Clusterin in Normal Brain Functions and During Neurodegeneration (Neuroscience Intelligence Unit)

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NEUROSCIENCE I N T E L L I G E N C E U N I T

2

Caleb E. Finch, Ph.D.

Clusterin in Normal Brain Functions and During Neurodegeneration

R.G. LANDES C O M P A N Y

NEUROSCIENCE INTELLIGENCE UNIT 2

Clusterin in Normal Brain Functions and During Neurodegeneration Caleb E. Finch, Ph.D. University of Southern California Los Angeles, California

R.G. LANDES COMPANY AUSTIN, TEXAS U.S.A.

NEUROSCIENCE INTELLIGENCE UNIT 2 Clusterin in Normal Brain Functions and During Neurodegeneration R.G. LANDES COMPANY Austin, Texas, U.S.A. Copyright © 1999 R.G. Landes Company All rights reserved. No part of this book may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Printed in the U.S.A. Please address all inquiries to the Publishers: R.G. Landes Company, 810 South Church Street, Georgetown, Texas, U.S.A. 78626 Phone: 512/ 863 7762; FAX: 512/ 863 0081

ISBN: 1-57059-583-6

While the authors, editors and publisher believe that drug selection and dosage and the specifications and usage of equipment and devices, as set forth in this book, are in accord with current recommendations and practice at the time of publication, they make no warranty, expressed or implied, with respect to material described in this book. In view of the ongoing research, equipment development, changes in governmental regulations and the rapid accumulation of information relating to the biomedical sciences, the reader is urged to carefully review and evaluate the information provided herein.

Library of Congress Cataloging-in-Publication Data

Clusterin in normal brain functions and during neurodegeneration / [edited by] Caleb E. Finch p. cm. -- (Neuroscience intelligence unit) Includes bibliographical references and index. ISBN 1-57059-583-6 1. Clusterin--Physiological effect. 2. Neurochemistry. 3. Nervous system--Degeneration--Molecular aspects. I. Finch, Caleb Ellicott II. Series. [DNLM: 1. Brain--physiology. 2. Nerve Degeneration--physiopathology. 3. Neurodegenerative Diseases--physiopathology. 4. Glycoproteins--physiology. WL 300 C649 1999] QP552.C56C575 1999 612.8'14--dc21 DNLM/DLC 98-50223 for Library of Congress CIP

NEUROSCIENCE INTELLIGENCE UNIT 2 PUBLISHER’S NOTE

Clusterin in Normal Brain Functions and During Neurodegeneration

R.G. Landes Company produces books in six Intelligence Unit series: Medical, Molecular Biology, Neuroscience, Tissue Engineering, Biotechnology and Environmental. The authors of our books are acknowledged leaders in their fields. Topics are unique; almost without exception, no similar books exist on these topics. Our goal is to publish books in important and rapidly changing areas of bioscience for sophisticated researchers and clinicians. To achieve this goal, we have accelerated our publishing program to conform to the fast pace at which information grows in bioscience. Most of our books are published within 90 to 120 days of receipt of University of Southern California the manuscript. WeLos would like to thank our readers for their Angeles, California continuing interest and welcome any comments or suggestions they may have for future books.

Caleb E. Finch, Ph.D.

Stephanie Stewart Production Manager R.G. Landes Company

R.G. LANDES COMPANY AUSTIN, TEXAS U.S.A.

