PrP c

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domain are like beads on a string, where each bead is a Cu-HGGGW segment separated by intervening Gly-Gln-Pro links. Interestingly, glycine and proline often participate in p-turns. Thus, the intervening links may provide a mechanism that allows the Cu-binding segments to fold and, perhaps, collapse together. Co-ordination dominated by a single histidine would make a PrP c molecule bound to Cu pH sensitive, supporting the idea that it may function in vivo as a Cu transporter, taking Cu into the acidic environment of lysosomes and releasing it there. Although there is now little doubt that Cu binds in the octameric repeat region, the "fifth Cu binding site" (Brown et al., 1997a; Jackson et al., 2001) is still a matter of controversy. Some authors propose that Cu binds in the form of Cu(II) in the C-terminal domain of PrP c . A mutation in amino acid residue 198 abolishes this binding, suggesting that the histidine involved in binding might be at the amino acid residue 187. Other authors suggest that the site of Cu binding is in the region of the toxic peptide PrP106 to 126. If the latter is true, the histidine within the peptide would be the one involved in binding, with additional co-ordination from the appropriate nitrogen atoms in the vicinity. It would also require two peptides to create this site. Recent work by Qin et al. (2002) used mass spectroscopic-based footprinting techniques to attempt to position the histidine-dependent metal co-ordination sites in Cu bound to PrP c . This technique allowed the total number of histidines involved in metal co-ordination to be determined by measuring the mass differences between apo- and metal co-ordinated proteins or peptides. They confirmed the Cu co-ordination sites at the four octarepeat histidines PHGGG/SWGQ (residues 60,68,76, and 84 of mouse PrPc) and also suggested a second type of site involving histidine 95, in the related sequence GGGTHNQ, possibly in conjunction with histidine 110 (equivalent to histidine 111 in human PrP). (Jackson et al., 2001; Hasnain et al., 2001). They reported that these sites had estimated binding affinities ranging from 2.2 X 10 - 6 M to 10" 14 M for the octarepeats and 5 X 10~6 M to 4 X 10~14 M for the site involving histidine-95 (Qin et al., 2000).

2.4. Crystal Structure of Copper-Bound PrPc Following the work reported by Aronoff-Spencer et al. (2000), the group of Glenn Millhauser made further studies on the ability of the sequence

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HGGGW to bind Cu. The first crystal structure of Cu binding to this region of the protein was recently reported (Burns et al., 2002). The binding of Cu to this peptide segment is consistent with EPR and other spectroscopic studies. The HGGGW-Cu complex included six ordered water molecules (Fig. 3). Thus, Cu(II) is found in a penta-co-ordinate environment with equatorial ligation from the dl nitrogen of the imidazole ring and deprotonated amide nitrogens of the next glycine residues. The second glycine residue contributes its amide carbonyl oxygen. Except for the histidine backbone nitrogen and a-carbon, all atoms from the histidine through to the nitrogen of the third glycine lie approximately in the equatorial plane and the Cu lies just above this plane, which is consistent with a pentaco-ordinated complex. The tryptophan indole ring also participates in the co-ordination environment in a rather unusual fashion. The indole nitrogen from the tryptophan side-chain is 3.9 A from the oxygen of water bound axially to the Cu(II), suggesting the presence of hydrogen bond. This arrangement places the plane of the indole ring above the Cu, such that it is nearly parallel to the equatorial plane. Two additional water molecules make hydrogen bonds to the axial water to form a network extending from the backbone carbonyl, preceding the histidine, to the carbonyl of the third glycine. There are no equatorial water molecules involved in this model.

Fig. 3. Model of the crystal structure of Cu(II) binding to a fragment of a single octarepeat based on Burns et al. (2002).

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Examinations of the intermolecular contacts found in the crystal structure reveal a potentially important docking interaction between HGGGW-Cu units that may explain previously observed co-operative binding of Cu to PrP c . However, there is still some controversy about this co-operativity as this was not evident from previous studies (Jackson et al., 2001) and the suggestions remain to be confirmed.

3. FUNCTION OF COPPER-BOUND PrP The in vivo function of the prion protein has been investigated, but remains an enigma. Nevertheless, the interaction of PrP c with Cu may hold clues about its physiological role. Much of the evidence discussed above shows that PrP c sequesters Cu. Possible functions for the Cu-bound prion protein might be either in the distribution of Cu in the cellular environment or, alternatively, the Cu might be essential to a specific physiological activity that the protein displays.

3.1. Copper Transport Experiments designed to look at the possible function of PrP c in Cu transport employed the use of Cu67. The studies involved three strains of mice: those overexpressing PrP c , those deficient in PrP c , and wild-type controls. Transport of unchelated Cu67 by cerebellar cells was recorded in all three strains, but kinetic studies showed that histidine-chelated Cu67 was taken up at a rate proportional to PrP c expression (Brown, 1999a). Kinetic parameters for Cu67 transport were also determined, and the Vmax values increased with higher expression of PrP c while the Km values (in the nM range) were not greatly different. These results suggested that there might be an increase in the number of Cu binding sites within the cerebellar cells, which can be related to PrP c expression. Further experiments in which cytosolic enzyme SOD-1 was immunoprecipitated from cells loaded with Cu67 showed that the Cu could be incorporated into SOD-1 in proportion to the level of PrP c expression in the cells studied. Thus, PrP c may participate in the incorporation of Cu into other cellular proteins (Brown and Besinger, 1998). In the brain, the highest concentrations of PrP c are found at the synapses and synaptosomes of PrP0/0 knockout mice demonstrated a strong reduction in Cu concentration. Also, Cu binding in the synaptic

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cleft showed a significant influence on synaptic transmission. Cu is released from the synapse during quantal release and it has been proposed that PrP c may have a function in regulating Cu at levels at neuronal synapses. Several studies have been performed to study this phenomenon. In one set of experiments, neuronal cells were loaded with Cu67 and allowed to release Cu spontaneously. Subsequently, they were treated with the depolarizing agent, veratridine, which blocks Na + channels in neurons. Veratridine released Cu from cells at levels corresponding to the expression of PrP c in those cells. Cells which did not express PrP demonstrated almost no veratridine-induced Cu release, while cells which overexpressed PrP c released higher quantities of Cu compared to wild-type cells (Brown and Besinger, 1998). Furthermore, electrophysiological experiments indicated that Cu applied to cerebellar slices inhibited the amplitude and frequency of inhibitory currents measured on Purkinje cells of PrP c deficient cells, but not wild-type cells, suggesting that some protection against Cu is absent from the synapses lacking PrP c . One proposal has been that PrP c itself performs this protective function (Brown et al., 1997b).

3.2. Copper-Dependent Antioxidant Function of PrP It has been demonstrated that both recombinant and brain-derived PrP c have SOD-like activity when bound to Cu (Brown et al., 1999b, 2001). The depletion of PrP c from cell extracts results in a lower SOD activity in the extract (Wong et al., 2000). Also, when PrP c converts to PrPSc, its SOD function is abolished (Thackray et al., 2002). Thus, it is possible that the normal function of PrP c is to act as a SOD-like enzyme and control oxidative stress. It has, however, been demonstrated that PrP c is different from cellular Cu/ZnSOD, especially in the way that it forms its complex with Cu (van Doorslaer et al., 2001). In addition, a very current question concerns oxidative stress as a factor causing neuronal damage in prion diseases and it is, therefore, of considerable interest to consider the SODlike function of PrP in this context. Recombinant PrP c , purified from E.coli expression systems and refolded with Cu can be used in quantitative assays designed to measure SOD activity. Experiments showed that both chicken and mouse rPrP c could catalyze the dismutation of the superoxide radical at a rate equivalent to one-tenth of SOD-1. SOD-1 is a very potent enzyme which

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catalyzes the reaction at around 100,000 times the spontaneous rate of superoxide degradation (Fridovich, 1975). It was, therefore, concluded that PrP c had significant SOD activity. This was also confirmed for native protein immunoprecipitated from mouse brain (Brown et al., 1999b). Strict controls were used to ensure that the prion protein SOD activity was a real enzymatic activity, and not simply due to Fenton chemistry arising from Cu ligation within the protein. The deletion of the specific octameric repeats of the N-terminal region abolished the SOD activity, despite there still being Cu bound at the C-terminal domain (Brown et al., 1999b). In addition, a peptide based on the octarepeat region with Cu bound to it had no SOD activity. When rPrP c was refolded without Cu and the Cu subsequently added to the refolded protein, the mixture did not demonstrate SOD activity to the level of the co-ordinated Cu in the protein (Brown et al., 1999b). It is, therefore, clear that regions outside the N-terminal octarepeat domain are required for SOD activity of the PrP c . Further evidence for this comes form amino acid analysis of rPrP c after Cu binding. It was found that methionine residues were oxidized in rPrP c with Cu incorporated during refolding (Wong et al., 1999). This is characteristic of certain antioxidant Cu binding enzymes, such as SOD-1 (Chowdhury et al., 1995). Thus, it could be concluded that the observed catalytic SOD activity was not due to the presence of Cu alone, but was more likely due to a true enzymatic activity. It would appear that the dismutation of superoxide could, indeed, be one of the normal functions of the prion protein. At least two Cu atoms per molecule of PrP c were necessary to endow the protein with SOD activity. Binding of Cu also induced a more ordered •structure on the molecule. On binding of two more Cu atoms, a further increase in SOD activity was observed and an additional molecular ordering was found (Miura, 1996, 1999). There are currently three known SODs in mammals (Fridovich, 1997): the cytosolic Cu/ZnSOD-1, mitochondrial MnSOD or SOD-2, and extracellular SOD or SOD-3. The former two are found in all cells at varying concentrations and often show increased expression under conditions of oxidative stress. Extracellular SOD exists in three different isoforms, binds one atom of Cu per molecule, and is either released into the extracellular matrix or remains bound at the cell surface. In brain tissue, the expression of SOD-3 is very low (Ookawara et al., 1998), although it is elevated in PrP knockout mice (Brown et al., 2001). It is interesting to

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correlate these observations with the in vivo expression of PrP c , considering its SOD activity. The expression of PrP c is highest in the brain and particularly abundant at synapses. It is also present at neuromuscular junctions (Gohel et al., 1999). Thus, it has been proposed that PrP c may serve as a synaptic SOD and may be released during transmission at the synapse. Superoxide is known to inhibit synaptic transmission and the presence of SOD activity in these regions of the nervous system may have a protective role. The possible protective role of PrP c against oxidative stress was confirmed in cell culture using PC 12 rat tumor cells, which can be differentiated into neurons using nerve growth factor (NGF). It was also observed that PrP c expression increased in PC 12 cell cultures on exposure to oxidative stress (Brown et al., 1997b).

4. ALTERED METAL BINDING IN THE OCTAREPEAT REGION OF PrPc It was recently demonstrated that recombinant PrP c also had the capacity to bind metal ions other than Cu (Brown et al., 2000). Most significantly, the bivalent anion manganese was found to bind in both the octarepeats and C-terminal sites (Collinge, 2001). In vitro metal ion occupancy experiments showed that when manganese replaced the Cu ion in the prion protein, PrP c altered its structure and took on a more PrPSc-like conformation (Brown et al., 2000). Manganese-bound prion protein also lost its SODlike function (Brown et al., 2000). Several lines of investigation have been taken to see if the metal binding of PrP c is altered in TSEs, if metal imbalances also correlate with the loss of antioxidant function in PrP c , and whether these alterations correlate with the disease phenotype, such as PrPSc and the PrP genotype at codon 129, which influences the manifestation of the disease (Worrall et al., 1999). Investigations on alterations in metal ion concentrations were carried out using mouse scrapie models (Thackray et al., 2002) and in samples from sporadic CJD cases (Wong et al., 2001). Changes in the levels of Cu and manganese were detected in the brains of scrapie-infected mice early in the disease, prior to the onset of clinical symptoms. In addition, a major increase in blood manganese was also noted in the early stages of the disease. Analysis of purified PrP from the brains of scrapie-infected mice also showed a reduction in Cu binding to the protein and a proportional decrease in antioxidant activity between 30 to 60 days post-infection.

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A striking elevation of manganese and, to a lesser extent, Zn accompanied by a significant reduction in Cu binding to purified PrP were found in subtypes of sporadic CJD the most common type of human prion disease. Studies were made using brain tissues and affinity purified PrP preparations (that is, PrP c , PrPSc, and possibly other abnormal PrP species) obtained from four major subtypes of sporadic CJD. These were identified according to the genotype at codon 129 of the PrP gene and the PrPSc type established by Parchi et al. (1999). Both Zn and manganese were undetectable in PrP c preparations from control brain preparations. However, Cu and manganese changes were pronounced in sporadic CJD subjects homozygous for methionine at codon 129 and carrying PrPSc type 1. It was also found that a decrease of up to 50% of Cu and an approximately ten fold increase in manganese occurred in the brain tissues of sporadic CJD subjects. Antioxidant activity of purified PrP was dramatically reduced by up to 85% in the sporadic CJD variants, and correlated with an increase in oxidative stress markers in sporadic CJD brains. These results clearly point to the fact that metal ion occupancy alterations in PrP play a pivotal role in the pathogenesis of prion diseases. Since the metal changes differed in each sporadic CJD variant, they may contribute to the diversity of PrPSc and disease phenotypes in sporadic CJD (Wong et al., 2001). These fascinating and significant results could also have a bearing on potential approaches to the diagnosis of CJD. The increase in brain manganese associated with prion infection is potentially detectable by magnetic resonance imaging, and the binding of manganese by PrP in sporadic CJD might represent a novel diagnostic marker.

5. PLASMINOGEN ACTIVATION BY PrP Pericellular proteolytic activity plays an important role in many physiological situations in various organs, including the brain. This proteolytic activity can act to degrade components of the extracellular matrix or activate bioactive molecules, such as growth factors, making it a key regulator of cellular behavior (Werb, 1997). The broad specificity serine protease plasmin is one of the principal activities involved in these processes. Plasmin is generated from the abundant zymogen plasminogen by a single proteolytic cleavage catalyzed by either of the two plasminogen activators, uPA and tPA. At the functional level, the activity of the plasminogen activation system is largely regulated by mechanisms that enhance the generation of

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plasmin (Ellis and Whawell, 1997). Thus, uPA-catalyzed plasminogen activation is stimulated by the binding of uPA to its cell surface receptor, uPR (Ellis et al., 1991), and tPA-catalyzed plasminogen activation juxtaposed to the plasminogen activator, either on the same co-factor molecule (as with fibrin) or to discrete cellular binding sites. Interactions with these molecules are mediated by "lysine-binding sites" in the kringle modules of plasminogen, which can be antagonized by lysine, such as eACA (Marti et al., 1997). These kringle modules preferentially bind C-terminal lysine residues (that is, those with a free carboxylate group), which can either be present in the native proteins or generated by the proteolytic action of plasmin. It was reported that plasminogen, which can act as a pro-protease and is implicated in neuronal excitotoxicity, is bound to PrPSc, but not PrP c (Fischer et al., 2000). Therefore, plasminogen represents the first endogenous factor that can discriminate between normal and pathological prion proteins. Binding was abolished if the conformation of PrPSc was disrupted by 6M urea or guanidine, but the isolated lysine-binding site of plasminogen (kringle modules I to III) retained the binding activity. Ellis et al. (2002) tested the hypothesis that, due to its reported ability to bind plasminogen, PrPSc is a regulator of plasminogen activation. They observed the effect of recombinant PrP, either containing Cu (holo-PrP) or devoid of it (apo-PrP), on plasminogen activation by both uPA and tPA. PrP was found to have no effect on plasminogen activation by uPA, but the activity of tPA was stimulated by up to 280 times. This was observed only with the apo-PrP isoforms. Thus, they demonstrated that PrP can, indeed, regulate plasminogen activation, but that a critical determinant of the interaction between PrP and tPA was related to the absence of Cu in the PrP. Failure of Cu to bind PrP may be an early event in the conversion of PrP c to PrPSc and in the stimulation of plasminogen activation. The similarity in the interaction of PrPSc or Cu-free rPrP with plasminogen or plasminogen activators suggests that PrPSc may be a form of PrP devoid of Cu.

6. METAL ION BINDING AND NEURODEGENERATIVE DISEASES Research has suggested that Cu has an important role to play in the prevention or moderation of a number of neurodegenerative diseases, including

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the polyglutamine diseases such as Friedrich ataxia and Huntington's disease, Parkinson's disease (PD), Wilson's disease, Menkes' diseases, amyotrophic lateral sclerosis (ALS), and Alzheimer's disease (AD) (Strausak et al., 2001). Cu is an essential metal that serves as a co-factor for a number of proteins and enzymes. Many proteins associated with neurodegenerative diseases have metal-binding properties and/or metal-responsive expression (Bush, 2000). Besides PrP c , the ectodomain of the Alzheimer precurser protein (Multhaup et al., 1996), amyloid-f} peptide (Atwood etal., 2000), and SOD-1 (Fridovich, 1975) exist in Cu-bound forms. In these examples, metal binding relates to pathogenesis via an impact on aggregation or production of oxidative damage. Cells rely on a number of transition metals to regulate a wide range of metabolic activities and signaling functions. The diversity and efficiency of their physiological functions are derived from the specific atomic properties of transition metals, most notably an incomplete inner valence subshell. These properties enable the metals to fluctuate among a variety of positively charged ionic forms, allowing for a chemical flexibility that can ultimately impose conformational changes on the proteins to which they bind. By this means, transition metals can serve as catalytic centers of enzymes for redox reactions involving, for example, molecular oxygen and endogenous peroxides (Hamai et al., 2001). In general, neurodegenerative diseases display two commonly recognized metal-dependent reactions. First, there are the metal-protein associations causing abnormal aggregation of proteins. These can involve both redox-inert metal ions, such as Zn 2+ , or redox-active ions, such as Cu 2+ or Fe 3+ . Secondly, there are the metal-catalyzed protein oxidations leading to protein damage and denaturation. These reactions involve only the redox-active metal ions such as Cu 2+ , Fe 3+ , or Mn 2+ . Both reactions can lead to the functional demise of their target protein. It has been proposed that certain neurodegenerative diseases are caused by the abnormal interaction of specific, susceptible target proteins in neural tissues enriched in metals with metal ions to which they do not normally bind. The interaction can then cause aggregation and/or oxidation of neural tissue mediated by the abnormal redox-active metal ion associating with the target protein, and can lead to the loss of function of the protein. This important mechanism of oxidative damage can involve the following reactions of the bound redox-active transition metals: an

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initial reduction of the metal, such as Cu 2+ or Fe 3+ , by electron donors like the superoxide radical (0 2 ), catecholamines, L-ascorbate, and mercaptanes, as well as the subsequent generation of hydroxyl (OH*) radical through the reduction of H 2 0 2 by the reduced metals. This highly reactive free radical attacks neighboring amino acid residues, producing carbonylcontaining derivatives on the proteins. Carbonyls can also be introduced into the protein as a result of oxidative cleavage of the peptide backbone. Thus, the carbonyls produced are signs of oxidative damage in proteins (Berlett and Stadtman, 1997; Stadtman and Levine, 2000; Butterfield and Kanski, 2001). Cu is a transition metal with multiple valencies, Cu(I) and Cu(II). In a free, unbound state, it is a powerful catalyst of auto-oxidation reactions, such as oxidation of ascorbic acid, thiols, and catecholamines, that generate various reactive oxygen species (ROS) (Halliwell and Gutteridge, 1990). In view of this, Shiraishi et al. (2000) reasoned that PrP may sequester Cu(II) and may prevent oxidative damage induced by the coexistence of Cu(II) and auto-oxidizable compounds. For this to occur, the Cu(II) bound to PrP c should preferably be maintained in the redox-inactive state. Otherwise, the protein itself would be subjected to oxidative degradation. Shiraishi et al. (2000) tested the octarepeat region for inhibitory effects on Cu catalyzed oxidations of L-ascorbic acid or glutathione and generation of ROS, such as OH*. Their results showed that the catalytic activity of the first Cu(II) ion bound to the octarepeat region was completely suppressed. The valence state of the Cu under reducing conditions was Cu(II). Thus, they suggested that the in vivo function of the prion protein might be to sequester Cu ions in the redox-inactive state, rendering Cu-induced generation of ROS impossible. From the preceding discussion, it can be seen that some of the symptoms of prion diseases may be caused by an abnormality in the active Cubinding site on the PrP protein. A similar phenomenon can also occur within other proteins, such as amyloid-fS in AD or the Cu/Zn SOD in familial ALS, and can be a potential factor in other neurological diseases. Under normal conditions, Cu binding to appropriate proteins is essential to oxidative stress homeostasis. Such Cu-active sites are very likely exposed to constitutively high concentrations of ROS, such as 0 2 and H 2 0 2 . H 2 0 2 can react with Cu + , which is produced transiently at the active site of these proteins, and generate the highly reactive and detrimental ion,

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OH*. In the normally folded proteins (PrP c in TSEs, Alzheimer precusor protein in AD, and SOD-1 in Familial ALS), the Cu 2+ active site is probably shielded and, therefore, does not undergo this abnormal reaction. However, changes in the conformation of these proteins may expose the active site and make it more prone to react to produce the OH* radical.

7. COPPER AND THE ENVIRONMENT Transition metal ion pollution in the environment has been an issue now for some years. Recently, it has become a question in epidemiological studies of prion disease distribution (Purdey, 2000). For example, chronic wasting disease (CWD) is a sporadic prion disease in deer and elk and is found in specific regions of the United States. The pathology is similar to spongiform degeneration induced by Cu deficiency, as seen in cuprizonetreated mice (Pattison and Jebbett, 1971a, 1971b). The soil in regions where CWD is high apparently have a deficiency in Cu and this disease was proposed to be related to environmental Cu deficiency (Purdey, 2000). CWD was first described as a spongiform encephalopathy when plaques were detected in the brains of a large percentage of afflicted mule deer (Bahmanyar et al., 1985). Later, this was confirmed to contain PrPSc by immunodetection (Guiroy et al, 1991), thus confirming CWD as a prion disease, though some authors still regard it as a Cu deficiency disease (Yashikawa et al., 1996). Thus, it has been proposed that environmental Cu deficiency may exacerbate or even cause prion diseases (Purdey, 2000). However, despite certain coincidental observations, there is still no direct evidence linking Cu deficiency to prion diseases. It is worth noting that when metal ions are implicated in neurological syndromes associated with abnormally folded proteins, the source of the metal ions is thought to be environmental exposure. Ingestion, or some other method of internalizing metal ions, is often thought to be the original cause of the abnormal interaction between metal ions and proteins. This, however, may be a misconception because even the brain, under normal conditions, has more than enough metal ions to cause abnormal metal binding, damage, and a general malfunctioning of metabolism. Take Zn as an example. Zn 2+ is released during neurotransmission at concentrations of about 300 pJVL This concentration is sufficient to be lethal to neuronal cell cultures (Frederickson, 1989). It is, therefore, necessary for

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the brain to have efficient homeostatic mechanisms and buffers that prevent the decompartmentalization of these metal ions. The blood-brain barrier is relatively impermeable to fluctuating levels of plasma metal ions. Some metal regulatory transport systems are energy-dependent, one of these being the Cu-ATPase, which functions in Wilson's disease (Tanzi et al., 1993). It is possible that damage to the blood-brain barrier or energy compromise in the brain perturbs metal levels and lead to abnormal binding in proteins. Thus, it is not necessary to hypothesize that neurological conditions, such as prion diseases, are caused by environmental exposure to metals alone. Clearly, other factors may play a primary role in these diseases.

8. CONCLUSION There is now a steadily increasing body of evidence confirming that the function of the normal prion protein, PrP c , is that of an antioxidant and/or a Cu-sequestering protein active in the prevention of Cu toxicity in neurons. The implication for prion diseases is that the loss of function of PrP c through conversion to, or interaction with, PrPSc may be responsible for some of the pathological changes seen in this family of diseases. Though PrP c unquestioningly binds Cu ions, it is still unclear whether PrPSc can associate with Cu in the same way. There is evidence that Cu, possibly binding at an inappropriate site, can convert PrP to a protease-resistant form (Fig. 4). In the course of the disease, the amount of PrP c generated by cells increases to a very high level and much of the protein is converted to the protease-resistant form, which then probably remains in the brain until the death of the individual. If all the generated PrP c initially bound Cu then, on conversion, the accumulated PrPSc would represent a huge sink of Cu. It is also possible that Cu might become trapped nonspecifically within the PrPSc aggregates. However, initial experiments with scrapie-infected mice showed no large increases in brain Cu suggesting that the conversion of PrP c to PrPSc actually results in a protein that either does not bind Cu or that there is a reduction in Cu within the cells. The evidence suggesting that alternative metal ions bind in the place of Cu in prion diseases seems to support this idea. In the coming years, the changes in Cu metabolism during prion diseases will become clearer. Indeed, if it is consistently shown that Cu is

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Cu fails to bind during synthesis

Extracellular movement

Normal Cu binding induces protective change in conformation

Cu binds to exposed "wrong" sites in conformationally immature protein

Cu causes conformational corruption

(A)

Fig. 4. Alternative interactions of Cu with prion protein that cause either (A) protective activity or (B) conversion to a toxic species.

reduced during prion diseases, this may lead to treatments designed to enhance Cu uptake by cells in affected individuals. Alternatively, if Cu bound to PrPSc is acting as an oxidant generating H 2 0 2 or hydroxyl radicals, it might be possible to reverse the entombment of Cu or other cations in PrPSc and, thus, alleviate the disease. Whatever advances may come in the future, one thing remains clear: the pathogenesis of prion diseases is tightly linked to the function and distribution of metal ions, particularly Cu, in the brain.

ACKNOWLEDGMENTS We acknowledge the support of the BBSRC and DEFRA towards our research fund.

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CHAPTER

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Metallothioneins in Neurodegeneration Michael Aschner, William F Silverman, Israel Sekler, Paolo Zatta

ABSTRACT Metallothioneins are ubiquitous low molecular weight proteins characterized by an abundance of the thiol-containing amino acid, cysteine. Metallothionein I and metallothionein II, the most widely expressed isoforms, are co-ordinately regulated in all mammalian tissues, while a third variant, metallothionein III, is predominantly expressed in zinc-containing neurons and absent from non-neural tissue. Metallothionein proteins have been implicated as regulators of gene expression in homeostatic control of cellular metabolism of metals and in cellular adaptation to stress, including oxidative stress. They regulate transcription, replication, protein synthesis, metabolism, and other zinc-dependent biological processes. Because the intracellular concentration of zinc is buffered by complexing with apothionein to form metallothionein, and disordered metallothionein homeostasis results in changes in brain zinc levels, there has been great interest in the potential role of metallothionein regulation in the etiology of neurodegenerative disorders. Abnormalities in metallothionein and/or zinc homeostasis have been reported in a wide variety of neuropathologies, including Alzheimer's disease, epilepsy, Friedrich's ataxia, Pick's disease, amyotrophic lateral sclerosis, schizophrenia, hepatic encephalopathy, multiple sclerosis, Guillaine-Barre syndrome, Parkinson's disease, retinitis pigmentosa, retinal dystrophy, Wernicke-Korsakoff syndrome, and alcoholism, though a direct association with the etiology of neurodegenerative diseases has yet to be established. This chapter commences with a brief discussion of the various brain metallothionein isoforms, followed by a survey of the evidence that metallothioneins are involved in neurodegeneration. Keywords: Metallothionein; neurodegenerative disorders; Alzheimer's disease; prion diseases.

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1. METALLOTHIONEIN ISOFORMS AND ABUNDANCE IN THE CENTRAL NERVOUS SYSTEM The metallothionein (MT) family belongs to a class of low molecular weight, intracellular, cysteine-rich proteins devoid of aromatic amino acids with a high affinity to metal ions (Oz et al., 1999). MTs are multifunctional proteins and their roles remain elusive after more than 40 years of intense investigation. They are known to regulate the release of gaseous mediators, such as hydroxyl radical and nitric oxide, apoptotic signaling, and the binding and exchange of heavy metals (such as copper (Cu), zinc (Zn), cadmium, and silver) (Simpkis, 2000). A relationship between MT and several diseases, including cancer, cardiovascular diseases, septic shock, and immunological alterations, has been widely investigated. The involvement of MTs in the physiopathology of disorders of the central nervous system (CNS) has been recently reviewed by Hidalgo et al. (2001). MTs are referred to as "housekeeping" proteins. Their basal mRNA expression level in the CNS is low and induction is not always apparent even with potent peripheral MT inducers, most likely because the brain is protected by the restrictive properties of the blood-brain barrier (Saijoh et al., 1994). Four major isoforms of MTs have been described: MT-I, MT-II, MT-HI, and MT-IV. MT-I and MT-II are found in all vertebrate tissues, differing at neutral pH by a single negative charge. Each represents a number of different isoproteins designated as MT-Ia, MT-Ib, MT-Ic, and so forth. MT-I and MT-II are single-chain proteins containing between 61 and 68 amino acids (species-dependent), about 20 of which are cysteines. Noncysteine amino acids serve as flexible spacers connecting the metalchelating cysteine residues. The formation of stable clusters is accomplished by the folding of the peptide backbone in a way that minimizes the strain on the conformation of the protein (Kille et al, 1994; Vasak et al., 1999). MT-III, a brain-specific MT isomer, was originally described as a growth inhibitory factor (GIF) (Uchida et al., 1991). It contains 68 amino acids; thirty-eight of the amino acids are identical both in alignment and type to the human MT-I and MT-II isoforms. MT-III possesses close structural homology to other mammalian MTs. Indeed, when aligned with MT-I and MT-II, the position of all 20 constituent cysteine residues is completely conserved (Kille et al., 1994; Aschner, 1996).

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The most abundant MT mRNA in the CNS is MT-I, followed by MT-III (Aschner, 1996). Both MT-I and MT-II mRNA are highly expressed in glia in the cerebellum, olfactory bulb, and eye. MT-III transcripts are particularly abundant in neurons in regions with high concentrations of vesicular Zn, such as the hippocampus, piriform cortex, and amygdala, though strong expression is also observed in the cerebellum, an area with little synaptic Zn (Masters et al., 1994).

2. THE ROLE OF METALLOTHIONEIN IN NEURODEGENERATIVE DISORDERS 2.1. Amyotrophic Lateral Sclerosis Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by loss of motor neurons in the spinal cord, brainstem, and motor cortex. Only 10% of cases are of a familial form that is linked to point mutations in the gene encoding for cytosolic Cu, Zn, and superoxide dismutase (SOD) (Sillevis-Smitt et al., 1994). In earlier studies of sporadic ALS, the amount of MTs appeared higher in the liver, kidney, and spinal cord of ALS patients than in control subjects (Blaauwgeers et al., 1996). In ALS, MTs are highly expressed in the gray matter of protoplasmatic astrocytes from the spinal cord. This likely reflects an increase in metal exposure or enhanced oxidative stress (Sillevis-Smitt et al., 1994). In wildtype mouse spinal cord, expression of MT-I-II has been observed in ependymal cells and in a subset of astrocytes of the white matter. MT-III is limited to neurons within the gray matter (Gong and Elliott, 2000). Compared to wild-type mice, transgenic mice (G93A) carrying the SOD1 mutation show a significant increase in expression of MT-I-II within astrocytes, both in the white and gray matter. Furthermore, MT-III in the neurons of G93A mice is also significantly upregulated (Gong and Elliott, 2000).

2.2. Multiple Sclerosis Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the CNS. Experimental autoimmune encephalitis (EAE) has been extensively used to study the human autoimmune disease MS. EAE is characterized by significant macrophage activation, T-lymphocyte infiltration, astrogliosis

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in the spinal cord, brainstem, and cerebellum with maximum manifestation at two weeks post-immunization (Penkowa and Hidalgo, 2000). Exogenous administration of Zn-MT-II significantly decreased the clinical symptoms, mortality, and leukocyte infiltration of the CNS during EAE (Penkowa and Hidalgo, 2001). The same authors also observed a significant decrease in the expression of interleukin (IL)-6 and tumor necrosis factor (TNF)-a in the CNS of EAE animals, which might have also played a causal role in the decrease in clinical manifestations and reduced rate of neuronal apoptosis. None of these effects could be ascribed to Zn content, suggesting that MT-I-II might be used as a pharmacotherapy in EAE/MS.

2.3. CNS Immune Responses and Seizures A recent study examined the role of MT-I and MT-II during experimental EAE (Penkowa et al., 2001a). In genetically altered mice that are MTI- and MT-II-deficient (MT knockout; MT-I/II-KO), a significantly higher susceptibility to the development of EAE relative to wild-type mice was noted. Furthermore, the inflammatory responses elicited by EAE were significantly altered in these mice. This was characterized by diminished rates of astrocytosis and increased macrophage and T-lymphocytes infiltration. Additional alterations in MT-I/II-KO mice included increased expression of proinflammatory cytokines (IL-lbeta, IL-6, and TNF-a) and increased oxidative stress and apoptosis in response to EAE. An earlier study by the same authors established that MT-I/II-KO mice were dramatically deficient in their ability to induce IL-6-induced angiogenesis (Penkowa et al., 2000a). Increased epileptiform activity and hippocampal degeneration following kainic acid-induced seizures have also been reported in MT-I/II-KO mice (Carrasco et al., 2000). These results support the notion that MT-I and MT-II modulate inflammatory responses in the CNS, thereby playing a neuroprotective role during EAE ontogeny and inflammation (Penkowa et al., 2001a), as well as during kainic acidinduced seizures (Carrasco et al., 2000). MT-I and MT-II have also been suggested to enhance neuronal survival during kainic acid-induced seizures, either via impaired Zn regulation or compromised antioxidant activity. Significantly, compared to wild-type controls, IL-6 deficient mice also demonstrate increased hippocampal neuronal injury and impairment in inflammatory responses following kainic acid-induced seizures,

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an effect that is also associated with reduced MT-I and MT-II protein levels (Penkowa et al., 2001b). The changes in neuronal tissue damage and brain regeneration of IL-6-KO mice were associated with an IL-6-dependent decrease in MT-I and MT-II expressions (Penkowa et al., 2000b). Another study, conducted on aged animals under constant stress conditions, observed MT-induced depletion of Zn from plasma and tissues. This mechanism was invoked to explain increased MT levels and a concomitant decrease in free Zn ion bioavailability, thereby perturbing immune responses known to be Zn-dependent (Mocchegiani et al, 2000, 2001). Recently, a chimeric fusion protein consisting of MT flanked by the cyan and yellow forms of the green fluorescent protein has been generated and expressed in endothelial cells. This paradigm has been used to explore metal interactions with MTs (Pearce et al., 2000). By measuring changes in fluorescent resonance energy transfer (FRET), changes in protein conformation resulting from release of metal may be followed, such as Zn and MT. These experiments indicate that interactions of Zn and Cu with MTs are dynamic and are regulated by cell signal transduction, such as the nitric oxide pathway.

2.4. Parkinson's Disease Parkinson's disease (PD) is a neurodegenerative disorder characterized by a preferential loss of the dopaminergic neurons of the substantia nigra pars compacta. The etiology of PD is unknown, though oxidative stress and mitochondrial inhibition have been suggested to be involved (see review by Jenner, 2001). Oxidative stress results either from excess generation or reduced scavenging of reactive oxygen species. Thus, in rodents and primates the systemic or intracranial application of the synthetic heroin analog, l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP), leads to the rapid loss of nigrostriatal dopaminergic neurons and the development of a syndrome virtually indistinguishable from idiopathic PD (Davis et al., 1979). Several studies have examined MT-I and MT-II in the context of oxidative stress. Rojas et al. (1996), for example, examined the effect of 6-hydroxydopamine (6-OHDA), an oxygen-radical generator and dopaminergic neurotoxin, on levels of Zn and MTs in the brain. They reported that 6-OHDA, introduced at a concentration producing a nearly

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complete lesion of the dopaminergic innervation of the striatum, reduced Zn and MT-I levels there. A lower concentration, sufficient to cause oxidative stress but not lethal to dopaminergic neurons, resulted in a robust upregulation of MT-I mRNA in various brain regions, though not in the striatum. The authors suggest that Zn or MT are altered in conditions where oxidative stress has occurred, and propose that areas of the brain, such as the striatum, which contain high iron levels but low levels of inducible MT, are particularly vulnerable to oxidative stress. Nevertheless, the idea that MT is neuroprotective against free radicals should be viewed with caution, since the study did not take into account key mediators of cell death, presumably involved in the degeneration of nigral dopaminergic neurons and their striatal projections. Furthermore, little data exist which directly link Zn or MT with mechanisms believed to underlie the development of PD. Interestingly, these same authors have demonstrated that another free radical generator, MPTP, reduces MT-I mRNA and protein and MT-II protein content in the striatum of mice (Rojas et al., 2000), and does not protect against MPTP toxicity (Rojas et al., 1996), further weakening the contention that MT is a principal antioxidant in the context of PD. Little information is available regarding MT-III and PD. MT-III has been shown to possess free radical scavenging ability (Uchida et al., 1991). Indeed, a study on a glial cell line suggested that oxidative stress might be a key factor in modulating MT-III mRNA expression (Sogawa et al., 2000). However, another study which looked at MT-III in the context of free radicals and dopaminergic neurotoxicity (Miyazaki et al., 2000) found that MT-III transcripts decreased in the striatum after exposure to 6-OHDA. More data is needed before the question of a role for MT-III as an antioxidant in the CNS can be answered. Another important question that has not, to our knowledge, been addressed is the role of Zn and MT in Lewy body formation. These are eosinophilic inclusion bodies present in the cytoplasm of nigral dopaminergic neurons in PD (Forno, 1969). They are formed as a result of disordered metabolism and/or transport of neurofilaments, and contain both amyloid-|3 (A3) and the non A(3 components of Alzheimer's disease (AD) amyloid-P protein precursor (A(3PP) or alpha-synuclein. Because of recent insights into the role of Zn in Alzheimer's type inclusion bodies (Cherny et al., 2001), it seems likely that regulators of intracellular

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Zn-like MTs will influence, or be influenced by, cellular changes that result in Lewy body formation. Indeed, alpha-synuclein has been shown to undergo precipitation and conformational change by heating in the presence of Zn (but not calcium or manganese) in vitro (Kim et al., 2000). Such a change could be involved in the abnormal aggregation of the protein observed in PD.

2.5. Alzheimer's Disease Alzheimer's disease (AD) is a chronic neurodegenerative disorder whose pathogenic mechanisms are poorly understood. In normal human brain, MT-I and MT-II are expressed in astrocytes, but not in neurons. Using antibodies to detect cells expressing MT-I-II in AD brain sections, a high level of expression of these proteins has been noted in the cortex, cerebral white matter, and cerebellum. In particular, MT-I-II have been observed in astrocytes and microcapillaries, as well as in the granular, but not in the molecular, layer of the cerebellum in AD brains (Zambenedetti et al., 1998). A role for the MT-III isoform in the etiology of AD has been proposed (Richarz and Bratter, 2002). Initially purified as a component of normal human brain extract, MT-III was originally postulated to be secreted by astrocytes and to suppress neurotrophic activity within the CNS (Uchida et al., 1991). These authors demonstrated that extracts prepared from AD brains increased the survival of rat cortical neurons in vitro. Additional studies indicated that this neurotrophic activity of AD brain extracts was due to a reduction of GIF, which was subsequently shown to be a new member of the MT gene family, and designated MT-III. Subsequent studies established that MT-III is predominantly expressed in the hippocampus and, specifically, within neuronal populations that sequester Zn in synaptic vesicles (Masters et al., 1994; Erickson et al., 1995). Additional analyses of MT-III protein and its mRNA revealed that its expression was decreased up to tenfold in AD brains (Yu et al., 2001), leading to speculation that downregulation of MT-III and aberrations in Zn homeostasis could lead to excessive neuritic sprouting and the formation of neurofibrillary tangles characteristic of the disease. In addition, MT-III was shown to protect cortical neurons in culture from the toxic effect of A3 peptides (Irie and Keung, 2001).

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An important pool of Zn in the CNS is present in synaptic vesicles in a subgroup of glutamatergic neurons in the hippocampus. "Free" or synaptic Zn is readily released by electrical stimulation, serving to modulate responses at receptors for a number of different neurotransmitters, including the excitatory and inhibitory receptors, N-methyl-D-aspartate and 7-aminobutyric acid, respectively (Cuajungco and Lees, 1997). Although this pool of Zn is the only one that is readily stained by histochemistry, it represents only about 5% of the total Zn content in the CNS (Frederickson et al., 2000). Given that Zn is readily released from MT by disulfides, which occurs under conditions of oxidative stress, consideration should be given to the possibility that changes in MT protein expression can alter intracellular Zn homeostasis. This would be very significant since even small increases in intracellular Zn, such as those which follow the massive release of extracellular Zn accompanying some neuropathological events such as stroke (Cuajungco and Lees, 1997), could result in neuronal death. The pathophysiological role of Zn has been highlighted recently by the demonstration that oral treatment of Tg2576 transgenic mice (overexpressing human mutant AfSPP) with clioquinol, an antibiotic and Cu/Zn chelator, reduced A(3 deposition by almost 50% (Cherny et al., 2001). Reduction in deposition of Ap protein in the clioquinol-treated animals was also accompanied by a significant improvement in the health of the mice. There was no monitoring, however, of MT expression in the antibiotic-treated subjects. Another recent study tying Zn to amyloid plaques showed that synaptic Zn is an important component of amyloid deposition in hApPP(+) mice (Lee et al., 2002). Indeed, female mice exhibited higher levels of synaptic Zn in soluble A(3 and plaques than males, suggesting a mechanism for the predominance of AD in females (Katzman et al., 1989). This difference disappeared in mice lacking synaptic Zn, supporting a direct role for this pool of Zn in promoting the formation of amyloid plaques. Evidence for altered Zn metabolism in AD includes the following (Bush et al., 1994; Cuajungco and Lees, 1997): decreased levels of Zn in the temporal lobe; increased cerebrospinal fluid concentration of Zn; increased hepatic Zn with reduced Zn bound to MT; increased extracellular Zn2+-metalloproteinase activity in the hippocampus; hippocampal cholinergic differentiation, a neurochemical deficit associated with AD which leads to elevation of Zn concentrations within this

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region; a pervasive abnormality of Zn metabolism is a common complication of Down's syndrome, a condition that is frequently associated with the development of premature AD; desferoxamine, a drug which chelates iron and aluminum has been reported to arrest the clinical progression of AD; Zn has been shown to aggregate A(3 amyloid, a form which is potentially neurotoxic, the principal component of the cerebral amyloid-containing plaques characterizing AD, specifically and saturably binds Zn; Zn increases the peptide's adhesive properties and resistance to proteolytic digestion, potentially representing the putative upstream lesion in the pathogenic cascade of amyloid formation; the Zn-dependent transcription factors NF-kappa B and Spl bind to the promoter region of the ApPP gene; and Zn inhibits enzymes that degrade A(3PP to nonamyloidogenic peptides which, in turn, degrade the soluble form of Ap (Cuajungco and Lees, 1997). A number of studies have examined but failed to find an association of MT-III with AD. For example, Erikson et al. (1994) examined the association between neurotrophic activity and MT-III expression in the frontal cortices of eight AD and five control brains, focusing on the proposed role of MT-III (that is, GIF) in inhibiting cell proliferation. They reported that extracts from the cortex of AD brains stimulated the survival of approximately twofold more rat cortical neurons than control extracts, demonstrating that the AD brain possesses elevated neurotrophic activity. Furthermore, when recombinant MTs were added to cultures grown in the presence of brain extract, MT-III, but not MT-I, had a negative effect on neuron survival, further supporting the idea that MT-III is a specific inhibitory factor in this assay. However, in contrast to previous reports, neither MT-III mRNA nor MT-III protein levels were significantly decreased in the AD group. Therefore, the authors concluded that the difference in neurotrophic activity between the AD and control brain samples examined was not directly mediated by MT-III, suggesting that MTIII downregulation is not a key pathogenic event in the etiology of AD. Similarly, the fate and significance of MT-III in the AD brain was evaluated in both the temporal and frontal cortices, hippocampus, and cerebellum of 11 AD patients and two groups of five and six control subjects, respectively (Amoureux et al., 1997). Reverse transcription-polymerase chain reaction was used to quantify the levels of MT-III mRNA in these brains. The distribution of MT-III was found to be similar to that of

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control, constitutive RNAs, that is, beta-actin, glyceraldehyde-3-phosphate dehydrogenase (G3PDH), and ribosomal RNA 18S (rRNA 18S), measured simultaneously. This shows that downregulation of MT-III mRNA in the frontal and temporal cortices and hippocampus is not associated with AD. The content of MT-III mRNA in the brain of AD patients is also not detectably altered (Amoureux et al., 1997) compared to neurologically unimpaired individuals. It is noteworthy that the contradictory results obtained on MT-III expression in AD may derive from the utilization of antibodies that face problems in targeting small epitopes due to steric hindrance by their surface peptide chains (Yanagitani et al., 1999). Mice lacking MT-III mRNA (MT-III-KO), following targeted gene inactivation, have decreased concentrations of Zn in several brain regions, including the hippocampus (Erickson et al., 1997). The pool of histochemically reactive Zn, however, does not appear to be altered in these animals. Indeed, MT-III-KO displayed normal spatial learning in the Morris water maze and were insensitive to systemic Zn or cadmium exposure. Furthermore, neuropathology or behavioral deficits were undetected in two-year-old MT-III-KO mice. However, the MT-III-deficient mice were more susceptible to kainic acid-induced seizures and exhibited an increase in neuronal injury in the CA3 field of the hippocampus. Conversely, mice overexpressing MT-III were more resistant to seizureinduced neuron injury in CA3, leading Erickson et al. (1997) to propose a role for MT-III in Zn regulation during neural stimulation.