CONTENTS 1. Clusterin Gene Locus Structure and Function in Development, Homeostasis, and Tissue Injury ............................................................... 1 Guang Zhu, Arthur Barrie III, Catherine Ebert, Mark E. Rosenberg, David P. Witte, Judith A.K.Harmony and Bruce J. Aronow Introduction ............................................................................................. 1 Clusterin Expression ............................................................................... 2 Gene Structure and Regulation ............................................................... 5 Summary ................................................................................................ 11 2. Clusterin and Apolipoprotein E Gene Expression in the Adult Brain .................................................................................... 17 Marc Danik, Jean-Guy Chabot, Denis Michel and Rémi Quirion Introduction ........................................................................................... 17 Clusterin Expression in the Adult Brain of Mammals ........................ 18 Apolipoprotein E Expression in the Adult Brain of Mammals ........... 25 Concluding Remarks ............................................................................. 27 3. Regulation of apoJ and apoE by Ovarian Steroids in the Brain .......... 35 David J. Stone and Irina Rozovsky Introduction ........................................................................................... 35 Estrogen and Apolipoproteins E and J in the Periphery ..................... 35 Steroids and Lipoproteins in the CNS .................................................. 36 Estrogen, AD, and Possible Mechanisms of Estrogen-Induced Neuroprotection ............................................ 40 Estrogen and Synaptic Sprouting: The Role of Apolopoprotein E ..... 41 Possible Compensatory Role of Apolopoprotein J .............................. 44 Summary ................................................................................................ 44 4. Lipoprotein Receptors in Brain .............................................................. 49 G. William Rebeck and Bradley T. Hyman Lipoproteins in the CNS—Source and Structure ................................ 49 Lipoprotein Receptors in the CNS ....................................................... 49 Functions of Lipoproteins and Their Receptors in the CNS ............... 53 Relationship to Alzheimer’s Disease ..................................................... 54 5. Clusterin-apoE Lipoprotein Particles .................................................... 61 David M. Holtzman, Mary Jo LaDu and Anne M. Fagan Clusterin/apoJ: An Apolipoprotein in Plasma and CSF ...................... 61 apoE and apoJ: Presence and Potential Roles in CNS ......................... 62 Potential Roles for apoE and apoJ in CNS Disease .............................. 65 Summary ................................................................................................ 66 6. Neurovascular Interactions of Alzheimer’s Amyloid β Peptide with Apolipoproteins J and E ................................................................. 71 Berislav V. Zlokovic, Blas Frangione and Jorge Ghiso Amyloid β ...................................................................................................... 71 Apolipoprotein J .................................................................................... 74

Apolipoprotein E ................................................................................... 75 Conclusions ........................................................................................... 79 7. Apolipoprotein E and Apolipoprotein J (Clusterin) in the Brain in Alzheimer’s disease ........................................................ 89 Edith G. McGeer, Claudia Schwab and Patrick L. McGeer Introduction ........................................................................................... 89 Association with Lesions in Alzheimer Brains ..................................... 89 Specificity to Alzheimer’s Disease ......................................................... 93 Possible Mechanisms for Changes in Clusterin and Apolipoprotein E in AD ............................................................. 94 8. Clusterin in Models of Central and Peripheral Injury and for Ischemia and Trauma ................................................................ 99 Håkan Aldskogius, Li Liu and Mikael Svensson Introduction ........................................................................................... 99 Peripheral Nerve Injury Produces an Increased Expression of Clusterin in CNS Neuronal Perikarya ....................................... 100 Peripheral Nerve Injury Produces an Increased Expression of Clusterin in Perineuronal Astrocytes ......................................... 100 Injury of Central Neural Pathways Also Produces an Increased Expression of Clusterin in Axotomized Neuronal Perikarya and Perineuronal Astrocytes ........................................................... 102 Wallerian Degeneration of Nerve Fibers in the CNS Is Accompanied by Increased Expression of Clusterin in Astrocytes and Oligodendrocytes ............................................... 102 Neuronal and Glial Cell Structures at a Lesion Site in the CNS Express High Levels of Clusterin ................................. 103 Clusterin Expression Following CNS Ischaemia Appears to Be Primarily Astrocyte–Associated ............................................ 106 Possible Functional Implications of Clusterin Expression Following Acute Insults of the Nervous System ............................ 106 9. Clusterin as a Neuroprotectant ............................................................ 109 Todd Morgan and Patrick May Introduction ......................................................................................... 109 Clusterin Expression and Localization in the Brain .......................... 109 Elucidation of Clusterin’s Role in the Brain ...................................... 110 Concluding Remarks ........................................................................... 115 Index ................................................................................................................ 121

EDITOR Caleb E. Finch School of Gerontology Division of Biogerontology Ethel Percy Andrus Gerontology Center University of Southern California Los Angeles, California 90089-0191 [email protected]

CONTRIBUTORS Håkan Aldskogius Department of Neuroscience Neuroregeneration Biomedical Center Uppsala, Sweden SE-751 23 [email protected] chapter 8 Bruce J. Aronow Children’s Hospital Research Foundation University of Cincinnati Cincinnati, Ohio 45229 [email protected] chapter 1 Arthur Barrie III Children’s Hospital Research Foundation University of Cincinnati Cincinnati, Ohio 45229 [email protected] chapter 1 Jean–Guy Chabot Douglas Hospital Research Centre Department of Psychiatry Faculty of Medicine McGill University Montreal, Quebec, Canada H4H 1R3 [email protected] chapter 2