2.6. Prion Diseases Prion protein (PrP) is a plasma membrane glycosylphosphatidylinositol (GPI)-anchored protein prevalently expressed in neurons. Its conservative presence among species suggests its relevant physiological role(s). Prion diseases are genetic or sporadic neurodegenerative and transmissible disorders characterized by an overdeposition of an abnormal isoform of PrP. These diseases include the sporadic form of Creutzfeld-Jacob disease (CJD), Gerstmann-Straussler-Scheinker syndrome (GSSS), and fatal insomnia. Analogous to other Cu-binding proteins, PrP has high a affinity to Cu (Km = nM) (Brown, 2001; Wong et al., 2001) and Cu binding to the PrP modifies its biochemical properties (Lehmann, 2002). However, PrP also binds other metal ions, such as manganese and Zn, albeit at a lower affinity (Brown, 2001).

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Changes in the concentration of Cu and manganese in the brain of scrapie-infected mice before the onset of clinical symptoms have been observed and, interestingly, a major increase in manganese in the blood in the early stage of the disease has been reported (Thackray et al, 2002). The Cu content of the PrP in the brains of scrapie-infected mice has been seen to reduce with a reduction in the antioxidant properties at between 30 and 60 days post-inoculation (Thackray et al., 2002). Several properties have been ascribed to PrP, such as Cu uptake, protection against oxidative stress, cell adhesion, differentiation, signaling, and cellular survival (Martins et al., 2002). Cu binding and the ensuing changes in the tertiary structure of PrP are pH-dependent (Gustiananda et al, 2002). The possible link between prion disease and Cu dismetabolism has been reviewed by Brown (2001). Few papers report data concerning the expression of MT in prion disease. These are summarized in Table 1. Expression of MT in the brain of human prion disease patients, with and without PrP gene mutation and polymorphisms, has been studied by Kawashima et al. (2000). MT immunoreactivity was positive in the cytoplasm and processes of astrocytes from the cerebral cortex and white matter, both in normal and prion diseased brains. However, in CJD brains, immunoreactivity varies from case to case and apparently depends on the duration of the disease. In CJD with a long disease course, MT-I-II-III immunoreactivity was significantly reduced in astrocytes, which is

Table 1. MTs and prion diseases. Disorder

MT I and MT-II

CJD

Induced in reactive astrocytes in the cerebral cortex Depends on the duration of the disease Varies from case to case mRNA increased Preserved

Uchida, 1991

Reduced

Kawashima et al, 2000

GSSS Kuru

MT-III

Preserved

Reduced in the vicinity of plaques

CJD = Creutzfeld-Jakob MT = metallothionein.

disease,

Reference

Kawashima et al, 2000 Kawashima et al., 2000

Kawashima et al., 2000

GSSS = Gerstmann-Straussler-Scheinker

syndrome,

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analogous to observations of MT-I-II in GSSS, while MT-III was unmodified. Conversely, in the vicinity of the Kuru plaques, astrocytes showed a weak or negative immunoreactivity for MT. Increased expression of MT-II in scrapie has been reported (Duguid et al., 1988), where a high level of MT-II mRNA has been observed only at the terminal stage of the disease (Dandoy-Dromn et al., 1998). Finally, to the best of our knowledge only one paper deals with the high expression of MT in Pick's disease (Duguid et al., 1989).

3. SUMMARY Given the prominent role played by MTs in modulating genetic processes through redistribution of Zn and regulation of reactive oxygen species, it is not surprising that a number of different MT genes are expressed in mammalian cells. This may also represent a type of redundancy that may serve as a "safety valve," providing a reserve capacity of MTs under stressful conditions. Functionally identical MTs might be regulated independently, thus permitting cell-specific MT expression. Alternatively, the distinct MT isoforms may have distinct functions, such as that of MT-III in regulating glutamatergic neurotransmission in the hippocampus. Though MT knockout and overexpressing animal models show permutations in normal immune responses, direct evidence for the role of MTs in neurodegenerative disorders is lacking. Nevertheless, given the cardinal role played by MTs in buffering intracellular Zn, additional studies of MT gene and protein expression are necessary to assess their contribution to both neuroprotection and neurodegeneration.

REFERENCES Amoureux MC, Van Gool D, Herrero MT, Dom R, Colpaert FC, Pauwels PJ. Regulation of metallothionein-III (GIF) mRNA in the brain of patients with Alzheimer's disease is not impaired. Mol Chem Neuropathol 1997; 32:101-121. Aschner M. The functional significance of brain metallothioneins. FASEB J 1996; 10:1129-1136. Blaauwgeers HG, Anwar-Chand M, van den Berg FM, Vianney de Jong JM, Troost D. Expression of different metallothionein messenger ribonucleic acids in motor cortex, spinal cord and liver from patients with amyotrophic lateral sclerosis. J Neurol Sci 1996; 142:39^4. Brown DR. Copper and prion disease. Brain Res Bull 2001; 55:165-173.

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Bush AI, Pettingell WH, Multhaup G, Paradis MD, Vonsattel J-P, Gusella JF, Beyreuther K, Masters CL, Tanzi RE. Rapid induction of Alzheimer's A(3 amyloid formation by zinc. Science 1994; 265:1464-1467. Carrasco J, Penkowa M, Hadberg H, Molinero A, Hidalgo J. Enhanced seizures and hippocampal neurodegeneration following kainic acid-induced seizures in metallothioneinI + II-deficient mice. Eur J Neurosci 2000; 12:2311-2322. 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. Cuajungco MP, Lees GJ. Zinc metabolism in the brain: Relevance to human neurodegenerative disorders. Neurobiol Dis 1997; 4:137-169. Dandoy-Dromn F, Guillo F, Bendoudjema L, Deslys JP, Lasmezas C, Dormont D, Tovey MG, Dron M. Gene expression in scrapie. Cloning of a new scrapie-responsive gene and the identification of increased levels of seven other mRNA transcripts. J Biol Chem 1998; 273:7691-7697. Davis GC, Williams AC, Markey SP, Ebert MH, Caine ED, Reichert CM, Kopin IJ, et al. Chronic Parkinsonism secondary to intravenous injection of meperidine analogues. PsychiatrRes 1979; 1:249-254. Duguid JR, Bohmont CW, Liu NG, Tourtellotte WW. Changes in brain gene expression shared by scrapie and Alzheimer's disease. Proc Natl Acad Sci USA 1989; 86: 7260-7264. Duguid JR, Rohwer RG, Seed B. Isolation of cDNAs of scrapie-modulated RNAs by subtractive hybridization of a cDNA library. Proc Natl Acad Sci USA 1988; 85:5738-5742. Erickson JC, Hollopeter G, Thomas SA, Froelick GJ, Palmiter RD. Disruption of the metallothionein-III gene in mice: Analysis of brain zinc, behavior, and neuron vulnerability to metals, aging, and seizures. J Neurosci 1997; 17:1271-1281. Erickson JC, Masters BA, Kelly EJ, Brinster RL, Palmiter RD. Expression of metallothionein-III in transgenic mice. Neurochem Int 1995; 27:35^-1. Erickson JC, Sewell AK, Jensen LT, Winge DR, Palmiter RD. Enhanced neurotrophic activity in Alzheimer's disease cortex is not associated with downregulation of metallothionein-III (GIF). Brain Res 1994; 649:297-304. Forno LS. Concentric hyalin intraneuronal inclusions of Lewy type in the brains of elderly persons (50 incidental cases): Relationship to Parkinsonism. J Am Geriatr Soc 1969; 17:557-575. Frederickson CJ, Suh SW, Silva D, Frederickson CJ, Thompson RB. Importance of zinc in the central nervous system: The zinc-containing neuron. J Nutr 2000; 130(5s suppl):1471s-1483s Gong YH, Elliott JL. Metallothionein expression is altered in a transgenic murine model of familial amyotrophic lateral sclerosis. Exp Neuro 2000; 162:27-36. Gustiananda M, Harris PI, Milburn PJ, Gready JE. Copper-induced conformational changes in a marsupial prion protein repeat peptide probed using FTIR spectroscopy. FEBS Lett 2002; 512:38^12.

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Hidalgo J, Aschner M, Zatta P, Varsak M. Roles of the metallothionein family of proteins in the central nervous system. Brain Res Bull 2001; 55:133-146. Irie Y, Keung WM. Metallothionien-III antagonizes the neurotoxic and neurotrophic effects of amyloid beta peptides. Biochem Biophys Res Commun 2001; 282(2):416^420. Jenner P. Parkinson's disease, pesticides and mitochondrial dysfunction. Trends Neurosci 2001;24:245-247. Katzman R, Aronson M, Fuld P, Kawas C, Brown T, Morgenstern H, Frishman W, Gidez L, Eder H, Ooi WL. Contribution by synaptic zinc to the gender-disparate plaque formation in human Swedish mutant A(3PP transgenic mice. Ann Neurol 1989; 25: 317-324. Kawashima T, Doh-ura K, Torisu M, Uchida Y, Furuta A, Iwaki T. Differential expression of metallothioneins in human prion diseases. Dement Geriatr Cogn Disord 2000; 11:251-262. Kille P, Hemmings A, Lunney EA. Memories of metallothionein. Biochim Biophys Acta 1994;1205:151-161. Kim TD, Paik SR, Yang C, Kim J. Structural charges in a-synuclein affect its chaperonelike activity in vitro. Protein Sci 2000; 9(12):2489-2496. Lee JY, Cole TB, Palmiter RD, Suh SW, Koh JY. Contribution by synaptic zinc to the gender-disparate plaque formation in human Swedish mutant AfJPP transgenic mice. Proc Natl Acad Sci USA 2002; 99:7705-7710. Lehmann S. Metal ions and prion diseases. Curr Opin Chem Biol 2002; 6:187-192. Martins VR, Linden R, Prado MAM, Walz R, Sakamoto AC, Izquierdo I, Brentani R. Cellular prion protein: On the road of functions. FEBS Lett 2002; 512:25-28. Masters BA, Quaife CJ, Erickson JC, Kelly EJ. Froelick GJ, Zambrowicz BP, Brinster RL, Palmiter RD. Metallothionein III is expressed in neurons that sequester zinc in synaptic vesicles. J Neurosci 1994; 14:5844-5857. Miyazaki I, Sagawa CA, Asanuma M, Higashi Y, Tanaka KI, Nakanishi T, Ogawa N. Expression of metallothionien-III mRNA and its regulation by levodopa in the basal ganglia of hemi-parkinsonian rats. Neurosci Lett 2000; 293(l):65-68. Mocchegiani E, Giacconi R, Cipriano C, Muzzioli M, Fattoretti P, Bertoni-Freddari C, Isani G, Zambenedetti P, Zatta P. Zinc-bound metallothioneins as potential biological markers of aging. Brain Res Bull 2001; 55:147-153. Mocchegiani E, Muzzioli M, Giacconi R. Zinc, metallothioneins, immune responses, survival and aging. Bio gerontology 2000; 1:133-143. Oz G, Pountney DL, Armitage IA. Metallothionein structure uptake. In Klaasen CD, editor. Metallothionein IV. Basel: Birkhauser Verlag, 1999:37^13. Pearce LL, Gandley RE, Han W, Wasserloos K, Stitt H, Kanai AJ, McLaughlin MK, Pitt BR, Levitan ES. Role of metallothionein in nitric oxide signaling as revealed by a green fluorescent fusion protein. Proc Math Acad Sci USA 2000; 3:477^4-82. Penkowa M, Espejo C, Martinez-Caceres EM, Poulsen CB, Montalban X, Hidalgo J. Altered inflammatory response and increased neurodegeneration in metallothionein I+11 deficient mice during experimental autoimmune encephalomyelitis. J Neuroimmunol 2001a; 119:248-260.

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Penkowa M, Carrasco J, Giralt M, Molinero A, Hernandez J, Campbell IL, Hidalgo J. Altered central nervous system cytokine-growth factor expression profiles and angiogenesis in metallothionein-I+II deficient mice. J Cerebral Blood Flow Metabolism 2000a; 20:1174-1189. Penkowa M, Giralt M, Carrasco J, Hadberg H, Hidalgo J. Impaired inflammatory response and increased oxidative stress and neurodegeneration after brain injury in interleukin-6deficient mice. Glia 2000b; 32:271-285. Penkowa M, Hidalgo J. Metallothionein-I-II expression and their role in experimental autoimmune encephalomyelitis. Glia 2000; 32:247-263. Penkowa M, Hidalgo J. Metallothionein treatment reduces proinflammatory cytokines IL-6 and TNF-alpha and apoptotic cell death during experimental autoimmune encephalomyelitis (EAE). Exp Neurol 2001; 170:1-14. Penkowa M, Molinero A, Carrasco J, Hidalgo J. Interleukin-6 deficiency reduces the brain inflammatory response and increases oxidative stress and neurodegeneration after kainic acid-induced seizures. Neuroscience 2001b; 102:805-818. Richarz AN, Bratter P. Speciation analysis of trace elements in the brains of individuals with Alzheimer's disease with special emphasis on metallothioneins. Anal Bioanal Chem 2002; 372:412^117. Rojas P, Cerutis DR, Happe HK, Murrin LC, Hao R, Pfeiffer RF, Ebadi M. 6-hydroxydopamine-mediated induction of rat brain metallothionein I mRNA. Neurotoxicology 1996; 17:323-334. Rojas P, Hidalgo J, Ebadi M, Rios C. Changes of metallothionein I + II proteins in the brain after l-mefhyl-4-phenylpyridinium administration in mice. Progress NeuroPsychopharmacol Biol Psychiatr 2000; 24:143-154. Saijoh K, Katsuyama H, Sumino K. Brain metallothionein gene expression and regulation. Biol Signals 1994; 3:150-156. Sillvetis-Smitt PA, Mulder TP, Verspaget HW, Blaauwgeers HG, Troost D, de Jong JM. Metallothionein in amyotrophic lateral sclerosis. Biol Signals 1994; 3(4): 193-197. Simpkis CO. Metallothionein in human disease. Cell Mol Biol 2000; 46:465-488. Sogawa CA, Miyazaki I, Sogawa N, Asanuma M, Ogawa N, Furuta H. Antioxidants protect against dopamine-induced metallothionein-III (GIF) mRNA expression in mouse glial cell line (VR-2g). Brain Res 2000; 853(2):310-316. Thackray AM, Knight R, Haswell SJ, Bhjdoso R, Brown DR. Metal imbalance and compromised antioxidant function are early changes in prion disease. Biochem J 2002; 362:253-258. Uchida Y, Takio K, Titani K, Ihara Y, Tomonaga M. The growth inhibitory factor that is deficient in Alzheimer's disease is a 68 amino acid metallothionein-like protein. Neuron 1991;7:337-347. Vasak M, Bogumil R, Faller P, et al. Structural and biological studies on native bovine Cu, Zn-metallothionein-3. In: Klaasen CD, editors. Metallothionein IV. Basel: Birkhauser Verlag, 1999: 15-22. Wong BS, Brown DR, Sy MS. A ying-yang role for metals in prion disease. Panminerva Med 2001; 43:283-287.

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Yanagitani S, Miyazaki H, Nakahashi Y, Kuno K, Ueno Y, Matsushita M, Naitoh Y, Taketani S, Inoue K. Ischemia induces metallothionein III expression in neurons of rat brain. Life Sci 1999; 64(8):707-715. Yu WH, Lukiw WI, Bergeron C, Niznik HB, Fraser PE. Metallothionein III is reduced in Alzheimer's disease. Brain Res 2001; 894(1):37^15. Zambenedetti P, Giordano R, Zatta P. Metallothioneins are highly expressed in astrocytes and microcapillaries in Alzheimer's disease. J Chem Neuroanat 1998; 15:21-26.

CHAPTER 13

Iron and Neurodegeneration Stacey L Grab, James R Connor

ABSTRACT Iron-related pathology is present in many neurodegenerative diseases, and the effects of iron mismanagement can serve as either primary or secondary causes of neurodegeneration. There are many mechanisms by which iron mismanagement can precipitate neurodegeneration, including misregulation of iron import and export, iron deficiency or accumulation, and oxidative damage resulting from loss of iron homeostasis. While the crucial role of iron in neurodegeneration is, in general, beginning to be appreciated, the mechanisms by which loss of iron homeostasis in the brain occurs are still unclear and questions regarding opportunities for therapeutic intervention involving iron chelation remain unanswered. Keywords: Iron transport; brain iron accumulation; neurodegeneration; oxidative stress.

1. INTRODUCTION Many neurodegenerative disorders share several general pathogenic processes, including oxidative stress and free radical activity, accumulation of intracellular aggregates, and mitochondrial dysfunction. An imbalance of iron (Fe) concentration is perhaps a common mediator of these effects, thus making an understanding of its regulation essential to attaining a complete picture of neurodegeneration. Fe is physiologically essential, a fact readily demonstrated by the syndromes associated with Fe-deficient states, including Fe deficiency anemia and cognitive deficits induced by developmental dietary Fe deficiency. The capacity of Fe to interconvert between ferric and ferrous states by readily accepting and donating electrons makes it quite useful to 323

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cytochromes and hemoglobin, and as a co-factor for enzymes. This property of Fe, however, illustrates the need for its tight regulation, as it can also catalyze free radical formation (Andrews, 1999). In the brain, Fe is the most abundant trace metal (Beard et al., 1993), with its concentration varying by region. Systemically, Fe circulates bound to transferrin (Tf). Tf is largely produced in the liver and has a half-life of eight days (Finch and Huebers, 1982). Within cells, Fe is stored as ferritin (Ft), which is a large, 24-subunit protein consisting of light and heavy chains that can store up to 4,500 atoms of Fe (Harrison and Arosio, 1996). The amount of Fe uptake into a cell is largely dependent upon the amount of transferrin receptor (TfR) on the membrane, with each TfR molecule having the ability to bind two diferric Tf molecules (four Fe 3+ atoms). TfR is regulated by cellular Fe and cytoplasmic ribonucleic acid (RNA) iron regulatory proteins (IRPs) (Rouault, 2001). IRPs interact with specific elements in messenger RNA (mRNA) transcripts referred to as iron response elements (IREs). Ft mRNA contains an IRE in the 5' untranslated region (5'UTR) and Tf contains five IREs in the 3' untranslated region (3'UTR) (Mullner and Kuhn, 1988; Leibold and Munro, 1988). There exist two distinct IRPs, IRP1 and IRP2. The presence or absence of Fe-sulfur clusters determines the functionality of IRP1 (Emerit et al., 2001). When the cluster is present, which occurs in conditions of sufficient Fe supply, IRP1 acts as an aconitase, interconverting citrate, and isocitrate. In states of Fe depletion, the cluster is absent and IRP1 takes on the role of a high-affinity binding site for IREs. IRP2 does not contain an Fe-sulfur cluster, and does not have aconitase activity. IRP2 is rapidly degraded under conditions of adequate Fe supply (Kuhn, 1998). Under low Fe conditions, IRPs bind to the 5'UTR of Ft and prevent its translation. Concurrently, IRPs bind to the 3'UTR of TfR and act to stabilize its RNA for receptor translation. In the presence of sufficient Fe, IRPs are released from both Ft and TfR, allowing for ribosomal attachment and subsequent translation of Ft and degradation of TfR mRNA (Rouault and Klausner, 1997). This elegant system of IRP-IRE Fe regulation allows for close control of cellular Fe homeostasis by the Fe molecule itself. The factors governing certain aspects of this system, such as Fe-sulfur cluster synthesis, are not fully understood, but may be affected by oxidative stress. It is also possible that dysfunction of any aspect of the system by various mechanisms may occur or result in

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neurodegenerative disease. IRP-IRE interaction in the brain is disrupted in some individuals with Alzheimer's disease (AD) (Connor et al., 1992), and targeted deletion of the IRP2 gene in mice may cause misregulation of Fe metabolism and a neurodegenerative disease characterized by neuronal Fe accumulation (LaVaute et al., 2001).

2. BRAIN IRON TRANSPORT AND NEURODEGENERATION Much is known about the regulation of Fe uptake and transport within the systemic circulation (Andrews, 2000), but less is understood about those compartments within organs that are physically separate from the systemic circulation and, therefore, cannot acquire Fe directly from serum Tf. Among these compartments is the central nervous system (CNS). Unlike endothelial cells of the systemic organs, the cells of the brain blood vessels are joined by tight junctions that present a physical barrier to the passage of proteins and molecules. Little is definitively known about the uptake and export of Fe within the brain, or about its carrier state and method of distribution within the CNS, but it seems clear that the behavior of Fe within the CNS is quite different from that within the systemic organs. These differences will be presented in this section. The role of Tf in CNS Fe regulation is not as straightforward as Tf in the systemic circulation. Fe and Tf distribution in the brain are seemingly paradoxical, in that the areas of highest Fe concentration — the globus pallidus, substantia nigra, red nucleus, dentate gyrus, thalamus, and putamen — have relatively low Tf concentrations. The highest Tf concentrations are found in the hippocampus and cortical region, and these areas demonstrate relatively low Fe levels. Notably, the areas of highest Fe concentration are associated with several neurodegenerative diseases, including Parkinson's disease (PD), AD, Huntington's disease (HD), and amyotrophic lateral sclerosis (ALS). Recent data from Zerpa et al. (2000) may help in understanding this apparent paradox. They note that while Tf is synthesized in oligodendrocytes, it does not appear to be secreted by oligodendrocytes. Furthermore, Tf was localized in the cytosol and not in the secretory compartment, as would be expected for a secreted protein. Zerpa et al. (2000) propose that such findings suggest that Tf synthesized in the brain is functionally different than Tf elsewhere in the body,

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and that Tf plays a role other than Fe transport in oligodendrocytes and myelination. The role of additional Fe transport proteins in the CNS is one of growing importance, as it appears that proteins such as ceruloplasmin (CP), lactotransferrin (Lf), melanotransferrin (MTf), and divalent metal transporter 1 (DMT1) exert a substantial influence on Fe trafficking. The uneven distribution of these proteins among brain regions implies that Fe transport is specific and regional within the brain. Hence, increased or decreased production of some of these proteins is not surprisingly implicated in some neurodegenerative diseases. It has long been observed that Fe accumulations are often present in the brains of sufferers of neurodegenerative disease, yet only recently have the mechanisms of these accumulations begun to be understood and, with this knowledge, the realization of the primary importance of errors in Fe metabolism and transport.

2.1. Dysfunctional Iron Import 2.7.7.

Lactotransferrin

Lactotransferrin (Lf) is found normally in breast milk and saliva, where it serves as a potent antibacterial agent by binding free Fe 3+ and denying it to invading bacteria. Lf is also found in the lesions of some neurodegenerative disorders, most dramatically in Guamian ALS-Parkinsonismdementia complex (Levuegle et al., 1994). This complex refers to the specific geographic isolate of motor neuron disease localized to Guam. Betz cells are immunoreactive for Lf in the affected areas of the ALS brain (Levuegle et al., 1994), suggesting the importance of increased Fe deposition. Nevertheless no evidence has, to date, convincingly suggested a purpose for Lf in the brain, nor a mechanism by which Lf functions in the brain or gains access to the brain. The overproduction of Lf and its receptor has also been associated with the degeneration of dopaminergic neurons in the substantia nigra in PD (Faucheux et al., 1995). Lactotransferrin receptor (LfR) immunoreactivity in the mesencephalon of PD brains was most pronounced in those areas with severe loss of dopaminergic neurons, and immunoreactivity intensity in the substantia nigra was greatest in those brains with the greatest dopaminergic loss. Expression of Lf-positive neurons was found to be decreased in PD, but

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of those neurons surviving, immunolabeling demonstrated higher Lf levels compared to control cases. Similarly, increased Lf expression has been found in AD, but LfR expression has not been studied, again suggesting the importance of further investigation focusing on Lf and LfR functions, mechanisms, and transport in both normal and diseased brains.

2.7.2.

Melanotransferrin

Melanotransferrin (MTf) is also associated with AD via overexpression and may contribute to excess Fe accumulation (Jeffries et al., 1996). MTf is an Fe-binding protein that was first identified on melanoma cells. The protein is encoded on chromosome 3 in humans. While it is structurally similar to Tf and Lf, its function has not been well determined. MTf has been shown to be expressed on reactive microglial cells in AD patients and on associated amyloid plaques in post-mortem tissue. MTf was not expressed on those microglia not associated with plaques, and was not found in tissue from PD, HD, or ALS brains, indicating that MTf is a marker for AD. In addition, a correlation has been shown between increasing serum MTf concentration and disease progression, suggesting the role of MTf as a provider of the excess Fe found in senile plaques (Kennard et al., 1996). The distribution and expression of MTf and its physiological role warrant further investigation.

2.2. Dysfunctional Iron Export 2.2.7. Ceruloplasmin Ceruloplasmin (CP) is serum glycoprotein belonging to a family of blue copper oxidases. Its primary role appears to be as a ferroxidase, aiding in Fe efflux from cells before binding to apotransferrin, but it also functions as an amine oxidase, an antioxidant, and a copper transporter (Qian and Wang, 1998). CP is mainly synthesized in hepatocytes, with a smaller amount being produced in the CNS most likely by astrocytes (Klomp et al., 1996). Mutations in the CP gene cause an autosomal recessive disease known as aceruloplasminemia. The disease is characterized by Fe accumulation, and is often clinically manifested by diabetes and retinal and basal ganglia degeneration. The cerebrospinal fluid contains not only increased Fe levels, but also increased peroxidation products and

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superoxide dismutase (SOD) activity. Though it is widely accepted that CP functions in Fe efflux, there is also evidence to support a dual role for CP, with functions both in Fe efflux and influx (Qian and Shen, 2001). Alteration of Fe efflux has been described in aceruloplasminemia, and it is likely that defects in other Fe transport mechanisms are responsible for other diseases that have not yet been elucidated.

2.2.2. Secondary iron accumulation Other diseases of Fe accumulation do not directly affect Fe storage and transport molecules, but are the result of mutations in molecules that interact closely with Fe and can affect its homeostasis. Hallervorden-Spatz syndrome (HSS) is a rare neurodegenerative disease of childhood in which extrapyramidal dysfunction is characterized by rigidity, dystonia, and choreathetosis. The globus pallidus and substantia nigra of these patients have substantial Fe accumulation. The underlying mutation in a small number of patients with this disease appears to affect the PANK2 pantothenate kinase, an enzyme essential to co-enzyme A synthesis and catalysis of the phosphorylation of vitamin B5 (pantothenate) in a cysteine-consuming reaction (Zhou et al., 2001). PANK2 is specifically expressed in the brain, and its absence in these patients likely results in the observed cysteine accumulation in degenerating areas of the brain. A possible mechanism for Fe-mediated damage implicates initial cysteine accumulation in the brain and resultant Fe chelation by cysteine. It is notable that the causative mutation is a loss-of-function mutation. Thus, possible therapeutic strategies for this subset of HSS sufferers may target delivery of phosphopantothenate or co-enzyme A. Beyond those diseases for which a specific means of Fe mishandling has been described, there exist several other potential mechanisms for production of Fe-induced pathology. The condition known as neuroferritinopathy provides an example of a defect not in Fe transport, but rather in Fe storage (Curtis et al., 2001). In this dominant condition, the insertion of an adenine within the gene that encodes for the Ft light polypeptide results in Fe accumulation in the basal ganglia. Identification of this gene mutation provides a suggestive therapy of Fe chelation with desferrioxamines. Accumulation of the pigment neuromelanin is known to occur in the brain with normal aging, and it is particularly abundant in the substantia

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nigra. Initially termed neuromelanin because of its similarity in appearance to cutaneous melanin, neuromelanin has recently been established as a true melanin by electron paramagnetic resonance and metal analysis studies (Enochs et al., 1993; Zecca et al., 1994). Neuromelanin can interact with several heavy metals, but binds Fe particularly strongly. Neuromelanin has been implicated in the development of PD, as an increased rate of degeneration of highly pigmented neurons has been observed in the substantia nigra. The concentration of Fe in the substantia nigra in PD increases by 30% to 35%. This accumulation seems to occur within neuromelanin granules, as the concentration of Fe in these granules is higher in PD patients than in normal subjects (Sofic et al., 1991; Good et al., 1992). In normal subjects, neuromelanin may play a protective role by sequestering redox-active Fe atoms, thus preventing oxidative stress and neuronal damage. When free Fe increases such that neuromelanin is saturated, however, neuromelanin may become cytotoxic, thereby catalyzing the production of free radicals. Hydrogen peroxide could potentially be produced in excess in such a situation and, because hydrogen peroxide can degrade neuromelanin, neuromelanin could be lost altogether, releasing more Fe and accelerating neuronal death (Zareba et al., 1995). As more is discovered about each of the Fe transport molecules and mechanisms of Fe storage, it is likely that their role in neurodegenerative diseases will become better understood. Two new animal models, the Belgrade rat in which a defect in the DMT protein is associated with decreased brain Fe acquisition (Burdo et al., 2001; Zywicke et al., 2002) and a Ft knockdown mouse that has normal Fe concentrations but a decrease in Ft concentration (Thompson et al., 2002), have considerable promise in elucidating the contribution of Fe mismanagement to neurodegenerative processes.

3. IRON AND INFLAMMATION The effect of inflammation on iron metabolism is widely accepted, especially in the example of anemia of chronic disease, but the processes underlying the relationship are poorly understood and represent an area in need of further investigation. One proposed mechanism of the role of iron in system-wide inflammation suggests that the TfR located on monocytes facilitates iron entry into the cell in early inflammation, while H ferritin-specific

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binding sites have a prolonged iron sequestration effect in macrophages (Scaccabarozzi, 2000). Inflammation can cause alterations in the bloodbrain barrier (BBB) that could result in increased iron influx. The mechanisms governing iron and inflammation in the CNS are not understood. It is known that inflammation increases serum ferritin, and that CSF ferritin is elevated in Multiple Sclerosis (MS) patients. Such observations suggest that CSF ferritin could be a marker of CNS inflammation. At the cellular level, cytokines secreted during inflammation may modify IRP function, altering cellular iron homeostasis and possibly catalyzing further iron accumulation. Inflammation also appears central to plaque formation in AD. AD is characterized by the formation of plaques throughout the brain. The principle component of these plaques is amyloid-beta (AfS), a protein derived by cleavage of amyloid-beta precursor protein (ApPP). ApPP is a transmembrane glycoprotein expressed in several types of mammalian cells that, when cleaved according to its major pathway, does not produce A(3. Evidence suggests that Fe availability, which itself appears to be affected by inflammation, may affect processing of ApJPP, as A(3PP mRNA contains a putative IRE based on sequence homology (Bodovitz et al., 1995). Neuritic plaques in the AD hippocampus demonstrate strong immunoreactivity for Ft, with the Ft accumulation being associated largely with reactive microglia (Grundke-Iqbal et al., 1990; Connor et al., 1992a, 1992b). The increased expression of IRP2, in association with neurofibrillary tangles, senile plaque neuritis, and neuropil threads, supports the role of altered Fe metabolism in the pathogenesis of AD (Smith et al, 1998). In addition, IRP-IRE interaction is altered in some AD brains. In the latter case, IRP is more difficult to dissociate from the IRE in AD brain tissue. The result of this abnormally tight association would be increased Tf receptor expression, decreased Ft expression, and increased cellular Fe accumulation. All of these are consistent observations in AD brain tissue (Pinero and Connor, 2000). Further related evidence for the role of Fe in AD is provided by magnetic resonance imaging of AD brains, which demonstrate increased Fe levels in the basal ganglia (Bartzokis et al., 2000). Alzheimer's plaques also contain oxidized and nitrated proteins of neuroinflammation, as well as a surrounding layer of activated microglia and astrocytes. Inflammatory response markers, including cytokines, acute phase reactants, and proteases, are also present in the AD brain. In addition, epidemiologic studies have demonstrated that anti-inflammatory

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agents and corticosteroids are associated with slowing the progression or delaying the onset of AD (Markesbery and Carney, 1999). Microglia cells, which serve as immunological scavengers in the brain, may be central to the inflammatory response in AD. Activated microglia cells are significantly increased in the AD brain, and are capable of releasing several interleukins (IL), including IL-1, IL-6, and the widely implicated tumor necrosis factor alpha (TNF-a) (Carpenter et al., 1993). Another related mechanism of damage in AD focuses on the ability of IL-1 to upregulate the expression of A|3PP. Such an upregulation could increase A@ and, in turn, cause hydrogen peroxide accumulation, thereby resulting in an IL-1-induced self-propagation of free radicals and neuron degeneration secondary to inflammation (Goldgaber et al., 1989). Recently, two studies have considered the possibility that carrying a mutation in the protein that causes hemochromatosis, an Fe overload disease, may place individuals at greater risk for AD or at least influence the age of onset of AD (Moalem et al., 2000; Sampietro et al., 2001). Although the brain has historically been considered protected from Fe accumulation in hemochromatosis, this theory has not been systematically studied (Sheldon, 1935) and the hemochromatosis (Hfe) protein that is associated with hemochromatosis is expressed on the brain microvasculature (Connor et al., 2001). Thus, there is reason to suspect that the presence of the Hfe mutation, the most common genetic mutation in Caucasians, could influence Fe accumulation in the brain (Connor et al., 2001).

4. BRAIN IRON DEFICIENCY Many of the neurodegenerative diseases, including AD, PD, and HD, share Fe accumulation as a common pathology. The converse situation of Fe deficiency can also have dire consequences. Systemic Fe deficiency adversely affects over half a billion people worldwide, with consequences ranging from anemia to cognitive deficits (Andrews, 1999). Fe is especially crucial during development, with the effects of early dietary Fe deficiency on the brain being largely irreversible. Given that Fe is required for proper myelination of the spinal cord and white matter of cerebellar folds, and is also a co-factor for enzymes in neurotransmitter synthesis (Larkin and Rao, 1990), it is not surprising that early Fe deficiency can have longterm effects. The oligodendrocyte is the predominant Fe-containing cell in

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the brain and is also the cell responsible for myelination (Hill, 1988; Connor and Menzies, 1996). In Fe deficiency, oligodendrocytes appear immature (Erikson et al., 1997). Disruption of oligodendrocyte maturation (as caused by some gene mutations) results in Fe accumulation that is only 50% of normal levels (Connor and Menzies, 1990). Restless legs syndrome (RLS) is an example of a disease of Fe deficiency. It is characterized by an irresistible urge to move the legs and, occasionally, the arms (Earley et al., 2000a). Symptoms are worst at night and when the sufferer is at rest, with the sensations being lessened by voluntary movement of the extremity. The majority of RLS sufferers demonstrate periodic limb movements of sleep and, consequently, suffer from sleep deprivation. A magnetic resonance imaging analysis has suggested that brain Fe levels in the substantia nigra are below normal in individuals with RLS (Allen et al., 2001). Both the substantia nigra and the putamen showed Fe losses in proportion to RLS severity. Cerebrospinal fluid levels of Ft, Fe, and Tf further support the theory that brain Fe is deficient in RLS. A 65% decrease in cerebrospinal fluid ferritin, as well as a threefold increase in cerebrospinal fluid Tf, was reported in RLS patients when compared to controls, despite normal serum levels of Ft and Tf in both populations (Earley et al., 2000b). A recent histopathological analysis also provides support that the RLS brain is Fe-deficient (Connor et al, under review). Finally, pregnancy, as an example of a secondary cause of RLS, supports the causative role of Fe deficiency in RLS, as there is an increased incidence of RLS in pregnancy attributable to Fe deficiency (Ekbom, 1960).

5. OXIDATIVE STRESS AND NEURODEGENERATION Beyond misregulation of Fe trafficking as a primary cause of neurodegeneration, consideration must also be given to defects in antioxidant defense mechanisms. The etiology of neurodegenerative diseases remains elusive, yet the body of evidence supporting the crucial role of oxidative damage is increasingly convincing. As the knowledge of the various mechanisms of oxidative stress grows, so too does the list of diseases in which oxidative stress has implications. Oxidative stress has gained recognition as a powerful mechanism of both primary and secondary pathology in a growing list of neurodegenerative diseases.

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Oxidative stress refers to the damage that can ensue in the presence of excess free radical production, either endogenous or exogenous, or from a reduced capacity to neutralize free radicals. A free radical, by definition, can be any independent molecule containing an unpaired electron. This unpaired electron makes such molecules highly reactive with macromolecular structures, potentiating tissue injury and homeostatic disruption. The most important mechanism of radical production in vivo is likely the decomposition of superoxide and hydrogen peroxide as catalyzed by transition metals (Young and Woodside, 2001). Normal cellular function involves the production of free radicals, yet these free radicals represent a precarious balance between health and disease. Any failure in the decomposition or scavenging of these radicals can result in a wide array of consequences, with neurodegenerative diseases being perhaps the most devastating of these. HD is a neurodegenerative disorder related to defective free radical detoxification. It is caused by a trinucleotide (CAG) repeat expansion, and is characterized by degeneration of the striatum and disturbances in motor and cognitive functions (Huntington's Disease Collaborative Research Group, 1993). While the generation of a toxic N-terminus fragment of unknown function known as huntingtin is likely the primary mechanism of pathogenesis, striatal Fe accumulation has been noted in presymptomatic Huntington's sufferers. It is thought that generation of free radicals, via this mechanism, plays a role in the neurodegeneration (Dexter et al., 1991). Further evidence supporting the crucial role of oxidative damage in neurodegeneration is demonstrated by both AD and PD. Alzheimer brains have an increase in Fe without an accompanying increase in Ft in certain regions of the brain, including the superior temporal gyrus and the frontal cortex. Such an abnormal Fe accumulation could put cells at an increased risk for oxidative stress; perhaps not coincidentally, these areas of the brain often demonstrate severe histopathology in AD. A recently developed animal model, in which the Ft protein is only 20% of normal despite normal Fe concentrations in the brain, is likely to provide insight into the contribution of Fe and oxidative stress to the neurodegeneration seen in AD brains (Thompson et al., 2002). Similarly, in PD, the primary site of neurodegeneration and the death of dopaminergic neurons in the substantia nigra pars compacta, a site that also often demonstrates increased Fe in individuals with severe PD. As in AD, Ft expression is also decreased in

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PD. Of the surviving neurons in PD, some may contain Lewy bodies, which are intracytoplasmic inclusions of neurofilaments, ubiquitin, and alpha-synuclein. It has been shown that alpha-synuclein aggregation can be induced in vitro by Fe-related oxidative stress, providing further evidence for the linkage between impaired Fe metabolism and pathologic intracellular aggregates in PD (see Chapter 14). ALS is a type of motor neuron disease characterized by progressive muscle weakness and wasting. Its prevalence is about five in 100,000, with approximately 10% of cases being familial autosomal dominant ALS. Mutations in the copper-zinc SOD1 gene are responsible for some forms of familial ALS, with greater than 50 mutations having been described in the SOD1 gene on chromosome 21. SOD1 normally converts superoxide anion to hydrogen peroxide, but can also form toxic hydroxyl radicals. The SOD1 mutations support an excitotoxicity and free radical damage basis of disease in familial ALS. The presence of Fe in spinal motor neurons may make them especially susceptible to ALS-type degeneration by facilitating the generation of free radicals (Kasarskis et al., 1995). Familial ALS represents only a fraction of all ALS cases, however, and a causal relationship for sporadic ALS is less clear.

5.1. Mitochondrial Dysfunction In considering Fe metabolism and oxidative stress in the context of neurodegeneration, the intermediary role of mitochondria is a crucial consideration. Mitochondria are the major intracellular source of free radicals, and thus any damage to mitochondrial deoxyribonucleic acid (DNA) or to nuclear DNA coding for mitochondrial proteins often has neurodegenerative implications. The primary role of mitochondria is to provide adenosine triphosphate (ATP) by aerobic metabolism. Thus, these organelles are found in particularly high concentrations in tissues with high aerobic activity, including the skeletal and cardiac muscles and the brain. In addition to ATP production, mitochondria play a key role in the regulation of apoptosis. Mitochondria have their own circular, doublestranded DNA, with a given mammalian mitochondria containing between two to 10 molecules of mitochondrial DNA (mtDNA). Of this DNA, 13 proteins form components of the respiratory chain and oxidative phosphorylation system (OXPHOS), with the remaining 70 proteins being

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supplied by nuclear genes. Neurodegenerative disorders involving mitochondria can accordingly be divided into those caused by OXPHOS abnormalities of either mtDNA or nuclear proteins (pure myopathies, complex I and II deficiencies), those resulting in OXPHOS abnormalities caused by nuclear mutations' coding for nonmitochondrial proteins (HD), and those caused by nuclear genes encoding mitochondrial non-OXPHOS proteins (Wilson's disease, Freidreich's ataxia (FA), and hereditary spastic paraplegia) (Orth and Schapira, 2001). Still, other diseases have unassigned mitochondrial involvement, including PD, AD, and ALS, as discussed in the previous section. FA is a dramatic example of the consequence of oxidative stress in the context of mitochondrial dysfunction. It is an autosomal recessive disorder with a prevalence of one in 30,000 live births. It is characterized by progressive ataxia, neuropathy, and cardiomyopathy resulting from the loss of the mitochondrial protein frataxin with resultant mitochondrial Fe overload. An expanded GAA trinucleotide repeat on chromosome 9 is responsible for the decreased frataxin levels (Campuzano et al., 1996). Neurons and cardiac muscle are affected to the greatest degree, a finding that correlates with the strong dependence of these tissues on a continuous demand for Fe uptake and a high level of mitochondrial energy production. Mouse and yeast models of FA demonstrate decreased aconitase activity, suggesting a role for frataxin in mitochondrial-cytosolic Fe cycling. Antioxidant co-enzyme Q and free radical scavenger idebenone represent potential cardiac therapeutics for FA patients (Rustin et al., 1999). In a cellular model to determine the intracellular events associated with oxidative stress and Fe, mitochondrial membrane potential and ATP production were both decreased when Fe-loaded astrocytes were exposed to a pro-oxidant, and were conserved in the presence of an Fe chelators (Robb et al., 1999).

6. POTENTIAL THERAPEUTICS There remain a great number of unanswered questions regarding the mechanisms of neurodegenerative diseases and, accordingly, there are few truly effective treatments for these diseases, but many potential therapeutic targets. Little is known about the impact of dietary antioxidants on the development and progression of neurodegenerative diseases, and past studies have been insufficient in design and methodology. The natural

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antioxidants, including vitamin E, carotenoids, and flavonoids, do not readily cross the blood-brain barrier in adults. Thus, other antioxidants, such as spin traps and low molecular mass oxygen scavengers, are being investigated (Halliwell, 2001). Another possibility to be investigated is that some drugs already in therapeutic use, such as selegiline, apomorphine, and nitecapone, may owe some of their effects to antioxidant action. Current treatment of Wilson's disease represents a prototype of neuroprotective therapy. It is a rare autosomal recessive disturbance of copper incorporation and resultant accumulation of copper in the brain, liver, kidney, and eyes. Decoppering therapy in such patients can slow or reverse the neurologic deterioration associated with copper accumulation. Such success suggests the importance of developing protective and preventive therapies for other neurodegenerative diseases associated with elemental Fe accumulation, including AD, PD, and HD. One obstacle to overcome involves a primary difference between copper and Fe excretion: there is an active, regulated mechanism of excretion for excess copper, but no such mechanism has been identified for Fe (Andrews, 2002). The delivery of nonlipophilic compounds to the brain is limited by the blood-brain barrier. The TfR itself possesses a potential route of therapy in neurodegenerative and other diseases, as antibodies that bind the TfR have been shown to selectively target blood-brain barrier endothelium. Specifically, the OX26 antibody against rat TfR has shown potential for brain drug and gene delivery when conjugated to immunoliposomes (Shi and Pardridge, 2000). In addition, conjugates between OX26 and a variety of therapeutic agents, including neuropeptides, polyamide nucleic acids, and nerve growth factor, have shown markedly increased delivery to the brain compared to intravenous administration alone (Kordower etal., 1994; Park et al., 1998). To significantly improve the clinical management of neurodegenerative diseases, genetic risk, susceptibility factors, and prodromal symptoms must be better characterized in order that preventive strategies can be targeted toward healthy subjects to postpone illness onset. In addition, it is plausible that effective treatment of many neurodegenerative diseases would involve a bimodal approach, with both a reduction in brain Fe levels and inhibition of free radical formation. Such a method could involve, for example, combination therapy with an antioxidant and an Fe chelator. Both the antioxidants and the chelators must have high penetrability of the blood-brain barrier.

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CHAPTER

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Iron, Neuromelanin, and ot-Synuclein in Neuropathogenesis of Parkinson's Disease Kay L Double, Kurt Jellinger, Luigi Zecca, Moussa BH Youdim, Peter Riederer, Manfred Gerlach

ABSTRACT Parkinson's disease is a common neurodegenerative disorder characterized clinically by motor dysfunction. The primary pathological changes in the Parkinsonian brain are the degeneration of pigmented dopaminergic neurons of the substantia nigra and the development of pathological inclusions called Lewy bodies. Progressive cell loss in Parkinson's disease is suggested to result from self-sustaining mechanisms related to oxidative mechanisms and mitochondrial dysfunction. A common factor linking oxidative damage, mitochondrial dysfunction, and the development of Lewy bodies in Parkinson's disease is the presence of a significant and pathological increase in the amount of iron in the degenerating substantia nigra. The role of iron in biochemical pathways proposed to mediate these mechanisms and their association with the etiology of Parkinson's disease is discussed. Keywords: Iron; Parkinson's disease; neuromelanin; alpha-synuclein; neurodegeneration; oxidative stress.

1. INTRODUCTION In 1817, James Parkinson (Parkinson, 1918) first described the disease which came to be known as Parkinson's disease (PD). The most common form of this disease is the idiopathic form, which is clinically diagnosed 343

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as distinct from the rarely encountered genetic forms of Parkinsonian syndromes accompanied by multisystem degeneration on the basis of its clinical presentation and progression. The primary pathological change in the Parkinsonian brain is the progressive degeneration of the neuromelanin-containing neurons of the substantia nigra pars compacta in the ventral midbrain (Jellinger, 1989). The loss of these neurons, and the consequent loss of dopamine in the striatum (putamen and caudate nucleus), disrupts the normal control of motor activity (Gerlach and Riederer, 1993). The pathological changes characterizing this disease are well documented, but the cellular mechanisms responsible for the progressive death of these neurons have yet to be established. The greatest risk factor for PD is age (Le Couteur et al., 2002). The reason for this association is not well understood, although a range of subtle changes in cellular systems are common to both normal aging and neurodegenerative diseases. As for other neurodegenerative conditions, it is thought that multiple triggering events may initiate degeneration in PD. The disease trigger, or triggers, in any one individual may combine a variety of genetic and environmental influences. The initiation of neurodegeneration may occur many years prior to the development of symptomology (Le Couteur et al., 2002). Once initiated, however, the evidence suggests that a small number of self-sustaining mechanisms result in ongoing cell loss. Two primary, and interrelated, mechanisms implicated in the etiology of PD are oxidative pathways and mitochondrial dysfunction. Increments in indices of oxidative damage to proteins, lipids, and deoxyribonucleic acid (DNA) and correlated changes in the endogenous antioxidant systems are associated with cell damage in the Parkinsonian brain (reviewed in Double et al., 2000). Interestingly, age-related changes include alteration that favor increases in oxidative damage, such as pro-oxidative changes in mitochondrial pathways, decreases in antioxidant protective systems such as the glutathione system, and changes in cytoplasmic calcium concentrations and membrane potential (Nicholls, 2002). Changes in mitochondrial function, in particular inhibition of complex I activity, have also been proposed to be causally related to cell death in PD (Jenner and Olanow, 1998). Complex I activity is readily restricted by oxidative damage, and the resulting changes in respiratory function further stimulate oxidative mechanisms (Nicholls, 2002). Oxidative mechanisms are also believed to underlie the

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development of Lewy bodies, pathological inclusions characterizing the Parkinsonian substantia nigra. A common factor linking oxidative damage, mitochondrial dysfunction, and the development of Lewy bodies is the presence of a significant and pathological increase in the amount of iron (Fe) in the degenerating substantia nigra in PD (Fig. 1). This article briefly reviews the role of Fe in the biochemical pathways proposed to mediate these mechanisms and their association with the etiology of PD.

2. REGIONAL AND SELECTIVE INCREASE IN BRAIN IRON IN PARKINSON'S DISEASE Fe is unevenly distributed within the brain, with the highest levels found in the basal ganglia (substantia nigra, putamen, caudate nucleus, and globus pallidus), red nucleus, and dentate nucleus (Sourander and Hallgren, 1958; Youdim, 1985; Riederer et al., 1989). Brain Fe is deposited early in life. Subsequent entry of Fe into the mature brain is tightly regulated and, unlike other tissues, the turnover of brain Fe is extremely slow and serum Fe has little access to this organ (Youdim, 1985). Once inside the brain, Fe is highly sequestered within organic storage forms, such as ferritin (Octave et al., 1983), with relatively little in a free and reactive form. Despite the highly developed regulatory systems for the control of Fe in the mature brain, increases in brain Fe are associated with a variety of neurodegenerative diseases and with neurotoxin-induced neurodegeneration, suggesting an association with the neurodegenerative process (reviewed in Gerlach et al., 1994). Increased regional brain Fe has been identified in Parkinsonian syndromes, such as PD, progressive supranuclear palsy, and multisystem atrophy, in trinucleotide repeat disorders, such as Huntington's disease and dentatorubral pallidoluysian atrophy, and in dementia disorders, such as Alzheimer's disease (AD) and dementia with Lewy bodies. In all of these disorders, the striking feature of this change is the close association between the increased tissue Fe and the degenerating brain region. In movement disorders, Fe levels are increased in the basal ganglia, the brain regions controling movement (Table 1), while in AD increased Fe is associated with the pathological hallmarks of this disease in the vulnerable cortical regions (Connor et al., 1992; Smith et al., 1997). Furthermore, the number of regions affected also parallel the pattern of degeneration seen in each disease. Thus, in PD significantly

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Changes to blood brain barrier

Disease triggers (unknown, but probably includes both genetic and environmental factors) Dysregulation of Fe buffering systems (ferritin, neuromelanin, and /Lewy bodies)

Changes in buffering/ antioxidant systems (TSOD/MAO-B activity \> glutathione peroxidase activity 4 catalase activity iGSH)

Increased nigral Fe (source/timepoint?)

Oxidative pathways Toxic metabolic products

(1) Fe 2+ +H 2 0 2 ->'OH+T)H+H 2 0+Fe 3+ (Fenton reaction) (2) 20 2 -»20 2 ->H 2 0 2 -?°OH+~OH+2H 2 0 (Mitochondrial respiration)

Neuronal death in substantia nigra

Parkinsons's disease

Fig. 1. Interacting molecular mechanisms underlying neurodegeneration in Parkinson's disease.

increased Fe levels are only found in the degenerating substantia nigra (Berg et al., 2001), while multisystem atrophy and progressive supranuclear palsy are characterized by increased Fe not only in the substantia nigra, but also in the degenerating caudate nucleus and putamen (Dexter et al., 1991, 1993). Hence, the topographical distribution of the increased Fe suggests a direct relationship with the disease state.

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Total Fe content in most brain areas do not differ between control and Parkinsonian brains (Riederer et al., 1989; Sofic et al., 1988; Dexter et al., 1989). However, an increase in total Fe levels of approximately 77% was measured in the substantia nigra of patients with PD compared to control subjects (Sofic et al., 1988). Furthermore, this increase was shown to be confined to the substantia nigra pars compacta, the tissue region containing the vulnerable dopaminergic neurons, but does not occur in the pars reticulata (Sofic et al., 1991). The presence of increased nigral Fe levels in PD has been confirmed by many research groups (Gerlach et al., 1994; Jellinger, 1999) using different quantitative methods (such as metal ion detection by inductively coupled plasma mass spectroscopy, X-ray microanalysis) and histochemical techniques. Magnetic resonance imaging studies using specialized sequences are also often quoted as supporting the accumulation of Fe in the nigrostriatal system in patients with PD (Gorell et al., 1995; Bartzokis et al., 2000; Graham et al, 2000). The topographic association between hypointensity on T2-weighted magnetic resonance images (bulk water proton spin-spin relaxation time) and localization of Fe has led many investigators to conclude that Fe is the factor primarily responsible for the reduced signal intensity observed on such images in PD.

3. POTENTIAL SOURCE OF INCREASED IRON The source of the localized increase Fe in PD is unknown, but several possibilities present themselves. First, the increase might result from increased entry of peripheral Fe into the substantia nigra via an alteration in the blood-brain barrier. In the adult brain, the entry of Fe into brain is highly regulated. However, circumstances such as local inflammation may disrupt this control and allow the access of Fe. Alternatively, rather than being the result of some pathological process increased Fe might also enter the substantia nigra via normal Fe regulatory systems, such as an increase in Fe transport sites. Studies have shown that the major Fe transport site, transferrin, is not upregulated in the substantia nigra in the PD brain (Morris et al., 1994; Faucheux et al., 1995a), although specific polymorphisms in the transferrin gene have recently been associated with PD (Borie et al., 2002). Another Fe-binding protein, lactoferrin, however, is reported to be upregulated in surviving neurons in the Parkinsonian

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substantia nigra (Leveugle et al., 1996) and increased numbers of receptors for this protein are present on neurons in the Parkinsonian nigra (Faucheux et al., 1995b). The translation of many proteins involved in Fe regulation is controled by two cytoplasmic proteins called iron regulatory protein 1 (IRP1) and iron regulatory protein 2 (IRP2). These proteins bind to stem loop structures called Fe-responsive elements of the protein's mRNA. If Fe is depleted, IRP binding results in decreased synthesis of proteins involved in Fe storage and mitochondrial metabolism, such as ferritin and mitochondrial aconitase, and increased synthesis of proteins, such as the transferrin receptor, involved in Fe uptake. While IRPmediated control systems are currently an area of intensive research and changes in these systems have been associated with Fe accumulation in animal models (LaVaute et al, 2001), recent data suggest that these systems remain unchanged in the substantia nigra in PD (Faucheux et al., 2002). A body of evidence indicates that disturbances in Fe metabolism occur at multiple levels PD (reviewed in Berg et al., 2001) and may reflect genetic differences in Fe regulatory systems in these individuals (Borie et al., 2002). Nevertheless, to date no convincing explanation for the selective increase in Fe in the Parkinsonian substantia nigra is available. Furthermore, while no clear environmental cause of PD has been recognized, epidemiological studies have implicated several environmental factors associated with an increased risk of PD. Of these, the association between long-term exposure to heavy metals, such Fe, copper, and manganese, and an increased risk of PD is one of the best described, suggesting that exposure to external sources of metals may be involved in the etiology of PD (reviewed in Gorell et al., 1999). Alternatively, rather than entering from outside the brain, increased levels of nigral Fe might be derived from within the brain itself. The substantia nigra is directly connected to the globus pallidus, one of the most Fe-rich regions of the brain, via afferent 7-aminobutyric acid pathways systems. To date, however, no know mechanisms exist to explain the movement of Fe from one brain region to another. A third possibility is that the measured increases in Fe reflect not an increase in total Fe content, but rather a redistribution of Fe from other cellular sources. One possible source of the measured increased in Fe is an increase in nigral glial cells. Glial cells, predominantly oligodendroglia, but also microglia and astrocytes, are the major source of the Fe storage protein

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ferritin. An increase in the number of Fe-positive glial cells is associated with nigral degeneration in PD (Jellinger et al., 1990, 1992). Despite increased numbers of ferritin-positive glial cells, total nigral ferritin concentrations appear to be unchanged in PD (Dexter et al., 1990; Mann et al., 1994; Connor et al., 1995). More recently, it has been demonstrated that expression of ferritin mRNA is increased in glial cells adjacent to neurons in the Parkinsonian substantia nigra. Despite increased nigral Fe content, no increased expression of ferritin mRNA nor changes in binding activity of associated IRPs were detected within the Parkinsonian dopaminergic neurons (Faucheux et al., 2002). The co-localization of Fe and ferritin in the Parkinsonian substantia nigra is supported by histochemical studies. Perl's staining demonstrated increased Fe(III) in Parkinsonian substantia nigra pars compacta localized in macrophages, astrocytes, and reactive microglia (Perl and Good, 1992), as well as in the cytoplasm of occasional nonpigmented neurons. Histochemically, Fe could not be demonstrated within the neuromelanin-containing neurons of the substantia nigra pars compacta, intracytoplasmic melanin, or extracellular melanin granules in the neuropil (Perl and Good, 1992), although Lewy bodies contain Fe (Hirsch et al., 1991; Perl and Good, 1992; Castellani et al., 2000; Jellinger et al., 1990). Ferritin-bound Fe can be released by a variety of physiological (Biemond et al., 1984; Monteiro and Winterbourn, 1989; Rief and Simmons, 1990; Yoshida et al., 1995; Double et al., 1997) and nonphysiological stimuli (Double et al., 1997), resulting in oxidative, mediated membrane damage (Double et al., 1997). Moreover, a dysregulation of ferritin homeostatic systems appears to occur in PD (Connor et al., 1995). A neurodegenerative syndrome exhibiting neuropathological and symptomatic similarities with PD, associated with a mutation in the ferritin gene, has also been described (Curtis et al., 2001). The question of the temporal association between the reported increase in Fe and neurodegeneration in PD is also nuclear. Early reports suggest that the increase in Fe might be a late or secondary event in the disease process. In 1994, Dexter et al. reported unchanged nigral Fe levels in cases of so-called incidental Lewy body disease characterized by mild neuronal loss and Lewy bodies in nigral neurons. The authors suggested that these pathological findings may represent an early preclinical state of PD. They concluded that increased Fe only occurs in advanced stages of neurodegeneration. This is supported by another report that nigral Fe concentrations are

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unchanged in "mild" cases PD, assessed semi-quantitatively as a mild nigral neuronal loss (Riederer et al., 1989). In contrast, more recent work using transcranial ultrasound indicates that increased Fe can be demonstrated in the substantia nigra of patients with PD even prior to the onset of clinical symptomology (Berg et al., 2002), suggesting the increase in Fe might occur earlier than previously thought. While both the source and timing of the apparent increase in Fe substantia nigra is still controversial, the presence of increased quantities of this metal in this brain region appears to be involved in the two primary cellular pathways associated with neurodegenerative changes in PD: oxidative stress and formation of Lewy bodies.

4. ROLE OF IRON IN OXIDATIVE PATHWAYS IN PARKINSON'S DISEASE The "oxidative stress" hypothesis of PD specifically proposes an imbalance between the formation of cellular oxidants and the antioxidative processes. Decreased activity of the endogenous antioxidant molecules, glutathione peroxidase and catalase, and reduced concentrations of reduced glutathione (GSH) are reported in the Parkinsonian substantia nigra. The significant and apparently early loss of systems designed to protect the brain, together with site-specific increases of superoxide dismutase and monoamine oxidase activity, may play an important role in priming the substantia nigra for oxidative damage in PD (reviewed in Beard et al., 1994; Gotz et al., 1994; Gerlach et al., 1995a; Bharath et al., 2002). Oxidative stress, resulting from increased formation of hydrogen peroxide and oxygen-derived free radicals, can damage biological molecules and initiate a cascade of events, including dysfunction of mitochondrial respiration, excitotoxicity, and a fatal rise in cytosolic calcium (Gerlach et al., 1995a; Nicholls, 2002). Hydrogen peroxide is produced in human tissues by several enzymes, such as superoxide dismutase, 1-amino acid oxidase, glycollate oxidase, xanthine oxidase, and monoamine oxidase. In dopaminergic nerve cells, it is mainly generated by monoamine oxidase via deamination of dopamine, and nonenzymatically by autoxidation of dopamine. Hydrogen peroxide is relatively inert and is not toxic to cells. The interaction of hydrogen peroxide with the reduced forms of transitional metal ions, such as Fe(II) or copper(I), however, decomposes

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hydrogen peroxide to the highly reactive hydroxyl free radical via the Fenton reaction (Eq. [1] in Fig. 1). In addition, hydroxyl radicals are produced in the mitochondria of nerve cells during oxidative phosphorylation (Eq. [2] in Fig. 1). Hydroxyl radicals have a strong reactivity with almost every molecular species found in living cells. Such reactions include breakage of single- and double-stranded DNA, chemical alterations of the deoxyribose purine and pyrimidine bases, membrane lipids, and carbohydrates, leading to a cascade of events with subsequent damage to the mitochondial electron transport system, disturbance of intracellular calcium homeostasis, induction of proteolysis by proteases, increased membrane lipid peroxidation, release of excitotoxic amino acids (glutamate and aspartate), and finally cell death (reviewed in Gotz et al., 1994; Gerlach et al., 1995a). While measurements of oxidative load within the degenerating human Parkinsonian substantia nigra are impractical, a considerable body of indirect evidence from experimental models and post-mortem studies support the hypothesis that oxidative stress contributes to the loss of dopaminergic neurons in patients with PD (reviewed in Beard et al., 1994; Gotz et al., 1994; Gerlach et al., 1995a). Not only is total nigral Fe content increased in PD, but the ratio of Fe(II) to Fe(III) shifts from two to one in control brains to one to two in the brains of PD patients (Sofic et al., 1988). This indicates an increased rate of synthesis of hydroxyl radicals. Post-mortem studies also report increased basal levels of thiobarbituric acid-reactive substances in the substantia nigra of PD patients (a measure of secondary products of lipid peroxidation), coupled with a decrease in the levels of polyunsaturated fatty acids, the substrates for lipid peroxidation (reviewed in Beard et al., 1994; Gotz et al., 1994; Gerlach et al, 1995a). In addition, there may be DNA damage as indicated by raised 8-hydroxydeoxyguanosine (Sanchez-Ramos et al., 1994), a product of free radical attack on guanine in DNA. Further evidence for the occurrence of oxidative stress in PD comes from studies on experimental models of this disease. For example, the Fe chelator desferrioxamine (desferal) and vitamin E protect rats against the 6-hydroxydopamine (6-OHDA)-induced reduction in striatal dopamine content and decrease of dopamine-related spontaneous locomotor activity (Ben-Shachar et al., 1991a). These findings indicate the prevention of 6-OHDA-induced degeneration of nigrostriatal dopaminergic neurons. 6-OHDA is thought to induce nigrostriatal dopaminergic lesions via

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generation of hydrogen peroxide and hydroxyl radicals derived from it, presumably initiated by a transition metal such as Fe. In fact, it has been shown by magnetic resonance imaging (Hall et al., 1992) and neurochemical and histochemical studies (such as Oestreicher et al., 1994) that Fe is increased in the striatum of 6-OHDA-lesioned rats. Furthermore, it has been shown that 6-OHDA releases Fe from ferritin in vitro (Monteiro and Winterbourn, 1989). Finally, intranigral injections of Fe(III) produce neurotoxic effects similar to those observed with 6-OHDA (Ben-Shachar and Youdim, 1991).

5. ROLE OF IRON IN FORMATION OF LEWY BODIES One of the primary neuropathological criteria for a confirmed postmortem diagnosis of PD is the presence of cytosolic filamentous inclusions known as Lewy bodies and Lewy neurites in some surviving dopaminergic nigral neurons. The major fibrillar material of these inclusion bodies is a presynaptic protein of unknown function, a-synuclein (Spillantini et al., 1997, 1998). While no prominent genetic cause of sporadic or idiopathic PD has been identified, specific mutations in the a-Synuclein gene have been correlated to the onset of Parkinsonism in certain forms of autosomal dominant early onset PD in humans (Polymeropoulos et al., 1997; Kriiger et al., 1998) and animal models of the disorder (Masliah et al., 2000; Feany and Bender, 2000). a-synuclein naturally aggregates (Conway et al., 2000) and in PD, as well as in other disorders such as dementia with Lewy bodies, the protein aggregates into the filamentous structures present in Lewy bodies. The reason for this aggregation is unknown, but it has been shown that even small amounts of di- and tri-valent metals, including Fe, increase the rate of a-synuclein fibrillation (Uversky et al., 2001). An analysis of Lewy bodies in the Parkinsonian nigra demonstrated that these pathological inclusions contain redox-active Fe (Castellani et al., 2000), suggesting that this mechanism may also occur in vivo. Recently, it was shown that, in the presence of Fe, a-synuclein stimulated the production of hydrogen peroxide in vitro via Fenton chemistry (Turnbull et al., 2001), possibly via the binding of Fe(II) to the protein (Golts et al., 2002). This effect was not seen for P- and -y-synuclein, which are not associated with neurodegenerative disease (Turnbull et al., 2001). While the relationship between the aggregation

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state of the protein and free radical production is unclear, this may represent an Fe-mediated mechanism by which hydroxyl radicals may be produced inside the nigral neurons. Oxidative stress is reported to induce the aggregation of a-synuclein (Hashimoto et al., 1999), leading to fibril formation (Jellinger, 2002). Aggregated a-synuclein in diseased brains displays evidence of oxidative damage (Giasson et al., 2000), suggesting a mechanistic link between Fe, oxidative stress, protein aggregation, and cell death in PD and other synucleionopathies (Fig. 1). An unresolved question is whether the formation of Lewy bodies within a neuron exerts a protective or toxic influence on the cell (Goldberg and Lansbury, 2000). The concentration and aggregation of proteins have been demonstrated in model systems to be detrimental to the cell (for example, see Ostrerova-Golts et al., 2000; Feany and Bender, 2000). On the other hand, there appears to be no correlation between the density of Lewy body formation and cell loss (Kremer and Bots, 1993; Gomez-Isla et al., 1999; Gomez-Tortosa et al., 1999; Henderson et al., 2000), and the low number of cells containing Lewy bodies in any brain region (less than 5% of the total neuronal number) would not be expected to result in a significantly altered synaptic function. Indeed nucleolar size, an indicator of RNA synthesis, does not vary in substantia nigra cells containing Lewy bodies compared to those who do not (Gertz et al., 1994), suggesting that Lewy bodies do not disturb cell metabolism. Lewy bodies have also been proposed to represent a protective reflex within the cell. Fibrillar neuronal inclusions may sequester toxic species, diverting asynuclein from toxic assembly pathways (Goldberg and Lansbury, 2000), thereby protecting the cell (Saha et al., 2000). Nevertheless, it is clear that significant intracellular protein aggregation and Lewy body formation are pathological processes, reflecting changes in the cellular environment.

6. PUTATIVE ROLE OF NEUROMELANIN IN PARKINSON'S DISEASE Histochemical studies ascribe the localization of Fe in the substantia nigra primarily to the glial compartment and changes in glial-associated ferritin have been described in PD. Fe-mediated oxidative pathways are difficult to attribute primarily to glial-associated Fe, however, because highly active free radicals produced in glial cells are unlikely to diffuse across the

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extracellular space to neurons prior to being inactivated. A more plausible source of free radicals is, thus, likely to come from a physically less remote source, for example, free radicals produced by an intraneuronal source. While some Fe sequestration appears to occur in a-synuclein in Lewy bodies, the importance of this Fe per se for neurodegeneration is unknown. A more plausible source of intraneuronal Fe that may be involved in oxidative pathways is the neuromelanin, which characterizes the dopaminergic neurons of the human substantia nigra (reviewed in Gerlach et al., 1994, 1995a). Histological studies have demonstrated that neuromelanin occurs as granules, possibly inactivated lyosomes, within the neuronal perikaryon (Gerlach et al., 1995a). Neuromelanin appears to be an efficient binder of a variety of transition metals, of which its interaction with Fe is of particular physiological interest. Studies using synthetic dopamine-melanin have demonstrated that the binding of Fe to neuromelanin is pH- and concentration-dependent and appears to be relatively specific, as flunitrazepam did not bind to dopamine-melanin and spiperone demonstrated low affinity. Moreover, Fe(III) could only be displaced from dopamine-melanin by compounds with Fe-chelating capacity, such as the 21-aminosteroid U74500A (lazaroid) and desferoxamine, but not by dopamine, spiperone, MPTP, MPP + , and apomorphine (Ben-Shachar et al., 1991b). Also, a high affinity (KD = 13 nM) and low affinity binding site for Fe(III) (KD = 200 nM) were demonstrated on synthetic dopamine-melanin (Ben-Shachar et al., 1991b). These data, obtained from a melanin model, concurs with data from purified human neuromelanin isolated from the substantia nigra of control individuals, which exhibit Mossbauer spectra corresponding to high-spin Fe(III), but not Fe(II) (Gerlach et al., 1995b). The capacity of neuromelanin to bind Fe appears to be considerable, and is estimated to represent up to 20% of the total Fe content of the substantial nigra (estimated to be up to 12 |xg/mg) (Zecca et al., 1996, 2001; Shima et al., 1997). Energy-dispersive X-ray analysis in the scanning transmission electron microscopy mode also demonstrated weak, but significant, Fe peaks in intraneuronal neuromelanin granules of dopaminergic neurons in the substantia nigra pars compacta of Parkinsonian patients post-mortem. No such peaks were evident in neuromelanin granules from the brains of matched control substantia nigra (Jellinger et al., 1992). No peaks were seen in nonmelaninized cytoplasm or adjacent neuropil of nigral neurons

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in both Parkinsonian patients and controls, in Lewy bodies within these neurons, or in synthetic dopamine-melanin uncharged with Fe (Jellinger et al., 1992). Similar results were also reported by Good et al. (1992). In substantia nigra tissue, neuromelanin is only about 50% saturated with Fe (III), suggesting that it maintains an important residual chelating capacity which can protect against Fe toxicity (Zecca et al, 1996; Shima et al., 1997). It appears that Fe is bound to neuromelanin via catecholic groups. Electron paramagnetic resonance studies indicate that ferric Fe in the substantia nigra is bound to neuromelanin as a high spin complex with an octahedral configuration (Zecca and Swartz, 1993; Zecca et al., 1996; Shima et al., 1997). Mossbauer spectroscopy also demonstrates the chelation of ferric Fe by the neuromelanin polymer and suggests that the Fe sites are arranged in a ferritin-like ironoxyhydroxyde cluster form (Gerlach et al, 1995b; GalazkaFriedman et al., 1996; Lopiano et al., 2000; Zecca et al., 2001). X-ray absorption fine structure spectroscopy (Kropf et al., 1998) and infrared spectroscopy (Bridelli et al., 1999) studies confirm that Fe in neuromelanin is bound by oxygen-derived phenolic groups in a octahedral configuration. The data to date suggests that Fe bound to neuromelanin represents a significant pool of intraneuronal Fe inside the vulnerable dopaminergic neurons (Zecca et al., 2001). While the physiological role of neuromelanin is unclear, it is thought to participate in scavenging metal-induced free radicals within the healthy brain (Gerlach et al., 1994). Thus, neuromelanin would play a role analogous to that played by melanin in the skin, where epidermal melanin acts as a protection against ultraviolet light-induced damage to the skin, probably by involvement of hydroxyl radical-induced melanin degradation. Indeed, both native and synthetic dopamine-melanin have been shown to diminish basal lipid peroxidation in rat brain tissue, as suggested by lower levels of thiobarbituric acid reactive substances (Ben-Shachar et al., 1991b; Double et al., 1999). Under pathological conditions, however, such as increased availability of Fe(III) occurring in PD, neuromelanin is suggested to potentiate the formation of oxygen-derived radicals (Ben-Shachar and Youdim, 1990). The production of hydroxyl radicals in the presence of melanin is significantly greater when Fe(III) is predominant (Pilas et al., 1988; Zareba et al., 1995), and significantly enhanced cerebral membrane damage can be demonstrated in vitro in the presence of a neuromelanin Fe(III) complex (Double et al., 1999). Accordingly, neuromelanin appears to be able to potentiate or

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inhibit the formation of oxygen-derived free radicals, thereby playing a dichotomous role, depending on the cellular environment. The selective increase of Fe(III) in the substantia nigra pars compacta (Sofic et al., 1988, 1991) and the susceptibility of the melaninized dopaminergic neurons in the nigra to degeneration in PD (Hirsch et al., 1988) have led Youdim et al. (1989) to suggest that this disorder could be a progressive siderosis of the substantia nigra. The increased formation of a neuromelanin/Fe(III) complex (Jellinger et al., 1992) is consistent with the hypothesis that neuromelanin acts as a intraneuronal pool of Fe, which subsequently contributes to free radical-producing or other pathways. The absence of significant quantities of alternative Fe-binding molecules, such as ferritin, within pigmented neurons, together with a decreased antioxidant capacity in the Parkinsonian substantia nigra, suggests that an increase in the amount of Fe available to interact with neuromelanin may result in such interactions exerting a substantial influence upon oxidative-mediated pathways and, thus, cell survival. Thus, although Fe levels are increased in areas of pathological change in a variety of neurodegenerative disorders, the presence of neuromelanin in the human substantia nigra may contribute to the vulnerability of these neurons in the Parkinsonian brain. While the cause of the increased Fe concentration in the Parkinsonian substantia nigra is unclear, the interaction of this metal with cellular constituents, such as a-synuclein and neuromelanin, appears to be important for the development of the characteristic neuropathology characterizing the disease and, possibly, oxidative-mediated neurodegeneration. These pathways may not represent the initial trigger of the disease processs, as suggested by recent work investigating the time course of dopaminergic cell death and Fe accumulation in animals models of PD (He et al., 2003). They may, however, reflect as yet unidentified alterations in Fe homeostasis and represent secondary, but important, mechanisms involved in the progressive nature of the disease. Recent unpublished data from one of the current authors and colleagues demonstrated that the novel brain permeable Fe chelator, VK-28, protects against striatal dopamine depletions induced by 6-OHDA toxicity in the rat (Youdim, unpublished observations). These data suggest that the development of Fe-chelating substances suitable for use in the central nervous system may provide novel points for therapeutic intervention in PD and other neurodegenerative disorders associated with increases in central Fe levels.

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Iron and Epilepsy Wei-Yi Ong*, Benjamin Kian-Chung Ong, Akhlaq A Farooqui, Chuang-Chin Chiueh, James R Connor

ABSTRACT Injections of iron salts into the sensorimotor cortex, hippocampus, and amygdala of experimental animals results in chronic recurrent focal paroxysmal electroencephalographic discharges, behavioral convulsions, and electrical seizures. The induction of epilepsy may be related to generation of free radicals, lipid peroxidation of neuronal membranes, increased intracellular calcium concentrations through reverse action of sodium-calcium exchanger/reduced activity of plasma membrane or endoplasmic reticulum calcium ATPases, increased release of excitatory neurotransmitters, including aspartate and glutamate, and increased influx of ions through glutamate receptors. Some of the above effects of iron can be abrogated by inhibitors of phospholipase A2 (PLA2) indicating that the damaging effects of iron may be due to perturbation of the lipid environment essential to normal functioning of membrane proteins. Iron in hemoglobin, or by itself, is also likely to be the cause of human epilepsy, in instances where there is increased iron load in the brain. These include subarachnoid hemorrhage, intraparenchymal hemorrhages due to head injury and stroke, malaria, human immunodeficiency virus encephalitis, and possibly, neuroleptic drug use. A reduced level of haptoglobin, a hemoglobin-binding protein, has also been observed in select kindred relatives affected with familial idiopathic epilepsy. An accumulation of iron has been observed in the motor cortex with age, and it is possible that this might contribute to the increased incidence of epilepsy among the elderly. Iron accumulates with time in rat hippocampus after kainateinduced epilepsy. The accumulation occurs in oligodendrocytes, and is likely to be a reflection of the high levels of iron in the extracellular space. The accumulation of iron is correlated with increased expression of the divalent metal transporter-1 in astrocytes in the glial scar and increased expression of heme

"•"Corresponding author. 365

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Ong W-Yetal. oxygenase-1 in reactive astrocytes and microglia, as well as degenerating neurons at the edge of the scar. The increased divalent metal transporter-1 expression could lead to increased uptake of iron, followed by redistribution to the extracellular space. In this model, iron is the consequence of epilepsy, although it is possible that it can also be a cause of epilepsy. Further work is necessary to elucidate the effects of lipid peroxidation of the cellular membranes on function of membrane proteins and the role of phospholipases, including PLA2, in perturbing the lipid environment. The possible presence of iron in the human brain after epilepsy also needs to be elucidated. The causes of dysregulation of iron in the glial scar after neuronal injury need to be studied. In addition, possible beneficial effects of iron chelators, antioxidants that cross the blood-brain barrier, or neuroprotective gene induction on epilepsy, need to be evaluated. Keywords: Iron; epilepsy; free radicals; glutamate; GABA; phospholipase A2; kainate; excitotoxicity.

1. INTRODUCTION A seizure is a transient disturbance of the cerebral function caused by an abnormal, sudden, excessive, and disorderly discharge of cerebral neurons. Seizures can result from either primary central nervous system dysfunction or an underlying metabolic derangement or systemic disease (Simon et al., 1999). They can be classified into two types: partial (in which a focal or localized onset can be discerned) and generalized (in which the seizures appear to begin bilaterally). Partial seizures are classified as simple when consciousness is undisturbed, and complex when consciousness is impaired. Generalized seizures are of two types: convulsive and nonconvulsive. The common convulsive type is the tonic clonic (grand mal) seizure. The classic nonconvulsive, generalized seizure is the brief lapse of consciousness or absence (petit mal) (Adams et al., 1997). Epilepsy is a group of disorders in which seizures recur, usually spontaneously. Cerebral contusion, cortical laceration, intracerebral hematoma formation, and hemorrhagic cortical infarction cause extravasation of red blood cells, followed by hemolysis, decompartmentalization of iron (Fe), and increased incidence of epilepsy (Triggs and Willmore, 1984; Willmore et al., 1990). Fe is a key component of cytochromes a, b, and c cytochrome oxidase and the Fe sulfur complexes of the oxidative chain. It is, therefore,

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important for the production of ATP. Fe is also a co-factor for tyrosine hydroxylase and tryptophan hydroxylase, which are enzymes involved in the synthesis of neurotransmitters (Chen et al., 1995), ribonucleoside reductase, the rate-limiting enzyme of the first metabolic reaction committed to DNA synthesis, and succinate dehydrogenase and aconitase of the TCA cycle (reviewed in Connor et al., 2001). The highest levels of Fe are found in the motor system, and the globus pallidus, substantia nigra zona reticulata, red nucleus, and myelinated fibers of the putamen show the highest rate of staining reactivity. Fe is predominantly accumulated in glial cells (Connor et al., 1992; Morris et al, 1992). Fe can cross the blood-brain barrier in at least two ways. In the first, Fe transferrin complex from the blood is transported intact across the capillary wall by receptor-mediated transcytosis. In the second, Fe transport is the result of receptor-mediated endocytosis of Fe-transferrin from the blood by capillary endothelial cells. This is followed by release of Fe from transferrin within the cell, recycling of transferrin to the blood, and transport of Fe into the brain (reviewed in Moos and Morgan, 2000). Metal transporters, such as the divalent metal transporter 1 (DMT1) and metal transport protein 1 (MTP-1), also play a role in brain Fe transport. DMT1 has been localized to astrocytic processes and end feet around blood vessels in the rat and monkey cerebral cortex (Wang et al., 2001, 2002a), as well as glial cells and neurons in the striatum, cerebellum, and thalamus in the rat brain (Burdo et al., 2001). The presence of DMT1 in astrocytic end feet in contact with blood vessels suggests that DMT1 may be important in the uptake of ferrous Fe presented on the abluminal membrane of endothelial cells of brain capillaries. The importance of DMT1 in Fe transport into the brain is shown in Belgrade rats, in which a defect in DMT1 is associated with lower levels of Fe in the brain. MTP-1 expression is robust in pyramidal neurons of the cerebral cortex, but is not detected in vascular endothelial cells and ependymal cells (Burdo et al., 2001). An increase in Fe without proper sequestration could increase the vulnerability of cells to oxidative stress. An important form of antioxidant defense is the sequestration of Fe in organic storage forms, such as ferritin (Hallgren and Sourander, 1958; Octave et al, 1983). Ferritin is highly expressed in oligodendrocyte lineage cells, including oligodendrocytes

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and oligodendrocyte precursors cells. The latter have the ability to internalize ferritin by receptor-mediated endocytosis (Hulet et al., 2000). It has been suggested that ferritin may form part of the cellular antioxidative stress proteins (Lin and Girotti, 1997; Garner et al., 1998; Oberle et al., 1998). Intracellular Fe concentration regulates the gene expression of ferritin, transferrin receptor, and heme, thus controlling Fe metabolism in brain cells. Fe regulates these proteins post-transcriptionally, through the regulation of the binding of the iron regulatory protein (IRP) to iron response elements (IRE) in the mRNA of proteins (Rouault et al., 1992; Crichton and Ward, 1995). A concordant regulation of ferritin and transferrin receptors exists, such that a cell can obtain Fe when it is needed, and sequester Fe when it is in excess (Pinero et al., 2000). In the presence of Fe and copper, hydrogen peroxide (H 2 0 2 ) is converted into reactive hydroxyl radicals (OH*) via the Fenton reaction: Fe 2+ + H 2 0 2 -> intermediate complexes -> Fe(III) + OH* + OH". The rate of generation of free radicals by Fe can be accelerated by ascorbate since ascorbate promotes the redox cycling of Fe complexes (Rauhala et al., 1998). Ferrous citrate complexes can generate reactive radicals through either the Haber Weiss reaction or Fenton reaction (Mohanakumar et al., 1994, 1998; Rauhala et al., 1996). Brain tissue contains a large amount of polyunsaturated fatty acids, including arachidonic acid, which are sensitive to Fe-induced oxidative stress and readily undergo lipid peroxidation, a free radical-induced chain reaction: - C H 2 - + OH*-* -C*H- + H 2 0 -C*H- + 0 2 -» -COO*H-. Lipid peroxidation of neural membranes and subsequent decomposition of lipid peroxides lead to increased membrane fluidity, generation of reactive lipid products (such as malondialdehyde and 4-hydroxynonenal), disturbance of calcium homeostasis, and finally cell death (Youdim et al., 1989; Halliwell and Gutteridge, 1999).

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2. IRON-INDUCED EPILEPSY (Fig. 1) 2.1. Animal Model Studies Most studies on Fe and epilepsy have focused on examining whether Fe could be a cause of epilepsy. A single injection of 5 |JLL to 10 |JLL of ferrous or ferric chloride into rat or cat sensorimotor cortex resulted in chronic recurrent focal paroxysmal electroencephalographic discharges, as well as behavioral convulsions and electrical seizures. Fe-filled macrophages, ferruginated neurons, and astroglial cells surrounded the focus of epileptic discharge (Willmore et al., 1978a, 1978b). Intracortical implantation of blood and blood products, including whole blood, hemolyzed erythrocytes, methemoglobin, ferritin, ferrous chloride, and ferric chloride, were also observed to result in the development of focal paroxysmal discharges which became more prominent and more frequent after several months (Hammond et al., 1980). As in the sensorimotor cortex, microinjection of ferric chloride solution in the frontal cortex induced epileptic discharges recorded by electrocorticography. The seizures lasted up to 60 days after the injection (Hattori et al., 1983). The features of the epileptic discharges in electrocorticograms were studied, and were found to include isolated spikes and spike and wave complexes. They lasted for more than 12 months after the injection. The isolated spike activity appeared unilaterally, while the spike and wave complexes appeared bilaterally (Moriwaki et al., 1990,1992). The amount of neuronal damage produced by Fe deposition increased with time. One day after injection, the mean number of fluoro-Jade-labeled degenerating neurons in the center of ferric ammonium citrate injection sites was five-fold higher than at ammonium citrate injection sites. This difference increased to 56-fold by day three. At five days post-injection, few dying neurons were observed at the control sites, but neurodegeneration continued beyond a week at the Fe-injected sites. Thus, Fe released during a brief episode of hypoxiaischemia or during a stroke may be neurotoxic for a protracted period (Bishop and Robinson, 2001). Fe deposition in the hippocampus also resulted in increased epileptic activity. Injection of 3 |JL1 of 100 mM ferrous chloride into the dorsal hippocampus resulted in convulsive seizures in rats (Willmore et al., 1986). Electrolytic deposition of Fe ions in the hilus of the dentate gyrus of rats

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(and not merely hilus tissue destruction) produced electrographic seizure activity within one to two hours of current passage. Seizures recurred most intensely for two to three hours, and sporadic epileptiform activity was detected for up to 12 hours. Motor seizures were observed in all seizing rats and always coincided with hippocampal seizure discharges. The dentate gyrus appeared to be the most epileptogenic site tested, since hippocampal Fe deposition that did not include the dentate gyrus, or Fe deposition in the entorhinal cortex, was significantly less epileptogenic (Pico and Gall, 1994). Injection of Fe into the amygdala were also epileptogenic. Rats microinjected with a 100 mM aqueous solution of ferric chloride into the amygdala showed epileptiform discharges from the ipsilateral amygdala soon after injection. Within five days, epileptiform discharges were also detected in the contralateral amygdala, and behavioral seizures were observed. These spontaneous seizures occurred with rearing and bilateral forelimb clonus. Seizures persisted during the 30 days of the experiment (Ueda et al., 1998). Free radicals generated by the action of Fe are important causes of damage, resulting in epilepsy. Free radicals could be formed as a result of the Fenton reaction, and could attack cell membranes, leading to lipid peroxidation and generation of lipid peroxidation products. An increase in levels of the lipid peroxidation product, malondialdehyde, was observed in the cortex of rats treated with Fe. The increase was reduced in rats treated with Fe and the Fe chelator desferrioxamine (Suzer et al., 2000). The effect of free radicals appears to be dependent on their ability to produce lipid peroxidation, and a correlation between lipid peroxidation and epileptiform discharges after intracortical injections of ferric chloride was demonstrated (Singh and Pathak, 1990). In contrast to injections of ferrous chloride, which caused an increase in fluorescent products of lipid peroxidation and permanent seizures, injection of cobalt chloride had no effect on fluorescent products, and caused only transient seizures. Since cobalt chloride injections failed to cause formation of lipid peroxidation products, whereas injections of Fe caused significant increase in fluorescent products, it was suggested that the occurrence of Fe-induced lipid peroxidation is of importance in the initiation of recurrent seizures (Triggs and Willmore, 1984). The link between free radicals, lipid peroxidation, and

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epileptic discharges was demonstrated by experiments, in which treatment with alpha tocopherol or 2 ppm selenium prevented the development of Feinduced epileptiform activity (Willmore and Rubin, 1981). Rats pretreated with 500 mg/kg DL-alpha-tocopherol acetate prior to the injection of 3 |JLL of 100 mM ferrous chloride into the dorsal hippocampus showed significantly less lipid peroxidation and fewer convulsive seizures than those not treated with tocopherol (Willmore et al., 1986). The free radical scavenger, melatonin, also decreased the concentration of thiobarbituric acid reactive substances, an index of oxidative damage, and suppressed or delayed the development of ferrous chloride-induced epileptic discharges (Kabuto et al., 1998). The species of radicals formed after Fe injections were examined using electron spin resonance, and found to include the superoxide radical (Willmore et al., 1983). The hydroxyl radical is an even more reactive free radical species than the superoxide radical, and scavengers of hydroxyl radicals, in particular, were shown to be effective in suppressing or delaying the occurrence of the ferrous Fe-induced epileptic discharges. Treatment of rats with epigallocatechin or a phosphate diester of vitamins E and C, which are potent hydroxyl radical scavengers, significantly inhibited the formation of malondialdehyde and epileptic discharges in the Fe-induced epileptic focus (Mori et al, 1990). Intraperitoneal injections of the hydroxyl radical scavengers, adenosine and 2-chloroadenosine, also suppressed or delayed the occurrence of epileptic discharges induced by ferric chloride injections into the sensorimotor cortex of rats (Yokoi et al., 1995). Oral administration of the hydroxyl radical scavanger, alpha-tocopheryl-L-ascorbate-2-O-phosphate diester, was recently shown to protect against oxidation of neural membranes and prevented the induction of epileptiform activities after ferric chloride injection into the rat motor cortex (Yamamoto et al., 2002). In addition to superoxide and hydroxyl radicals, other free radical species, such as the l,l-diphenyl-2-picrylhydrazyl radical, may also be involved in Fe-induced epilepsy (Hiramatsu et al., 1994). In contrast, the nitric oxide radical does not seem to be a prominent feature of Fe-induced damage. Intracortical injections of Fe have instead been reported to result in a significantly decreased nitric oxide synthase activity at the injection site (Kabuto et al., 1996). Nitric oxide has been shown to scavenge

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short-lived, highly reactive free radicals, such as superoxide anion, hydroxyl radical, peroxyl lipid radicals, and thiyl radicals (Chiueh, 1999). Although destructive at high concentrations, the presence of nitric oxide at the time of hydroxyl radical production can lead to HN0 2 , thus inhibiting the damaging effects of hydroxyl radicals. The hydroxyl radical scavenging actions of nitric oxide donors have been shown to provide protection against ferrous citrate-induced neurotoxicity (Rauhala et al., 1996, 1998; Mohanakumar et al., 1998). When added with 1 mM ferrous chloride, nitric oxide donors including NOC-18, SNAP, and NOR-4 (0.1-1 mM) have been shown to inhibit lipid peroxidation and suppress cell death in a concentration-dependent manner (Nara et al., 1999). In view of these observations, it is possible that the decreased nitric oxide synthase activity after Fe treatment might result in reduced level of nitric oxide, and could further contribute to hydroxyl ion-induced damage. The free radicals catalyzed by Fe could affect neuronal membranes and result in increased excitation or decreased inhibition of neurons and epilepsy. These possible effects could be direct effects of free radicals on receptor or transporter proteins, or indirect effects of Fe on lipid components of the cell membrane, changes in its composition and structure, and consequent interference with the function of membrane proteins. The functions of excitatory amino acids, including glutamate and aspartate have been reported to be affected by Fe-induced free radical formation. An increase in both luminol and lucigenin enhanced chemiluminescence was observed in the cortex, hypothalamus and hippocampus of rats that had received Fe injection into the cortex. In contrast, no increases in chemiluminescence was observed in Fe-injected rats pretreated with MK-801, an antagonist of the N-methyl-D-aspartate (NMDA) class of glutamate receptors, suggesting that these receptors play important roles in the development of post-traumatic epilepsy (Kucukkaya et al., 1998). Fe treatment may result in increased activity of ionotropic glutamate receptors. The maximal release of [3H] y-aminobutyric acid (GABA) from cultured retinal cells, evoked by NMDA under potassium-induced depolarization, was significantly higher in peroxidized cells compared with control cells. The change in the intracellular sodium concentration evoked by saturating concentrations of NMDA under depolarizing conditions was also significantly higher in peroxidized cells than in controls (Agostinho et al., 1996).