Marc Danik Douglas Hospital Research Centre McGill Centre for Studies in Aging McGill University Montreal, Quebec, Canada H4H 1R3 [email protected] chapter 2 Catherine Ebert Children’s Hospital Research Foundation University of Cincinnati Cincinnati, Ohio 45229 [email protected] chapter 1 Anne M. Fagan Dept. of Neurology and Center for the Study of Nervous System Injury Molecular Biology & Pharmacology Washington University School of Medicine St. Louis, Missouri 63110 [email protected] chapter 5 Blas Frangione Department of Pathology New York University School of Medicine New York, New York 10016 chapter 6

Jorge Ghiso Department of Pathology New York University Medical Center New York, New York 10016 [email protected] chapter 6 Judith A.K. Harmony Children’s Hospital Research Foundation University of Cincinnati Cincinnati, Ohio 45267 [email protected] chapter 1 David M. Holtzman Dept. of Neurology and Center for the Study of Nervous System Injury, Molecular Biology & Pharmacology Washington University School of Medicine St. Louis, Missouri 63110 [email protected] chapter 5 Bradley T. Hyman Alzheimer Research Unit Massachusetts General Hospital Boston, Massachusetts 02129 [email protected] chapter 4 Mary Jo LaDu Scios Inc. 820 W. Maude Ave Sunnyvale, California 94086 [email protected] chapter 5 Li Liu Department of Neuroscience, Neuroregeneration Biomedical Center Uppsala, Sweden SE-751 23 [email protected] chapter 8

Patrick May CNS and Molecular Biology Research Lilly Research Laboratories A Division of Eli Lilly and Company Lilly Corporate Center Indianapolis, Indiana 46285 [email protected] chapter 9 Edith G. McGeer Kinsmen Laboratory of Neurological Research University of British Columbia Vancouver, B.C., Canada V6T 1ZE [email protected] chapter 7 Patrick L. McGeer Kinsmen Laboratory of Neurological Research University of British Columbia Vancouver, B.C., Canada V6T 1ZE [email protected] chapter 7 Denis Michel Endocrinologie moléculaire de la Reproduction Université de Rennes Rennes, France 35043 [email protected] chapter 2 Todd Morgan School of Gerontology Division of Biogerontology Ethel Percy Andrus Gerontology Center University of Southern California Los Angeles, California 90089-0191 [email protected] chapter 9

Rémi Quirion Douglas Hospital Research Centre Department of Psychiatry Faculty of Medicine McGill University Montreal, Quebec, Canada H4H 1R3 [email protected] chapter 2 G. William Rebeck Alzheimer Research Unit Massachusetts General Hospital Boston, Massachusetts 02129 [email protected] chapter 4 Mark E. Rosenberg Department of Medicine University of Minnesota Minneapolis, Minnesota 55455 [email protected] chapter 1 Irina Rozovsky School of Gerontology Division of Biogerontology Ethel Percy Andrus Gerontology Center University of Southern California Los Angeles, California 90089-0191 [email protected] chapter 3 Claudia Schwab Kinsmen Laboratory of Neurological Research University of British Columbia Vancouver, B.C., Canada V6T 1ZE [email protected] chapter 7 David J. Stone Harvard Medical School Lab for Structural Neuroscience Belmont, Massachusettes 02178 [email protected] chapter 3

Mikael Svensson Department of Clinical Neuroscience Section of Neurosurgery Karolinska Hospital Stockholm, Sweden SE-171 76 [email protected] chapter 8 David P. Witte Children’s Hospital Research Foundation University of Cincinnati Cincinnati, Ohio 45267 [email protected] chapter 1 Guang Zhu Children’s Hospital Research Foundation University of Cincinnati Cincinnati, Ohio 45267 [email protected] chapter 1 Berislav V. Zlokovic Departments of Neurological Surgery and Division of Neurosurgery Childrens Hospital Los Angeles USC School of Medicine Los Angeles, California 90033 [email protected] chapter 6

PREFACE

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his volume was assembled in response to the expanding role of clusterin (apoJ) in research on neural systems. In 1995, the R.G. Landes Company did a great service to the clusterin research community through publication of “Clusterin: Role in Vertebrate Development, Function, and Adaptation”, edited by Judith A.K. Harmony. This volume contained one chapter on neural systems (CE Finch and PC May). During the past five years, work on clusterin neural systems has advanced rapidly, as may be judged from the present articles. Overall, publications on clusterin in neural systems represent about 20% of the 500 papers in the literature since the molecule was first identified more than 15 years ago. The earliest isolation of this polynomial protein was in 1982 by Fischer– Colbrie, Schachinger, Zangerle, and Winkler1 as the so–named “glycoprotein III” (GPIII) from chromaffin granules of the bovine adrenal medulla. This isolation from bovine adrenal slightly preceded an independent isolation from ram testis fluid in 1983, named “clusterin” by Fritz, Burdzy, Setchell, and Blaschuk2 and one from rat Sertoli cells in 1984, named “dimeric acidic glycoprotein” by Sylvester, Skinner, and Griswold.3 Much later, the sequence of GPIII was identified as clusterin by Palmer and Christie in 1990.4 The CNS side began about ten years ago, when the mRNA was cloned from an Alzheimer brain (pADHC-9) in 1988 by May, Lampert–Etchells, Johnson, Poirier, Masters, and Finch;5,6 in1989, clones were derived from scrapie infected hamster brains by Duguid, Bohmont, Liu, and Turtellotte7 and from a quail neuroretinal cell line (T64) by Michel, Gillet, Volovitch, Pessac, Calothy, and Brun;8 in 1991, from a human glioma (TB16) by Danlik, Chabot, Mercier, Benabid, Chauvin, Quirion, and Suh;9 and in 1992, from human retinitis pigmentosa retinas by Jones, Meerbux, Yeats, and Neal.10 The diverse roles of clusterin are surveyed in the following chapters. Although all warm blooded vertebrates examined have clusterin homologues (chapter 1), no related molecule has been reported for any invertebrate. Much is known about the regulation of clusterin expression in neural systems during normal (chapters 2–5) and pathological conditions (chapters 6–9). Besides dynamic responses to intracellular signals, clusterin is regulated by steroid hormones and cytokines (chapter 3). Roles of clusterin in cholesterol transport (chapters 4 and 5) and as a molecular chaperone, of the amyloid β–peptide for example (chapter 6) could be common to processes that occur in many organ systems. Clusterin shows a Janus face, with both neuroprotective (chapter 9) and neurodegenerative activities (chapters 6–9). In large part, the excitement of working on this molecule lies in its connections to many areas of biology and medicine which do not usually interface at scientific meetings. The development of the clusterin field has been stimulated by periodic workshops which were attended by most clusterin researchers. Clusterin Workshop I was held in 1992 at Pembroke College, Cambridge UK (Irving Fritz and Brendan Murphy, Co–organizers); Workshop II, in 1994 at Coeur d’Alene Idaho (Michael Griswold, Caleb Finch, and Judith Harmony, Co–organizers), and