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Kainate, used at a subsaturating concentration, also evoked significantly greater increases of the intracellular sodium concentration in peroxidized cells, than in control cells. These results suggest that the activity of ionotropic glutamate receptors is increased during conditions of oxidative stress and lipid peroxidation (Agostinho et al., 1996). Interestingly, the effect of oxidative stress on the NMDA receptor was significantly reduced by a phospholipase A2 (PLA2) inhibitor (Agostinho et al., 1996). This suggests that the lipid environment of the NMDA receptor could be crucial to its function. The peroxidized cell membrane is more susceptible to hydrolysis by PLA2 (McLean et al., 1993). In contrast to hypoxia alone (which generates arachidonic acid leading to feedback inhibition of PLA2 and minimal losses of phospholipids), treatment of kidney tubule cells with Fe and peroxidation of cell membranes resulted in early phospholipid losses (Zager et al., 1999). The breakdown of phospholipids could produce a "packing defect" in the membrane, which could then affect the function of membrane proteins (Farooqui et al., 2000). There is evidence that the channel region of the NMDA receptor may be affected by this form of Fe-induced damage. Treatment with ferrous chloride led to marked inhibition of MK-801 binding to the channel of the NMDA receptor, in a concentration-dependent manner in rat brain synaptic membranes (Ogita et al., 1999). Of the various saturated and unsaturated free fatty acids, both oleic and arachidonic acids exclusively decreased the potency of ferrous chloride to inhibit MK-801 binding when rat brain membranes were first treated with fatty acids, followed by treatment with ferrous chloride. These results suggest that changes in membrane phospholipids may be responsible for the effect of ferrous ions on the NMDA channel (Ogita et al., 1999). The observation that fatty acids inhibited the ferrous chloride induced-changes in the NMDA receptor argues against a direct effect of fatty acids or their metabolites in effecting these changes. An accumulation of fatty acids could, on the other hand, result in the inhibition of PLA2 (Sevanian and Rashba-Step, 1997) and prevent the formation of "packing defects" in the membrane. The above observations support the notion that perturbation of the physical structure of the cell membrane may be critical in effecting changes to the NMDA receptor. The importance of an intact cell membrane in NMDA function is further supported by the

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observation that pretreatment with the detergent Triton X-100 resulted in potentiation of the ability of ferrous chloride to interfere with MK-801 binding (Shuto et al., 1997). Fe may also produce changes in glutamate uptake and glutamate transporters. The uptake velocities of glutamate (and GAB A) from synaptosomal fractions was severely reduced in the presence of Fe and other metals (Gabrielsson et al., 1986; Keller et al., 1997). Injection of ferric cations into rat hippocampus resulted in bilateral elevations in the expression levels of the neuronal glutamate transporter EAAC-1, but a decrease in levels of the glial glutamate transporter GLAST at 30 days after injection, at a time when experimental animals were experiencing spontaneous limbic behavioral seizures. The decreased expression of GLAST may result in increases in extracellular glutamate, which could lead to increased excitation of neurons and epilepsy (Ueda and Willmore, 2000). Fe may also cause an increase in glutamate or aspartate release. Fe and peroxide treatment increased the basal release of D-[3H] aspartate from cerebral cortical synaptosomes (Gilman et al., 1994). A significant increase in potassium-stimulated aspartate release, but no significant differences in the release of glutamate or in the uptake of glutamate and aspartate between Fe-injected and saline-injected cortex was also described, in the rat cerebral cortex (Janjua et al., 1990). Injection of ferrous chloride was reported to result in increased levels of both aspartate and glutamate in epileptic brain tissue in another study (Engstrom et al., 2001). Fe-induced free radical damage may also affect the function of the inhibitory neurotransmitter, GABA. As in excitatory amino acid transmission, GABA receptors, transporters, and release may be affected. Addition of H 2 0 2 to rat hippocampal slices resulted in decreased function of the GABAA receptor, as evidenced by decreased intracellular chloride concentration and reduced ability of a GABA agonist, muscimol, to increase intracellular chloride concentrations in CA1 hippocampal neurons (Sah et al., 2002). A superoxide radical-generating system consisting of xanthine plus xanthine oxidase also decreased muscimol-induced chloride uptake in rat cerebral cortical synapsosomes (Schwartz et al., 1988). This effect of free radical damage on decreasing the function of an inhibitory transmitter receptor contrasts with the facilitatory effect of radicals on the NMDA receptor described above, and may result in an imbalance in excitation/inhibition, and epilepsy.

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In addition to the GABAA receptor, a decrease in GABA transaminase activity has been reported after ferric chloride-induced epilepsy (Pathak et al., 1984). GABA transaminase is both a key synthetic enzyme for GABA and a degrading enzyme. A decrease in GABA uptake by rat brain synaptosomes was shown after lipid peroxidation, induced with xanthine and xanthine oxidase (Braughler et al., 1985) or Fe/ascorbate-induced treatment (Gabrielsson et al., 1986; Rafalowska et al., 1989; Palmeira et al., 1993). The effect of decreased uptake would be to increase GABA concentration at GABAergic synapses. It is not known whether this could result in increased inhibition of neurons, since oxidative injury also concomitantly reduced GABAA receptor function (Sah et al, 2002). As mentioned above, lipid peroxidation of neuronal cell bodies produced an increase in GABA release in response to NMDA (Agostinho et al., 1996). In contrast, peroxidation appeared to have the opposite effect on synaptosomes, and inhibited GABA release. Fe/ascorbate-induced lipid peroxidation produced a decrease in calcium-dependent and calcium-independent efflux of accumulated [3H] GABA in response to potassium pulses (Palmeira et al., 1993). Peroxidation also caused a significant inhibition of veratridine-dependent release of GABA from synaptosomes, although potassium-dependent release of these neurotransmitters was not affected (Rafalowska et al., 1989). Comparison of GABA uptake and release after peroxidation suggested that GABA release was more severely affected than GABA uptake. The difference between release and uptake may result in a reduction of GABAergic transmission, leading to decreased inhibition of neurons and seizures (Zhang et al., 1989). An increase in excitatory neurotransmitter release following Fe-induced oxidative stress could be due to an effect of Fe in increasing intracellular calcium concentrations. This could occur via increased influx of calcium ions or reduced efflux of calcium. Peroxidation of rat brain synaptosomes by ferrous Fe and H 2 0 2 was coupled with a rapid and large (two- to seven-fold) uptake of calcium by synaptosomes (Braughler et al., 1985). Fe-induced calcium uptake was blocked by high concentrations of either desferrioxamine or methylprednisolone. In contrast, calcium channel blockers did not affect calcium uptake induced by Fe (Braughler et al., 1985). The effect of Fe on calcium influx, therefore, does not appear to occur via increased calcium influx through voltage-dependent calcium channels. Instead, Fe appeared to inhibit the function of these channels.

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Fe and H 2 0 2 significantly reduced potassium (depolarization) induced calcium influx, and this reduction was abolished by hydroxyl radical scavengers. Hydroxyl radicals were also found to suppress calcium influx induced by BAY K8644, an activator of L-type voltage-dependent calcium channels (Shirotani et al., 2002). An alternative way in which Fe could increase intracellular calcium concentration is by affecting the sodium-calcium exchanger (Amoroso et al., 2000). The exchanger usually functions to remove calcium from the cell but, when working in reverse, can lead to calcium accumulation in the cell. An increase in sodium efflux and intracellular calcium concentration was observed in C6 glioma cells treated with sodium nitroprusside, a nitric oxide donor and an Fe-containing molecule. The effect was blocked by bepridil, a specific inhibitor of the sodium-calcium exchanger, indicating that the calcium influx occurred through reverse action of the exchanger (Amoroso et al., 2000). A hydroxyl radical-induced calcium influx in rabbit and rat myocardium due to reverse mode of the sodium-calcium exchanger has also been described (Zeitz et al., 2002). Fe may also reduce the activity of the sodium-potassium ATPase and the plasma membrane or endoplasmic reticulum sodium/calcium ATPases (Kaplan et al., 1997). This could lead to depolarization (and possibly calcium influx through voltage-dependent calcium channels, though these may be damaged as well) and/or reduced extrusion or sequestration of calcium. The calcium-dependent ATPase activity of synaptosomal plasma membranes was significantly depressed following peroxidation of membrane lipids, and the calcium ATPase activity of endoplasmic reticulum was also affected during ascorbate/Fe-induced oxidative stress (Pereira et al., 1996). As in the Fe-induced increase in NMD A receptor activity, the lipid environment of the membrane protein appears important, and the effect of Fe on ATPase activity was abrogated by PLA2 inhibitors. These observations support the concept that increased synaptosomal calcium concentration due to oxidative stress may result from inhibition of plasma membrane and endoplasmic reticulum membrane calcium ATPase activities, as a result of the alteration of the lipid environment required for activity of these proteins (Pereira et al., 1996). A calcium-independent, nonvesicular, release of neurotransmitters after Fe treatment has also been described, but is controversial. Sodiumdependent amino acid transporters normally function to remove amino

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acids from the synaptic cleft, but when working in reverse, can lead to the release of neurotransmitters. Treatment with peroxide, Fe alone, or peroxide with Fe was reported to significantly increase the calciumindependent basal release of D-[3H] aspartate (Gilman et al., 1994). However, another study reported that release of aspartate, glutamate, and GABA, which occurs through reversal of the sodium-dependent amino acid transporters was reduced following ascorbate/Fe-induced oxidative stress (Rego et al., 1996).

Ca2*

Membrane

Native phospholipid

•

4

Peroxidized phospholipid

cPLA, •*-

Depolarization

Accumulation of arachidonicacid Increase in eicosanoids VitC ROS OH"

•

Dama 9 e t 0 Phospholipids, proteins,and nucleic acids

Neuroinflammation

Neural cell injury Surface transport system and Fe-binding proteins

Membrane

Fe3*

Fig. 1. Hypothetical diagram showing the relationship among Fe, phospholipids, and phospholipase A2 during kainic acid (KA)-induced neurotoxicity. Membrane phospholipids are hydrolyzed by cytosolic phospholipase A2 (cPLA2). Peroxidized phospholipids are a better substrate for cPLA2 than the native phospholipids. In KA-induced neurotoxicity, increased cPLA2 activity after epileptic seizure results in loss of essential phospholipids and accumulation of arachidonic acid. This triggers off an uncontrolled arachidonic acid cascade with alterations in ion homeostasis and cellular redox (Farooqui et al., 2000). The reduced iron (Fe2+) catalyzes the formation of hydroxyl radicals in the presence of H 2 0 2 through the Fenton reaction, leading to more oxidative damage to native membrane phospholipids, proteins, and nucleic acids. ROS = reactive oxygen species, SOD = superoxide dismutase.

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It has also been suggested that Fe can affect ion channels by virtue of its magnetic properties (Dobson and St Pierre, 1996). Such interactions may lead to changes in channel properties that might underlie some forms of epilepsy.

2.2. Human Studies The presence of an intracranial hemorrhage, especially those occurring in the brain tissue itself, has a robust association with the development of epilepsy. When hemoglobin or myoglobin is exposed to low levels of H 2 0 2 (such as a one-to-one molar ratio of H 2 0 2 to protein), they are converted into Fe(IV) (heme ferry 1) species: Heme Fe(III) protein + H 2 0 2 —> heme [Fe(IV) = O] protein* + H+ + H20. The ferryl species can stimulate peroxidation of lipids and oxidize other molecules. When myoglobin or hemoglobin is exposed to an excess (more than a 10-to-one molar ratio) of H 2 0 2 in vitro, they are degraded, releasing both heme and Fe ions (from heme ring breakdown). The heme so released can stimulate lipid peroxidation. When added to lipids, heme proteins, in the absence of added H 2 0 2 , could stimulate peroxidation by a mechanism that probably involves the decomposition of pre-existing traces of lipid peroxides in the lipids to alkoxyl and peroxyl radicals (Hogg et al, 1994; Rice-Evans et al, 1993; reviewed in Halliwell and Gutteridge, 1999): Heme Fe(III) + LipOOH -» heme Fe 2+ + LOO* + H + , Heme Fe 2+ + LipOOH -* heme Fe(III) + LipO* + OH". As is evident from the above reactions, Fe in the heme molecule plays a key role in radical generation even before heme breakdown. Heme is broken down by the action of heme oxygenase (HO). This enzyme is found in the endoplasmic reticulum. It catalyzes the breakdown of heme to biliverdin, with the release of carbon monoxide and Fe ions (Maines, 1997, 2000). Two isoforms of HO have been characterized. A constitutive isoform (HO-2; predominant under physiological conditions) and a stress-induced (Keyse and Tyrrell, 1989) isoform (HO-1; identical to heat shock protein 32). The biliverdin produced by the action of HO is converted into bilirubin by the enzyme biliverdin reductase in the

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cytosol (Tenhunen et al., 1968; reviewed in Elbirt and Bonkovsky, 1999). Bilirubin is an antioxidant, whereas the Fe formed is a pro-oxidant (reviewed in Halliwell and Gutteridge, 1999). Intracranial hemorrhages can be classified into extradural, subdural, subarachnoid (based on their relationship with the dura and arachnoid layers of the meninges covering the brain), and intraparenchymal (within the brain tissue). The potential for epileptogenesis appears related to the location of the extravasated blood. Extradural hemorrhages occur between the dura mater and the skull. They commonly occur as a result of a temporal or parietal fracture with laceration of the middle meningeal artery or vein. Less often, there is a tear in a dural venous sinus. Though the brain tissue may be compressed as a result of the hematoma, extravasted blood does not come into direct contact with the brain tissues. There is no increased incidence of epilepsy following hemorrhage in this location. Subdural hemorrhages occur between the dura mater and the arachnoid mater. They commonly occur as a result of acceleration/deceleration injury following a blow to the head, and tearing of the cerebral veins before the point of entry into the dura mater. In this situation, blood may compress the brain tissue, but is separated from the brain tissue itself by the arachnoid mater and the subarachnoid space. Although convulsions are occasionally seen in patients with associated cerebral contusions, they cannot be regarded as a cardinal sign of subdural hematoma (Adams et al., 1997). Subarachnoid hemorrhages occur between the arachnoid mater and pia mater covering the brain tissue. There are many causes of subarachnoid hemorrhages, but the most common cause is a ruptured aneurysm in one of the constituent vessels forming the arterial circle of Willis at the base of the brain. The characteristic of this condition is the tendency for the hemorrhage to recur from the same site. In this location, blood is separated from the brain tissue by only a thin layer of pia mater. Convulsive seizures, usually brief and generalized, are rare and occur in fewer than 10% of cases of subarachnoid hemorrhage. They are not recurrent and occur in relation to acute bleeding or rebleeding from an aneurysm. These early seizures do not correlate with the location of the aneurysm (Adams et al., 1997). Seizures are, however, observed if subarachnoid hemorrhage is associated with intraparenchymal hemorrhage, such as that following rupture of an arteriovenous malformation (AVM) which partly involves the

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brain parenchyma, and bleeding into the subarachnoid space (see below). Fe released by action of HO-1 in the arterial walls could catalyze the formation of free radicals, which lead to sustained arterial contraction and vasospasm following subarachnoid hemorrhage (Findlay et al., 1991; Ono et al., 2000). The vasospasm was strongly associated with fixed ischemic deficits, but not epilepsy (Ildan et al., 2002). Intraparenchymal hemorrhages occur in the brain tissue itself. They can be the result of trauma to the head and contusion of the brain tissue, chronic hypertension and degenerative changes in cerebral arteries (primary/hypertensive intracerebral hemorrhage), or rupture of AVMs in the brain. The presence of parenchymal blood appears to be an important element in the pathogenesis of seizures (Mori et al., 1991; Ueda et al., 1998). Post-traumatic seizures are either focal in type or generalized with loss of consciousness (grand mal). Petit mal is, rarely if ever, due to trauma. The risk for seizures is directly related to the severity of the injury. For instance, it has been observed that seizures occur within one year in about 7% of civilian and 34% of military head injuries. This difference has been correlated with the much higher proportion of penetrating wounds in military cases which, in turn, may be related to the amount of bleeding and Fe released into the brain tissues (Evans, 1962). In the Vietnam Head Injury Study, increased risk for epilepsy was associated with retained metal fragments, intracranial hematoma, persistent neurologic deficits, and degree of brain volume loss (Salazar et al., 1985). The extravasated blood clots within hours and the hematoma is surrounded by petechial hemorrhages from torn arterioles and venules. The red blood cells are phagocytosed by macrophages within 24 hours and hemosiderin is first observed around the margins of the clot in five to six days. In two to three months, the clot is slowly absorbed, leaving a smooth-walled cavity or a yellow brown scar. The Fe pigment in macrophages becomes dispersed and stud adjacent astrocytes and neurons, and may persist well beyond the border of the hemorrhage for years (Adams et al., 1997). Following traumatic brain injury, accumulation of HO-1 positive microglia/macrophages was detected as early as six hours post-trauma, and was still pronounced at six months. HO-1 was also weakly expressed in astrocytes in the perifocal penumbra (Beschorner et al., 2000). The Fe released by the HO could have an epileptogenic effect. Electrocorticograms of the brain in regions adjacent to the old traumatic foci reveal a number of electrically

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active zones adjacent to scars. The microscopic pathology of these foci shows neuronal loss and an increase in astrocytes (Adams et al., 1997). Primary (hypertensive) intracerebral hemorrhage is due predominantly to chronic hypertension and degenerative changes in cerebral arteries. Hemorrhages are described as massive, moderate, small, slit, and petechial. Slit refers to an old collapsed hypertensive or traumatic hemorrhage that lies just beneath the cortex. The bleeding can occur in various places in the brain. The most common sites are the putamen and adjacent internal capsule, central white matter of the temporal parietal or frontal lobes, thalamus, cerebellar hemisphere, and pons. Seizures, usually focal, occur in the first few days in some 10% of supratentoral hemorrhage. Seizures have been reported especially in association with subcortical slit hemorrhages (Adams et al., 1997). A recent large-scale survey of seizures after stroke has shown that seizures occurred in 8.9% of patients with stroke, and the only risk factor for seizures after hemorrhagic stroke was cortical location (Bladin et al., 2000). A greater incidence of seizures in hemorrhagic stroke (4.3%) compared to ischemic stroke (2%), as well as an increased incidence of seizures in strokes with cortical involvement, was also found in a separate study (Arbiox et al., 1997). Thus, hemorrhage in or near the cerebral cortex appears to be most epileptogenic among the various sites of intracerebral hemorrhages. As in hemorrhagic stroke, cortical location was found to be a risk factor for epilepsy after ischemic stroke (Bladin et al., 2000). An AVM consists of a tangle of dilated vessels that form an abnormal communication between the arterial and venous systems. It is a developmental abnormality, representing persistence of an embryonic pattern of blood vessels. The tangled blood vessels interposed between arteries and veins are abnormally thin and do not have the structure of normal arteries or veins. Most AVMs are clinically silent for a long time, but sooner or later they bleed. When hemorrhage occurs, blood may enter the subarachnoid space, but since the AVM lies within cerebral tissue, bleeding is likely to be partly intracerebral. Seizures are commonly associated with AVMs and, in 30% of cases, it is the first and only manifestation (Adams et al, 1997). An increase in brain Fe accumulation can also result from infectious agents. These include cerebral malaria and human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) encephalopathy.

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Both diseases are associated with increased incidence of epilepsy. In cerebral malaria, capillaries and venules are packed with parasitized erythrocytes and the brain is dotted with many foci of necrosis surrounded by glia (Durck nodes). Cerebral malaria is fatal in 20% to 50% of affected patients. It is characterized by headache, seizures, and coma. A survey of Nigerian children with fevers and malaria parasitemia showed that approximately two-thirds had seizures. Of these, 69% had generalized seizures, 21% had partial and generalized seizures, and 10% had partial seizures (Akpede et al., 1993). An induction of HO-1 has also been observed in the brain in lesions of human cerebral malaria. A close association was observed between HO-1 expression with areas of bleeding, suggesting that released hemoglobin and heme, known inducers of HO-1, are mainly responsible for induction of monocytic HO-1 expression (Schluesener et al., 2001). It is possible that the Fe released by action of HO-1 on parasitized erythrocytes may at least be partly responsible for the seizures in cerebral malaria. The progression of HIV infection towards its more advanced stages is accompanied by increasing body Fe stores. Fe accumulates in macrophages, microglia, endothelial cells, and monocytes. The Fe burden is especially heavy in bone marrow, brain white matter, muscle, and liver (reviewed in Boelaert et al., 1996). The caudate nucleus and globus pallidus appeared to be particularly affected, based on magnetic resonance imaging studies (Miszkiel et al., 1997). At autopsy, the number of hemosiderin-laden macrophages was found to be significantly increased in the brains of patients with AIDS compared with age-matched control subjects (Gelman et al., 1992). Seizures have been reported in 4% to 11% of HIVinfected patients (Wong et al., 1990; Van Paesschen et al., 1995). The seizures may occur at any stage of the disease, and may even be the presenting symptom of HIV infection (Holtzman et al., 1989). An increased Fe uptake across the blood-brain barrier might also occur in patients undergoing neuroleptic drug treatment, resulting in epilepsy. Antipsychotic drugs, such as haloperidol and chlorpromazine, can alter the blood-brain barrier and enhance the normally restricted Fe transport into the brain in rats (Ben-Shachar et al., 1993). A common side effect of these drugs is epilepsy, and the increased brain Fe content has been suggested to relate to the toxic effects of these drugs (Ben-Shachar et al., 1993). Other neuroleptics, including chlorpromazine, thioridazine,

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and fluphenazine, showed a stimulatory effect on Fe uptake from Fe donors in synaptosomal preparations of rat cerebral cortex (Penatti et al., 1998). It is possible that the increased Fe might interfere with ionic homeostasis in nerve terminals, resulting in epilepsy. An increased amount of Fe/transferrin saturation in the blood does not appear to be the cause of epilepsy in the majority of patients. In a study of 130 patients with epilepsy, abnormally high transferrin saturations were observed in only 10 patients (Ikeda, 2001). In contrast, in select kindred relatives affected with familial idiopathic epilepsy, most individuals suffering seizures also have low levels of the plasma hemoglobin-binding protein, haptoglobin (Panter et al., 1984). Binding of hemoglobin to haptoglobin decreases the effectiveness of hemoglobin in stimulating lipid peroxidation. The hemoglobin-haptoglobin complexes are cleared from the circulation by the liver, and at least part of the complex is excreted intact into the bile (Halliwell and Gutteridge, 1999). It has been suggested that hypohaptoglobinemia, either inherited or acquired through traumatic processes, may prevent efficient clearing of interstitial hemoglobin from the central nervous system (Panter et al., 1985). There are several inborn errors of metabolism that could result in abnormal brain accumulation of Fe. These include Hallervorden-Spatz syndrome, aceruloplasminemia characterized by Fe overload in basal ganglia (Gitlin, 1998), and Friedreich's ataxia characterized by neuronal and myocardial mitochondrial Fe accumulation (Puccio and Koenig, 2000). In Hallervorden-Spatz syndrome, imaging and pathologic studies demonstrated that excess Fe accumulates in the globus pallidus and substantia nigra, the exact nuclei in which neurons are lost (Swaiman, 1991). Although the symptoms of Hallervorden-Spatz syndrome, such as dystonia, rigidity, and choreoathetosis, are mostly associated with damage to the basal ganglia, some patients have hard-to-control seizures (Elejalde et al., 1979; reviewed in Chiueh, 2001). Whether Fe might be a factor in childhood epilepsies is not clear. The immature nervous system is especially enriched in Fe, particularly around the time of myelination (Connor et al., 1995; Cao et al., 2001). The increased Fe is associated with increased amounts of the lipid peroxidation product, 4hydroxynonenal, in the supraventricular corpus callosum and overlying deep layers of the rat motor cortex during this time (Cao et al., 2001). It has been suggested that free Fe and the presence of lipid peroxidation products could

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set up the immature nervous system for increased cytotoxicity (Ferriero, 2001). This idea is supported by observations that children with febrile illness and seizures were less frequently Fe-deficient than those with febrile illness without seizures (Kobrinsky et al., 1995). It is possible that Fe could play a role in increased incidence of epilepsy among the elderly. There is an increase in Fe in the human brain with age (Connor et al., 1990; Samson and Nelson, 2000). The increase appeared particularly in the basal ganglia (Drayer, 1988; Pujol et al., 1992; Floyd and Carney, 1993; Schenker et al, 1993), but was also observed in the cerebral cortex (Imon et al., 1995; Hirai et al., 1996; Korogi et al., 1997). Fe is detected as low intensity areas by T2 weighted magnetic resonance imaging studies. In the cortex, low intensity areas were most frequently observed in the aged motor cortex, followed by the visual and sensory cortices (Imon et al., 1995; Hirai et al., 1996; Korogi et al, 1997). The amount of Fe in the cortex has been reported to be higher than that in the basal ganglia, based on results of studies on Tl relaxation rate (Ogg and Steen, 1998). The increase of Fe in the aging brain coincides with an increased incidence of epilepsy in the elderly. Seizures are the third most frequently encountered neurological problem in the elderly population, and the incidence of recurrent unprovoked seizures peaks in older patients (reviewed in van Cott, 2002). It is possible that Fe accumulation, particularly in the motor cortex, could be one of the causes of increased incidence of epilepsy among the elderly.

3. IRON AS A CONSEQUENCE OF EPILEPSY Unlike studies on increase of Fe as a cause of epilepsy, fewer studies have been carried out on the increase of Fe as a consequence of epilepsy. These have mostly been carried out on laboratory animals, and little published information is available on Fe as a consequence of human epilepsy. In animals, focal epilepsy is induced by the topical application of aluminum hydroxide or systemic or intracerebroventricular application of tetanus toxin, kainate, and pilocarpine. Most of these models share the characteristic of producing acute seizures followed, often after a latent period, by the development of chronic epilepsy with seizures. These behaviors are similar to human complex partial seizures. While the early seizures may arise from different mechanisms, the chronic models

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seem to depend largely on resultant neuropathological change. Another method of inducing epilepsy in animals is by kindling. In this model, repeated subconvulsive electrical stimulation to the amygdala leads to increasing after-discharge and, ultimately, behavioral seizures (Goddard et al., 1969). A study to elucidate the distribution of ferric and ferrous Fe in the dorsal hippocampus after epilepsy induced by intracerebroventricular injection of kainate in rats showed that very light staining for Fe was observed in the hippocampus in normal, saline injected, or 1-day postkainate injected rats. At one week post-injection, a number of ferric Fepositive cells, but very few ferrous Fe-positive cells, were present in the degenerating CA fields. At one-month post-injection, large numbers of ferric Fe-positive glial cells (Figs. 2A and 2B) and some ferrous Fepositive blood vessels were observed. At two months post-injection, large numbers of ferric and ferrous Fe-positive glial cells were present. The labeled cells had light and electron microscopic features of oligodendrocytes and were double-labeled with CNPase, a marker for oligodendrocytes. In addition, the study showed a shift in the oxidation state of the accumulated Fe, with more cells becoming positive for ferrous Fe at a late stage (Wang et al, 2002a). The observation of an increasing number of ferric and ferrous Fepositive cells in the degenerating hippocampus with time is consistent with the results of a nuclear microscopic study. This technique utilizes a variety of high energy (MeV) ion beam techniques at submicron spatial resolutions to provide structural and quantitative elemental analysis of biological tissue, down to the parts per million level of analytical sensitivity (Watt, 1995). An increased amount of Fe was detected in the degenerating hippocampus after intracerebroventricular kainate injection (Ong et al., 1999). The increase occurred continuously throughout the four-week post-lesion interval (the longest post-lesion time interval in which samples were scanned in the experiment). The majority of the increased Fe was distributed at the edge of the glial scar (Ong et al., 1999). A likely consequence of the high amounts of Fe in the hippocampus after kainate injection is that it could promote free radical damage in the adjacent neurons. Any Fe present is also likely to be more harmful to neurons than glial cells. This is due to the increased antioxidant defenses in glial cells, such as elevated levels of the antioxidant glutathione, observed

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Fig. 2. Light micrographs of normal and kainate-injected rats. The normal hippocampus is shown in A,C, and E. B and D show the hippocampus from rats that had been injected with kainate at one month, and F at 3 weeks, previously. (A and B) Perls' stained sections show light staining in the normal hippocampus (asterisk in A), but an increase in staining in oligodendrocytes (arrows) in the lesioned CA field (asterisk in B). (C and D) DMT1 stained sections show light staining of astrocytic end feet in the normal hippocampus (arrow in C), but dense staining of reactive astrocytes in the lesioned CA field (arrows in D). (E and F) HO-1 stained sections show only occasional labeled nonpyramidal neurons in the stratum oriens in the normal CA fields (arrow in E), but dense staining of reactive astrocytes in the lesioned CA field (arrows in F). Scale= 100 Jim.

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in astrocytes after kainate treatment (Ong et al., 2000). A chemiluminescence analysis study has also shown that despite greater accumulation of Fe and aluminum in glial cells than neurons, reactive oxygen species were twice as high in neurons than glial cells, indicating that the antioxidant defenses are greater in glial cells than neurons (Oshiro et al., 2000). A casual role for Fe in hippocampal damage after kainate treatment was shown by the observation that Fe deprivation resulted in decreased damage, whereas Fe supplementation resulted in increased damage and microgliosis in rats (Shoham and Youdim, 2000). It is therefore possible that although Fe may not be the initiator of epilepsy in the kainate model, it is accumulated consequent to neuronal injury and might contribute to neuronal injury and epilepsy. Besides kainate-induced injury, Fe deposition has also been observed after transient forebrain ischemia in the rat brain (Danielisova, 2002). An increase in Fe in the brain after epilepsy could be the result of increased transport of Fe across the blood-brain barrier. A recent study on DMT1 expression in the hippocampus of rats after kainate-induced epilepsy has shown a significant increase in the density ratios of DMT1/ 3-actin bands observed in Western blots in the one week, one month, and two months post-kainate injected hippocampus compared to uninjected and one-day post-kainate injected hippocampus (Wang et al., 2002b). The increase in DMT1 protein was paralleled by an increase in DMT1 immunoreactivity in astrocytes. Light staining for DMT1 was observed in the uninjected, saline-injected, and one-day post-kainate injected rat hippocampus. In contrast, an upregulation of DMT1 was observed in reactive glial cells at one week, one month, and two months post-kainate injection (Figs. 2C and 2D). Electron microscopy confirmed that the glial cells had morphological features of astrocytes. DMT1 is a cellular Fe transporter responsible for transport of metal ions from the plasma membrane to endosomes, and the observation that DMT1 is present on astrocytic end feet in contact with blood vessels suggests that astrocytes may be involved in uptake of Fe from endothelial cells (Wang et al., 2002b). In addition to increased Fe transport, it is also possible that focal deposits of Fe could result from a breakdown of Fe containing heme proteins, followed by Fe efflux and redistribution of the Fe. These activities could occur through HO-1. Transfection of HO-1 into mammalian cells stimulates Fe efflux, whereas Fe efflux is greatly diminished in fibroblasts from HO-1

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knockout mice (Ferris et al., 1999). An essential role for HO-1 in Fe efflux is supported by evidence of low serum Fe and accumulation of Fe in the tissues of HO-1 knockout mice (Poss and Tonegawa, 1997a, 1997b). The distribution of HO-1 mRNA and protein has been studied in the hippocampus of rats after intracerebroventricular injections of kainate (Matsuoka et al., 1998; Nakaso et al., 1999; Lu and Ong, 2001). At post-injection weeks one to three, HO-1 was observed in glial cells in the center of the lesion (Figs. 2E and 2F) and in neurons at the perifocal region of the glial scar. The glial cells were found to have features of viable astrocytes and microglia, while the neurons contained discontinuous cell membranes and nuclear outlines and had features of degenerating neurons. Recent in vitro results on fibroblasts transfected with HO-1 cDNA showed that, despite cytoprotection with low (less than fivefold compared with untransfected cells) HO-1 activity, high levels of HO-1 expression (more than fifteen fold) were associated with reactive Fe and toxicity (Suttner and Dennery, 1999). It is therefore possible that the high levels of expression of the enzyme could lead to excessive Fe accumulation and toxicity to neurons. Little is known about the source of heme proteins that form the substrate of HO. One possibility is that heme in prosthetic moiety of hemoproteins, such as hemoglobin, catalase, soluble guanalylate cyclase, cytochrome b5, cytochromes P450, and nitric oxide synthase, are released or made available in degenerating neurons and form the substrate of HO. Although it remains to be demonstrated whether the increased expression of HO-1 protein expressed after epilepsy is accompanied by increased activity, it has been shown in Alzheimer's disease patients that levels of cerebrospinal fluid bilirubin derivatives are increased significantly compared with those of controls. This increase is not due to the increased permeability of the blood-brain barrier, and is presumably due to increased HO activity (Kimpara et al., 2000). Heme itself crosses the blood-brain barrier poorly and negligible amount of radioactivity was recovered from the brain, following intravenous or intramuscular injections of 59Fe labeled heme arginate (Linden et al., 1987).

4. CONCLUSION AND DIRECTIONS FOR FUTURE RESEARCH Injections of Fe salts into the sensorimotor cortex, hippocampus, and amygdala of experimental animals have been shown to result in chronic

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recurrent focal paroxysmal electroencephalographic discharges and behavioral convulsions and electrical seizures. The induction of epilepsy is related to the generation of free radicals, lipid peroxidation of neuronal membranes, increased intracellular calcium concentrations through reverse action of sodium calcium exchanger/reduced activity of plasma membrane or endoplasmic reticulum calcium ATPases, increased release of excitatory neurotransmitters including aspartate and glutamate, and increased influx of ions through glutamate receptors. Some of the above effects of Fe can be abrogated by inhibitors of PLA2, but not arachidonic acid, indicating that the damaging effects of Fe may be due to perturbation of the lipid environment essential to normal functioning of membrane proteins. Fe in hemoglobin, or by itself, is also likely to be the cause of human epilepsy in instances where there is increased Fe load in the brain. These include subarachnoid hemorrhage, intraparenchymal hemorrhages due to head injury and stroke, malaria, HIV encephalitis, and possibly neuroleptic drug use. A reduced level of haptoglobin, a hemoglobin-binding protein, has also been observed in select kindred affected with familial idiopathic epilepsy. An accumulation of Fe has been observed in the motor cortex with age. It is possible that this might contribute to the increased incidence of epilepsy among the elderly. Fe accumulates with time in the rat hippocampus after kainate-induced epilepsy. The accumulation occurs in oligodendrocytes and is likely to be a reflection of the high levels of Fe in the extracellular space. The accumulation of Fe is correlated with an increased expression of DMT1 in astrocytes in the glial scar, and an increased expression of HO-1 in reactive astrocytes and microglia, as well as degenerating neurons at the edge of the scar. The increased DMT1 and HO-1 expression in astrocytes could lead to increased uptake of Fe followed by efflux of Fe and redistribution to the extracellular space. In this model, Fe is the consequence of epilepsy, although it is likely that it can also be a cause of epilepsy. Further work is necessary to elucidate the effects of lipid peroxidation of the cellular membranes on the function of membrane proteins and the role of phospholipases, including PLA2 in perturbing the lipid environment. The possible presence of Fe in the human brain after epilepsy also needs to be elucidated. The causes of dysregulation of Fe in the glial scar after neuronal injury need to be studied, and the possible beneficial effects of Fe chelators or antioxidants that cross the blood-brain barrier or neuroprotective gene induction on epilepsy need to be evaluated.

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Puccio H, Koenig M. Recent advances in the molecular pathogenesis of Friedreich ataxia. Hum Mol Genet 2000; 9:887-892. Pujol J, Junque C, Vendrell P, Grau JM, Marti-Vilalta JL, Olive C, Gili J. Biological significance of iron-related magnetic resonance imaging changes in the brain. Arch Neurol 1992;49:711-717. Rafalowska U, Liu GJ, Floyd RA. Peroxidation induced changes in synaptosomal transport of dopamine and gamma-aminobutyric acid. Free Rad Biol Med 1989; 6:485^-92. Rauhala P, Khaldi A, Mohanankumar KP, Chiueh CC. Apparent role of hydroxyl radicals in oxidative brain injury induced by sodium nitroprusside. Free Rad Biol Med 1998; 24:1065-1073. Rauhala P, Mohanakumar KP, Sziraki I, Lin AM, Chiueh CC. S-nitrosothiols and nitric oxide, but not sodium nitroprusside, protect nigrostriatal dopamine neurons against iron-induced oxidative stress in vivo. Synapse 1996; 23:58-60. Rego AC, Santos MS, Oliveira CR. Oxidative stress, hypoxia, and ischemia-like conditions increase the release of endogenous amino acids by distinct mechanisms in cultured retinal cells. J Neurochem 1996; 66:2506-2516. Rice-Evans C, Green E, Paganga G, Cooper C, Wrigglesworth J. Oxidized LDL induce iron release from activated myoglobin. FEBS Lett 1993; 326:177-182. Rouault TA, Haile DJ, Downey WE, Philpott CC, Tang C, Samaniego F, Chin J, Paul I, Orloff D, Harford JN, Klausner RD. An iron-sulfur cluster plays a novel regulatory role in the iron-responsive element binding protein. Biometals 1992; 5:131-140. Sah R, Galeffi F, Ahrens R, Jordan G, Schwartz-Bloom RD. Modulation of the GABA(A)-gated chloride channel by reactive oxygen species. J Neurochem 2002; 80:383-391. Salazar AM, Jabbari B, Vance SC, Grafman J, Amin D, Dillon JD. Epilepsy after penetrating head injury. I. Clinical correlates: A report of Vietnam Head Injury Study. Neurology 1985; 35:1406-1414. Samson FE, Nelson SR. The aging brain, metals and oxygen free radicals. Cell Mol Biol 2000; 46:699-707. Schenker C, Meier D, Wichmann W, Boesiger P, Valavanis A. Age distribution and iron dependency of the T2 relaxation time in the globus palldius and putamen. Neuroradiology 1993; 35:119-124. Schluesener HJ, Kremsner PG, Meyermann R. Heme oxygenase-1 in lesions of human cerebral malaria. Acta Neuropathol 2001; 10:65-68. Schwartz RD, Skolnick P, Paul SM. Regulation of gamma aminobutyric acid/barbiturate receptor-gated chloride ion flux in brain vesicles by phospholipase A2: Possible role of oxygen radicals. J Neurochem 1988; 50:565-571. Sevanian A, Rashba-Step J. Phospholipase A2 activation: An early manifestation of oxidative stress. In: Forman HJ, Cadenas E, editors. Oxidative Stress and Signal Transduction. New York: Chapman and Hall, 1997: 77-107. Shirotani K, Katsura M, Higo A, Takesue M, Mohri Y, Shuto K, Tarumi C, Ohkuma S. Suppression of Ca 2+ influx through L-type voltage-dependent calcium channels by hydroxyl radical in mouse cerebral cortical neurons. Brain Res Mol Brain Res 2002; 92:12-18.

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Shoham S,Youdim MB. Iron involvement in neural damage and microgliosis in models of neurodegenerative diseases. Cell Mol Biol 2000; 46:743-760. Shuto M, Ogita K, Minami T, Maeda H, Yoneda Y. Inhibition of [3H]MK-801 binding by ferrous (II) but not ferric (III) ions in a manner different from that by sodium nitroprusside (II) in rat brain synaptic membranes. J Neurochem 1997; 69:744-752. Simon RP, Aminoff MJ, Greenberg DA. Clinical Neurology, 4th ed. Comecticut: Appleton and Lange, 1999. Singh R, Pathak DN. Lipid peroxidation and glutathione peroxidase, glutathione reductase, superoxide dismutase, catalase, and glucose-6-phosphate dehydrogenase activities in FeCl3-induced epileptogenic foci in the rat brain. Epilepsia 1990; 31: 15-26. Suttner DM, Dennery PA. Reversal of HO-1 related cytoprotection with increased expression is due to reactive iron. FASEB J 1999; 13:1800-1809. Suzer T, Coskun E, Demir S, Tahta K. Lipid peroxidation and glutathione levels after cortical injection of ferric chloride in rats: Effect of trimetazidine and desferoxamine. Res Exp Med 2000; 199:223-229. Swaiman KF. Hallervorden-Spatz syndrome and brain iron metabolism. Arch Neurol 1991;48:1285-1293. Tenhunen R, Marver HS, Schmid R. The enzymatic conversion of heme to bilirubin by microsomal heme oxygenase. Proc Natl Acad Sci USA 1968; 61:748-755. Triggs WJ, Willmore LJ. In vivo lipid peroxidation in rat brain following intracortical Fe 2+ injection. / Neurochem 1984; 42:976-980. Ueda Y, Willmore LJ. Sequential changes in glutamate transporter protein levels during Fe3+-induced epileptogenesis. Epilepsy Res 2000; 39:201-209. Ueda Y, Willmore LJ, Triggs WJ. Amygdalar injection of FeCl3 causes spontaneous recurrent seizures. Exp Neurol 1998; 153:123-127. Van Cott AC. Epilepsy and EEG in the elderly. Epilepsia 2002; 3:94-102. Van Paesschen W, Bodian C, Maker H. Metabolic abnormalities and new-onset seizures in HIV-seropositive patients. Epilepsia 1995; 36:146-150. Wang XS, Ong WY, Connor JR. A light and electron microscopic study of the iron transporter protein DMT-1 in the monkey cerebral neocortex and hippocampus. J Neurocytol 2001;30:353-360. Wang XS, Ong WY, Connor JR. Increase in ferric and ferrous iron in the rat hippocampus with time after kainate-induced excitotoxic injury. Exp Brain Res 2002a; 143:137-148. Wang XS, Ong WY, Connor JR. A light and electron microscopic study of divalent metal transporter-1 distribution in the rat hippocampus after kainate-induced neuronal injury. Exp Neurol 2002b; in press. Watt F. Nuclear microscopy in the life sciences. Nucl Instr Meth B 1995; 104:276-284. Willmore LJ. Post-traumatic epilepsy: Cellular mechanisms and implications for treatment. Epilepsia 1990; 31:S67-S73. Willmore LJ, Hiramatsu M, Kochi H, Mori A. Formation of superoxide radicals after FeCl3 injection into rat isocortex. Brain Res 1983; 277:393-396. Willmore LJ, Rubin JJ. Antiperoxidant pretreatment and iron-induced epileptiform discharges in the rat: EEG and histopathologic studies. Neurology 1981; 31:63-69.