Workshop III, Villars sur Ollon, Switzerland (Lars French, Claudia Hoch–Brandt, and Jürg Tschopp, Co–organizers). In January 1999 in Ventura CA, Workshop IV will take place under expanded auspices, as a Gordon Conference Workshop on Clusterin and ApoE (Caleb Finch, and Judith Harmony, Co–organizers). The expansion of the Clusterin Workshop to include apoE was stimulated by interactions of these lipoproteins, including a novel lipoprotein particle secreted by astrocytes, which contains both clusterin and apoE (chapter 5) and lipoprotein receptors which can endocytose both clusterin and apoE (chapter 4). Future research on clusterin will be facilitated by the increasing availability of transgenic mice (clusterin–KO and clusterin overexpressors) which are being characterized (chapter 1). This book was assembled with the skillful administrative assistance of Linda A. Mitchell. Caleb E. Finch Los Angeles, CA August 31, 1998 References 1. Fischer–Colbrie R, Schachinger M, Zangerle R, Winkler H. Dopamine β-hydroxylase and other glycoproteins from the soluble content and the membranes of adrenal chromaffin granules. Isolation and carbohydrate analysis. J Neurochem 1982; 38:725-732. 2. Fritz IB, Burdzy K, Setchell B, Blaschuk O. Ram rete testis fluid contains a protein (clusterin) which influences cell–cell interactions in vitro. Biol Reprod 1983; 28:1173-1188. 3. Sylvester SR, Skinner MK, Griswold MD. A sulfated glycoprotein synthesized by Sertoli cells and epididymal cells is a component of the sperm membrane. Biol Reprod 1984; 31:1087-1101. 4. Palmer DJ, Christie DL. The primary structure of glycoprotein III from bovine adrenal chromaffin granules. J Biol Chem 1990; 265:6617-6623. 5. May PC, Johnson SA, Lampert–Etchells MA, Finch CE. In situ mapping of pADHC–9: A poly(A)RNA sequence overexpressed in Alzheimer’s disease hippocampus. Soc Neurosci Abstracts 1998; 14:897. 6. May PC, Lampert–Etchells MA, Johnson SA et al. Dynamics of gene expression for a hippocampal glycoprotein elevated in Alzheimer’s disease and in response to experimental lesions in rats. Neuron 1990; 5:831-839. 7. Duguid JR, Bohmont CW, Liu N, Turtellotte WW. Changes in brain gene expression shared by scrapie and Alzheimer’s disease. Proc Nat Acad Sci (USA) 1989; 86:7260-7264. 8. Michel D, Gillet G, Volovitch M, Pessac B, Calothy G, Brun G. Expression of a novel gene encoding a 51.5 kD precursor protein is induced by different retroviral oncogenes in quail neuroretinal cells. Oncogene Res 1989; 4:127-136. 9. Danik M, Chabot J–G, Mercier A–L, Benabid C, Chauvin C, Quirion R, Suh M. Human gliomas and epileptic foci express high levels of a mRNA related to rat testicular sulfated glycoprotein 2, a purported marker of cell death. Proc Nat Acad Sci (USA) 1991; 88:8577-8581. 10. Jones SE, Meerabux JMA, Yeats DA, Neal MJ. Analysis of differentially expressed genes in retinitis pigmentosa retinas. FEBS Lett 1992; 300:279-282.

CHAPTER 1

Clusterin Gene Locus Structure and Function in Development, Homeostasis, and Tissue Injury Guang Zhu, Arthur Barrie III, Catherine Ebert, Mark E. Rosenberg, David P. Witte, Judith A.K.Harmony and Bruce J. Aronow

S

trong induction of clusterin (apolipoprotein J, apoJ) gene expression and its accumulation at sites of injury has been observed by numerous investigators studying apoptotic and tissue injury processes such as those resulting from chemical injury, hormone-withdrawal, or ischemic tissue damage. While the function(s) of clusterin remain unclear, these models may provide clues into its potential roles during pathophysiologic processes. Similarly, the study of clusterin gene activation mechanisms may also provide a window into the regulatory mechanisms and genetic circuitry that are responsible for controlling physiologic and pathophysiologic responses to tissue injury. In particular, clusterin activation occurs at intermediate to late stages in the injury response; this distinguishes it from immediate-early types of response genes. Thus, the hunt for regulatory mechanisms responsible for the control of clusterin gene expression represents an exciting and important goal.

Introduction The clusterin gene encodes a protein polypeptide thought until recently to be strictly a secretory glycoprotein with hydrophobic and heparin binding domains. Recently, however, an intracellular or nuclear form ofclusterin has also been implicated. Neither the function nor the determinants of the relative production of either form are known. However, their expression and induction in interesting locations and in a variety of important pathophysiological processes associated with cell death and injury has inspired thought and tempted much speculation. Since the ability of a tissue and the organism as a whole to withstand injury depends on selective apoptosis, synthesis of adaptive gene products and repair, clusterin regulation serves as aprototypical model for an injury response gene. Clusterin is expressed both within and surrounding pathological lesions and thus has the opportunity to modify extracellular fluids and surfaces at critical biological interfaces. Protective secretory molecules induced at dynamic biointerfaces and injury sites are likely to be a fundamental requirement for all complex multicompartment organisms. However, the roles and regulation of these types of molecules in response to injury is a largely unexplored biological territory. Our hypothesis for the secreted form of clusterin is that it represents an extracellular tissue repair gene induced in jeopardized boundary zones. It may

Clusterin in Normal Brain Functions and During Neurodegeneration, edited by Caleb E. Finch. ©1999 R.G. Landes Company.

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Clusterin in Normal Brain Functions and During Neurodegeneration

serve as an extracellular chaperone of bioactive hydrophobic molecules and denatured proteins/peptides released at sites of high tissue turnover, thus promoting the survival of bystander cells. Based on these observations and hypotheses, we are trying to understand the clusterin gene cis-regulatory sequences that control constitutive and cell type-specific clusterin gene expression in response to cellular injury. There is little knowledge of the genetic mechanisms operative in animal tissues for initiating, propagating, and inhibiting the injury cascade. Ultimately, knowledge of clusterin’s control mechanisms will lead us to an improved understanding of the transacting factors and receptor/signal transduction machinery that allows tissues and cells to withstand harsh environments and injuries.