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Role of Iron Metabolism in Multiple Sclerosis Maritha J Kotze, J Nico P De Villiers, Monique G Zaahl, Kathryn J H Robson

ABSTRACT Multiple sclerosis is a chronic inflammatory disease of the central nervous system caused by a complex interaction between genetic and environmental factors. Support for a role of iron metabolism in multiple sclerosis was obtained by the analysis of the gene encoding the solute carrier family 11 (proton-coupled divalent metal ion transporters) member 1 (SLC11A1), formerly known as NRAMP1, in the genetically homogenous Afrikaner population of South Africa. Under-representation of allele 2 of the functional Z-DNA forming promoter polymorphism in patients compared with population-matched controls largely excluded the hypothesis that multiple sclerosis is primarily caused by a virus infection, since this allele has previously been linked to various infectious diseases and appears to protect against autoimmune disease. Differentially increased expression of alleles 3 and 5 upon stimulation with ferric ammonium citrate (p < 0.05) provided a direct link between the regulation of iron and susceptibility to autoimmune disease, especially since co-existence of these alleles is associated with multiple sclerosis in the South African population. Keywords: Iron metabolism; multiple sclerosis; SLCllAlgene; transcriptional activity.

1. INTRODUCTION Elucidation of the etiology of neurological diseases where iron (Fe) has been implicated would require a better understanding of the role of genes involved in Fe metabolism and their involvement in oxidative damage and heme biosynthesis. Although a melange of immunologic abnormalities have been documented in patients with Fe deficiency and in Fe overload 399

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states, the clinical importance of such abnormalities remains largely undefined (Bryan, 1991). Fe is essential to many metabolic processes, including DNA, RNA, and protein synthesis, as a co-factor of many heme and nonhaem enzymes, the formation and maintenance of myelin, and the development of the neuronal dendritric tree (Gerlach et al., 1994; Connor and Menzies, 1996). Since hemproteins are involved in electron transport, their optimal functioning is critical to axonal and myelin integrity. The complexity of Fe metabolism pathways is much greater than is presently appreciated and, although the transport of Fe into mitochondria represents an important step in Fe metabolism, this is often overlooked. Mitochondria utilize most of the cellular Fe to produce heme- and Fe-sulphur cluster-containing proteins, such as the cytochromes. An imbalance in the compartmentalization of Fe, rather than a decrease or increase in the absolute amount of body Fe, may result in neurological deficit. This is evident in patients with aceruloplasminemia where, unlike other Fe overload syndromes, neurological manifestations appear to dominate (Harris et al., 1998). Reduction in Fe binding to transferrin, as a consequence of defective oxidation of ferrous Fe to ferric Fe, results in impaired transport of Fe from intracellular stores to plasma. This leads to decreased serum Fe and microcytic anemia. Affected patients eventually die from effects of Fe accumulation in the basal ganglia, while the initial problem lies with the supply of Fe for key synthesis processes required for cell growth. These may include optimal expression of the ceruloplasmin gene implicated in neuronal survival in the retina and basal ganglia (Gitlin, 1998) and, although not investigated, interference of Fe delivery for heme biosynthesis involved in energy production and neurological integrity (Meyer et al., 1998). Although Fe deficiency-induced anemia is the most common Fe metabolism-related disease globally, much is still to be learned about the potential consequences of low availability of Fe for essential cellular processes, particularly in genetically predisposed individuals. Fe deficiency can be caused by inadequate dietary Fe absorption, loss or dysfunction of absorbing enterocytes, increased blood losses associated with malignant and inflammatory diseases, and abnormally high gastric pH. Fe withholding of the host upon infection with pathogens requiring Fe for proliferation might also result in hypoferremia and/or anemia of inflammation. It is generally assumed that these conditions are a consequence of

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the blockage of the macrophage release of senescent red blood cellsderived Fe (Roesser, 1980; Lee, 1983). Since Fe modulates cytokine activities, nitric oxide production, and immune cell proliferation (Weiss, 2002), this metal has to be immediately available for mounting a successful immunological defense and, at the same time, be effectively unavailable to the invading pathogen. The continuous battle for Fe between the host and invading pathogens may result in cytokine-mediated responses, which are involved in various infectious and autoimmune diseases. In this overview, the role of the solute carrier family 11 (protoncoupled divalent metal ion transporters) member 1 (SLC11A1), formerly known as the natural resistance-associated macrophage protein 1 (NRAMP1), will be discussed in relation to Fe homeostasis and disease susceptibility. This gene has been linked to various autoimmune and infectious diseases, which led to speculation that regulation of Fe by SLC11A1 may be of major importance in this context (Blackwell et al., 2000). Support for a role of Fe homeostasis in the pathogenesis of multiple sclerosis (MS), known to involve an autoimmune process, was obtained from recent detection of an association with the SLC11A1 gene (Kotze et al., 2001).

2. IRON AND MULTIPLE SCLEROSIS Defining the cause of MS represents a major challenge because little is certain about the pathogenesis of this disease. The varied clinical picture raises the possibility that MS is not a well-defined etiology, but consists of genetically different subtypes in addition to subtypes with no genetic contribution (phenocopies) (Rasmussen and Clausen, 2000). Different genes and environmental factors may be involved in the induction and progression of MS, with external factors affecting the population risk and genetic susceptibility accounting for familial risk. The proposed role of Fe in the etiology of MS could explain this complexity. Fe represents the most abundant metal in the human body and maintenance of Fe homeostasis involves a complex interplay between Fe receptor cells (erythrocyte precursors, duplicating and growing cells, and hepatocytes) and Fe donor cells (macrophages, intestinal mucosal cells, and hepatocytes). The regulatory process occurs within the context of different Fe transport proteins that are responsible for influx or efflux of Fe, and reductases with extracellular or intracellular activity.

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The high demand of Fe in the brain and the central nervous system, as well as their sensitivity to Fe-induced peroxidative damage, suggests the need for stringent regulation of Fe availability in these organs. Hulet et al. (1999) have demonstrated that the normal pattern of transferrin and ferritin binding is disrupted in the brain tissue of MS patients. This finding provided evidence of ferritin binding in human brain and suggests that loss of ferritin binding is involved in, or is a consequence of, demyelination associated with MS. Differences in Fe parameters measured in the cerebrospinal fluid (CSF) of MS patients have been reported by several groups (LeVine et al., 1999; Weller et al., 1999; Zeman et al., 2000). Zeman et al. (2000) have highlighted the potential role of transferrin determination in CSF as a means to distinguish between relapsing-remitting, secondary progressive, and primary progressive MS subtypes. They noted that transferrin is also a growth factor of importance in the proliferation of activated T lymphocytes. It has been hypothesized that cytokines from T cells play a functional role in regulating the expression of transferrin and in modulating Fe status through intestinal Fe absorption and transport (Lieu et al., 2001). The functional linkage between immune function and Fe absorption became evident when the major histocompatibility complex (MHC)encoded hemochromatosis (HFE) protein was identified. This finding provided evidence that MHC-encoded class I molecules might play a role in Fe metabolism (Salter-Cid et al., 2000). Although increased mean serum ferritin levels were reported by Valberg et al. (1989) in MS patients, no subjects with clinically manifested hereditary hemochromatosis (HH) were recognized by these authors among 1,700 patients with MS. Since MS and HH affect the same ethnic group, these findings may indicate interaction of the HFE gene with Fe-related genetic and/or environmental factors involved in the MS phenotype. Identification of a South African family where two sisters with MS were found to be homozygous for the common HH mutation C282Y has excluded the likelihood of a linkage disequilibrium effect with a putative MS-related gene on chromosome 6. Lack of clinical manifestation of HH in the MS patients, despite the presence of high transferrin saturation and ferritin levels (Kotze et al., 1999), raises the possibility that MS and clinically manifested HH may be mutually exclusive.

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2.1. Association Between Multiple Sclerosis and SLC11A1 Gene The first molecular study performed in MS patients to investigate the potential role of Fe metabolism, in association with infectious and/or autoimmune disease susceptibility linked to the SLCllAlgene, was performed in the genetically homogenous Afrikaner population of South Africa (Kotze et al., 2001). The unique genetic background of this population representing Dutch, German, French, and British origins provides the advantage to study the genetics of complex diseases on the basis of an expected limited number of disease-associated genes/mutations introduced from Europe. The SLC11A1 gene has been linked to various infectious and autoimmune diseases (Hofmeister et al., 1997; Marquet et al., 1999; Graham et al., 2000), which may relate largely to the functional activity of the Z-DNA forming GT-repeat polymorphism in the promoter region of the gene (Searle and Blackwell, 1998). The different alleles of the GT-repeat polymorphism detected in the South African Afrikaner population is shown in Table 1 (modified from Kotze et al., 2001), together with the allelic distribution observed in patients and controls. The allelic distribution differed significantly between the MS patients and population-matched controls, with underrepresentation of allele 2 previously implicated in susceptibility to infectious diseases. Analysis of a second age- and population-matched control group of 336 South African Caucasians in comparison with the MS patients confirmed this association (p < 0.03) (data not shown).

Table 1. Comparison of allelic distribution of the SLC11A1 GT-repeat polymorphism between South African MS patients and controls.

Alleles 2 [t(gt)5ac(gt)5ac(gt)10g] 3 [t(gt)5ac(gt)5ac(gt)9g] 5 [t(gt)4ac(gt)5ac(gt)10ggcaga(g)]

Multiple sclerosis (n=208)*

Controls (n= 1,076)*

41 (20%) 160 (77%) 7 (3%)

320 (30%) 752 (70%) 4 (0.4%)t

*From Kotze et al., 2001. tMS versus control group: p < 0.01, 2 df, %2 = 35.2.

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Samples were obtained from the parents and siblings of one of the MS patients with SLC11A1 genotype 3/5, whose aunt on the paternal side has also been diagnosed with definite MS. The pedigree is illustrated in Fig. 1. The finding that allele 5 was inherited from the unaffected mother of the index case, while her aunt with MS is from the paternal side of the family, highlights the complexity of the disease mechanism, which appears to be dependant on the co-existence of appropriate environmental and genetic factors. Allele 5 was associated with different haplotypes as defined by intragenic SLC11A1 polymorphisms in the two MS patients, probably due to a crossover or recurrent mutational event (data not shown). The presence of genotype 3/5 in several family members without MS in all three generations is in accordance with a polygenic genetic basis for this disease. Recently, Vitale et al. (2002) have demonstrated that even in a MS family with an autosomal dominant inheritance pattern, co-inheritance of two loci are required for the development of the disease. It seems likely that different combinations of gene-environment interactions may cause MS in diverse population groups. SLC11A1, which regulates Fe and is also regulated by Fe (Atkinson and Barton, 1998), may serve as a model for various diseases where interaction of the gene with Fe as a modifying factor may have a relatively small effect on familial risk, but a large effect on population risk. Similar effects can be expected for other genes involved in Fe metabolism, such as the hemochromatosis gene which has been identified as a modifier

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Fig. 1. Pedigree of the index patient (arrow) with SLC11A1 genotype 3/5 who was diagnosed with relapsing-remitting MS. Allele 5 was inherited from her unaffected mother. The same genotype was detected in her father's sister who was diagnosed with secondary progressive MS. MS=multiple sclerosis.

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locus for various conditions. These may include chronic liver disease (George et al., 1999), porphyria (Roberts et al., 1997), inherited anemias (Yaouanq et al., 1997), cystic fibrosis (Rohlfs et al, 1998), diabetes (Kwan et al., 1998), cardiovascular disease (Tuomainen et al., 1999; Roest et al., 1999), and Alzheimer's disease (Moalem et al., 2000). Even in conditions where Fe may not have a primary action, its potential secondary role cannot be negated. Hemochromatosis is an important paradigm for medical genetics because it offers an opportunity to explore the complexity of gene-gene and gene-environment interactions, and the same may apply to SLCllAl-associated disease.

2.2. Differential Expression of SLC11A1 Alleles The association detected between MS and the SLC11A1 gene does not exclude the possibility that the multiple pleiotropic effects associated with macrophage activation (Blackwell, 1996), and not the regulation of Fe transport per se, account for disease susceptibility. The effects on macrophage function include enhanced chemokine KC, tumour necrosis factor a, interleukin 1(3, inducible nitric oxide synthase, and MHC class II expression; all have potential importance in the induction/maintenance of autoimmune diseases and are crucially to the resistance to intramacrophage pathogens. Searle and Blackwell (1998) have used reporter gene constructs to demonstrate that the Z-DNA forming dinucleotide repeat in the promoter region of the SLC11A1 gene has endogenous enhancer activity. Different alleles showed a similar degree of enhancement of reporter gene expression in the presence of interferon-7 (IFN7), while addition of bacterial lipopolysaccharide (LPS) caused significant reduction in expression driven by allele 2 and enhanced expression driven by allele 3. These results were in accordance with the hypothesis that chronic hyperactivation of macrophages associated with allele 3 is functionally linked to autoimmune disease susceptibility, while the poor level of SLC11A1 allele 2 expression contributes to infectious disease susceptibility. The likelihood that allele 3 would conversely protect against infectious disease, and allele 2 against autoimmune disease, was supported by data from Kotze et al. (2001) who compared the allele frequencies of the promoter variant between different age groups. SLC11A1 alleles considered to be detrimental in relation to autoimmune disease susceptibility

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appear to be maintained in the population as a consequence of improved survival to reproductive age following infectious disease challenge. Ebers (1998) has highlighted the possibility that, within the context of susceptibility alleles being advantageous in one context and deleterious in another, no single allele sufficient for the development of MS may be found. In an attempt to demonstrate a possible direct relation of SLC11A1 alleles with cellular Fe status, functional studies were performed to test for differential expression of allelic variants of the promoter polymorphism upon Fe loading. We hypothesized that the expression of alleles 3 and 5 shown to be associated with autoimmune disease susceptibility would respond to treatment with ferric ammonium citrate (FAC). Figure 2 demonstrates that this, indeed, is the case. Similar to the results of Searle and Blackwell (1998), the addition of IFN7 and LPS as exogenous stimuli, separately or in combination, caused

n No stimuli • IFN^ m IFN-y+LPS • LPS

m

Allele 2

Allele 3 SLC11A1 promoter variants

FAC

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Fig. 2. SLC11A1 promoter variants transfected into U937 cells. The effect of ferric ammonium citrate (FAC) on levels of luciferase reporter gene activity driven by three polymorphic alleles for SLC11A1 detected in the study cohort, in relation to interferon-7 (IFN7) and bacterial lipopolysaccharide (LPS) enhanced expression, is illustrated. The mean normalized values obtained over three independent experiments, including triplicate transfections for each experiment, are graphed for SLC11A1 constructs. The following comparisons were statistically significant: allele 2 = no stimuli versus IFN7, p < 0.05; no stimuli versus IFN7 + LPS, p < 0.05; allele 3 = no stimuli versus FAC, p < 0.05; IFN7 versus FAC, p < 0.015; IFN-v + LPS versus FAC, p < 0.03; allele 5 = no stimuli versus IFN7, p < 0.05; no stimuli versus IFN7 + LPS, p < 0.05; allele 2 versus allele 3 = IFN7 + LPS, p < 0.03; FAC, p < 0.006; LPS, p < 0.04. Similar results were obtained for these variants transfected into THP-1 cells.

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an enhancement of luciferase reporter gene expression for all three constructs. The differences in baseline activity between alleles 2 and 3 were statistically significant with the addition of LPS (p < 0.04), LPS superimposed on the background of IFN7 (p < 0.03), and FAC (p < 0.006). Notably, the most significant effect between different alleles is observed with FAC. While this results in enhanced promoter activity for alleles 3 and 5, which is shown to be over-represented in MS patients compared with population-matched controls (Table 1), reduced expression in relation to LPS and 1FN7 is obtained with respect to allele 2. With the addition of FAC to the U937 compared with no stimuli, only alleles 3 Q?<0.05) and 5 (p<0.05) showed significantly increased expression. Low expression of allele 2, in the presence of high cellular Fe, may represent a mechanism whereby it exerts protection against autoimmune disease. Conversely, high expression of allele 3 upon Fe loading may be related to the body's response of withholding Fe from invading pathogens. The biological consequences of the SLC11A1 allelic response in relation to Fe loading (or depletion) in individuals genetically predisposed to various diseases warrant further studies. It is well-known that a significant inflammatory process may interfere with Fe delivery for heme biosynthesis. The Fe transport (NRAMP) proteins, SLC11A1 and the divalent metal iron transporter 1 (DMT1), are associated with the macrophage response to invading bacteria and may play a central role in this process. Both proteins are upregulated following bacterial infection (Govoni et al., 1999; Wardrop and Richardson, 2000). SLC11A1 is only expressed in macrophages and a few other tissues, such as liver, while DMT1 has been detected in most tissues.

2.3. Inter-Relationship between Iron Metabolism and Other Heavy Metals Both Fe deficiency and Fe overload are associated with an increased risk of infection. The natural resistance-associated proteins (SLC11A1 and DMT1) and regulation of the labile Fe pool appears to be central to this conundrum. Both SLC11A1 and DMT1 are divalent cation (Fe 2+ , Zn 2+ , and Mn 2+ ) transporters, providing a model for metal ion homeostasis in macrophages. SLC11A1 transports Fe and other divalent metal ion across membranes and regulates both intracellular pathogen proliferation and the

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macrophage inflammatory response (Govoni et al., 1997). SLC11A1 is also involved in the regulation of phagosomal pH (Hackam et al., 1998), which may be related to Fe deficiency resulting from abnormally high gastric pH. In the context of this review on the role of Fe metabolism in MS, it is important to note that this neurological disease and hyperuricemia (gout) appears to be mutually exclusive (Hooper et al., 1998). It seems likely that the significantly reduced levels of serum uric acid reported in MS patients, compared with controls, may be related to interference with Fe release/uptake, especially since nitric oxide implicated in this process seems to be involved in the pH-related regulatory pathways (Mulero and Brock, 1999). Further studies are, however, required to determine whether the findings of Hooper et al. (1998) are related to Fe release, since it is uncertain whether the pH effect is relevant to the exact location or environment of Fe uptake. The role of heavy metals in the pathogenesis of MS was supported by the finding that the frequency distribution of transferrin phenotypes differed from controls in a cohort of MS patients working at a manufacturing plant that uses zinc as a primary metal (Schiffer et al., 1994). These authors hypothesized that low-level chronic zinc exposure in the workplace increased the risk of MS in genetically susceptible individuals, resulting in an increased incidence of MS in these workers. DNA analysis of the CI (P570P) and C2 (P570S) variants of transferrin in South African MS patients and population-matched controls revealed similar allelic distributions. Further studies are warranted to determine whether variant C3 corresponds with mutation G277S in the transferrin gene as suggested by Lee et al. (2001), in order to eventually determine whether the association between MS and variant C3 reported by Schiffer et al. (1994) can be confirmed at the DNA level. This allele represents a risk factor for Fe deficiency anemia in menstruating women (Lee et al., 2001). Transferrin is involved in myelinogenesis and neuronal development (Lin and Connor, 1989; Espinosa de los Monteros et al, 1989), and it seems possible that different transferrin alleles may affect remyelination or immunologic processes differently. Transferrin plays a critical role in binding and transporting Fe, thereby reducing the toxic side effects of Fe. Although transferrin might be involved in the transport of a number of metals, such as aluminum, manganese, cadmium, copper, and zinc, Fe has the highest affinity to

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transferrin and will displace other metals. The affinity of transferrin for Fe is pH-dependent, in which Fe is released from transferrin when the pH is lower than 6.5. The primary function of transferrin is to accept Fe from plasma and to transport it to various cells and tissues. After Fe is released from transferrin, it passes through the endosomal membrane via the Fe transporter, DMT1, into the cytoplasm. Fe released in the cell can be utilized for the synthesis of heme or incorporated to Fe-containing molecules. Intracellular Fe can modulate the activity of Fe regulatory proteins or can be stored in ferritin complexes. Fe metabolism has also been functionally linked to the metabolism of copper, which can mediate DNA damage induced by hydrogen peroxide in the presence of low Fe conditions (Almeida et al., 2000). Further elucidation of the inter-relationship between the metabolism of Fe and other metals in the nervous system will increase our understanding of the pathophysiology and treatment of neurodegenerative diseases.

3. CONCLUSIONS As an essential nutrient and a potential toxin, Fe poses an exquisite regulatory problem in biology and medicine. Fe serves as a metal co-factor for many enzymes, including hemoproteins that are involved in a broad spectrum of crucial biological functions, such as oxygen binding (hemoglobins), oxygen metabolism (oxidases, peroxidases, catalases, and hydroxylases), and electron transfer (cytochromes). It has, therefore, became clear that the control of Fe metabolism is interconnected with other cellular pathways, and further analysis of these aspects are likely to provide a better understanding of the pathological processes in which Fe may participate. The enhanced expression of SLC11A1 alleles 3 and 5 of the Z-DNA forming promoter polymorphism in the presence of high cellular Fe reported in this study, in relation to allele 2, may represent a mechanism whereby protection against infectious agents known to play a role in MS could be achieved. Our findings raise the possibility that individuals who are genetically predisposed to MS may suffer serious consequences due to chronic Fe withholding following invasion by pathogens. If one or more of the several factors that can cause Fe deficiency/dysregulation could trigger off autoimmune MS, then it is likely that MS subtypes (Reich et al., 1998; Rooney et al., 1999) involving major Fe-related genes may

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exist. It would appear that most of the previous hypotheses formulated for the etiology of MS are linked together by the proposed dysregulation of Fe metabolism, which may disturb the heme biosynthesis pathway. If this hypothesis is proven, the challenge would be to administer Fe supplementation in such a way that poor expression of allele 2 is obtained, thereby protecting against autoimmune disease, while sufficient Fe is delivered for optimal functioning of cellular processes without compromising the immune defense against invading pathogens (which may trigger off porphyria). It has previously been shown that some of the autoimmune diseases associated with SLC11A1 may be linked to decreased Fe absorption or defective Fe supply for erythropoiesis, which can be effectively treated by intravenous Fe saccharate (Weber et al., 1988; Cazzola et al., 1996). The use of advanced technologies, such as DNA microarrays, to evaluate the effects of Fe deficiency and overload on gene expression could uncover novel genes whose expression is affected by cellular Fe levels and would eventually provide a clearer picture of the significance of Fe metabolism in health and disease. It seems possible that sequence variation in a variety of Fe-related genes, underlying defective proteins or differences in expression levels of alleles at these loci, may contribute to individual variability in Fe metabolism which, in turn, may contribute to genetic predisposition to many common diseases that may be treatable or preventable by manipulation of Fe status.

ACKNOWLEDGMENTS The study was supported financially by the Harry and Doris Crossley Foundation and the University of Stellenbosch. The South African Multiple Sclerosis Society is acknowledged for their support. Professor Louise Warnich and Dr. Lana du Plessis are acknowledged for helpful discussion and Dr. Armand V Peeters for technical assistance. Roberta N Rooney is thanked for her insight and valuable input in the study. Monique Zaahl received a Commonwealth Split-site Fellowship and bursaries from the University of Stellenbosch, South African Medical Research Council, and the Harry and Doris Crossley Foundation. Nico de Villiers received student bursaries from the University of Stellenbosch, Freda and Becker Trust, and the Harry and Doris Crossley Foundation.

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Harris ZL, Klomp LW, Gitlin JD. Aceruloplasminemia: An inherited neurodegenerative disease with impairment of iron homeostasis. Amer J Clin Nutr 1998; 67:972-977. Hofmeister A, Neibergs HL, Pokorny RM, Galandiuk S. The natural resistance-associated macrophage protein gene is associated with Crohn's disease. Surgery 1997; 122:3-178. Hooper DC, Spitsin S, Kean RB, Champion JM, Dickson GM, Chaudhry I, Koprowski H. Uric acid, a natural scavenger of peroxynitrate, in experimental allergic encephalomyelitis and multiple sclerosis. Proc Natl Acad Sci USA 1998; 95:675-680. Hulet SW, Powers S, Connor JR. Distribution of transferrin and ferritin binding in normal and multiple sclerotic human brains. J Neurol Sci 1999; 165:48-55. Kotze MJ, de Villiers JNP, Carr J, Schmidt S, Warnich L, Hillermann R. Lack of clinical expression of mutation C282Y implicated in haemochromatosis and porphyria in two sisters diagnosed with multiple sclerosis. Eur J Hum Genet 1999; 7:S59. Kotze MJ, de Villiers JNP, Rooney RN, Grobbelaar JJ, Mansvelt EPG, Bouwens CSH, Carr J, Stander I, du Plessis L. Analysis of the NRAMP1 gene implicated in iron transport: Association with multiple sclerosis and age effect. Blood Cells Mol Dis 2001; 27:44-53. Kwan T, Leber B, Ahuja S, Carter R, Gerstein HC. Patients with type 2 diabetes have a high frequency of the C282Y mutation of the hemochromatosis gene. Clin Invest Med 1998; 21:251-257. Lee GR. The anemia of chronic disease. Sent Hematol 1983; 20:61-80. Lee PL, Halloran C, Trevino R, Felitti V, Beutler E. Human transferrin G277S mutation: A risk factor for iron deficiency anaemia. Brit J Haematol 2001; 115:329-333. LeVine SM, Lynch SG, Ou CN, Wulser MJ, Tam E, Boo N. Ferritin, transferrin and iron concentrations in the cerebrospinal fluid of multiple sclerosis patients. Brain Res 1999; 821:1-515. Lieu PT, Heiskala M, Peterson PA, Yang Y. The roles of iron in health and disease. Mol Aspects Med 2001; 22:1-87. Lin HH, Connor JR. The development of the transferrin-receptor system in relation to astrocytes, MBP and galactocebroside in normal and myelin deficient rat optic nerves. Brain Res 1989; 49:265-274. Marquet S, Sanchez FO, Arias M, Rodrigues J, Paris SC, Skamene E, Schurr E, Garcia LF. Variants of the human NRAMP1 gene and altered human immunodeficiency virus infection susceptibility. J Infect Dis 1999; 180:1521-1525. Meyer UA, Schuurmans MM, Lindberg RLP. Acute porphyrias: Pathogenesis of neurological manifestations. Sem Liver Dis 1998; 18:43-52. Moalem S, Percy ME, Andrews DF, Kruck TP, Wong S, Dalton AJ, Mehta P, Fedor B, Warren AC. Are hereditary hemochromatosis mutations involved in Alzheimer's disease? Amer J Med Genet 2000; 93:58-66. Mulero V, Brock JH. Regulation of iron metabolism in murine J774 macrophages: Role of nitric oxide-dependent and independent pathways following activation with gamma interferon and lipopolysccharide. Blood 1999; 94:2383-2389. Rasmussen HB, Clausen J. Genetic risk factors in multiple sclerosis and approaches to their identification. J Neurovirol 2000; 2:S23-S27.

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Reich B, Ehlers I, Will H, Heesen C, de Villiers JNP, Rooney RN, Kotze MJ. Autoantibodies against nucleolar structures correlate significantly with an unusual association between multiple sclerosis and porphyria symptoms. J Neuroimmunol 1998; 90:S77. Roberts AG, Whatley SD, Morgan RR, Worwood M, Elder GH. Increased frequency of the haemochromatosis Cys282YTyr mutation in sporadic porphyria cutanea tarda. Lancet 1997; 349:321-323. Roesser HR Iron metabolism in inflammation and malignant disease. In: Jacobs A, Worwood M, editors. Iron in Biochemistry and Medicine. Academic Press, 1980: 605-640. Roest M, van der Schouw YT, de Valk B, Marx JJ, Tempelman MJ, de Groot PG, Sixma JJ, Banga JD. Heterozygosity for a hereditary hemochromatosis gene is associated with cardiovascular death in women. Circulation 1999; 100:1268-1273. Rohlfs EM, Shaheen NJ, Silverman LM. Is the hemochromatosis gene a modifier locus for cystic fibrosis? Gene Test 1998; 2:85-88. Rooney RN, Kotze MJ, de Villiers JNP, Hillermann R, Cohen JA. Multiple sclerosis, porphyria-like symptoms and a history of iron deficiency anemia in a family of Scottish descent. Amer J Med Genet 1999; 86:194-196. Salter-Cid L, Brunmark A, Peterson PA, Yang Y. The major histocompatibility complexencoded class I-like HFE abrogates endocytosis of transferrin receptor by inducing receptor phosphorylation. Genes Immun 2000; 1:409-417. Schiffer RB, Wetkamp LR, Ford C, Hall WJ. A genetic marker and family history study of the upstate New York multiple sclerosis cluster. Neurology 1994; 44:329-333. Searle S, Blackwell JM. Evidence for a functional repeat polymorphism in the promoter of the human NRAMP1 gene that correlates with autoimmune versus infectious disease susceptibility. 7 Med Genet 1998; 36:295-299. Tuomainen TP, Kontula K, Nyyssonen K, Lakka TA, Helio T, Salonen JT. Increased risk of acute myocardial infarction in carriers of the hemochromatosis gene Cys282Tyr mutation: A prospective cohort study in men in eastern Finland. Circulation 1999; 100:1274-1279. Valberg LS, Flanagan PR, Kertesz A, Ebers GC. Abnormalities in iron metabolism in multiple sclerosis. Canad J Neurol Sci 1989; 16:184-186. Vitale E, Cook S, Sun R, Specchia C, Subramanian K, Rocchi M, Nathanson D, Schwalb M, Devoto M, Rohowsky-Kochan C. Linkage analysis conditional on HLA status in a large North American pedigree supports the presence of a multiple sclerosis susceptibility locus on chromosome 12pl2. Hum Mol Genet 2002; 11:295-300. Wardrop SL, Richardson DR. Interferon-gamma and lipopolysaccharide regulate the expression of Nramp2 and increase the uptake of iron from low relative molecular mass complexes by macrophages. Eur J Biochem 2000; 267:6586-6593. Weber J, Werre JM, Julius HW, Marx JJ. Decreased iron absorption in patients with active rheumatoid arthritis, with and without iron deficiency. Ann Rheumatic Dis 1988; 47:404-409.

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Weiss G. Iron and immunity: A double-edged sword. Eur J Clin Invest 2002; 32:S70-S78. Weller M, List U, Schabet M, Melms A, Dichgans J. Elevated CSF lactoferrin in superficial siderosis of the central nervous system. J Neurol 1999; 246:3-945. Yaouanq J, Grosbois B, Jouanolle AM, Goasguen J, Leblay R. Haemochromatosis Cys282Tyr mutation in pyridoxine-responsive sideroblastic anemia. Lancet 1997; 349:1475-1476. Zeman D, Adam P, Kalistova H, et al. Transferrin in patients with multiple sclerosis: A comparison among various subgroups of multiple sclerosis patients. Acta Neurol Scand 2000; 101:89-94.

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Manganese Toxicity: A Critical Reappraisal Patrizia Vernole, Maria Morello, Giuseppe Sancesario, Alessandro Martorana, Giorgio Bernardi, Antonella Canini, Palma Mattioli

ABSTRACT Manganese is an essential trace element for many living organisms. It is mostly known as a co-factor for the activity of some important enzymes, like manganese-dependent superoxide dismutase in the mitochondria of neurons and glial cells, and glutamine synthetase in the cytoplasm of astrocytes. The use of electron microscopy imaging and electron energy loss spectroscopy, associated with a transmission microscope, revealed the distribution of manganese in specific cellular structures, emphasizing its localization not only in mitochondria and lysosomes, but also in the nucleus, nucleolus, and likely ribosomes. The functional role of this distribution is not yet clear. We summarize some studies on the involvement of manganese in nucleic acid functions. In the nucleus, manganese is involved in chromatin and chromosomes' condensation, and can be a physiological stimulating co-factor of DNA and RNA polymerases. Increased manganese concentrations in vitro can induce errors in DNA synthesis. Keywords: Manganese; nucleic acids; toxicity; nervous system; enzymes.

1. INTRODUCTION The metal manganese (Mn) is a trace element, that is, its amount in living cells is quite low. In liver, the total concentration of Mn is about 35 |xM (based on cell water content), but free Mn 2+ is less than 1 (xM (Ash and Schramm, 1982). It has been known for a long time that chronic excess of Mn in mammals induces an atypical Parkinsonian syndrome, which is often accompanied by severe dystonia of the trunk and limbs, and can be 415

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preceded by behavioral changes and psychiatric manifestations (Pal et al., 1999; Barbeau, 1984). Such signs and symptoms indicate selective impairment of basal ganglia functions. The mechanisms of such specific Mn toxicity are, however, still poorly understood. Analogous to Parkinsonisms produced by deficient dopaminergic neurotransmission in the striatum, the toxic effects of Mn on the brain were first thought to be dependent on its interaction with dopamine metabolism, emphasizing the potential role of Mn 3+ or its complexes for dopamine oxidation in dopamine-rich nigrostriatal nerve terminals (Archibald and Tyree, 1987; Donaldson 1987). However, positron emission tomography studies have not demonstrated any significant presynaptic or post-synaptic nigrostriatal dopaminergic dysfunction in patients with chronic Mn intoxication (Wolters et al., 1989). Unlike idiopathic Parkinson's disease sustained by loss of neurons in the substantia nigra, the atypical Parkinsonian features in chronic Mn intoxication is accompanied by a loss of neurons and the proliferation of astrocytes in the globus pallidus, nucleus caudate, and putamen, with little or no evidence of histological damage in the substantia nigra (Banta and Markesbery, 1977; Barbeau, 1984; Feldman, 1994; Olanov et al., 1996). The ability of Mn to induce such selective lesions correlates with marked regional differences in the distribution of Mn in the monkey brain, with the putamen and the globus pallidus showing the highest Mn concentrations after acute or chronic injections (see Feldman, 1994). Fractionation biochemical studies suggest that the increased accumulation of Mn at the whole tissue level may be accounted for by a selective distribution of Mn in mitochondria and lysosomes, which appear to absorb the bulk of Mn overload (Aschner and Aschner, 1990; Gavin et al., 1990; Suzuki et al., 1983). Accordingly, several studies have pointed out the role of Mn in disrupting mitochondrial Ca 2+ homeostasis, inhibiting oxidative phosphorylation, and decreasing cellular energy supplies (Aschner and Aschner, 1990). To date, Mn toxicity does not seem to be dependent on a single metabolic dysfunction. Instead, different detrimental events have been hypothesized: coupling Mn uptake with coincident transport of aluminum and iron in the brain, altered calcium kinetics in mitochondria, increased production of reactive oxygen species, and reduced energy status of neurons (Verity, 1999). The wide range of Mn biological activities could be

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explained by its ability, as a divalent cation, to bind negatively charged nitrate, phosphate, and carboxylate groups so that, like other transition metals, it is able to react with proteins and, in particular, can inhibit or activate various enzymes. Mn, however, is also a necessary co-factor for some metalloenzymes or manganoproteins (Christianson, 1997), such as the Mn superoxide dismutase (MnSOD) and the glutamine synthetase (GS) (Prohaska, 1987; Wedler and Denman, 1984). MnSOD is present in the mitochondria of neurons and glial cells (Medina et al. 1996; Inagaky et al., 1991), where it catalyzes by redox cycles the dismutation of superoxide anion into molecular oxygen and hydrogen peroxide. Unlike MnSOD, GS is a cytosolic enzyme specifically contained in glial cells. It accounts for approximately 80% of total Mn in the brain, where it catalyzes the conversion of glutamic acid into glutamine (Wedler and Denman, 1984; Feldman, 1994).

2. SUBCELLULAR LOCALIZATION OF MANGANESE Lai et al. (1999) performed a study on rat after chronic oral intake of MnCl. They found accumulation of Mn not only in the mitochondria, but also in synaptosomes and nuclei, suggesting that these latter structures, along with the mitochondria, constitute putative subcellular targets of Mn toxicity. All previous studies describing the physiology and toxicology of Mn were performed by means of biochemical analyses on tissue homogenates. This was due to a lack of cytochemical methods that could resolve Mn content in subcellular structures. The combined use of electron spectroscopy imaging (ESI), electron energy loss spectroscopy (EELS), and transmission electron microscopy provides an opportunity to detect directly the spatial distribution of Mn in fixed brain tissue without separating cells or fractionating their intracellular organelles (Morello et al., 2002). An ongoing study in our laboratory with ESI and EELS has shown, in normal rat, the detailed subcellular distribution of Mn in the neurons and glial cells of the putamen and globus pallidus. It also emphasized the higher density of Mn bound not only to the mitochondria and lysosomes, but also to the cytoplasm, nuclei, and nucleoli, suggesting that the nucleus and nucleolus can be among the main sites of Mn activity. We briefly summarize these results.

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2.1. Manganese in Cytoplasm of Neurons and Glia Except for mitochondria and lysosomes, Mn present in the cytoplasm could not be associated with certainty to specific organelles in our study, since the cytoplasm has a sponge-like appearance in ESI microphotographs. Therefore, Mn can presently be considered to be bound to unidentified cytoplasm structures. It is conceivable, however, that this Mn could be partly associated with the membrane of endoplasmic reticulum, and mainly with ribosomes, due to the similar distribution and approximate diameters of Mn granules and ribosomes in the cytoplasm.

2.2. Manganese in Nucleus Mn binds, to the same extent, wherever heterochromatin is present in the nucleus of neurons and glial cells, according to obvious differences in their nuclear morphology. The high density of Mn in the nuclear heterochromatic regions suggests that Mn binding can be related to the functional state of DNA, since heterochromatin consists of the inactive regions of the chromosomes.

2.3. Manganese in Nucleolus The very high content of Mn in the nucleolus of striatal and pallidal neurons was a new finding. At present, its role is open to speculation. The nucleolus contains the genes of r-RNA in an active state, cytologically corresponding to the so-called nucleolar organizer region where ribosomal RNA synthesis and ribosome assembly take place (Schwarzacher and Mosgoeller, 2000). The accumulation of ribosomal RNA in glial cells cannot be discerned from the heterochromatin regions under the electron microscopy, but we suppose that high levels of Mn are bound to neuronal and glial nuclei wherever the ribosomal DNA regions of different chromosomes are clustered.

3. MANGANESE AND NUCLEIC ACIDS It is evident that the distribution of Mn does not simply match that of manganoproteins or other Mn-sensitive enzymes. Mn binds tightly in the nucleus to heterochromatin and nucleoli, and in the cytoplasm to mitochondria and, possibly, ribosomes and other structures. This binding could be aspecific without a clear physiological relevance. Alternatively,

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Mn binding to such heterogenous structures may have a general role as a prosthetic group: keeping nucleic acids in a suitable conformation and affecting their activity considerably, as partly suggested by previous biochemical studies (Han et al., 1991; Pan et al., 1993; Xu and Bremer, 1997). The notion that Mn is constitutively bound to DNA and RNA molecules prompted us to summarize the most relevant studies on the relationship between Mn and nucleic acids. The ability of Mn to interact with the nucleotides of DNA, RNA, and ribozymes has been demonstrated by numerous biochemical experiments (Han et al, 1991; Jouve et al., 1975; Kimes and Morris, 1973; Mullen et al., 1990; Pan et al., 1993; Pyle, 1996; Vogtherr and Limmer, 1998; Xu and Bremer, 1997). These studies suggest that Mn ions can neutralize the negative charge of nucleotides and phosphate groups, thereby stabilizing the RNA and DNA filaments (Jouve et al, 1975). Xu and Bremer (1997) suggested that Mn, as a divalent cation, can form ionic bonds with the phosphate residues of adjacent nucleotides and, thus, cause an overwinding of the DNA helix.

3.1. Manganese and DNA Mn 2+ can influence the activity of enzymes responsible for DNA and RNA synthesis. Low concentrations of Mn 2+ can increase significantly the rate of deoxynucleotide triphosphate (dNTP) polymerization and, hence, DNA synthesis. This increase might be related to the observations that the presence of Mn 2+ can decrease the fidelity of DNA replication (Van de Sande et al, 1982; Beckman et al., 1985), by modifying the efficiency of DNA polymerases, such as the beta polymerase and Klenow fragment (Beckman et al., 1985; Pelletier et al., 1996). Moreover, Mn 2+ can substitute Mg 2+ to stimulate calf thymus primase (Grosse and Krauss, 1985); low concentrations of Mn significantly increase the rate of both initiation and elongation in a template-dependent manner by enhancing the ability of primase to incorporate NTPs, as already described for DNA polymerases. In vitro Mn can also alter the sequence specificity for primer synthesis, leading the enzyme to prefer sequences in the template similar to those generally used in vivo. Mn 2+ also decreases the sensitivity of primase to inhibitions by anions. All the effects can be observed in the presence of high concentrations of Mg 2+ , which indicates that Mn 2+ has

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specific sites of interaction with the enzyme that are not occupied by Mg 2+ (Kirk and Kuchta, 1999). However, the real importance of these observations has yet to be established, since concentrations used in vitro were in the range of 50|JLM to 100 (JLM (Kirk and Kuchta, 1999) while in vivo Mn 2+ concentrations are about 2 fxM to 30 |JLM (Brandt and Schramm, 1986).

3.2. Manganese and Chromatin MnCl2 and other divalent ions can induce compaction of chromatin (Borochov et al., 1984). Chromatin structure in cell nuclei, or superhelical DNA conformation in isolated chromatin, is maintained at cation concentrations of 5mM to 1 mM for Ca 2+ , Mn 2+ , and Mg 2+ . The same cations probably also have a role in modulating DNA-histones interactions (Darzynkiewicz et al., 1976). The rate of transcription of DNA is not significantly affected by the degree of chromatin condensation or the state of higher-order coiling influenced by the cations (Chegini et al., 1981). A significant decrease in the intrinsic optical anisotropy of DNA has been found in the presence of Mn 2+ and Cu 2+ , but not Mg 2+ . These observations are interpreted in terms of a specific organization of DNA in a compact rigid structure in the presence of Mn 2+ or Cu 2+ , but a nonspecific coil in the presence of Mg 2+ (Emonds-Alt et al., 1979). It is known that the state of phosphorylation of specific histones is also related to chromatin condensation and, possibly, to gene expression. Mn seems to be involved in these functions. In fact, the activity of rabbit muscle protein phosphatase-1 in histone phosphorylation is strongly stimulated by Mn 2+ (Zhao et al., 1994). In the rat, Mn 2+ stimulates phosphorylation of some histones and inhibits that of others, while other divalent cations do not have the same effects (Brockenbrough and Korc, 1990).