Clusterin Expression Clusterin Protein Clusterin is conserved in mammals, though more divergent in vertebrates as a whole. We first identified clusterin as a lipid binding and transport protein in human plasma,1,2 but we have been most impressed by its expression at critical boundary interfaces in both normal and pathophysiologic circumstances. Clusterin is expressed constitutively and developmentally in an interesting series of epithelial cells, frequently at the interfaces of fluidtissue boundaries.3,4 The induction or deposition of clusterin has been associated in humans and other species with a variety of pathological disorders, including neurodegeneration, viral infection, prostatic involution, renal injury, Alzheimer's disease, and atherosclerotic vascular disease.5-7 The secreted form of clusterin consists of disulfide-linked α and β subunits proteolytically derived from a 70 kDa glycoprotein precursor.8 Potential protein functional domains, such as the signal peptide, Cys-rich motifs involved in the interchain disulfide bonds, amphipathic helices and linear “heparin-binding” domains, distributed in each subunit, fall predominantly within single exons, and the α−β cleavage site is encoded within exon 5.9-12 No homologous sequence has been found in unicellular organisms. This seems particularly meaningful in view of the fact that all cells can regulate gene expression in response to the environment; however, only complex organisms establish tissue boundaries.

Nuclear Clusterin Recently Jin and Howe have detected a form of clusterin that accumulates in the nucleus that is induced by 16 h exposure to TGF-β in two fibroblast cell lines, 10T1/2 and 3TP, two epithelial cell lines, CCL64 and HeLa, and in primary BAEC.13 The protein appeared to lack a signal peptide and α/β cleavage site, but it is unclear whether this form represents an alternately initiated translation product or an alternately processed form. We have also detected uncleaved forms of the protein in a megakaryocytic cell line induced to differentiate with phorbol myristic acid.14 Akakura et al also observed the presence of strong nuclear staining in androgen-dependent mouse Shionogi carcinoma subjected to multiple cycles of androgen withdrawal in vivo.15 Nuclear staining occurred in both the apoptosing tumor cells in regressing tumors after androgen-withdrawal and in nonregressing cells after adaptation to multiple cycles of androgen withdrawal. The mechanism of nuclear clusterin accumulation remains to be determined. Alternate transcription, mRNA processing, translational initiation, preprotein processing, and intracellular trafficking are all possible mechanisms.

Structure and Regulation of Clusterin Genes

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Constitutive Clusterin Expression at Fluid-Tissue Interfaces We have previously reviewed sites of high constitutive clusterin expression (Aronow et al. In Clusterin: Role in Vertebrate Development, Function, and Adaptation. Landes 1995). These comprise a predominance of epithelial cells that line compartments containing biologically active fluids such as gastric, pancreatic, urinary, and bile secretions. Most of these cell types are highly secretory and include epithelial lining cells of the esophagus, biliary ducts, gallbladder, urinary bladder, ureter, kidney distal convoluted tubules, gastric glands, Brunner’s glands, choroid plexus, ependyma, ocular ciliary body, testis, epididymus, and visceral yolk sac. Several nonepithelial secretory cell types that express high-levels of clusterin also line fluid compartments such as synovial lining cells and ovarian granulosa cells. These examples suggest that there are exquisite local signals affecting the regulation of clusterin gene expression and also that high level secretory behavior of a cell type does not necessarily imply that the cell will exhibit high-level clusterin expression. However, there may be no examples of high level clusterin-expressing cells that do not exhibit considerable secretory activity. This raises the possibility that a key parameter affecting the induction of clusterin in injured tissues is similar to some of the factors that influence constitutive clusterin expression, perhaps with secretion against a hydrostatic or other type of gradient. It is interesting to consider the GI tract in this context: Epithelial cells of the stomach and the duodenal Brunner’s gland, perhaps the most active secretory cell types of the GI tract, express clusterin at a very high level, but the absorptive terminal small bowel and colon express virtually no clusterin. We would thus predict that sites of clusterin expression not known to be secretory are in fact highly secretory.