3.3. Manganese and RNA Synthesis and Processing As described above for primase, Mn can stimulate RNA polymerase activity. In particular, Mn 2+ and Mg 2+ are equally effective in activating RNA polymerase I, while RNA polymerase II is stimulated more effectively by Mn 2+ than by Mg 2+ . The third enzyme, RNA polymerase III, is stimulated slightly more efficiently by Mn 2+ than Mg 2+ . The relative sensitivity of

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polymerase activity to these cations was once used for identification purposes, although selective enzyme inhibitors are now preferred. For RNA polymerase I, Mn is a better effector than magnesium in starting the reaction; for elongation, both ions are good effectors. Moreover, a high concentration of Mn is inhibitory (Nagamine et al., 1978). Mn is also involved in the processing of RNA in eukaryotic cells. 2+ Mn can be useful in some splicing reactions. For example, two or more metal ions (such as Mn and Magnesium or Zinc and Magnesium) are necessary for the self-splicing of group I introns in the pre-mRNA from the nrdB gene in bacteriophage T4. Mn also seems necessary for the transesterification reactions of the first step of self-splicing of group I introns in tetrahymena (Sjogren et al., 1997). Also, spliceosoma ribonucleoprotein complex appears, in other cell types, to be a metalloenzyme with functional similarities to the system used in tetrahymena (Sontheimer et al., 1997). Anyway, most known RNA-catalyzed reactions either require or are greatly stimulated by divalent metal ions, which can play structural roles or participate directly in RNA catalysis (Pan et al., 1993). The capping reaction at the 5' extremity of eukaryotic mRNA can also involve Mn ions. The capping enzyme, 8GTP-RNA guanylyltransferase, which is responsible for the reaction of guanylation, requires GTP binding. The enzyme then undergoes a conformational change to become an active form, able to bind magnesium or Mn ions and promotes the guanylation of mRNA (Hakansson et al., 1997). Besides mRNA, the maturation of ribosomal RNA also requires Mn. Comparison of specific cleavage patterns of 16S and 23 S rRNA with different divalent cations has demonstrated the presence of sites specific to a particular metal, including Mn (Kramer 1995). The biogenesis of intronencoded small nucleolar RNAs (snoRNAs) requires Mn 2+ . In fact, the endoribonuclease that excises the snoRNAs from the larger RNA precursors in xenopus laevis needs Mn as a co-factor in a wide range of concentrations, from 0.5mM to 5-10mM (Caffarelli et al., 1997). Divalent metal ions are also essential to the structure and catalytic activities of ribosomes (Dorner and Barta, 1999).

4. CONCLUSIONS In vitro studies have described the ability of Mn and other divalent cations (that is, Mg 2+ and Ca 2+ ) to interact with different mitochondrial enzymes

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(Senior, 1981; Smith et al., 1985), as well as with DNA and RNA molecules and their metabolism (Kimes and Morris, 1973; Pan et al., 1993; Pyle, 1996; Vogtherr and Limmer, 1998). In the nucleus, Mn can be a stimulating co-factor of DNA and RNA polymerases, while increased Mn concentrations in vitro can induce errors in DNA synthesis. Mn is also involved in chromatin and chromosomes condensation. However, it is important not to confuse the ability of Mn 2+ to substitute for Mg 2+ in vitro with any normal in vivo function, since the concentration of Mg 2+ in vivo is much higher than the concentration of Mn. Our ESI study demonstrates that nucleic acids normally contain Mn, suggesting that this presence may be physiologically relevant to nucleic acid metabolism. Further studies should clarify whether the nucleus and the nucleolus participate in the accumulation of Mn overload and constitute, along with mitochondria, the putative subcellular targets of Mn toxicity.

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Morello M, Canini A, Grilli Caiola M, Martorana A, Mattioli P, Bernardi G, Sancesario G. Manganese detected by electron spectroscopy imaging and electron energy loss spectroscopy in mitochondria of normal rat brain cells. J Trace Microprobe Tech 2002; 20:481-491. Mullen GP, Serpersu EH, Ferrin LJ, Joeb LA, Mildvan AS. Metal binding to DNA polymerase I, its large fragment, and two 3'-5'-exonuclease mutants of the large fragment. J Biol Chem 1990; 265:14327-14334. Nagamine Y, Mizuno D, Natori S. Differences in the effects of manganese and magnesium on initiation and elongation in the RNA polymerase I reaction. Biochim Biophys Acta 1978; 519:440-446. Olanov CW, Good PF, Shinotoh H, Hewitt KA, Vingerhoets F, Snow BJ, Beal MF, Calne DB, Perl DP. Manganese intoxication in the rhesus monkey: A clinical, imaging, pathologic, and biochemical study. Neurology 1996; 46;492^498. Pal PK, Samii A, Calne DB. Manganese neurotoxicity: A review of clinical features, imaging and pathology. Neurotoxicology 1999; 20:227-238. Pan T, Long DM, Uhlenbeck OC. Divalent metal ions in RNA folding and catalysis. In: Gesteland RF, Atkins JF, editors. The RNA World. New York: Cold Spring Harbor Laboratory Press, 1993: 271-302. Pelletier H, Sawaya MR, Wolfe W, Wilson SH, Kraut J. A structural basis for metal ion mutagenisity and nucleotide selectivity in human DNA polymerase beta. Biochemistry 1996; 35:12762-12777. Prohaska JR. Functions of trace elements in brain metabolism. Physiol Rev 1987; 67:858-910. Pyle AM. Role of metal ions in ribozymes. Met Ions Biol Syst 1996; 32:479-520. Ram BP, Munjal DD. Galactosyltransferases: Physical, chemical and biological aspects. CRC Crit Rev Bioch 1985; 17:257-311. Schwarzacher HG, Mosgoeller W. Ribosome biogenesis in man: Current views on nucleolar structure and function. Cytogenet Cell Genet 2000; 91:243-252. Senior AE. Divalent metals in beef heart mitochondrial adenosine triphosphate. Demonstration of the metals in membrane-bound enzyme and studies on the interconversion of the "1-Mg" and "2-Mg" forms of the enzyme. J Biol Chem 1981; 256:4763-4767. Sjogren AS, Petterson E, Sjoberg BM, Stromberg R. Metal ion interaction with cosubstrate in self-splicing of group I introns. Nucl Ac Res 1997; 25:648-653. Smith RA, Latchney LR, Senior AE. Tight divalent metal binding to Escherichia coli Fladenosinetriphosphatase. Complete substitution of intrinsic magnesium by manganese or cobalt and studies of metal binding sites. Biochemistry 1985; 24:4490^4494. Snyder RD. Role of active oxygen species in metal-induced DNA strand breakage in human diploid fibroblasts. Mutat Res 1988; 193:237-246. Sontheimer EJ, Sun S, Piccirilli JA. Metal ion catalysis during splicing of premessenger RNA. Nature 1997; 388:801-805. Suzuki H, Wada O, Inoue K, Tosaka H, Ono T. Role of brain lysosomes in the development of manganese toxicity in mice. Toxicol Appl Pharmacol 1983; 71:422^429.

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Van de Sande JH, Mcintosh IP, Jovin TN. Mn 2+ and other transition metals at low concentrations induce the right-to-left helical transformation of poly d(G-C). EMBO J 1982; 1:777-782. Verity MA. Manganese neurotoxicity: A mechanistic hypothesis. Neurotoxicology 1999; 20:489^198. Vogtherr M, Limmer S. NMR study on the impact of metal ion binding and deoxynucleotide substitution upon local structure and stability of small ribozyme. FEBS Lett 1998; 433:301-306. Wedler FC, Denman RB. Glutamine synthetase: The major Mn(II) enzyme in mammalian brain. Curr Top Cell Regul 1984; 24:153-169. Wolters EC, Huang CC, Clark C, Peppard RF, Okada J, Chu NS, Adam MJ, Ruth TJ, Li D, Calne DB. Positron emission tomography in manganese intoxication. Ann Neurol 1989;26:647-651. Xu YC, Bremer H. Winding of the DNA helix by divalent metal ions. Nucleic Acid Res 1997;25:4067-4071. Zhao S, Zhang Z, Lee YC. Comparison of the enzymatic activities of native and recombinant protein phosphatase-1 towards histone. Biochem Mol Biol Int 1994; 34:1027-1033.

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CHAPTER

18

Cupric and Mercuric Ions Affect the Structure and Functions of Cell Membranes Mario Suwalsky, Fernando Villena, Hernan Cardenas, Beryl Norris, Carlos Patricio Sotomayor, Paolo Zatta

ABSTRACT Copper and mercury play a toxic role in several pathological processes, including neurodegeneration. The structural effects of Cu 2+ and Hg 2+ on cell membranes were studied through their interactions with human erythrocytes, isolated resealed membranes, and molecular models. Both ions induced shape changes in erythrocytes, which took the form of echinocytes due to location of the ions in the outer monolayer of the erythrocyte membrane. Fluorescence spectroscopy analysis revealed that the interactions occurred in the polar region of me membrane, that is, at the hydrophobic-hydrophilic interface. X-ray experiments indicated that both ions interacted with the polar groups of phospholipids located in the outer monolayer of the erythrocyte membrane. Finally, electrophysiological measurements of toad skin showed that Cu 2+ and Hg 2+ inhibited the active transport of ions. The experimental results confirmed the toxicity of both heavy metals on membrane structure and functions. Keywords: Copper; mercury; membranes; metal ions; erythrocytes.

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1. INTRODUCTION The contamination of cells by metallic ions may be described as a sequence of processes starting with interactions between the cations and some components of the cell membrane, followed by transport across the membrane and involvement with different cytoplasm and/or nuclear components. The cell membrane is a diffusion barrier that protects the cell interior. Therefore, its structure and functions are susceptible to alterations as a consequence of interactions with heavy metals. This chapter describes the molecular mechanisms of the interaction of Cu 2+ and Hg 2+ ions with cell membranes. Copper dismetabolism in the brain has not received sufficient attention considering that the main forms of copper in living organisms are present as copper proteins (such as ceruloplasmin, metallothioneins, superoxide dismutase, and cytochrome oxidase). Free copper in the brain is toxic and leads to neuronal and cellular damage through free radical generation (Parmar and Day a, 2001). Copper has been described as a relevant etiopathogenic co-factor in several neuropathologies, such as Menkes's and Wilson's disease (Strausak et al., 2001), and amyotrophic lateral sclerosis (Hartmann and Evenson, 1992). Besides copper accumulation, a neuronal copper deficiency has been invoked as a possible etiological factor in the more common neurodegenerative diseases, such as Alzheimer's disease, amyotrophic lateral sclerosis and, more recently, prion diseases (Brown, 2001, 2003). The toxic effects of Cu2+ ions on membrane structure and functions have been reported, such as changes in the permeability (Gwozdzinski, 1991) and direct hemolysis of erythrocytes (Gwozdzinski, 1991; Sansinanea et al., 1994), conductance changes in ionic channels and pumps of different cell membranes (Kiss and Osipenko, 1994a), decrease of neuronal membrane fluidity (Ohba et al., 1994), and serious destruction of the cell membranes of human peripheral lymphocytes and monocytes (Steffensen et al., 1994). These effects have been ascribed to Cu2+ interaction with membrane proteins (Gwozdzinski, 1991; Kiss and Osipenko, 1994a), to changes in the lipid region of the membrane without involving proteins (Rock et al., 1995), and to lipid peroxidation (Kiss and Osipenko, 1994b). Cu2+ has also been demonstrated to play a crucial role in the pathogenesis of various neurodegenerative diseases (Perry et al., 2002). Several neurotoxic effects induced by Hg 2+ ions have been described at the cellular and molecular levels. Hg 2+ modulates 7-aminobutyric acid-activated and voltage-activated chloride channels, increases the

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release of neurotransmitters at the frog neuromuscular junction, reduces the Na+/K+-ATPase activity in the brain, prevents phosphorylation, modulates the metabolism of mRNA, and affects the calcium fluxes of synaptosomes (Kiss and Osipenko, 1994b; Biisselberg et al., 1994). Mercury chloride at nanomolar levels inhibits glutamate uptake and stimulates glutamate release in cultured astrocytes (Albrecht and Matyja, 1996). It has been reported that mercury is elevated in Alzheimer's disease (Ely, 2001; Saxe et al., 1999) in most regions studied (Cornett et al., 1998) and has been implicated in amyotrophic lateral sclerosis and Parkinson's disease (Carpenter, 2001). It accumulates in brain cells, especially in astroglia (Tiffany-Catiglion and Qian, 2001). In particular, inorganic mercury could remain within neurons indefinitely; in female mice, for instance, mercury was seen in motor neurons at half the exposure times of male mice (Pamphlett and Coote, 1998). On the other hand, it has been reported that Hg 2+ caused serious destruction of human lymphocyte and monocyte membranes (Steffensen et al., 1994), and decreased human placenta (Boadi et al., 1992) and rat red cell membrane fluidity (Delnomdedieu and Allis, 1993).

2. A MODELLISTIC APPROACH Membranes are often involved in neuropathological processes. Therefore, membrane modellistic approaches are widely used to shed light on the interaction between metal ions and biological membranes (Zatta and Suwalsky, 2001). With the aim to better understand the molecular mechanisms of this kind of interaction, we utilized three well-established, paradigmatic models in neurotoxicological studies. Such models regard human erythrocytes (Shohet and Mohandas, 1988), molecular models of biomembranes, and the isolated toad skin. The erythrocyte membrane is primarily a lipid protein structure, where lipids exist in a bilayer crossed by several intrinsic membrane proteins and are closely associated with a matrix of extrinsic membrane proteins. The composition of the lipids in human erythrocytes within the bilayer is well documented and consists of about 50% cholesterol and 50% phospholipids by weight. The major class of phospholipids is phosphatidylcholine (29%), sphingomyelin (24% to 26%), phosphatidylethanolamine (29% to 31%), and phosphatidylserine (12% to 14%) (Lubin et al., 1989). The molecular models of biomembranes consisted of multilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representing the

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phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively (Devaux and Zachowsky, 1994). Chemically, these two phospholipids only differ in their terminal amino groups, being + NH 3 in DMPE and + N(CH 3 ) 3 in DMPC. Moreover, both molecular conformations are very similar: their acyl chains are mostly parallel and extended with the polar groups lying perpendicular to them. However, DMPE packs tighter than DMPC (Suwalsky, 1996). This effect, due to smaller DMPE polar groups and higher effective charge, makes for very stable bilayer arrangement that is not perturbed by water or by a number of compounds (Suwalsky et al., 1994). On the other hand, the gradual hydration of DMPC leads to water filling the highly polar interbilayer spaces. These conditions promote the incorporation of foreign molecules into DMPC bilayers, their interaction with the lipid chemical groups, and the ensuing perturbation of DMPC molecular arrangement. The capacity of the metal ions to perturb the multilayer structures of DMPC and DMPE was determined by X-ray diffraction. Intact human erythrocytes incubated with the metal ions were observed by scanning electron microscopy (SEM) whereas their membranes, consisting of resealed ghosts, were studied by fluorescence spectroscopy. In the epithelia, such as the toad skin, the two-membrane hypothesis has served as an excellent model in the study of Na + transport (Rytved et al., 1995; Ussing, 1994). According to this hypothesis, Na + diffuses into cells at the outer (mucosal or apical) membrane and is actively extruded across the inner (serosal or basolateral) membrane by Na+/K+-ATPase in exchange for K + . The short circuit current is the amount of current necessary to bring the potential difference across the skin down to zero and measures active Na + transport. All these systems and techniques have been used in our laboratories to determine the interaction with, and membrane-perturbing effects of metal ions (Suwalsky et al., 2002), pesticides (Suwalsky et al., 2001a) and drugs (Suwalsky et al., 2001b).

2.1. Scanning Electron Microscope (SEM) Studies of Human Erythrocytes 2.7.7. Effects of Cu2+ ions SEM observations (Fig. IB) revealed that human red blood cells incubated with 0.1 mM CuCl2 lost their normal discoid shapes. Figure 1C shows that

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Fig. 1. Scanning electron microscopy (SEM) images of human erythrocytes (magnification X 2,900). (A) Control. Incubated with (B) 0.1 mM CuCl2 and (C) 1 mM HgCl2.

Cu 2+ ions induced crenation, that is, abnormalities consisting of a spiny configuration. This effect was observed in various degrees of intensity, ranging from a few roundish blebs to a generalized shape change that took the form of echinocytes, that is, erythrocytes with numerous protuberances or spicules over their surfaces.

2.1.2. Effects of Hg2+ ions Human red cells were incubated with 0.1 mM and ImM HgCl2. The SEM observations indicated that the lower concentration did not induce

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a significant change in the shape of the erythrocytes. However, 1 mM Hg ions produced echinocytosis (Fig. 1C); a few cells also showed stomatocytosis, with evagination in one surface and depression in the opposite surface.

2.2. Fluorescence Measurements of Isolated Reseated Human Erythrocytes Membranes 2.2.1. Effects of Cu2+ ions The effect of Cu 2+ on the lipid matrix of the erythrocyte membrane was evaluated at the deep hydrophobic core and at the hydrophobic-hydrophilic interface, that is, the phospholipid polar group level, by means of 1,6diphenyl-l,3,5-hexatriene (DPH) steady-state fluorescence anisotropy and laurdan fluorescence spectral shifts (quantified through general polarization, GP), respectively. As shown in Table 1, increasing concentrations of Cu 2+ of up to 1 mM did not significantly affect the anisotropy of DPH. This result implied that Cu 2+ did not affect the hydrophobic acyl chain region of the membrane. However, the abovementioned Cu2+ concentration range produced an 18% increase of the laurdan GP value, which indicates a decrease in the molecular dynamics or water penetration at the phospholipid polar group level. This can be explained in terms of strong interactions of Cu 2+ ions with the phospholipid polar head groups, leading to a net ordering effect at the hydrophobic-hydrophilic interface both in the bulk and in the lipid-protein boundary.

2.2.2. Effects of Hg2+ ions The results are summarized in Table 1. Hg 2+ ions induced a moderate concentration-dependent increase in the fluorescence anisotropy of DPH Table 1. Effect of Cu 2+ and Hg 2+ on the anisotropy (r) of 1, 6-diphenyl-l,3,5-hexatriene (DPH) and the general polarization (GP) of laurdan embedded in human erythrocyte membranes. Cu 2+

Hg 2+

Concentration (mM)

r DPH

GP laurdan

r DPH

GP laurdan

0 0.1 1

0.267 0.267 0.265

0.384 0.428 0.454

0.230 0.233 0.258

0.268 0.271 0.336

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and a greater increase of laurdan GP; in the 0 to 1 mM range, they were of the order of 7% and 25%, respectively. These results can be explained in terms of strong interactions of Hg 2+ ions with phospholipid polar heads and/or protein groups located in the hydrophobic-hydrophilic interface, which induce tighter molecular packing.

2.3. X-Ray Diffraction Analysis of Phospholipid Bilayers 2.3.1. Effects of Cu2+ ions Figure 2A shows the results obtained after DMPC was mixed and had interacted with water and Cu 2+ solutions. As expected, pure water altered the structure of DMPC: its bilayer width expanded from about 55 A in the dry state to 65 A when immersed in water, and the reflections were reduced to only the first three orders of the bilayer width. On the other hand, a new and strong reflection of 4.2 A showed up. Its appearance was indicative of the fluid state reached by DMPC bilayers. In fact, it corresponds to the average separation of the fully extended hydrocarbon chains organized with rotational disorder in a hexagonal lattice. The effects of Cu 2+ ions were concentration-dependent: 10~5 M induced a slight weakening of the low angle reflections, which disappeared at 10~3M, an action pointing to a complete structural disturbance of DMPC polar head region. Hence, the hydrophobic chains were somewhat disordered, as indicated by the slightly decreased intensity of the 4.2 A reflection. Results of the interaction of DMPE with water and Cu 2+ solutions (Fig. 2B) show that a Cu 2+ concentration of as high as 0.1 M did not significantly perturb DMPE multilayers.

2.3.2. Effects of Hg2+ ions DMPC multilayers were incubated with water and Hg 2+ solutions. Figure 2C discloses that exposure to 10" 5 M Hg 2+ ions induced a marked decrease in the phospholipid low angle reflection intensities, which were about 75% lower than those of Hg2+-free DMPC. This result implied that the low concentration of Hg 2+ ions induced molecular disorder of the lipid bilayer, particularly of the polar head region. However, 10~4 M Hg 2+ increased the intensities of these reflections to values close to those of pure DMPC, indicating that some sort of molecular reordering had occurred. Higher Hg 2+ concentrations reversed the previous tendency

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once more, because the intensities again decreased. When the ion concentration was increased to 0.1M, no reflections were observed, indicating complete perturbation of the DMPC structure. Results from similar experiments with DMPE are exhibited in Fig. 2D. Increasing concentrations of Hg progressively decreased DMPE reflection intensities. This

65 32.5

51.4

4.2

4.04

Observed spacing (A) D

DMPE •

H-0

10"'M

HgCI 2

10'3M

HgCI 2

10'2M

HgCI 2

10~1 M HgCI 2 J

10"5M

HgCI 2

10"4M

HgCI 2

1(T3M

HgCI 2

10"2M

HgCI 2

10" 1 M

HgCI 2

l_

65 32.5

4.2

51.4

4.04

Observed spacing (A)

Fig. 2. Microdensitograms from X-ray diagrams of (A) DMPC, (B) DMPE with water and CuCl2 aqueous solutions, (C) DMPC, and (D) DMPE with water and HgCl2 aqueous solutions, (a) Low angle and (b) high angle reflections. DMPC=dimyristoy[phosphatidylcholine, DMPE=dimyristoylphosphatidylethanolamine.

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finding showed that both the polar and acyl chain regions were perturbed with equal intensity in a concentration-dependent manner.

2.4. Electrophysiological Measurements of Isolated Toad Skin as an Electrophysiological Study Model 2.4.1. Effects of Cu2+ ions Increasing CuCl2 concentrations applied in the Ringer's solution bathing the external (mucosal) surface of the toad skin elicited a dose-dependent and irreversible decrease of the electrical parameters of the skin, the potential difference, and short circuit current (Fig. 3A). It was found that 1.6 mM abolished the electrical activity. On the other hand, higher concentrations of Cu 2+ ions added to the inner (serosal) surface also produced a dose-dependent and irreversible decrease of these parameters (Fig. 3B); the electrical activity was abolished at 5 mM Cu 2+ . These results implied a severe perturbation of the activity of membrane-bound ion channels.

2.4.2. Effects of Hg2+ ions Figures 3C and 3D show that HgCl2 elicited a dose-dependent and partly reversible decrease of the electrical parameters in both the inner and outer surfaces of the toad skin. At the maximum concentrations (15 |xM and 10 |xM, respectively), the electrical activity of the skin was abolished.

3. CONCLUDING REMARKS The SEM observations indicated that both Cu 2+ and Hg 2+ ions reacted with the human erythrocyte membrane, changing its normal discoid shape to an echinocytic form, characterized by the formation of blebs or protuberances over the cell surface. According to the bilayer couple hypothesis of Sheetz and Singer (1974), this type of shape change may be due to an accumulation of Cu 2+ or Hg 2+ ions in the outer moiety of the red cell membrane. Insertion in the inner monolayer would induce a cup shape (stomatocytic) form. On the other hand, fluorescence spectroscopy analysis confirmed that both ions interacted with the human erythrocyte membrane. Moreover, it was found that the interactions occurred in the polar region of the membrane, that is, at the hydrophobic-hydrophilic

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oSCC •PD

100-

100-

50-

50-

a>

a.

— I

0

1 mM Cu Cl 2

1 mM Cu Cl 2

(A)

(B)

100-

100

"a

T3

50"

50

a)

Q.

£L

10 15 \iU Hg Cl2 (C)

8

20

10 12

pM Hg Cl 2 (D)

Fig. 3. Inhibitory effects of increasing concentrations of copper and mercury ions on the electrical properties of the isolated toad skin. Results are expressed as percentage decrease in control values. Each point represents the mean ± standard deviation. Cu 2+ effects on the (A) outer mucosal and (B) inner serosal surface; *p < 0.05 and **p < 0.01 (student's paired t test). Hg 2+ effects on the (C) outer mucosal and (D) inner serosal surface; *p < 0.01 and **p < 0.001. PD = potential difference, SCC = short-circuit current.

interface. X-ray experiments were performed in DMPC and DMPE bilayers, phospholipid classes located in the outer and inner monolayers, respectively, of the human erythrocyte membrane (Devaux and Zachowsky, 1994). The results also indicated that both ions interacted with the polar groups of phospholipids located in the outer monolayer of

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the erythrocyte membrane. As previously reported, it is likely that Cu 2+ ions preferentially bind to the lipid phosphates (Suwalsky et al., 1998), whereas Hg 2+ ions are linked to the lipid primary amino groups (Suwalsky et al., 2000). Finally, the electrophysiological measurements performed in toad skin showed that both Cu 2+ and Hg 2+ induced irreversible inhibition of the active transport of ions. The relatively greater inhibitory effect of Cu 2+ applied to the outer surface of the skin could be due to its affinity for a binding site of the amiloride-sensitive apical Na + channel (Flonta et al., 1998). These experimental results confirmed the toxicity of heavy metals on membrane structure and functions. However, despite the qualitative similarity of the effects induced by both ions, Cu 2+ seemed to be more toxic than Hg 2+ . In fact, in all experiments, except those performed on human erythrocyte membranes, similar results were attained with Hg 2+ concentrations 10 times higher than those of Cu 2+ . Most likely, the toxic effects of both ions arise from their interactions with proteins, either directly through the formation of stable bonds or indirectly. In the latter case, the interactions of these ions with membrane lipids would perturb their bilayer conformation. Therefore, cell membrane structure and physiological properties, such as fluidity, permeability, receptor, and channel functions, can be affected.

ACKNOWLEDGMENTS This work was supported by grants from FONDECYT (1020476) and CONICYT (Chile)-CNR (Italy) International Program of Scientific Cooperation (2000-5-02-099).

REFERENCES Albrecht J, Matyja E. Glutamate: A potential mediator of inorganic mercury neurotoxicity. Met Brain Dis 1996; 11:175-184. Boadi WY, Urbach J, Brandes JM, Yannai S. In vitro exposure to mercury and cadmium alters term human placental membrane fluidity. Toxicol Appl Pharmacol 1992; 116: 17-23. Brown DR. Copper on prion diseases. In: Zatta P, editor. Metal Ions and Neurodegenerative Disorders. Singapore: World Scientific, 2003: 263-289. Brown DR. Copper and prion disease. Brain Res Bull 2001; 55:165-74. Biisselberg D, Pekel M, Michael D, Piatt B. Mercury (Hg 2+ ) and zinc (Zn 2+ ): Two divalent cations with different actions on voltage-activated calcium channel currents. Cell Mol Neurobiol 1994; 14:675-687.

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Carpenter DO. Effects of metals on the nervous system of human and animals. Int J Occup Med Environ Health 2001; 14:209-218. Cornett CR, Markesbery WR, Ehmann WD. Imbalances of trace elements to oxidative damage in Alzheimer's disease brain. Neurotoxicology 1998; 19:339-345. Delnomdedieu M, Allis JW. Interaction of inorganic mercury salts with model and red cell membranes: Importance of lipid binding sites. Chem Biol Interactions 1993; 88;71-87. Devaux PF, Zachowsky A. Maintenance and consequences of membrane phospholipid asymmetry. Chem Phys Lipids 1994; 73:107-120. Ely JTA. Mercury-induced Alzheimer's disease: Accelerated incidence? Bull Environ Contam Toxicol 2001; 67:800-806. Flonta ML, Beir-Simaels JD, Mesotten D, Van Driessche W. Cu 2+ reveals different binding sites of amiloride and CDPC on the apical Na channel of frog skin. Biochim Biophys Acta 1998;1370:169-174. Gwozdzinski K. A spin label study of the action of cupric and mercuric ions on human red blood cells. Toxicology 1991; 65:315-323. Hartmann HA, Evenson MA. Deficiency of copper can cause neuronal degeneration. Med Hypoth 1992; 38:75-85. Kiss T, Osipenko O. Metal-ion induced permeability changes in cell membranes: A minireview. Cell Mol Neurobiol 1994a; 14:781-789. Kiss T, Osipenko ON. Toxic effects of heavy metals on ionic channels. Pharmacol Rev 1994b; 46:245-267. Lubin B, Kuypers F, Chiu D. Red cell membrane lipid dynamic. In: Brewer GJ, editor. The Red Cell. New York: Alan R Liss Inc., 1989: 507-524. Ohba S, Hiramatsu M, Edamatsu R, Mori I, Mori A. Metal ions affect neuronal membrane fluidity of rat cerebral cortex. Neurochem Res 1994; 19:237-241. Pamphlett R, Coote P. Entry of low doses of mercury vapor into nervous system. Neurotoxicology 1998; 19:39^7. Parmar P, Day a S. The effect of copper on (3H)-tryptophan metabolism in organ cultures of rat pineal glands. Metab Brain Dis 2001; 16:199-205. Perry G, Sayre LM, Atwood CS, Castellani RJ, Cash AD, Rottkamp CAS, Smith MA. The role of iron and copper in the etiology of neurodegenerative disorders: Therapeutic implications. CNS Drugs 2002; 16:339-352. Rock E, Gueux E, Mazur A, Motta C, Rayssiguier Y. Anemia in copper-deficient rats: Role of alterations in erythrocyte membrane fluidity and oxidative damage. Amer J Physiol 1995; 2 6 9 : 0 2 4 5 - 0 2 4 9 . Rytved KA, Brodin B, Nielsen R. Prostaglandin release from dermis regulates Na + permeability of frog skin epithelium. Acta Physiol Scand 1995; 153:263-270. Sansinanea A, Cerone S, Najle R, Auza N. Lipid peroxidation in erythrocyte membranes of sheep with chronic copper poisoning. J Clin Biochem Nutr 1994; 17:65-72. Saxe SR, Wekstein MW, Kryscio RJ, Henry RG, Cornett CR, Snowdon DA, Grant FT, Schmitt FA, Donegan SJ, Wekstein DR, Ehmann WD, Markesbery WR. Alzheimer's disease, dental amalgam and mercury. J Am Dent Assoc 1999; 130:191-199.

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Sheetz MP, Singer SJ. Biological membranes as bilayer couples. A molecular mechanism of drug-induced interactions. Proc Natl Acad Sci USA 1974; 75:4457^1461. Shohet SS, Mohandas N, editors. Red Cell Membranes. New York: Church Livingstone, 1988. Steffensen IL, Mesna OJ, Andruchow E, Namork E, Hylland K, Andersen RA. Cytotoxicity and accumulation of Hg, Ag, Cd, Cu, Pb and Zn in human peripheral T and B lymphocytes and monocytes in vitro. Gen Pharmacol 1994; 25:1621-1633. Strausak D, Mercer JFB, Dieter HH, Stremmel W, Multhaup G. Copper in disorders with neurological symptoms: Alzheimer's, Menkes's and Wilson's disease. Brain Res Bull 2001;55:175-186. Suwalsky M. Phospholipid bilayers. In: Salamone JC, editor. Polymeric Materials Encyclopedia. Boca Raton: CRC Press, 1996: 5073-5078. Suwalsky M, Norris B, Kiss T, Zatta P. Effects of Al(III) speciation on cell membranes and molecular models. Coord Chem Rev 2002; 228:285-295. Suwalsky M, Ramos P, Villena F, Cardenas H, Norris B, Cuevas F, Sotomayor CP. The organophosphorous insecticide parathion changes properties of natural and model membranes. Pest Biochem Phys 2001a; 70:74-85. Suwalsky M, Sanchez I, Bagnara M, Sotomayor CP. Interaction of antiarrhythmic drugs with model membranes. Biochim Biophys Acta 1994; 1195:189-196. Suwalsky M, Schneider C, Villena F, Norris B, Cardenas H, Cuevas F, Sotomayor CP. Dibucaine-induced modification of sodium transport in toad skin and model membrane structures. Z Naturforsch 2001b; 56c:614-622. Suwalsky M, Ungerer B, Quevedo L, Aguilar F, Sotomayor CP. Cu 2+ ions interact with cell membranes. J Inorg Biochem 1998; 70:233-238. Suwalsky M, Ungerer B, Villena F, Cuevas F, Sotomayor CP. HgCl2 disrupts the structure of the human erythrocyte membrane and model phospholipid bilayers. J Inorg Biochem 2000; 81:267-273. Tiffany-Catiglion E, Qian Y. Astroglia as metal depots: Molecular mechanisms for metal accumulation, storage and release. Neurotoxicology 2001; 22:577-592. Ussing HH. Does active transport exists? J Membr Biol 1994; 137:91-98. Zatta P, Suwalsky M. Aluminum, membrane and Alzheimer's disease. In: Exley C, editor. Aluminum and Alzheimer's Disease: The Science that Describes the Link. Amsterdam: Elsevier Science, 2001: 279-291.

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CHAPTER 19

Influence of Lead Exposure on Brainstem Functions Ombretta Mameli, Marcello Alessandro Caria

ABSTRACT The effects of exposure of low lead concentrations on the brainstem and the cerebellum are reviewed. Neurophysiological parameters relative to the neural pathways, auditory function, and vestibular functions were considered. Clinical and experimental data were examined in an attempt to identify a possible relationship between neurophysiological and morphological alterations and blood/tissue concentration of lead. The literature shows that brainstem functions are significantly impaired by lead exposure even at very low levels, and the vestibular system, is one of the most sensitive targets. Keywords: Lead exposition; peripheral nerve function; auditory function; vestibular function; cerebellum.

1. OCCUPATIONAL AND ENVIRONMENTAL LEAD EXPOSURE In the last 10 to 15 years, increased attention has been paid to subclinical, early, or subtle health damage induced by lead (Pb) exposure. Greater knowledge has been acquired on the effects induced by low level Pb exposure, particularly on the nervous, cardiovascular, endocrine, and immune systems, as well as on the reproductive function and some hematic parameters. However, several problems still remain such as a possible mutagenic and carcinogenic action of Pb on humans. Most of the abovementioned effects were observed for blood Pb (BPb) levels below the current limits proposed for workers and the 441

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general population. In addition, for certain effects, such as those on heme, central nervous system, and blood pressure, it has not been possible to establish a clear threshold for Pb concentration. In an attempt to limit, as much as possible, the effects induced by Pb exposure in the general population, several laws were issued both in Europe, by the European Community directives (77/312 and 98/24), and in Italy, by the Italian government (DPR 496/1982, 626/1994, and 25/2002), to reduce the Pb level considered to be neurotoxic. At present, the risk of increased Pb absorption is higher in workers employed in specific activities (Pb recovery, repair of radiators, bronze, and copper foundries) and in small factories where environmental or preventive measures are inefficient. More attention must be paid to preventive measures in higher risk activities, but the current environmental level and blood concentrations in Italy (35 mg/100 mL and 70 mg/ lOOmL, respectively) should be modified by taking into account the changes that have been introduced in working conditions. At present, in industrial activities where Pb is largely utilized (accumulators and ceramics), the mean BPb concentrations in workers range from 25 mg/100 mL to 35 mg/100 mL (Apostoli, 1998). The second Italian campaign on biological surveillance of the general population against the risk of Pb intoxication, which was carried out according to the regulations of DPR 496/82, showed a significant reduction (from 40% to 50%) in BPb concentration compared to the values detected during the decade 1985 to 1996 (Mendito et al., 1998). A total of 7,749 nonexposed subjects (4,346 women and 3,403 men, of which 559 and 611 were under 15 years of age, respectively) were examined in various Italian regional centers under the co-ordination of the Italian National Institute of Health (Istituto Superiore di Sanita). The median value of adult BPb concentration was 86 mg/L and 53.5 mg/L in men and women, respectively. In the young, it was 50 mg/L and 43 mg/L, respectively. This investigation pointed out that, in certain areas, more than 5% of individuals under the age of 15 had BPb levels above 100 mg/L. This concentration is known to be the safety limit indicated by the Center for Disease Control and Prevention in the USA to avoid irreversible injury to the central nervous system. In fact, it indicated that 10 mg/100 mL represents the limit beyond which children's learning capacity could be damaged (Ferri et al., 1998).

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Finally, even though legislation in the last three decades has effectively reduced the Pb exposure levels and blood concentrations in the developed countries (Pirkle et al., 1998), ongoing research has shown adverse effects at even lower concentrations; over the same period, the threshold levels associated with health risk have been revised downward. In particular, the Center for Disease Control and Prevention in the USA has reduced the blood Pb level from 60 ug/dL, prior to 1971, to 10 ug/dL in 1991 (Schaffer and Campbell, 1994). At lower levels, Pb is associated with a number of adverse effects on human health, including deficits in the central nervous system (Bellinger et al., 1992; Dietrich, 1999; Finkelstein et al., 1998; Goyer, 1990; Mayfield, 1983; Needleman et al., 1979, 1990). Clinical studies suggest that, in particular, the negative relationship between BPb concentration and cognitive performance extends from below 10 ug/dL up to the limits of measurement (Bellinger et al., 1992; Schwartz, 1994). In any case, many studies have demonstrated that the reference levels for the general population set out by the EEC Directive (77/312) is way too high.

2. NEUROTOXIC ACTIVITY OF LEAD In the last decade, the effects of Pb exposure on cognitive functions have been extensively investigated. Studies have demonstrated, beyond any reasonable doubt, that this heavy metal impairs neurobehavioral systems by reducing learning/memory capacities. Pb exposure also diminishes intelligence abilities and is associated with a deficient intelligence quotient score (Schwartz, 1994; Alfano and Petit, 1981; Needleman and Bellinger, 1991; Needleman et al, 1979; Baker et al., 1984, 1985; Hanninen et al., 1978; Mantere et al., 1984; Jeyaratnam et al., 1986; Yokoyama et al., 1988). Pb exposure has been demonstrated to disrupt the dopaminergic system (Shafiq-ur-Rehman, 1991; Cohn and Cory-Slechta, 1994; CorySlechta et al., 1992) by inducing, at presynaptic level, an alteration of the synthesis and release sequence of dopamine in the mesolimbic system (Lasley, 1992). It also induces an upregulation of the D2-D3 receptors (Cory-Slechta et al., 1992) with particular regard to those localized in the

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accumbens nucleus, which appears to be a preferential target of Pb damage (Widzowski et al., 1994). Recently, the N-methyl-D-aspartate (NMDA) receptor has been shown to mediate Pb-induced neurotoxicity (Brook et al., 1993; Guilarte et al., 1994; Jett and Guilarte, 1995; Schulte et al., 1995; Ma et al., 1997). This receptor is known to be directly involved in the plasticity phenomenon underlying learning and memory processes (Collingridge and Lester, 1989). It appears that Pb-induced neurotoxicity, such as memory deficits and intelligence deficiency, may be related to a disruption of the NMDA receptors' function in hippocampal formation and cerebral cortex (Ma et al., 1997), which represent the critical neural structures for learning and memory. The impairment of the glutamatergic system induced by Pb exposure is also involved in the deficiency of neurobehavioral abilities related to the prefrontal cortex functions, especially the visuo-spatial abilities and motor functions (Barth et al., 2002). Notwithstanding the large body of evidence demonstrating the neurotoxic effect of Pb on cognitive, behavioral, and psychological performance, to date relatively little information is available on the impact of Pb exposure on brainstem functions. The negative effects of Pb on the central and peripheral nervous systems have been documented, showing abnormalities in visual-, somatosensory-, and auditory-evoked potentials (Jeyaratnam et al., 1985; Otto et al., 1985; Holdstein et al., 1986), as well as a slowing of peripheral nerve conduction velocities (Seppalainen et al., 1975, 1983; Araki and Honma, 1976; Landrigan et al., 1976; Feldman et al., 1977; Buchthal and Behse, 1979; Araki et al., 1980; Ashby, 1980; Singer et al., 1983; Triebig et al, 1984) following exposure to Pb. As for the autonomic nervous system, it has been observed that workers with BPb concentration above 30 ug/dL had a significant decrease in ECG R-R variability during deep breathing (Teruya et al., 1991). A significant relationship between BPb concentration and R-R variability was also observed by Araki et al. (1990) in a follow-up study of a Pb-smelting worker. These observations were recently confirmed in a study demonstrating that Pb affects the autonomic nervous system function mainly through a depression of the parasympathetic activity (Murata et al., 1993). This chapter focuses on the effects exerted by low Pb exposure on brainstem and cerebellar functions.

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3. EFFECTS OF LEAD ON PERIPHERAL NERVE FUNCTION Seppalainen et al. (1972, 1975, 1979, 1983) and Araki and Honma (1976) were the first to describe a subclinical Pb-induced neuropathy by studying the nerve conduction velocity (NCV) in exposed workers. Their results were subsequently confirmed by several authors (Paurev et al., 1979; Bord et al., 1982; Singer et al., 1983; Ehele, 1986; He et al., 1988). At that time, the conventional methods for determining NCV in the peripheral nervous system allowed one to detect only two discrete values: the fastest and slowest conduction velocities. In fact, the function of the vast majority of axons within the nerve could not be directly evaluated. Nowadays, the availability of advanced computerized technologies, such as "long latency" evoked potential technique, have enabled researchers and clinicians to measure the distribution of NCV without using invasive methods (Barker et al., 1979; Hirata et al., 1980; Jeyaratnam et al., 1985). Furthermore, the effects of Pb exposure have been recently studied in the human somatosensory ascending pathways using short latency somatosensory evoked potential (SLSEP) (Araki etal., 1986a, 1986b, 1987). Using a modified Jones' method (Jones, 1977; Araki et al., 1986c, 1987), it was possible to record the N9 component of SLSEPs, at the Erb's point level (the supraclavicular fossa), after stimulation of the median nerve in the wrist. The Nil and N13 components could be recorded at the second and seventh cervical vertebrae levels, whereas the N20 and P23 components of SLSEP could be recorded on the scalp overlying the contralateral sensory cortex. Using this technique, Murata et al. (1993) showed that the radial and median NCVs were significantly slowed in 22 male workers in a gun metal foundry whose BPb concentration ranged from 12 ug/dL to 64 ug/dL. In particular, the N9 peak, N9 to N13, and N13 to N20 interpeak latencies of the SLSEPs, which represent the cervicospinobulbar and central conduction time, respectively, were prolonged as it was the V10 velocity of the distribution of nerve conduction (DCV). However, when considering the absolute values of these parameters, it is necessary to take into account the existence of daily variations in the SLSEP and DCV, with an occurrence falling within 4.4% and 4.2%, respectively (Murata et al., 1987a; Araki et al., 1986a).

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Hirata and Kosaka (1993), in their study of 41 Pb-exposed male workers from four Japanese factories involved in the manufacture of Pb glass-based colors, analyzed the correlation between a number of neurophysiological parameters and several Pb exposure indicators, including duration of exposure, current Pb concentration in whole blood, timeweighted average of Pb concentration in whole blood, and age. The current Pb concentration in the whole blood of these workers ranged from 13 ug/dL to 70 ug/dL and the time-weighted average of Pb concentration in the whole blood in the last five years ranged from 12.8 ug/dL to 70.1 ug/dL. The neurophysiological tests showed that Pb chronic exposure to Pb significantly reduced the conduction velocity in the radial nerve and slowed the onset latency of the N20 component of the SLSEP. These demonstrate that the nerve conduction in both the central and peripheral nervous systems is affected by Pb exposure. These observations confirmed the already large body of evidence on subclinical peripheral nerve dysfunction induced by Pb exposure (Araki and Honma, 1976; Buchthal and Behse, 1979; Ashby, 1980; Triebig et al, 1984; Jeyaratnam et al., 1985; Lockitch, 1993). A significant reduction of the maximum motor NCV of the radial nerve at proximal level (that is, arm segment) has been described in battery workers with BPb concentration of 60 ± 15 ug/dL (Ashby, 1980), in workers suffering from "clinical Pb poisoning" with BPb concentration of 72ug/dL (from 27ug/dL to 180 ug/dL) (Vasilescu, 1973), and in secondary Pb smelters with BPb concentration of 30 ug/dL to 110 ug/dL (Lilis et al., 1977). Murata et al. (1987) and Hirata and Kosaka (1993) also reported the reduction of motor NCV in Pb-exposed gun-metal and glass-based colors workers, respectively. Furthermore, the motor fibers of the ulnar nerve have been reported by Seppalainen et al. (1972, 1975) to be particularly sensitive to lead exposure. In fact, they showed a significant reduction in the motor fiber conduction velocity following Pb exposure. Therefore, a clear relationship between BPb levels and NCVs (Seppalainen et al., 1979) has been demonstrated beyond any reasonable doubt. A recent paper reported the results of a clinical and electrophysiological study of 46 workers with neuropathic features out of a total population of 151 workers who showed high blood and/or urinary Pb concentration (Rubens et al., 2001). The average duration of occupational exposure for the neuropathic group ranged from, eight to 47 years. The mean BPb

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concentration was 63.9 ug/dL. All 46 workers had distal paresthesiae, pain, impaired pin prick sensation, diminished or absent ankle jerks, and autonomic vasomotor or sudomotor disturbances. None of them had motor abnormalities. Analysis of the peripheral motor nerve function at the distal level showed that the conduction velocity and compound muscle action potential amplitudes were normal, with only slight prolonged latencies compared to the controls. Sensory nerve action potential amplitudes were found at the lower end of the normal range. At the distal level, the latencies were prolonged. No direct correlation was found between the biochemical variables and the clinical and electrophysiological data. Workers with unusually long-term Pb exposure showed moderate sensory and autonomic neuropathic features, rather than the motor neuropathy classically attributed to Pb toxicity. We proposed that the traditional motor syndrome associated with subacute Pb poisoning could most likely be a form of Pb-induced porphyria, rather than a direct neurotoxic effect of Pb. This hypothesis was supported by the observation that this cohort of workers showed a significant increase of urinary coproporphyrins (66.7 ug/g), urinary aminolevulinic acid (1.54 ug/g), and creatinine (20 ug/g to 80 ug/g). The results of this study raise the question of whether individuals enroled in a study on Pb neurotoxicity must also be investigated for these parameters. In conclusion, the analysis of the available literature shows that, with current knowledge, it is difficult to identify a threshold for BPb concentration that relates to the neurotoxic effect of the peripheral nerve function.