Induced Clusterin Expression The range of cell types in which clusterin is known to be inducible is rapidly expanding, but there is only limited information available to explain how this occurs. Clusterin is strikingly induced in some tissues that are undergoing apoptosis during development, or in the adult in tissues that undergo cycles of expansion and regression (e.g., uterus,16 mammary gland5,17), particularly tissues in which the gene is normally expressed at low levels or is undetectable. Clusterin gene expression is increased by 100- to 1000-fold during pathological apoptosis that accompanies castration-mediated prostatic involution;18-20 similar increases occur in vitamin A-deprived seminal vesicle epithelia,21 in several models of chemical and physical kidney injury,22-25 and in neurodegenerative conditions of the brain.9,26-29 In tissues where the clusterin gene appears to be expressed constitutively there is no further induction with apoptosis—e.g., the postpartum involution of the endocrine pancreas (clusterin expression in ascinar cells) with loss of beta cells of the islets,30 and the thymus in medullary epithelial cells during cortical lymphocyte involution in response to dexamethasone administration.31,32 Frequently, there is no correlation between the cells in which clusterin is upregulated and those which are undergoing apoptosis, as evident during renal6,23,33-35 and neuronal36,37 injury. In fact, clusterin mRNA is frequently localized to surviving cells.36,38 In light of this, and the extensive constitutive pattern of clusterin’s expression in diverse cells subject to apoptosis, it is unlikely that clusterin is a primary mediator of the cell death program. Thus, an attractive hypothesis is that clusterin induction is a reactive response to a specific environmental change that occurs in tissues during apoptosis. In support of this hypothesis, within a given tissue the upregulation of clusterin is an “all-or-none” process. When clusterin is upregulated in uterine glandular epithelial cells immediately preceding the time of blastocyst implantation,16 either all epithelial cells of a gland viewed in cross-section express clusterin message, or none do. In ischemia-injured kidneys,35,39 induced clusterin message is evident in every epithelial cell in a tubule cross-section or is not present at all.

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Clusterin in Normal Brain Functions and During Neurodegeneration

Clusterin Expression Induced at Pathological Boundaries Clusterin is expressed at high levels predominantly by cells situated at dynamic interfaces created during development and in response to injury.3 Clusterin gene induction, while restricted to areas of active tissue reorganization, occurs in a very wide range of tissue and cell types. The strongest induction of clusterin occurs in animal models of xenobiotic tissue injury. Neurotoxic chemicals induce clusterin upregulation only in astrocytes in the brain; nephrotoxic chemicals induce expression only in epithelial cells in the kidney; and antiandrogens induce clusterin mRNA only in the androgen-dependent cell types of the male reproductive tract. Cells showing high levels of clusterin mRNA are those in proximity to the injured or reorganizing tissue. Numerous cell types in those jeopardized zones can express the clusterin gene, including epithelial cells, cardiac and smooth muscle myocytes, and neurons. This regulatory pattern is in itself extremely interesting, and suggests that shared and likely fundamental genetic mechanisms control injury and repair in multiple tissues in response to independent xenobiotic agents. Rozovsky et al38 compared clusterin induction in the brain by two xenobiotic agents that injure neurons in distinct anatomical regions of the hippocampus. The excitotoxin kainic acid kills the pyramidal neurons of the CA3-CA4 region, with clusterin expression induced in the surrounding reactive atrocytes; no expression is detected in astrocytes in proximity to the kainic acid-resistant neurons of the dentate gyrus. In contrast, when colchicine is used to injure the granule neurons of the dentate gyrus, the surrounding astrocytes express abundant clusterin message with little or no message in distal astrocytes. Astrocytes themselves, which can be induced to express high levels of clusterin mRNA, can survive a wide range of neurotoxin-induced and other injuries, suggesting that clusterin expression is important in preserving cell viability. In the heart, strong clusterin gene induction occurs in ventricular cardiomyocytes surrounding myocardial tissue damaged from autoimmune myosin-induced myocarditis or pressure overload-induced ischemia, where neither obviously damaged myocytes nor inflammatory cells express the gene. However, pressure overload of the heart causing significant ventricular hypertrophy in the absence of cellular injury does not result in clusterin induction. In the absence of tissue injury, a clusterin response does not occur. We also have found no evidence of clusterin induction in dramatically dilated hearts from mice treated with phenylhydrazine, which induces acute high output heart failure without inflammation or myofibril degeneration. In the kidney, multiple models of acute renal failure cause clusterin induction in tubular epithelial cells, including ureter obstruction, ischemia/reperfusion, and nephrotoxic injuries such as folic acid nephropathy, gentamicin nephrotoxicity and myoglobinuric renal failure.41,42 Sawczuk et al43 found that clusterin induction occurs in tubular epithelial cells of the obstructed kidney, but not in the contralateral unobstructed kidney, which undergoes compensatory hypertrophy. However, during development, abundant clusterin mRNA is present in the metanephric kidney, initially in the ureteric bud and later in the tubular epithelial cells. However, in the adult kidney, expression only occurs in a limited number of medullary tubules.3 Two notable examples of clusterin induction in response to hormonal withdrawal are in prostate gland subjected to the loss of testosterone and in the lactating mammary gland subjected to weaning. There is some evidence that clusterin activation is a response to the loss of repression by hormone, but this is less clear in the mammary gland, as occasional tubules in the actively lactating gland are intensely positive for clusterin expression. This occurs in the exact same pattern as in the bulk of the mammary epithelial cells that become high expressers at day 3 to day 5 post weaning. Thus, an interesting question is the extent to which induction is primarily the result of stasis or obstruction, rather than the loss of trophic