4. EFFECTS OF LEAD ON AUDITORY FUNCTION The central auditory pathways travel from the cochlear nuclei to the medial geniculate nuclei into the midbrain before targeting the primary auditory cortex. The second order neurons, localized in the cochlear nucleus, send their axons to other centers of the brain via three pathways: dorsal acoustic stria, intermediate acoustic stria, and trapezoid body. The first binaural interaction occurs in the superior olivary nucleus which receives trapezoid body inputs. The medial and lateral divisions of the superior olivary nucleus are involved in the localization of sound in space. Post-synaptic axons from the superior olivary nucleus, along with axons from the cochlear nuclei, form the lateral lemniscus which ascends to the

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midbrain. Axons relaying input from both ears are found in each lateral lemniscus. The axons synapse in the inferior colliculus, and post-synaptic cells in the colliculus, in turn, send their axons to the medial geniculate body of the thalamus before finally terminating in the primary auditory cortex. Therefore, in this sensory system, the midbrain is strongly involved in the separation of timing and intensity of the acoustic signals, as well as in their spatial localization, while at the same time transmitting this elaborate information to the auditory cortex where the timing, intensity, and frequency of the sound are mapped. Based on its anatomy and functional organization, the auditory system represents an excellent model for the detection, at the brainstem level, of a possible neurotoxic effect induced by Pb exposure. It has been reported that hearing impairments occur in workers exposed to Pb, particularly in those with BPb levels above 70 ug/dL (Repko and Corum, 1979). In the last two decades, elevation of the hearing threshold has been described in children with very low BPb levels (Robinson et al., 1985; Otto et al., 1985; Rothenberg et al., 1994, 2000), including concentrations below 10 ug/dL (Schwartz and Otto, 1987, 1991). However, the nature of the effects on the different parameters affected by Pb (increase or decrease of latencies of wave I, wave V, and inter-wave intervals) was not consistent across these studies. On the contrary, other authors (Buchanan et al., 1999; Counter et al., 1997) have reported normal hearing threshold in two cohorts of Ecuadoran children with high BPb levels (40 ug/dL and 52 ug/dL, respectively). The neurotoxic action of Pb on the auditory system has been studied by analyzing the brain auditory evoked potential (BAEP) recorded by scalp electrodes. BAEP measures volume-conducted electrical potential to transient auditory stimuli generated by neural activity in the auditory nerve and brainstem. Using this technique, Otto et al. (1985) reported that the increased latencies of waves III and V of the BAEP were linearly correlated with the BPb levels. Increased latencies of these evoked potentials in children and adult workers exposed to Pb were also observed by Holdstein et al. (1986). On the contrary, Robinson et al. (1985) showed only a curvilinear relationship between these parameters and observed that the conduction velocity in the auditory pathway increased only above 25 ug/dL of BPb. Murata et al. (1987) and Lille et al. (1988) failed to find any significant relationship between BPb and BAEP latencies. The latter

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found that the BPb values ranged from 2 ug/dL to 24 ug/dL while the former discovered that the BAEP latency correlated better with hematocrit. Data from the literature on adults exposed to Pb in the workplace suggest that the effects are only observed at high BPb concentrations (Discalzi et al., 1992; Araki et al., 2000). Differences in the outcomes could also be explained by the fact that the histories of Pb exposure are often lacking in detail (Lasky et al., 2001). Confounding factors may vary from cohort to cohort. This explains why relatively high BPb levels have no apparent effect on hearing threshold in one cohort, while in another a light increase in the BPb level could induce an opposite effect. Furthermore, evaluation of the hearing threshold frequently reflects a combination of cognitive and noncognitive factors, as well as hearing sensitivity. Thus, the differences in hearing threshold sensitivity in children exposed to Pb could reflect their background abilities. Exhaustive descriptions of the experiments are found in the interesting reviews by Fox (1992) and Otto and Fox (1993). More recently, the effect of Pb on the auditory system has been studied by otoacoustic emissions (OAEs), which measure sound pressure variations, in the occluded ear canal, that reflect the movements of the cochlear partition probably induced by the outer hair cells. It has been reported that these evaluations allow one to identify the site lesion associated with Pb exposure along the auditory pathway. Furthermore, they enable one to differentiate true auditory deficits from attentional, cognitive, and motivational deficits that may interfere with the interpretation of the behavioral test. In a cohort of Ecuadoran children with elevated BPb levels (52 ug/dL), Buchanan et al. (1999) observed that OAEs were present from 1 KHz to 16 KHz, although at a diminished amplitude, and that this parameter did not correlate with the BPb concentrations. Experimental studies performed in guinea pigs showed that injections of high doses of Pb acetate were able to induce BPb concentrations ranging from 310 ug/dL to 420 ug/dL. Though unable to determine the histological changes in the gangliar spiral neurons, they did induce demyelination and axonal degeneration of the acoustic nerve (GozdzikZolnierkiewicz, 1969). This result agrees with the electrophysiological evaluation of the guinea pig's cochlear nerve (Yamamura et al, 1987, 1989), which showed a dose-dependent threshold increase in the primary component of the nerve action potential that, in turn, was associated with a

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dose-dependent decrease in its maximum amplitude. However, at the same BPb concentration of 80 ug/dL to 142 ug/dL, no changes in the endocochlear or cochlear microphonic potentials could be observed. The experimental studies on animal models provided an interesting complement to human studies. In fact, it has been demonstrated that Pb affects the conductive mechanisms of the middle ear (Lasky et al., 1998), sensory transduction in the cochlea (Lasky et al., 1995; Liang et al., 1999; Lilienthal and Winneke, 1996; Reeker et al., 1999), and neural transmission along the auditory nerve and brain stem pathways (Reeker et al., 1999; Yamamura et al., 1989). Recent observations in rhesus monkeys, which show an ontogenetic similarity with the human auditory function (Doyle et al., 1983; Eggermont, 1985; Kraus et al., 1985; Laughlin et al., 1999; Lasky et al, 2001) indicated that elevated Pb concentrations, corresponding to BPb of 35 ug/dL to 40 ug/dL, had little persisting effect on middle ear function, cochlear mechanical function, or neural function from the auditory nerve to the primary auditory cortex. Therefore, the results of these experiments confirm the hypothesis that acoustic impairment could only be determined following higher levels of Pb exposure.

5. EFFECTS OF LEAD ON VESTIBULAR FUNCTION The central vestibular pathways, like the auditory system, also extend into the midbrain from the vestibular nuclei to the thalamus before targeting the cerebral cortex. The cell bodies of the afferent fibers of the vestibular system lie in the vestibular ganglion and, through their myelinated axons, reach the vestibular nuclei complex within the brainstem. This nuclear complex occupies a substantial portion of the medulla and includes the following nuclei: lateral, medial, superior, and inferior vestibular nuclei. Each nucleus, distinguishable on the basis of its histological feature, has a distinctive set of connections with the periphery and central nervous systems, including the spinal cord, oculomotor nuclei (III, IV, and VI) and cerebellum. In particular, the lateral vestibular nucleus participates in posture control, the medial and superior vestibular nuclei mediate the vestibulo-ocular reflexes (VORs), and the inferior vestibular nucleus integrates inputs from the labyrinth and cerebellum. The vestibular system provides the brain with the following related information: dynamic information mainly mediated by the semi-circular canals which track the

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rotation of the head in space and evokes a reflex control of the eye movements, and static information mainly mediated by the utricle and saccule which enable are to define the actual position of the head in space. The latter function plays a pivotal role in postural control. In our opinion, the vestibular system represents the most complete model for the evaluation of possible neurotoxic effects induced by Pb exposure on the brainstem structures. In an attempt to investigate the adverse effects of Pb exposure on the vestibulocerebellar and spinocerebellar systems, the computerized technique of postural sway analysis was used in the last decade. In 60 workers who were exposed (84 months' exposure) to Pb, and who had a mean BPb concentration of 36 ug/dL (range, 6.4 ug/dL to 64.5 ug/dL), a significant derangement of postural stability was found compared to 14 controls (mean 6.3 ug/dL; range, 3.1 ug/dL to 10.9 ug/dL). Significant differences were found in a number of parameters: deviation of the co-ordinates from the pressure center, the path sway length, the mean velocity of the pressure center (Vel) along its path, and the area within the path of the pressure center (Ao). Romberg test results of the latter parameters (Vel and Ao) in exposed workers were also significantly different from those of controls'. On the contrary, no significant differences were observed in postural sway parameters between the groups when the eyes were kept open (Chia et al., 1994, 1996). This observation confirms the case report, described by Bhattacharya and Linz (1991), of a 15-year-old boy who showed an increased postural sway for postural tasks requiring input from higher centers. At superimposable BPb concentration (37.5 ug/dL), workers who stood upright, with their eyes open, and barefoot on the force platform showed postural sway parameters markedly different from those of controls. However, more movements were needed in the exposed workers compared to the controls in order to maintain stability, standing on the platform with their eyes closed and heads tilted. This observation suggests that Pb affects postural control in asymptomatic workers (Ratzon et al., 2000). Exposure to Pb stearate in 49 male workers from a chemical factory (aged between 27 and 43 years), who had an average BPb concentration of 48ug/100 g (range, 11 (xg/lOOg to 113 ug/100 g), induced significant changes in posturographic pattern. We concluded that these results could

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be due to an asymptomatic derangement of both the vestibulocerebellum (lower vermis) and anterior cerebellar lobe functions, as well as the spinocerebellar afferent pathway (Yokoyama et al., 2002). These results demonstrate that the control of posture is certainly affected by Pb exposure, although they do not allow one to understand the precise mechanisms by which the vestibular system is impaired by Pb. In fact, to maintain a stable position while standing requires a number of adjustments to be made in order to produce three main actions. First, support the head and body weight against gravity and other external forces. Second, maintain the center of the body's mass by aligning within its base of support on the ground. Finally, stabilize the supporting segments of the body while the others are being moved. These adjustments are induced by sensory information originating from various receptors: cutaneous, proprioceptive, and visual. As mentioned in the previous sections, one must bear in mind the fact that peripheral nerves and visual system functions can be impaired by Pb exposure. Furthermore, in order to integrate postural adjustments with voluntary movements, postural responses use the cortical circuits and spinal and brainstem reflex circuitries. In our opinion, the analysis of the postural sway is only useful for a general, nonspecific evaluation of asymptomatic subjects, who have been chronically exposed to occupational or environmental Pb, to detect clinical signs related to Pb neurotoxicity. It appears difficult, using this test, to distinguish the attributes of each of the previously mentioned structures in order to determine the impairment observed in subjects exposed to Pb. Therefore, we are convinced that the analysis of the vestibulospinal reflex would be more useful for this purpose. Human studies cannot conclusively determine a cause and effect relationship between Pb exposure and vestibular dysfunction. Instead, experimental studies on animal models provide a necessary complement to human studies. In neonatal rats, it was observed that chronic administration of Pb chloride solutions (PbCl2) via drinking water for three months, at a concentration of 0.05%, 0.1%, and 0.2%, induced an increase in the number and intensity of immunoreactive neurons of the locus coeruleus to tyrosine hydroxylase antibody. Degeneration phenomena, which include intra-axonal vacuole formation and widening of the extracellular spaces, were found by electron microscopy analysis in and around the tyrosine hydroxylase immunoreactive axons (Lee et al., 2002). Therefore, the

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norepinephrinergic neurons of the locus coeruleus represent a sure target for the neurotoxic action of Pb. This observation agrees with the results described in catecholaminergic neurons chronically exposed to Pb (Palmer et al., 1984; Nehru and Sidhu, 2001). The locus coeruleus is strongly influenced by labyrinthine inputs (Pompeiano et al., 1991). Furthermore, it has been demonstrated that noradrenergic coeruleospinal neurons exert a dynamic control on posture during extrinsecation of vestibulospinal reflexes (Pompeiano et al., 1988) by controlling the spinal motor output (Fung et al., 1991). Therefore, the impairment of the above nervous structure by Pb could explain the postural derangement observed in clinical studies on Pb exposed subjects tested on the force platform. The cerebellum also represents a favorite target for Pb accumulation and a main source of Pb-induced neuropathology (Krigman et al., 1977; McConnel and Berry, 1979; Petit and LeBoutillier, 1979; Berney, 1993). In rats intraperitoneally injected with 7.8 ug/g b.w. and 15.6 ug/g b.w., it was shown that Pb was concentrated in the cerebellar white matter two to three times more than in the cerebellar cortex for both low and high doses (Lindh et al., 1989). In their study on the cerebellum of rat pups exposed to Pb (BPb, 45 ug/dL), Hasan et al. (1989) were unable to detect alterations in the rate of cell acquisition and migration, as well as their final number, compared to age-matched controls. They concluded that a chronic low level of Pb exposure produced no significant effects in the early phases of the developing cerebellum. However, Pb has been demonstrated to induce a prodifferentiative effect in glial cells of the cerebellum of postnatal rats (Cookman et al., 1988), and to interfere with developmental gene expression (Zawia and Harry, 1996). In particular, it has been shown that Pb affects the gene expression of neuronal growth-associated protein 43, a glial myelin basic protein, and a glial fibrillary acid protein in the developing cerebellum of rat. Activator of protein 1 (API) is an important transcription factor during brain development and Pb has been demonstrated to increase the API DNA binding activity in PC 12 cells (Chakraborti et al., 1999). These data suggest that the long-term pathological effects, either evident or subclinical, induced in animals by Pb may be partly due to a disruption of the co-ordinated pattern of selective gene expression (Zawia and Harry, 1996). In a rat model of chronic early childhood, Patrick and Anderson (2000) showed that moderate Pb exposure (0.3 ug/100 mL to 1.2 ug/100 mL) induced, among other morphological

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differences of the features of Purkinje cells, an increased dendritic spine density and an altered pattern of dendritic branching in compared to the controls (reviewed in Patrick and Anderson, 1995). On the contrary, decreased synaptic contacts, dendritic branching, and spine density have been described in rats exposed to Pb (Pentschew and Garro, 1966; Lorton and Anderson, 1986). The inconsistency in experimental data could depend on a number of factors, including species type, developmental stage during exposure (such as maturation at birth), the dose and form of Pb preparation used, and duration of the exposure. However, all these experiments show that Pb exposure significantly alters the morphological pattern of Purkinje cells. This observation suggests another interpretation of the neurobehavioral symptoms described in Pb-afflicted children. The mechanism by which Pb induces morphological and neurobehavioral alterations remains unknown. The neurotransmitter systems that operate in the cerebellum include glutamate, -y-aminobutyric acid, 5-hydroxytryptamine acetylcholine, and norepinephrine. Some of these neurotransmitters, both in vivo and in vitro, were studied in relation to Pb neurotoxicity (Kolton and Yaari, 1982; Cooper et al., 1984; Shellenberg, 1984; Alfano and Petit, 1985; Spence et al., 1985; Minnema and Michaelson, 1986; Minnema et al., 1988; Lasley et al., 1984; Tomsig and Suszkiw, 1991; Markovac and Goldstein, 1988; Habermann et al., 1983; Goldstein and Ar, 1983; Sandhir and Gill, 1994; Nechay and Saunders, 1978; Rajanna et al., 1997; Boykin et al., 1991; Chetty et al., 1993; Vig et al., 1994). These studies demonstrated that Pb may gradually alter the voltage membrane parameters of a neuron with a number of mechanisms, such as a blockade of transmitter release, stimulation of a spontaneous release, interference with transmembrane ionic distribution, and activation of intracellular signals. It is, therefore, possible that even in adults these mechanisms operate to impair the cerebellar function, and that the postural sway derangement, clinically observed in asymptomatic workers, could at least be partly due to a cerebellar dysfunction. In an attempt to evaluate the effect of Pb on the vestibular system in an in vivo model, Mameli et al. (2001) recently studied the VOR in chronically Pb exposed rats. This reflex utilizes a neuronal circuit in which the sensory input generated by head displacement is compared with the output of oculomotor neurons producing the ocular movements. The VOR allows the eye movements to be adjusted until they coincide with

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the current head position, so that the image of an object is stabilized on the retina. The results of this experiment showed that low levels of chronic (three months) Pb exposure (50 ppm of Pb acetate in drinking water (determined significant changes in the post-rotatory nystagmus (PRN) parameters of all treated animals. The VOR was tested under basal conditions and repeated every 10 days throughout the whole period of Pb exposure. When the reflex showed significant and consistent alterations of the monitored parameters, the animals were sacrificed and Pb blood and brain concentration were determined by atomic absorption spectrophotometry. At the end of the experiment, following three months of exposure, the mean concentration of Pb was 21.52 ± 14.25 ug/L in blood, 2.16 ± 1.34 ug/g in the telencephalon, and 1.69 ± 1.09 ug/g in the brainstem cerebellum. The analysis of the VOR revealed that Pb treatment induced significant changes in the following PRN parameters: onset latency, duration, frequency, amplitude of nystagmic jerks, and slow phase velocity. These alterations were already detectable at very low concentrations of Pb in blood and nervous tissue. In particular, the patterns of PRN responses were characterized and classified into four types: progressively inhibitory (40%), prematurely inhibitory (25%), late inhibitory (25%), and excitatory-inhibitory (10%). In exposed animals, the VOR impairment was strictly related to the increase in Pb concentrations in the blood and brain. This was because, under matched experimental conditions, control animals showed a normal VOR. The results of this experiment demonstrated, for the first time in vivo, that the brainstem cerebellum, where the neural circuits for VOR induction and control are located, is a target of the neurotoxic action of Pb. The results also showed that it is not possible to assess a "general toxic thereshold" of Pb concentration in either blood and/or nervous tissue that will enable are to predict the appearance or signs of VOR derangement. In fact, concomitant to VOR impairment, Pb concentration showed a considerably high interindividual variability, even among animals which had the same pattern of VOR impairment. This observation indicates the existence of a largely variable "individual toxic threshold." In fact, in animals showing the "prematurely inhibitory" PRN response the VOR changes had an early appearance (after 45 days of treatment) and a BPb concentration only slightly higher than the basal values. On the contrary, animals which exhibited the "late inhibitory" response showed an impairment of PRN parameters only after three

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months of Pb acetate exposure. Nevertheless, even in this group the BPb and brain concentration could be consistently low. The fact that a delay in PRN onset latency was consistently observed in the majority of the animals suggests that Pb exerts a neurotoxic effect even at low concentrations, most likely by changing the membrane properties of the receptors and/or interfering with their depolarization. The effects of Pb exposure on the Ca + + ion channels (Hinkle et al., 1987; Markovac and Goldstein, 1988; Habermann et al., 1983; Goldstein and Ar, 1983; Sandhir and Gill, 1994; Tomsig and Suszkiw, 1991) could also affect the depolarization mechanism of vestibular receptors. However, since Pb exposure slows nerve conduction (Seppalainen, 1972, 1975; Araki et al., 1986a, 1986b, 1987; Murata et al., 1993; Hirata and Kosaka, 1993; Araki and Honma, 1976; Buchthal and Behse, 1979; Ashby, 1980; Triebig et al., 1984; Jeyaratnam et al., 1985; Lockitch, 1993), the delayed PRN response could also be a result of vestibular nerve conduction failure. It should also be considered that the cerebellum, which exerts a complete control on the vestibular system and oculomotor nuclei (Pompeiano, 1974), is a target, as we have previously seen, of Pb neurotoxicity. Impairment of Purkinje cells could, in fact, result in an increase in their direct inhibitory effect on both vestibular and deep cerebellar nuclei. The reduced excitatory output from deep cerebellar nuclei could, in turn, be responsible for the disruption in the activity of the extrinsic ocular muscle and vestibular nuclei. The direct inhibition of the vestibular nuclei could explain the changes in the vestibular components of the PRN and in other parameters, such as the slow phase velocity and amplitude of the jerks. In view of the extensive reciprocity between the vestibular nuclei of both sides, the cerebellum and the oculomotor nuclei (Baker and Highstein, 1975; Baker et al., 1969; Maciewicz et al., 1977; Maeda et al., 1971; Pompeiano, 1974), a reduction in the intensity of vestibular and cerebellar signals to the oculomotor nuclei could explain the fall in output to the extrinsic ocular muscles. This study demonstrated that Pb, even at low concentrations, impairs the vestibular system and reflex activity related to its function. It also confirmed that the brainstem and the cerebellum are definite targets of the negative action of this heavy metal. Finally, the analysis of the VOR seems to be more specific compared to an analysis of the postural sway in the investigation of the vestibular

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and cerebellar functions. Therefore, it should be considered as an elective test for screening environmentally or occupationally Pb exposed subjects for the detection of subclinical signs of neurotoxic Pb effects.

6. CONCLUSIONS To date, the data on Pb neurotoxicity have demonstrated that Pb exposure, even at low concentration, induces long-term dysfunction in brainstem structures, the magnitude of which is strictly related to its concentration and duration of the exposure. The cerebellar, auditory, and vestibular functions are all affected by Pb neurotoxicity, although these structures seem to show a wide range of sensitivity to Pb exposure. The vestibular system is the most responsive to Pb insult. The mechanisms by which Pb neurotoxicity impairs neuronal function include impairment of gene expression, ion channels, synaptic receptors, and neurotransmitter release. Morphological changes in dendritic arborization and synaptic contacts have also been described in animal models. These effects could all lead to changes in a number of electrophysiological parameters, which can be observed following Pb exposure in the acoustic, vestibular, and cerebellar systems. The results are, in large part, confirmed by clinical screenings on Pb exposed population that demonstrate, at even very low concentrations, this metal is often able to impair cognitive and sensorimotor functions without the subject showing any clinical signs. To what extent these changes are dose-dependent, species-specific, or related to Pb preparation and mode of administration still remains to be determined, including a safe Pb concentration level in the environment and/or working places in which no effect could be detected. It is, therefore, imperative that the prevailing environmental levels and blood concentrations of Pb could be reconsidered by the relevant authorities in all countries.

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CHAPTER 20

Neuroprotective Effects of Lithium Sophie Ermidiou-Pollet, Serge Pol let

ABSTRACT Multiple actions of lithium are critical to its therapeutic effects. These complex effects stabilize neuronal activities, support neuronal plasticity, and provide neuroprotection. Three interacting systems appear most critical. Modulation of neurotransmitters by lithium likely readjusts balances between excitatory and inhibitory activities, and so may contribute to neuroprotection. Lithium also modulates signals impacting on the cytoskeleton, a dynamic system contributing to neural plasticity. Finally, lithium adjusts signaling activities regulating second messengers, transcription factors, and gene expression. Neuroprotective effects may be derived from its modulation of gene expression. These findings suggest that lithium may exert some of its long-term beneficial effects in the treatment of mood disorders via underappreciated neuroprotective effects. Keywords: Lithium; neuroprotection; neurotransmitters; signaling networks; transcription factors; gene expression.

1. INTRODUCTION The introduction of lithium (Li) salts for the prevention of recurrences of affective illnesses has been hailed as a major advance in modern psychiatry. Nowadays, Li continues to be the most effective treatment for bipolar disorders, both for the acute manic phase and as prophylaxis for recurrent manic and depressive episodes (Goodwin and Jamison, 1990; Moore et al., 2000a). Clinical studies have attempted to uncover the biological factors mediating the pathophysiology of bipolar disorders. Many comprehensive reviews discuss the aspects of bipolar disorders (Duman et al., 1994; Lachman and Papolos, 1995; Manji and Lenox, 1999, 2000) and the use 467

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of Li (Wood and Goodwin, 1987; Mork et al., 1992; Price and Heninger, 1994; Jope and Williams, 1994; Belmaker et al., 1996; Soares and Gershon, 1998; Jope, 1999a, 1999b; Shaldubina et al., 2001), and provide detailed coverage of important earlier progress in this field. Recent findings suggest that Li may exert some of its long-term beneficial effects via neuroprotective effects (Manji et al., 1999). It is now wellestablished that, in the developing nervous system, programmed cell death is responsible for the intricate matching of neurons to their targets. Neuronal survival depends on specific "survival" factors and genetic programs. On the other hand, such pathways may also be involved in the cell death and atrophy that occur pathologically in certain neurodegenerative disorders. These changes may arise from aberrantly activated gene-directed process or the absence of critical trophic signals (Manji et al., 1999). According to Jope (1999b), three systems appear as critical sites of action for Li. First, Li seems to alter the balance among neurotransmitter activities and may contribute to neuroprotection. Second, it modulates signals impacting on the cytoskeleton at multiple levels, including signals from glycogen synthase kinase 3(3 (GSK-3|3), cyclic AMP (cAMP)dependent kinase, and protein kinase C (PKC). These actions may be critical for the neuronal plasticity involved in mood recovery and stabilization. Finally, lithium adjusts signaling activities regulating second messengers, transcription factors, and gene expression. The neuroprotective effects of Li may be derived from its modulation of the expression of genes involved in regulating degenerative and regenerative mechanisms.

2. LITHIUM AND NEUROTRANSMITTER EFFECTS The underlying biological processes responsible for the episodic clinical manifestation of mania and depression are consistent with a dysregulation within the limbic system and associated regions of the brainstem and prefrontal cortex. The biogenic amine neurotransmitter systems are distributed extensively in the limbic system. Thus, it is not surprising that most of the early research on the actions of Li have focused on modulation of neurotransmitter synthesis, release, and uptake. Lithium was found to influence, to some extent, every neurotransmitter investigated, although most research has focused on catecholamines and acetylcholine (Wood and Goodwin, 1987). Recent studies have pointed out

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the modulation of glutaminergic neurotransmission by Li. It has been found that Li acutely increased synaptic concentration of glutamate, which led to an increase and stabilization of glutamate uptake transporter capacity (Dixon and Hokin, 1998). This could mediate a reduction and stabilization in excitatory neurotransmission after Li treatment and contribute to the neuroprotective effect of Li. Chronic Li administration blocks the excitotoxicity induced by glutamate agonists (Nonaka et al., 1998). It inhibits glutamate-induced neuronal calcium uptake and cell death (Nonaka et al., 1998). This novel action of Li suggests that chronic Li treatment may be beneficial to brain damage induced by hypoxic insult. It has been shown that Li administration in rats reduced the size of the ischemic infarct 24 hours after occlusion of the left middle cerebral artery (Nonaka and Chuang, 1998). This neuroprotective effect of Li is probably due to its ability to attenuate excessive calcium influx mediated by N-mefhyl-D-aspartate (NMDA) receptors. NMDA receptor over-activation has been strongly implicated in ischemic brain injury (Albers et al., 1989). N-acetyl-aspartate (NAA) is a putative neuronal marker localized to mature neurons (Birken and Oldendorf, 1989). A relative decrease in this compound may reflect decreased neuronal viability, function, or neuronal loss (Tsai and Coyle, 1995). It has been shown that chronic Li administration at therapeutic doses increases NAA concentration in the human brain in vivo (Moore et al., 2000a). Therefore, neuronal viability and function are increased in the human brain. Investigations of other neurotransmitters have been performed. The overall consequences of these actions may be that Li readjusts the balance between excitatory and inhibitory activities. Hence, it is the adjusted balances, rather than actions on a single neurotransmitter, that facilitate mood recovery and stabilization (Jope, 1999b).

3. LITHIUM AND SIGNALING NETWORK The regulation of cell survival and cell death is a complex process involving multiple interacting signaling pathways, transcription factors, and gene expression. Thus, the effects of Li on signaling pathways and transcription factors (Jope, 1999b; Manji et al., 1995a) may also contribute to its neuroprotective effects.

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Li influences the phosphoinositide (PI) and cAMP pathway. Here it also appears that multiple sites are affected by Li, and that the overall outcome has to do with the balances achieved between negative and positive modulatory effects of Li rather than from an individual site of action (Jope, 1999a).

3.1. PI-PKC Signal Transduction Pathways Generally, changes in neuronal function that persist after the activation of neurotransmitter pathways are brought about by post-translational effects of specific protein kinases. PKC is one of the major intracellular mediators of signals, which appears to play a crucial role in the regulation of synaptic plasticity and various forms of learning and memory. It has been shown that Li decreases the activity of PKC as a result of its direct inhibition of the enzyme, inositol monophosphatase, in the PI signaling pathway (Lenox, 1987; Manji and Lenox, 1994, 1999; Wang et al., 1999; Soares et al., 2000). Moreover, in rats chronically treated with Li, there is a reduction in the hippocampus of the expression of a major PKC substrate, the myristoylated alanine rich C kinase substrate (MARCKS) (Lenox et al., 1992; Mork et al., 1992; Manji et al., 1996; Watson and Lenox, 1996). It seems that transcriptional inhibition of the Macs gene underlies the chronic Li-induced downregulation of MARCKS expression (Wang et al., 2001). MARCKS has been implicated in brain development, cytoskeletal remodeling, Ca/calmodulin signaling, and neuroplasticity. It is involved in cytoskeletal architecture and inhibits PI signaling (Rosen et al., 1990; Seykora et al., 1991; Blackshear, 1993; Glaser et al., 1996), suggesting that Li may reduce this inhibitory influence on PI signaling as a result of downregulating MARCKS. It can be hypothesized that the reduction in MARCKS protein, following long-term Li administration, may play a role in altering pre/post-synaptic membrane structure, thus stabilizing aberrant neuronal signaling in key regions of patients with bipolar disorders (Lenox, 1987; Lenox and Manji, 1998; Lenox and Hahn, 2000).

3.2. Lithium and cAMP Pathway Numerous studies have demonstrated that cAMP second messenger system is modulated by Li (Wood and Goodwin, 1987; Avissar and Schreiber, 1992; Jope and Williams, 1994). In general, Li increases basal

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levels of cAMP, but impairs receptor-coupled stimulation of cAMP production. These dual effects may be due to reduction in the activity of the inhibitory G; protein (Avissar and Schreiber, 1992; Manji et al., 1995), inhibitory effect on the activation of the stimulatory Gs protein (Mork et al., 1992; Avissar and Schreiber, 1992; Mork and Geisler, 1995), or direct inhibitory effects on adenyl cyclase (Mork and Geisler, 1995). Overall, it appears that these actions of Li reduce the magnitude of fluctuations in cAMP levels by increasing the lowest basal levels and decreasing maximal stimulated increases, thus stabilizing the activity of this signaling system. A primary action of cAMP is to stimulate the activity of cAMPdependent protein kinase A. It seems that Li inhibits protein kinase A-induced phosphorylation of cytoskeletal proteins (Mori et al., 1996; 1998), an action that may contribute to long-term modulation of the neuronal structure and function by Li.

3.3. Protein Phosphorylation of Cytoskeletal Proteins Protein phosphorylation of cytoskeletal proteins was recently found to be affected by a newly discovered inhibitory effect of Li on GSK-3(B (Klein and Melton, 1996; Stambolic et al., 1996; Hong et al., 1997; MunozMontano et al., 1997). GSK-3(3 is known to play a critical role in the central nervous system by regulating various cytoskeletal processes via its effects on tau (T), synapsin I, and long-term nuclear events via phosphorylation of c-jun and nuclear translocation of p-catenin (Dale, 1998; Willert and Nusse, 1998). Dephosphorylation of T caused by Li enhances the binding of T to microtubules, which promotes microtubule assembly (Lovestone et al., 1996). Therefore, Li may stabilize microtubule-based neuronal structure. However, inhibition of GSK-3(3 by Li also dramatically decreases mitogenactivated protein IB (MAP-IB) phosphorylation (Agam and Levine, 1998; Garcia-Perez et al., 1998; Lucas et al., 1998). Dephosphorylation of MAP-IB, in contrast to T dephosphorylation, decreases its ability to bind and stabilize microtubules. This response to Li treatment was also reported to cause increased axonal spreading and increased growth cone area (Lucas et al., 1998). These findings suggest that neuronal cytoskeletal rearrangements may be a significant consequence of Li administration. The opposing effect of dephosphorylation of T and MAP-IB, promoting

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microtubule disassembly and assembly, respectively, caused by Li's inhibition of GSK-3P, support the fact that Li acts at multiple sites to adjust the balance between opposing influences rather than have a unidirectional effect. On the other hand, intracellular neurofibrillary tangles found in Alzheimer's disease are composed of filaments that contain an abberantly hyperphosphorylated form of the microtubule-associated protein T. Hyperphosphorylation of T is an early event in the course of Alzheimer's disease that may precede the disruption of the microtubule cytoskeleton. Thus, inhibition of the GSK-3(3 by Li represents an attractive potential mechanism to reduce the accumulation of hyperphosphorylated T that is found in neurofibrillary tangles. GSK-3p also plays a major role in regulating p-catenin levels. P-catenin plays a central role in developmental and apoptotic processes via gene regulation (Agam and Levine, 1998). Recent studies have shown that presenilin-1 forms a complex with P-catenin (Zhang et al., 1998). Mutations in the presenilin-1 gene have been found in many patients with familial Alzheimer's disease, together with a reduced level of (3-catenin. Loss of p-catenin signaling seems to increase neuronal vulnerability to apoptosis induced by amyloid-p protein (Zhang et al., 1998). Thus, inhibition of GSK-3P by Li may serve to offset the P-catenin destabilizing effects of mutated forms of presinilin-1, thereby reducing the vulnerability of affected neurons to apoptosis induced by amyloid-P protein.

4. LITHIUM AND TRANSCRIPTION FACTORS, GENE EXPRESSION, AND PROTEIN LEVELS Transcription factors fulfil the critical functions of transmitting and regulating signals to the nucleus, allowing cells to respond to a changing environment through alteration in gene expression. Thus, transcription factors may be selectively influenced by Li, both as a result of Li's modulation of the activities of specific signaling systems and as targets for Li to modulate the expression of genes selectively. Studies on Li have focused on transcription factors coded by immediate early genes, those responding rapidly and transiently to cell signals, such as the AP-1 transcription factor comprising dimers of the fos and jun protein families.

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Stimulation of AP-1 DNA binding activity in rat brain was found to be attenuated by Li (Williams and Jope, 1995). This attenuation was confirmed in cultured cells, but elevated basal AP-1 DNA activity was noted as the concentration of Li increased (Jope and Song, 1997; Unlap and Jope, 1997). The decrease in receptor-coupled stimulation of AP-1 could result from inhibition by Li of PI hydrolysis, reduced second messenger activities, or other effects that regulate stimulation of AP-1. The increased basal level of AP-1 activity is likely due to inhibition by Li of GSK-3(3, an enzyme that is inhibitory towards AP-1. Hence, blockade by Li can elevate basal AP-1. These combined effects of Li on basal and stimulated AP-1 may model how multiple actions of Li are integrated to stabilize fluctuations in signaling systems. It seems likely that alterations in neuronal gene expression and, consequently, protein levels induced by Li may underlie the necessity for long-term treatment. This time delay may be required for neuronal proteins to reach new steady-state levels in the presence of Li, which mediates structural and/or functional neuronal alterations (Jope, 1999b).

4.1. Lithium and Bcl-2 Substantial progress has been made in identifying the genes that are responsive to trans-synaptic stimulation. One of the genes whose expression was markedly increased by Li treatment in rats is the transcription factor PEBP2P (Chen and Chuang, 1999). The major neuroprotective protein, Bcl-2, is regulated by PEBP2C3 (Klampfer et al., 1996). Extensive research aimed at elucidating the signaling pathways and proteins involved in regulating physiological and pathophysiological cell death has revealed critical roles for mammalian proteins, the Bcl-2 family of proteins. Bcl-2 is the acronym for the B-cell lymphoma/leukemia-2 gene. It is expressed in the rodent and mammalian nervous systems and is localized to the outer mitochondrial membrane, endoplasmic reticulum, and nuclear membrane. This protein inhibits both apoptotic and necrotic cell death induced by diverse stimuli (Merry and Korsmeyer, 1997; Adams and Cory, 1998; Bruckheimer et al., 1998; Sadoul, 1998; Wilson, 1998; Li and Yuan, 1999). A role for Bcl-2 in protecting neurons from cell death is now supported by abundant evidence, both in vitro and in vivo (Bonfanti et al.,

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1996; Lawrence et al., 1996; Chen et al, 1997; Kostic et al, 1997; Merry and Korsmeyer, 1997; Adams and Cory, 1998; Bruckheimer et al., 1998; Sadoul, 1998; Raghupathi et al., 1998; Yang et al., 1998; Li and Yuan, 1999). It has been shown that chronic Li greatly increases the level of the neuroprotective protein, Bcl-2, in areas of rodent frontal cortices, hippocampus, and striatum in vivo (Chen et al., 1999; Manji et al., 2000), as well as in cultured cells of both rodent and human neuronal origins in vitro (Manji et al., 1999). In cultured cell systems, Li has also been demonstrated to reduce the levels of the pro-apoptotic protein p53 (Chen and Chuang, 1999; Lu et al., 1999).

5. CONCLUSION Does Li have neuroprotective effects? Multiple actions of Li support neural plasticity and stabilize neurotransmitter balances, signaling activities, and gene expression. At the level of neurotransmission and signal transduction, Li seems to readjust the balance between excitatory and inhibitory activities. Several earlier studies have already tried to demonstrate the neuroprotective effects of Li (Volonte and Rukenstein, 1993; Di Mello et al., 1994; Li et al, 1994; Inouye et al., 1995; Pascual and Gonzalez, 1995; Dixon and Hokin, 1998). In vitro and in vivo studies dealing with the neuroprotective effects of Li have shown that, First, the modulation of glutaminergic neurotransmission by Li could mediate a reduction and stabilization in excitatory neurotransmission. The neuroprotective effect of Li is probably due to its ability to attenuate excessive calcium influx mediated by NMD A receptors. Secondly, chronic Li administration at therapeutic doses increases NAA concentration in the human brain in vivo, thereby increasing neuronal viability and function. Thirdly, Li reduces the magnitude of fluctuations in cAMP levels by increasing the lowest basal levels and decreasing maximal stimulated increases, thus stabilizing the activity of this signaling system. Fourthly, Li inhibits protein kinase A-induced phosphorylation of cytoskeletal proteins, an action that may contribute to long-term modulation of neuronal structure and function by Li. Fifthly, the reduction in MARCKS protein, following long-term Li administration, may play a role in altering pre/post-synaptic membrane structure. This stabilizes aberrant neuronal signaling in key regions of patients with bipolar disorders.

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Finally, it is estimated that inositol monophosphates constitute only about 10% to 15% of the total brain phosphomonoesters (Renshaw et al., 1987). The effects of Li on other lipids are not well documented. In our laboratory, we have shown that in rats submitted to pre- and/or post-natal stresses, by increasing the most unsaturated phospholipids and decreasing the less unsaturated (thereby modifying the ratio of saturated to unsaturated fatty acids towards an increase in fatty acid unsaturation), Li may counteract the effects of pre- and post-natal stresses on the saturation of the phospholipid fatty acids of the opioid receptor membrane (ErmidouPollet et al., 1999). On the other hand, we have shown that in mice receiving intracranial injections of Li, tremors and convulsions may occur depending on the concentration of Li injected. When, between zero and 30 minutes, depending on injected Li concentrations, tremors and convulsions occur and when, between 30 minutes and one hour, depending on injected Li concentrations, convulsions disappear and tremors subsist, a decrease in monosialo-gangliosides (GMj) and an increase in gangliosides with four sialic acid residues (GQ) can be observed. These changes in the distribution of gangliosides may be related to the presence of tremors. The disappearance of the tremors may be explained by a return to normal of the GM^nd GQ gangliosides level (ErmidouPollet and Pollet, 2000). Therefore, it seems that Li may also play a protective role via its action on the brain membrane lipids. At therapeutic concentration, Li also directly inhibits GSK-3J3. This inhibition may serve to offset the p-catenin destabilizing effects of mutated forms of presinilin-1, thereby reducing the vulnerability of affected neurons to apoptosis induced by amyloid-(3 protein. On the other hand, dephosphorylation of T caused by Li enhances the binding of T to microtubules. This may stabilize microtubule-based neuronal structure. The opposing effect of dephosphorylation of T and of MAP-IB, promoting microtubule disassembly and assembly, respectively, caused by Li's inhibition of GSK-3p, support the fact that Li acts at multiple sites to adjust the balance between opposing influences rather than having a unidirectional effect. The combined effects of Li on basal and stimulated AP-1 may model how multiple actions of Li are integrated to stabilize fluctuations in signaling systems. Li's robust upregulation of Bcl-2 levels at therapeutically relevant concentrations likely plays a major role in its protective effects. The increase in Bcl-2 levels, inhibition of GSK-3(3 and accompanying effects on T and (3-catenin, and clear evidence on neuroprotective

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effects all suggest that the potential efficacy of Li in the long-term treatment of various neurodegenerative disorders should be investigated. Actually, any data to support or refute the contention that chronic Li administration in bipolar patients results in a reduction in the incidence of severity of neurodegenerative disorders in this population can be found. Clearly, it is necessary to demonstrate, in a longitudinal study, that Li treatment does reduce or delay central nervous system cell death or atrophy in mood disorders patients. It has been found that Li-treated bipolar patients exhibit smaller reduction in frontal cortices volume than non-Litreated patients (Jope, 1999b). Moore et al. (2000b) and Lachman and Papolos (1995) found that after four weeks of treatment with Li, though there was no significant change in the composition of gray and white matter, there was a significant increase in the volume of gray matter in bipolar disorders patients. On the other hand, in animal experiments — Bcl-2 knockout mice are now available (Hochman et al., 2000) — do they show any behavioral effects ? However, clear evidence for the neuroprotective effects of Li, as well as a growing appreciation that mood disorders are associated with cell loss or atrophy, suggests that these effects may be very relevant to the long-term treatment of mood disorders. Increasing knowledge of the etiology and pathogenesis will provide future opportunities to develop specific therapies aimed at protecting neurons from underlying degenerative processes.

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Williams MB, Jope RS. Circadian variation in rat brain AP-1 DNA binding activity after cholinergic stimulation: Modulation by lithium. Psychopharmacology 1995; 122:363-368. Wilson MR. Apoptosis: Unmasking the executioner. Cell Death Differ 1998; 5:646-652. Wood AJ, Goodwin GM. A review of the biochemical and neuropharmacological actions of lithium. Psychol Med 1987; 17:579-600. Yang L, Matthews RT, Schulz JB, Klockgether T, Liao AW, Martinou JC et al. 1-Methyl4-phenyl-l,2,3,6-tetrahydropyride neurotoxicity is attenuated in mice overexpressing Bcl-2. JNeurosci 1998; 8145-8152. Zhang Z, Hartmann H, Do VM, Abramowski D, Sturchler-Pierrat C, Staufenbiel M et al. Destabilization of (3-catenin by mutations in presenilin-1 potentiates neuronal apoptosis. Nature 1998; 395:698-702.

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CHAPTER

21

Histopathological Changes in Brain of Uremic Patients on Chronic Hemodialysis Pamela Zambenedetti, Mario Andriani, Maurizio Nordio, Paolo Zatta

ABSTRACT Chronic renal failure and its treatment are often associated with neurologic disturbances that must be differentiated from generalized nervous system complications independent from it and related to other vascular or degenerative diseases. We report an immunohistochemical study on the brains of 13 uremic patients who underwent dialysis with the aim to find out, if any, relevant morphological alteration that could be related to the status derived from chronic renal disease. Indeed, morphological and histochemical alterations were observed in the controls. Activation of microglial cells and astrocytosis, with an overexpression of metallothioneins-I-II, is described. In some patients, the presence of senile plaques positive for amyloid-p was also observed. In addition, the microtubule-associated protein was detected in the neurons of six of the 13 cases studied; decreased expression of the presence of synaptobrevin and phosphorylated neurofilaments indicated alterations of neuron function. Keywords: Alzheimer's disease; uremia; nephropathy; metallothionein; renal failure; encephalopathy; glia; microglia.

1. INTRODUCTION Neurological derangements are common in chronic renal failure patients. In many instances, they are not typical of uremia, but are related to 483

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cerebrovascular disorders or aging (Savazzi et al., 2001); in other cases, they are present only in endstage renal disease (ESRD) patients, such as dialysis dementia (Alfrey et al., 1976), dialysis disequilibrium syndrome, and uremic encephalopathy (Loockwood, 1989; Kawahara et al., 1988). In the last decade, new ESRD patients are older, thus brain disorders occurred before the institution of dialysis treatment became common. Cerebral atrophy described in hemodialysis patients is dependent on the severity and duration of hypertension (Savazzi et al., 1999). Early atherosclerosis and related hypoperfusion can be considered the most important causes of parenchymal cerebral damage in uremia (Savazzi et al., 2001). In uremic rats, an increase in total calcium content of the cerebral cortex, with increased levels of cytosolic calcium in synaptosomes, has been found. Moreover, an inappropriate response to depolarization has been observed in synaptosomes, affecting neurotransmitter metabolism (Smogorzewski, 2001). While CAT and MRI abnormalities have been clearly documented in uremic patients and neurophysiologic derangements have been observed at the neuronal level in experimental animals, histological changes have been studied mostly in case reports and in an unsystematic manner. We studied, therefore, at the histological level the expression of markers of cellular activity in the brain of uremic patients.

2. EXPERIMENTAL PROCEDURES 2.1. Brain Sampling Brain tissues from 13 dialyzed subjects and four case controls were obtained from the Brain Bank at the General Hospital in Dolo-Venice, Italy. Sections were cut from the frontal, parietal, and occipital lobes. Tissues were fixed in 10% buffered formalin and then embedded in paraffin.

2.2. Histological Analysis Tissue sections measuring 6 urn were deparaffinized and hydrated according to standard procedures. The endogenous tissue peroxidases were blocked by incubation with 3% H 2 0 2 in phosphate buffer solution (PBS) for 10 minutes, followed by three washes in PBS thrice. Sections were preincubated for 30 minutes with 3% bovine serum albumin in PBS

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Changes in Brain of Uremic

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Patients

Table 1. Antibodies utilized in immunohistochemistry*.

Antigen

Supplier

Host

Dilution

Synaptophysin

Oncogene Research Product (Cambridge, UK)

Monoclonal Mouse

1:50

Synaptobrevin

Oncogene Research Product (Cambridge, UK)

Monoclonal Mouse

1:50

Metallothionein

Dako (Milan, Italy)

Monoclonal Mouse

1:50

Microtubuleassociated protein

Boehringer-Mannheim (Germany)

Monoclonal Mouse

1:400

Glial fibrillary acidic protein

Dako (Milan, Italy)

Polyclonal Rabbit

1:100

Ferritin

Dako (Milan, Italy)

Polyclonal Rabbit

1:100

Amyloid-(34

Boehringer-Mannheim (Germany)

Polyclonal Rabbit

1:10

Tau

Boehringer-Mannheim (Germany)

Monoclonal Mouse

1:20

Phosphorylated neurofilament

Affinity (Exeter, UK)

Monoclonal Mouse

1:1,000

*The secondary antibodies (Dako, Milan, Italy) used were anti-mouse or anti-rabbit, depending on the source of the primary antibodies. The chromogenic enzyme horseradish peroxidase was activated by incubation with Tris-HCl, pH 7.2 containing the substrate 3,3'-diaminobenzidine, 10mM CaCl2, and 0.03% H 2 0 2 for 10 minutes. Sections were then washed, counterstained with hematoxylin, rinsed in MilliQ water, dehydrated by serial passages in alcohol, and monted in Clearium medium (Surgipath).

and then incubated separately with the different antibodies overnight. The primary antibodies utilized in our study are reported in Table 1.

3. IMMUNOHISTOLOGICAL FINDINGS The uremic subjects included 10 males and three females, with a median age of 73 years (range, 57 to 80 years). They were on hemodialysis for a mean period of 21 months (range, six to 180 months). Six patients died of cardiovascular diseases, two of infectious diseases, two of neoplasms,

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P et al.

and one of dementia. The four control subjects included three females and one male with a mean age of 79 years (range, 65 to 86 years). All died of cardiovascular diseases. The data are reported in Table 2. Twelve of the 13 uremic brains showed an intense expression of metallothioneins (MTs), which were completely absent in nonuremic brains. A similar behavior was observed for ferritin (13/13 versus 0/4). On the contrary, synaptobrevin expression was reduced in all uremic brains compared to nonuremic brains. Also, phosphorylated neurofilament expression was reduced in 12 uremic patients. Amyloid-p4 (ApJ4) was present in four uremic brains and absent in control brains. The relevant immunohistochemical results are summarized in Table 2. Table 2. Synthesis of the immunohistochemical results. Cause Age of Subject (years) Sex death

4593

563 586 4306 4044

548 560 505 3772 2743

554 589 577

Mo M T I - I I

72 77 80 78 62 73 68 70 57 73 68 65 74

M M M M M M M M F F F M M

SS CA CA DE A T CA CA P CA CA A PN

25 18 78 180 10 34 27 25 6 14 15 20 21

++ +/+/++ +/+++ +/++ ++

65 71 87 86

F M F F

CA CA CA CA

-

A £ 4 Ferritin MAP Tau

AbPh

Synbr

+

++ + -

+ + ++ -

+ + + +

-

+ + + + +

+

+ -/+ +

+

+ + +

-

++ ++

-

+/+ + +

+ + +

-

-

-

-

+/-

+

+

+

+

+ +

+/-

+ +

+

+

+ + + +

++ ++ ++ ++

+ + + +

-

Cont

592 576 556 585

A=aneurysm, Ap4=amyloid-P4, AbPh=phosphorylated neurofilament, AC=adenocarcinoma, CA=cardiac arrest, Cont=control, DE=dialysis encephalopathy, MAP=microtubule-associated protein, Mo=months of dialysis treatment, MT=metallothionein, P=pneumonia, PN=pulmonary neoplasia, SS=septic shock, Synbr=synaptobrenin, T=thrombosis. Immunopositive cell for microscopic field (magnification 500X). ( - ) - » 0 - 3 ; ( - / + ) -> 4 - 6 ; ( + ) -¥ 7-10; ( + + ) - > 10-14; ( + + + ) - > > 15.

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Figure IA shows the white matter of a case control where astrocytes reacting with anti-MT-I-II were not observed. In all the case controls, the rare astrocytes present were not activated as expected. Figures IB, Ie, and ID show the white matter of uremic subjects treated with dialysis where diverse activated astrocytes can be seen. In Figure IB, a perivascular astrocyte is also present. Figure 2A shows a sample from the gray matter of a case control where microglial cells positive to anti-ferritin are absent. In contrast, many ferritin-positive microglial cells are present in a specimen from a uremic subject who underwent dialysis (Fig. 2B). While the case control scored negative using anti-microtubuleassociated proteins (MAPs) (Fig. 3A), a strong reactivity can be observed

Fig. 1. Cerebral white matter stained with anti-metallothionein I-II antibody. (A) Case control: (B and C) Metallothionein-I-II positive activated perivascular astrocytes. (D) Protoplasmatic astrocytes in an uremic subject. (Magnification 500X)

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*

A

..B

a

t

0

J'

Fig. 2. Cerebral gray matter stained with anti-ferritin antibody. (A) Control. (B) Activated microglial cells in an uremic subject. (Magnification 500X)

AJ

>. *•• .

B

Fig. 3. Cerebral gray matter stained with anti-microtubule-associated protein (MAP) antibody. (A) Case control negative for anti-MAP. (B) Positivity for MAP antibody in the pyramidal neurons of an uremic subject. (Magnification 500X)

in the pyramidal neurons of an uremic subject (Fig. 3B). Intra- and extra-neuronal amyloid is a characteristic feature of several neurodegenerative diseases. Likewise, anti-Ap4 antibody recognized senile plaques in some of the uremic patients who underwent dialysis, but scored negative in the case control. Figures 4A and 4B show two distinct types of A|34-positive senile plaques detected in an uremic patient.

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Fig. 4. Two examples of positivity for amyloid-f34 antibody in the gray matter of an uremic subject: presence of diffuse/immature plaque-like structures. (Magnification 500X)

4. REMARKS Renal failure commonly affects the nervous system, and the effects of kidney failure on the nervous system are more pronounced when failure is acute. The neurologic manifestations of renal failure are variable, nonspecific, and most likely result from multiple metabolic derangements. Dialysis itself is associated with different and distinct disorders of the central nervous system, and patients on maintenance hemodialysis are at risk for several neurological implications. Nervous system dysfunction remains a major cause of disability in patients with ESRD. In this connection, cerebral atrophy was observed in the majority of subjects on long-term hemodialysis treatment but, notably no significant correlation was found between the degree of atrophy and relevant hematoseric parameters (Savazzi et aI., 1999). Uremic encephalopathy is associated with problems in cognition and memory, and may progress to delirium, convulsions, and eventually coma (Burns and Bates, 1998). Although clinical, epidemiologic, toxicologic, and electroencephalographic features of dialysis encephalopathy have been throughly described, the neuropathologic and clinicopathologic aspects of the disease have remained rather obscure. In spite of an abnormal accumulation of aluminum, some uremic subjects treated by dialysis are not affected by dialysis encephalopathy

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(Andriani et al., 1995). The relevance of this observation, in terms of the connection between aluminum and neurological manifestations, remains unclear. In our study, the most typical aspects found in uremic brains were an enhanced expression of MT-I-II and a reduced expression of synaptobrevin and phosphorylated neurofilaments. Reactive astrocytosis is a well-recognized general phenomenon where astrocytes can change in phenotype. Glial changes are invariably accompanied by structural synaptic remodeling which result in an increased number of neurotransmitter afferents (Theodosis and Poulain, 1999). One of the functions attributed to the activation of astrocytes is the restoration of damaged tissue. However, the role played by astrocytes in inflammatory reactions in several neurological diseases associated with uremia has not received much attention, and hence our interest in establishing the status of astrocytes' activation in the brains of uremic patients. MTs are low molecular weight zinc-binding protein thiols whose function remains unclear. In primates and humans, MTs are mainly found in the astrocytes (Hidalgo et al., 2001; Mocchegiani et al., 2001). In the mammalian central nervous system, MTs are found in pia-arachnoid, ependymal cells and astrocytes, and both furnish essential elements such as Cu(II) and Zn(II) and protect neurons against toxic ions (see Chapter 11). It has been hypothesized that MTs can act as antioxidants intercepting free radicals by forming complexes with redox metals. MTs can be secreted by glial cells in response to the presence of xenobiotics and dismetabolic metal ions (Nordio et al., 1998). MTs may, thus, serve to protect cells from the toxic effects of heavy metals and oxidative stress. MTs are highly expressed in uremic subjects undergoing dialysis, indicating activation of astrocytes as a result of alterated metal ions' homeostasis and oxidative stress. Synaptic vesicles contain neurotransmitter molecules that are predominantly proteic in character. One set of these proteins, synaptophysin and synaptobrevin, appear to be specific to the formation of a fusion complex of small vesicles (Savazzi et al., 1995). Synaptophysin and synaptobrevin are integral proteins that cross the membrane of vesicles. Both can bind Ca 2+ and may be phosphorylated at a tyrosine residue. Alterations in the expression of these proteins are an important marker for the functionality of neurons and axonal current. While synaptobrevin does not appears

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to be altered in our uremic subjects (data not reported), synaptophysin levels appear to be modestly reduced with respect to the controls. Uremic encephalopathy may initially worsen with dialysis as a result of impaired synaptic functions and metabolic alterations, such as relevant ionic changes (Burns and Bates, 1998), probably related to ionized Ca 2+ cellular content. The positive expression of A(34 in four uremic brains, although not uniform, suggests some interesting considerations. Treatment of experimental animals with aluminum salts produces a striking and long-lasting accumulation of amyloid-(3 protein precursor (Af3PP) and an activation of microglial cells (Shigematsu and McGeer, 1992). In this connection, it is known that uremic subjects under dialysis are at risk for aluminum accumulation in the brain (see Chapter 4). It is worth noting that in Alzheimer's disease (AD), perivascular microglial cells are involved in the production of A(34 fibrils and accumulation of amyloid in the capillary wall. In addition, microglial cells are loosely arranged in primitive plaques in AD (Wisniewski and Wegiel, 1995). In our study, we observed that microglial cells are all activated to some extent (Fig. 1). Classical and primitive plaques are complex deposits that include amyloid, microglial cells, dystrophic neurons, and activated astrocytes. According to some authors, microglial cells represent the site of amyloid formation (Wegiel and Wisniewski, 1992). It is worth noting that, in our uremic subjects, microglial cells are not only activated (Fig. 2), but also tested positive for A(34 (Fig. 4). These findings confirm previous observations reported by other authors (Brun and Dictor, 1981). In addition, a study of patients with chronic renal failure revealed nuclear immunostaining with an antibody to the N-terminal region of the A(3PP in 14 of 15 cases studied (Candy et al., 1992), with five of these patients having amorphous senile plaques containing A(34. These plaques were of the same so-called "immature" type observed in AD. On the other hand, neurofibrillary tangles, a characteristic feature of AD, are never, or very rarely, observed in dialysis patients. In this regard, Edwardson et al. (1991) concluded that Al(III) may induce some AD-like neuropathological changes in uremic subjects through mechanisms involving transferrin-mediated uptake of Al(III) by neurons or decreased turnover of the A(3PP.

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5. CONCLUSIONS Histological modifications that occur in the brains of dialysis patients who died for reasons apparently not related to dialytic treatment are unspecific (Burks et al., 1976). Our study shows morphological alterations sometimes coherent with clinical and neurological manifestations. It is notable that morphological changes observed in neurons and glial cells are rather different from those observed in AD. This is an important issue in that some authors hypothesized that the abnormal accumulation of Al(III), observed in the brains of uremic subjects in dialysis, could be a high risk factor for AD. In agreement with other authors (Reusche and Seydel, 1993), we did not observe any neurofibrillary tangles or "mature" senile plaques in the brain tissue of uremic patients, and detected a limited number of plaques of the "immature" and diffuse type in only a few cases. Thus, the Ap4 detected in some of our patients could indicate that amyloidosis is more likely a general feature than a specific property. As dialysis itself is associated with a variety of neurological syndromes, including disequilibrium syndrome, subdural hematoma, and Werniche's encephalopathy, the involvement of neurologists in a tight collaboration with nephrologists is highly advised in order to prevent the aggravation of neurological, clinical symptomatology. Patients under dialysis show histochemical alterations that suggest the need for careful attention in the clinical practice in a collaborative approach among different medical specialties.

ACKNOWLEDGMENT The authors thank Ms Cristina Renesto for her excellent technical assistance.

REFERENCES Alfrey AC, LeGendre GR, Kaehny WD. The dialysis encephalopathy syndrome. Possible aluminum intoxication. N Engl J Med 1976; 22:184-188. Andriani M, Nordio M, Zambenedetti P, et al. L'emodialisi induce malattia? In: Saporiti E, Marchini E, editors. L'alluminio: Dalla Storia Alia Ricerca, Milan: Wichting Publishing, 1995: 9-21. Beecker A, Drenckhahn A, Pahner I, et al. The synaptophysin-synaptobrevin complex: A hallmark of synaptic vesicle maturation. J Neurosci 1999; 19:1922-1931.

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Brun A, Dictor M. Senile plaques and tangles in dialysis dementia. Acta Pathol Microbiol Scand 1981; 89:193-198. Burks JS, Alfrey AC, Huddlestone J, Norenberg MD, Lewin E. A fatal encephalopathy in chronic hemodialysis patients. Lancet 1976; 1:764-768. Bums DJ, Bates D. Neurology and the kidney. J Neurol-Neurosurg Psychiatr 1998: 810-821. Candy JM, McArthur FK, Oakley AE et al. Aluminum accumulation in relation to senile plaques and neurofibrillary tangles formation in the brains of patients with renal failure. Neurol Sci 1992; 107:210-218. Edwardson JA, Ferrier IN, McArthur FK et al. Alzheimer's disease and the aluminum hypothesis. In: Nicolini M, Zatta P, Coram B, editors. Aluminum in Chemistry, Biology and Medicine. New York: Raven Press, 1991: 85-96. Fraser CL, Arieff Al. Nervous system and complications in uremia. Ann Intern Med 1988; 109:143-153. Hidalgo J, Aschner M, Zatta P, Vasak M. Roles of the metallothionein family of proteins in the central nervous system. Brain Res Bull 2001; 55:133-146. Kawahara T, Markert M, Waiters JP. Neutrophil radical production by dialysis membrane. Nephrol Dial Transpl 1988; 3:661-665. Loockwood AH. Neurologic complications of renal disease. Neurol Clin 1989; 7:617-627. Mocchegiani E, Giacconi R, Cipriano C et al. Zinc-bound metallothioneins as potential biological markers of aging. Brain Res Bull 2001; 55:147-154. Nordio M, Andriani M, Gerotto M. et al. Serum concentration of trace elements during different stages of chronic renal failure. ItalJ Min Electrol Metabol 1998; 12:81-86. Reusche E, Seydel U. Dialysis-associated encephalopathy: Light and electron microscopic morphology and topography with evidence of aluminum by laser microprobe mass analysis. Acta Neuropathol 1993; 86:249-258. Savazzi GM, Cusumano F, Bergamaschi E et al. Hypertension as an etiopathological factor in the development of cerebral atrophy in hemodialysed patients. Nephron 1999; 81:17-24. Savazzi GM, Cusumano F, Musini S. Cerebral imaging changes in patients with chronic renal failure treated conservatively or in hemodialysis. Nephron 2001; 89:31-36. Savazzi GM, Cusumano F, Vinci S, Allegri L. Progression of cerebral atrophy in patients on regular hemodialysis treatment: Long-term follow-up with cerebral computed tomography. Nephron 1995; 69:29-33. Shigematsu K, McGeer PL. Accumulation of amyloid precursor protein in damaged neuronal processes and microglia following intracerebral administration of aluminum salts. Brain Res 1992; 593:117-123. Smogorzewski MJ. Central nervous dysfunction in uremia. Am J Kidney Dis 2001; 38:S122-S128. Suzuki Y. Genes, cells and cytokines in resistance against development of toxoplasmic encephalitis. Immunobiol 1999; 201:255-271. Theodosis DT, Poulain DA. Contribution of astrocytes to activity-dependent structural plasticity in the adult brain. Adv Exp Med Biol 1999; 468:175-182.

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Trznadel K, Lucinak M, Pawlicki L. Superoxide anion generation and lipid peroxidation processes during hemodialysis with reused cuprophan dialyzer. Free Rad Biol Med 1990; 8:429^132. Vukicevic S, Kracun I, Vukelic Z. 24R,25-dihydroxyvitamin D3 prevents aluminuminduced alteration of brain gangliosides in uremic rats by keeping the metal within perivascular astrocytes of the blood-brain barrier. Neurochem Intern 1992; 20:391-399. Wegiel J, Wisniewski HM. Tubuloreticular structures in microglial cells, pericytes and endothelial cells in Alzheimer's disease. Acta Neuropathol 1992; 83:653-658. Winkelman MD, Ricanati ES. Dialysis encephalopathy: Neuropathological aspects. Human Pathol 1986; 17:823-833. Wisniewski H, Wegiel J. Non-neuronal cells involved in (3-amyloid deposition in Alzheimer's disease. In: Zatta P, Nicolini M, editors. Non-neuronal Cells in Alzheimer's Disease. Singapore: World Scientific, 1995: 194-203.

CHAPTER 22

Clinical Neurotoxicity of Metals and Neurodegenerative Disorders Marcello Lotti

ABSTRACT While clinical neurotoxicity of metals rarely results in neurodegeneration, the pathophysiology of Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis is characterized by the accumulation of certain metal ions in the central nervous system, which eventually leads to free radical-mediated oxidative stress and neuronal death. However, it is not clear if metal accumulation is a primary or secondary event, whether it is pivotal in driving the progression of the disease, and how it may interact with genetic factors. Example only one suggests analogies between clinical neurotoxicity of metals and neurodegenerative diseases. Clinical, histopathological, and toxicological characteristics of manganese poisoning overlap with those of two metal accumulation diseases, Wilson's disease and Hallervorden-Spatz disease. The understanding of mechanistic analogies and differences among toxic and nontoxic diseases may help to clarify the role of metal accumulation in neurodegeneration. Keywords: Neurotoxicity; neurodegeneration; Alzheimer's disease; Parkinson's disease; amyotrophic lateral sclerosis.

1. INTRODUCTION The asymmetry between the way we understand etiology and pathophysiology of neurodegenerative disorders is striking. It is also one of the most disturbing. While heredity has been established in a few, the etiology of 495

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the vast majority of cases is still unknown. Conversely, various pathways of neurodegeneration have been explored and the key role of metal ions has been highlighted by several studies, as has been described in the various chapters in this book. In recent years, the search for the etiologies of neurodegenerative disorders has resulted in a focus on genetic factors known to play important roles in the pathogenesis of Alzheimer dementia (Lendon et al., 1997), Parkinson's disease (PD) (Polymeropoulus et al., 1997), and amyotrophic lateral sclerosis (ALS) (Rosen et al., 1993). However, only about 1% to 10% of such cases are hereditary often as autosomal dominant traits (Martin, 1999). A possible role of environmental toxins in causing or precipitating neurodegenerative disorders has been actively explored for a long time now, and the report that l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) causes Parkinsonism in humans has led the hunt, unsuccessful, so far for candidate environmental chemicals (Ben-Shlomo, 1996). In the case of metals, this possibility has revolved largely around the putative role of some ions and, in particular, that of aluminum in Alzheimer's disease (AD) (Alfrey et al., 1976; Neri and Hewitt, 1991; Exley, 2001). However, the pathological role of metals, if any, remains unclear and there is no compelling evidence that they serve as primary toxins which can initiate these diseases. The outbreak of a neurodegenerative disorder characterized by any combination of dementia, Parkinsonian features, and motor neuron disease, which occurred between 1945 and 1980 in the indigenous population of Guam and other islands in the Western Pacific (Spencer et al., 2000), is an example of the qualified failure to clarify genetic-environmental relationships in neurodegeneration. Investigators hoped that this syndrome would represent the Rosetta Stone of neurodegenerative disorders, and early suggestions indicated that the disease may have been related to the consumption of seeds of cycas circinalis (Kurland, 1963). Aluminum was also considered an etiological factor because the metal had accumulated in neurofibrillary tangles found in brains of affected subjects, but no association was found between the incidence of Guam disease and the geographical distribution of aluminum in the island's waters (Zhang et al., 1996). The disease has rapidly declined since, though some cases of dementia with rigidity continue to be seen. The epidemiological evidence

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supports a probable environmental cause, in particular that of Cycas consumption (Spencer, 2000), but it remains unsolved what role toxicity had in this cluster of atypical neurodegenerative disorders, considering that the possibility of a contributing genetic factor has not been excluded (Zhang et al., 1996; Bird, 2001). This book, as well as a large body of clinical and experimental literature, indicates that several metals are involved in the neuropathology of disorders such as AD, ALS, and PD (Campbell et al., 2001). Increased concentrations of metal ions have been found in the nervous tissues of these patients and the underlying cause of neuronal cell degeneration is thought to be a measurable increase in free radical-mediated oxidative stress at some stage during disease progression (Bains and Shaw, 1997). Chapter 1 summarizes the central role of metals in oxidative damage and response in neurodegenerative diseases. Nevertheless, fundamental questions remain unanswered, such as whether these mechanisms represent a primary initiating event or a secondary phenomena, if they are involved in the progression of the disease, and if and how metals may interact with genetic factors. Moreover, transition metals are normally tightly regulated to prevent free radical formation, and several additional factors have been identified which could either account or exacerbate the toxic potential of brain-associated metals in neurodegenerative disorders (Bains and Shaw, 1997; Patrick et al., 1999; Hironishi et al., 1999; Oshiro et al., 2000; Montoliu et al., 2000; Horning et al., 2000; White et al., 2001; Rajan et al., 2001; LaFerla, 2002; see Chapter 17). The vastness of the subject matter is daunting, the literature is monumental, and each week's journals publish many new items of interest. Consequently, the following commentary has to be selective. Its purpose is to discuss some of the above questions in light of the clinical characteristics of metal poisoning as they compare with those of neurodegenerative disorders.

2. SYSTEMIC DISEASES AND NEUROTOXICITY OF METALS As a result of changes in metal homeostasis, neurotoxicity of metals may be involved in some neurological complications of systemic diseases.

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Neurotoxicity of copper is thought to occur in Wilson's disease WD" which is a rare autosomal recessive disorder of copper metabolism, as indicated by a decrease in the rate of copper incorporation into ceruloplasmin, an altered hepatic copper uptake, a prolonged copper turnover, and its accumulation in several organs including the brain (Sternlieb, 1978). The deposition of copper in the basal ganglia, mainly putamen and pallidum, may result in the clinical manifestations of tremor, diskinesias, and rigidity, whereas wider deposition in the brain may account for mental deterioration and varied neurological signs (Menkes, 1989). The WD gene, ATP7B, is a membrane-bound P-type copper transporting ATPase containing six copper-binding sites (Bull et al., 1993; Tanzi et al., 1993). Mutations of ATP7B result in reduced biliary excretion of copper and low incorporation of copper into ceruloplasmin. It is postulated that copper toxicity in the brain may result from accumulation of copper in neurons whose ability to efflux copper is restricted by the loss of ATP7B activity (see Chapter 7). Another event in WD may be abnormal iron metabolism, as suggested by the linkages between iron and copper metabolism (Siegel et al., 1999). However, hemochromatosis, a disease caused by altered iron homeostasis, is not associated with basal ganglia pathology. Moreover, exogenous copper and iron, which are involved in the events connected with neurodegenerative diseases, do not affect the nervous systems of poisoned patients (Ellenhorn, 1997). Liver failure is thought to cause accumulation of manganese in the basal ganglia, thus explaining the extrapyramidal symptoms frequently encountered in cirrhotic patients, such as tremor and rigidity. Blood manganese concentrations are increased in these patients and a positive correlation was found between blood manganese concentrations and the magnitude of pallidal signals on magnetic resonance images (MRI) (Spahr et al., 1996). These patients display several clinical similarities with those poisoned by manganese (see below).

3. DIFFERENCES BETWEEN METAL POISONING AND NEURODEGENERATION Clinical features of neurotoxicities show some differences with those of neurodegenerative disorders. A dose-related selectivity is a cardinal tenet

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of neurotoxicity caused by chemicals triggering off structural damage to the nervous system (Schaumburg, 2000a). For instance, lower doses of lead and methylmercury may cause peripheral neuropathy, while higher doses are associated with acute encephalopathy and cerebellar ataxia, respectively (Ellenhorn, 1997). Even at higher doses, neurotoxicities of metals show a remarkable selectivity. Thus, high doses of methylmercury may cause selective degeneration of the calcarine cortex and cerebellum (Bakir et al., 1973), and high doses of manganese cause degeneration of certain basal ganglia where the metal accumulates (Yamada et al., 1986). On the contrary, most neurodegenerative disorders display much more complex manifestations due to a wide range of neuronal deaths and losses of connectivity. For instance, pure pyramidal syndromes are rare in neurology, since a few neurodegenerative diseases (such primary lateral sclerosis and hereditary spastic paraparesis) are confined to the upper motor neuron cell bodies or their descending cortico-spinal tracts. Examples of a more widespread involvement of the central nervous system include PD, which is often associated with dementia (Lang and Lozano, 1998a), and AD which may be associated in some cases with cortical blindness (Bird, 2001). While neurodegenerative diseases are progressive, most neurotoxic disorders improve or disappear after cessation of exposure, unless a significant neuronal loss or irreparable damage of the brain or spinal cord occurred. In such a case, the disease is usually not progressive and possible worsening of clinical signs over time may be due to the concurrent physiological loss of neurons during aging (Spencer et al., 1987). However, this general concept has recently been challenged by the findings of gliosis in the brains of MPTP-poisoned patients whose exposure halted several years before death (Langston et al., 1999). It is concluded that a self-limited toxic insult to the nigrostriatal system of these patients had triggered off a self-perpetuating process of neurodegeneration. Since manganese neurotoxicity may also be progressive, as shown in a controlled study of five patients (Huang et al., 1993), it is far from clear why some neurotoxicities are progressive while others are not.

4. METAL POISONINGS Neurotoxicity of metals of clinical relevance is nowadays a rare condition and most information derives from old case reports and epidemiological

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studies in work environments. Several metals have been associated with various forms of neurotoxicity, which either mimick or represent real neurodegenerative disorders. Examples include aluminum (Mahurkar et al., 1973), lithium (Smith and Kocen, 1988), bismuth (Jungreis and Schaumburg, 1993), mercury (Tsubaki and Takahashi, 1986; Adams et al., 1983), manganese (Chu et al., 2000), and lead (Boothby et al., 1974; see Chapter 18). Metals that have also been etiologically associated with neurodegenerative disorders in patients without clinical evidence of poisoning include selenium (Kilness and Hochberg, 1977), manganese (Mitchell et al., 1991), and lead (Boothby et al, 1974). Conclusive evidence of neurotoxicity associated with neurodegeneration is only available for a few metals and two opposite examples will be discussed. Correlations between exposure to manganese, a defined clinical syndrome, and basal ganglia pathology are well-established. Human manganism is characterized by extrapyramidal symptoms and signs, and was the consequence of occupational exposures in miners and industrial workers (WHO, 1981; Wang et al., 1989). The disease is now rare because of improved hygienic conditions in work places. The syndrome reveals some clinical features different from those observed in patients affected by PD and include psychotic symptoms (referred to as locura manganica), dystonia, rigidity associated with normal diadochokinesis, and failure of levodopa therapy (Beuter et al., 1994; Calne et al., 1994; Lu et al., 1994). Neuropathology is also different because lesions are selective to the globus pallidus, where the metal accumulates, whereas normal histology is observed in the substantia nigra (Yamada et al., 1986). Interestingly, the clinical and neuropathological features of manganese toxicity display striking similarities with Hallervorden-Spatz disease. This disease is a progressive autosomal recessive disorder characterized by extrapyramidal signs associated with changes of the medial segment of globus pallidus and less severe neuronal loss in the rest of the basal ganglia. Increased amount of iron and other metals, such as copper and zinc, are found in the affected tissues (Rapin, 1989). Extrapyramidal signs, histopathology, and selective metal accumulation in both WD and manganism are, therefore, overlapping with those of Hallervorden-Spatz disease. Given these similarities in the clinical features and regional distribution of metals, it was suggested that such selective kinetics of metal access to the basal ganglia may have a central

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role in the pathogenesis of metal accumulation diseases (Chu et al., 2000). This example suggests that information derived from commonality between diseases that is not induced by chemicals and that elicited by toxicants may help to understand both. Unlike manganism, the clinical and histopathological features of aluminum neurotoxicity indicate that the hallmark of the disease is not neurodegeneration. Dialysis encephalopathy was seen in patients after years of parenteral aluminum exposure from an aluminum-contaminated dialysate (Alfrey et al., 1976; Flendrig et al., 1976). No signs of neurological disease have been observed in individuals with normal renal function who were occupationally exposed to aluminum (Spencer et al., 2000). Dementia and other neurological disturbances, including seizures, characterize aluminum intoxication and evolve gradually over the course of the disease. However, it is not clear what the neurophysiological bases of dementia are and to what extent the encephalopathy is progressive. Histological examination of brains of patients who had died of dialysis encephalopathy revealed mild spongiform pathology in the cerebral cortex associated with nonspecific neurological changes (Burks et al., 1976) and without marked loss of neurons (Winkelman and Ricanati, 1986; see Chapter 4). Although aluminum has been associated with a variety of neurodegenerative diseases, the analysis of a wide range of neurological disorders has found elevated aluminum in some cases, but this was not a consistent finding within a given disorder (Traub et al., 1981; Zatta, 1993; Exley, 2001).

5. DISCUSSION Accumulating evidence suggests that copper and/or iron have an important role in the pathophysiology of neurodegenerative disorders (Campbell et al., 2001; see Chapters 12 and 14), including AD (Huang et al., 1999; see Chapters 6 and 8), PD (Riederer et al, 1989), ALS (Andrus et al., 1998; see Chapter 9), and prion disease (Wadsworth et al., 1999; see Chapter 10). Evidence includes increased redox-reactive copper and iron levels (Sayre et al., 2000), changes in the expression of metalbinding proteins (Aschner, 1996; Adlard et al., 1998; see Chapters 5 and 11), promotion of amyloid-3 aggregation (Atwood et al., 1998), and free radical generation by amyloid (Bondy et al., 1998), indicating that

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excessive production of damaging free radicals is associated with neurodegeneration. Nevertheless, exceptions have been observed and an exploration of the reasons for such discrepancies and the significance of metal accumulation in neurodegenerative disorders may be better understood. For instance, manganese has been shown to mediate opposite effects in the nigrostriatal system since it causes both neurodegeneration and an antioxidative effect, as opposed to the exclusively pro-oxidant properties of iron in brain tissues (Sziraki et al., 1998). Consequently, manganism may be produced with mechanisms other than those of oxidative stress (Graham, 1984; Brouillet et al., 1993; Desole et al., 1994; Lloyd, 1995; Chang and Liu, 1999; see Chapter 16). Lithium may also display opposite effects (Smith and Kocen, 1988; see Chapter 19). Whether metal accumulation and oxidative stress represent the primary event in neurodegeneration is unclear. Increased iron and reduced complex I activity were not found in the brains of patients with incidental Lewys body disease, suggesting that these may be later or secondary changes (Jenner and Olanow, 1996), a hypothesis which is also supported by evidence from animal experiments (Double et al., 2000). On the contrary, MRI studies indicated increased manganese in the basal ganglia of intoxicated patients, but the abnormal MRI signals decrease or disappear after exposure (Ejima et al., 1992), suggesting that neurodegeneration may be a sequela of the initial phase of manganism. Based on the mechanism of oxidative stress in triggering off neurodegeneration, the hypothesis was made that inadequate intake of antioxidant might have predisposed patients to PD. This was shown in a study where patients had an intake of vitamin E lower than the controls' (de Rijk et al., 1997). However, these studies were generally inconclusive (Lang andLozano, 1998a). Whether deposition of transition metals in the brain (see Chapter 1) increases the oxidative load on the cells, thereby driving neurodegeneration and its progression, is also unclear. In the attempt to slow the progression of neurodegeneration, clinical trials with antioxidant therapy were initiated. They have shown some benefits from treatment (Parkinson Study Group, 1996; Schneider et al., 1997), though, on the whole, these results are not convincing (Lang and Lozano, 1998b). Nevertheless, it should be stressed that the clinical course of neurodegenerative disorders

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may be quite bizarre because it is characterized by both slow progression and fast acceleration, thereby making the assessment of therapeutic effects quite difficult. Several links between genetic factors and metal toxicity in the development of neurodegeneration and other neurological diseases (see Chapter 15) have been suggested. Mutations of the copper/zinc superoxide dismutase gene in familial ALS are one example (Rosen et al., 1993; see Chapter 9). Superoxide dismutase mutations are thought to cause a gain-of-function that is selectively lethal to motor neurons. Thus, excessive levels of hydroxyl radical can be formed through the reaction of hydrogen peroxide with a transition metal, such as iron (Martin, 1999). An example of a loss-of-function disorder is Friedreich's ataxia, a hereditary neurodegenerative disease. It has been linked to a defect in the mitochondrial protein frataxin (Campuzano et al., 1997), eventually leading to abnormal accumulation of iron in the mitochondria (Babcock et al., 1997). Elucidation and characterization of the interactions between aberrant proteins and metals may help in understanding how affected cells die and whether chelation and other therapies may inhibit or prevent these diseases. In this respect, the discovery that deposition of amyloid-@ is accelerated by metals, notably copper and zinc (Atwood et al., 1998; Huang et al., 1999; Cuajungco et al., 2000) offers some promising perspectives on chelation therapy (see Chapter 4). However, chelation may remove unwanted metals and it seems somewhat paradoxical that a recent candidate for chelating therapy is iodochlorohydroxyquin (Cherny et al., 2001), an old and well-known antibiotic that was responsible for a myelopathy epidemic (Schaumburg, 2000b). In conclusion, the clinical toxicology of metals offers some, though limited, clues to understand neurodegeneration. Nevertheless, it is increasingly appreciated that the mechanisms of many forms of toxicity and the pathophysiology of common diseases may involve a few stereotypical reactions, such as the mechanism of apoptosis (Lotti and Nicotera, 2002). In this respect, the mechanisms of metal neurotoxicity and neurodegeneration could be explored within a common frame. One example of this approach is to study the analogies and differences in protein-protein interactions, as well as the complexes that proteins form at various stages of metal neurotoxicity and neurodegenerative diseases. The study of such interactions, called the sociology of proteins (Abbott, 2002), is now possible (Gavin et al., 2002;

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Ho et al., 2002) and may help in understanding the role of metal ions in the initiation and/or progression of various forms of neurodegeneration.

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Index 6-hydroxydopamine

5, 7

a-synuclein 357 •y-aminobutyric acid

82

A(3 aggregation 4 aceruloplasminemia 220 adenylate cyclase 149 alkyl peroxides 7 alpha synuclein 312, 313 aluminum 118 aluminosilicates 118 Alzheimer's disease 1, 100 Amnion's horn 18, 23 amygdala 23, 53, 370 amyloid (3 protein precursor 4 amyloid-beta 3, 101 amyotrophic lateral sclerosis 10 anorexia 158 anterograde transport 73 apomorphine 336 arteriovenous malformation 379 astrocytes 6,77 axonal transport 75 basal ganglia 22 Bcl-2 226,473 bilirubin 388 blood-brain barrier 88, 149, 208, 298, 308, 336, 367, 382 Bowman's glands 69 brain homeostasis 186 brainstem 20 bulimia 158 Camelford 120 canalicular membrane 220 catalase 2 catecholamines 77 centrum semiovale 21 cerebellum 17,75 cerebral malaria 382

ceruloplasmin 6, 186 Chamorro 46 chelation therapy 104 citrate 118 clioquinol 314 copper foundries 442 copper-zinc superoxide dismutase 2 corpus callosum 19 corpus mamillare 20 Creutzfeldt-Jacob syndrome 121 Creutzfeldt-Jakob disease 9 cuprizone 282 CuZnSOD 8, 10 CuZnSOD gene 10 CuZnSOD knockout mice 9 cytokines 101 desferrioxamine 100,315 diabetes 405 dialysis encephalopathy 120, 121 dopamine 7 electrocorticograms 380 electrocorticography 369 epiphysis 24,53 experimental autoimmune encephalitis 309 extrapyrimidal syndromes 7 familial AD 184 Fenton reaction 5, 352 Fenton 7 Feralex-G 100, 108 ferritin 72, 345, 354 flavonoids 336 fluidity 437 Freidreich's ataxia 335 gamma-(p-y) secretase 101 glial fibrillary acidic protein 77 globus pallidus 17 glutathione 7 509

510

Index

glycation 273 granulomas 81 Guam island 17, 45, 46, 49 Guamian dementia 326

Morin stain 122 Mossbauer 355 multiple sclerosis 309 muscimol 374 myelinogenesis 408

head injury 195 hemochromatosis 402,405 hemodialysis 120 hippocampus 16, 19, 22, 23, 52, 53,74 histocompatibility complex 402 Huntington's disease 9, 325, 345 hypothalamus 20, 24, 74

nerve growth factor 292 neurinoma 132 neurofibrillary tangles 3 neuromelanin 329, 354, 355, 356 New Guinea 46 nitecapone 336 nitric oxide 77 nucleus basalis of Meynert 32 nystagmic jerks 455

inductively coupled plasma mass spectroscopy 247 inflammation 4 inherited anemias 405 instrumental neutron activation analysis 36,247 interleukin-l(3 101 iron regulatory protein 3

olfactory pathways 68 olfactory bulb 22 olfactory mucosa 70 olfactory neuron 69 otoacoustic emissions 449 oxidative stress 2, 3, 4, 5, 6, 7, 8

kainic acid 310 Kii Peninsula 46 Kuru 318 lactotransferrin 6, 326 laser microprobe mass analysis (LAMMA) 82, 129, 132 Lewy bodies 334, 345, 355, 356 lipid peroxidation 6 lipofuscinosis 122 locus coeruleus 22 Lou Gehrig's disease 263 manganese superoxide dismutase 2 manganoproteins 417 Mclntyre powder 120 melanotransferrin 6 melatonin 152,371 mental lethargy 158 metalloproteinases 248 microglia 5 microparticle-induced X-ray emission MnSOD 8

Parkinson's disease 1 peroxidase 3 peroxynitrite 2 pesticides 430 PIXE 246 pons 18,24 porphyria 405 positron emission tomography 416 presenilin 5 presenilin-1 and -2 101 prion diseases 1 pro-oxidant 7 pulvinar 18 putamen 17, 18, 20, 22, 23, 45, 53 quinone

7

Romberg test

3

451

selegiline 336 senescent red blood senile plaques 3 silicon 118

401

511

Index somatostatin 151 spin trap 268 spinal cord 75 sprouting 147 substantia nigra 7, 76 synaptic plasticity 249 temporal lobe 17 thalamus 18,19,21,74

transferrin 76, 118, 186, 408 transgenic mice 10 transgenic mouse 5, 6, 8 veratridine

375

xenobiotics

88

ZnTl-4

144

PAOLO ZATTA is the Director of the "Metalloproteins" Unit of the CNR-lnstitute for Biomedical Technologies at the University of Padua, Italy. Graduated in Chemistry, he received his doctoral degree in Biological Sciences at the University of Padua. He was a post doctoral fellow at the University of California at San Francisco, and was a visiting scientist at the University of Leeds in UK, University of Hamburg in Germany, University of California at San Francisco and University of Georgia in USA, University of Tel Aviv in Israel and University of Concepcion in Chile. Prof. Zatta's scientific interests concern the Physiopathological Role of Metal Ions in Neurodegenerative Disorders. He has published about 200 full papers and several reviews and books as an editor. He is also the organizer and co-organizer of several international conferences, including Aluminum in Chemistry, Biology and Medicine, Padua, Italy, 1992-1994; Second International Conference on Alzheimer's Disease and Related Disorders, Padua, 1992; First and Second International Conference On Metal Ions and the B r a i n : From N e u r o c h e m i s t r y t o Neurodegeneration, Padua, Italy, 2000; Fez, Morocco, 2002. More information on Prof. Zatta's scientific activities can be found at http://www.bio.unipd.it/-zatta/aluminum.html

Numerous studies have established a clear connection between neuronal oxidative stress and several neurodegenerative diseases, with consequential damages to lipids, proteins, nucleic acids, etc. In addition, several modifications indicative of oxidative stress have been described in association with neurons, neurofibrillary tangles and senile plaques in Alzheimer's disease, including advanced glycation end products and free carbonyl oxidation. Oxidative damage and antioxidant responses are now well characterized, but sources of damaging free radicals are yet to be fully understood. Evidences of alteration in metal ions metabolism have been reported in various diseases like Alzheimer's, Wilson, Menkes, Prion, Pick, Huntington disease, epilepsy and other pathological events. Thus, metal ions play a pivotal role in neurodegenerative phenomena. Chelation therapy is still in the early days of its development, but research in this area could lead to new products that could revolutionize treatment. Two international conferences on "Metals and the Brain: From Neurochemistry to Neurodegeneration" (Padova, Italy, 2000 and Fez, Morocco, 2002) were recently held to discuss the role of metal ions in neurophysiopathology. A third will be held in 2005 in Johannesburg, South Africa. This book follows the same train of thought as those conferences, in order to highlight the unquestionable importance of metal ions in the research on the neurophysiopathology of neurodegenerative diseases. The excellent reputation of the scientists who have contributed to this project ensures the quality of the chapters presented here, and hopefully this will help spur new research initiatives in the field, which is still in its infancy.

Metal Ions and Neurodegenerative Disorders World Scientific

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