Structure and Regulation of Clusterin Genes

5

hormonal regulation per se. If it were obstruction that were the primary issue, could it be that an element of the prostatic involution induction also relates to a response to obstruction or reduced clearance of secretory material? The circuitry responsible for responding to these types of signaling pathways is unclear, but an important advance will be to delineate clusterin gene regulatory elements and identify transcriptional factors involved in the induction.

Gene Structure and Regulation Clusterin Gene Structure The clusterin molecule is encoded by a single gene responsible for both constitutive and induced expression and is present in the genomes of all vertebrate species examined. As shown in Figure 1.1, the rat,44 human,45 and mouse11 genes are divided into 9 exons, encompassing 15-23 kb. The intron-exon organization and sizes of both exons and introns are highly similar, with several notable expansions of the first and sixth introns in the human gene. Quail clusterin is similar to the mammalian genes in exonic organization, although the introns are dramatically smaller (Fig. 1.1). The avian gene can be transcribed from alternative promoters, P1 and P2, adjacent to alternative first exons. Both promoters are active in transient transfection assays; however, the possibility for developmental or tissue specific use cannot be eliminated. The potential regulatory elements identified in the proximal promoters of the mammalian genes are distributed between the two quail promoters.

Clusterin Gene Regulation Cis-elements responsible for clusterin induction are poorly understood. Sequence analysis for consensus cis-regulatory elements surrounding the promoters of the mouse, rat and human clusterin genes reveals classical TATAA and CAAT elements and a variety of other consensus cis-elements, including AP-1, AP-2, CRE/ATF and NF-κ2. An AP-1 site is very close to the CAAT site of the promoter in human, rat, mouse and quail genes. Direct genomic sequence comparison indicates that the degree of similarity between mammalian genes is extremely high (83%) in the region spanning the first 140 nucleotides upstream of the transcription start site. This similarity drops to 34% for the remainder of the upstream sequence, although the relative positions of several other motifs, including AP-1 and NF-e2 binding domains, are conserved. However, a demonstration of the functional significance of these elements is a difficult process. The CRE, AP-1 and AP-2 sites within the proximal promoter and first intron have been suggested to be responsible for cAMP-mediated suppression of clusterin expression in cultured Sertoli47-49 and Leydig50,51 cells. In primary cultures of rat astrocytes, clusterin is upregulated by IL-1β, as well as IL-2, IL-3 and IL-6, consistent with the presence of multiple IL-6 cis elements throughout the mammalian genes. However, these have yet to be shown to be functional.

Heat Shock Model for Clusterin Expression Induction Protection against numerous types of physical and chemical stress is mediated in all organisms by the upregulation of heat shock genes (hsp70, hsp90, hsp110, small hsps, etc.) expression. The majority of heat shock proteins appear to defend cells from injury by acting as molecular chaperones against denatured proteins and protein aggregates. Increased levels of heat shock proteins from an initial hyperthermic exposure have been shown to confer cellular thermotolerance to subsequent heat shocks. The activation of the heat stress response has been observed in a variety of central nervous system disease states ranging from inflammation to ischemia. Moreover, it has been suggested that heat shock proteins may be

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Clusterin in Normal Brain Functions and During Neurodegeneration

Fig. 1.1. Organizational and selected element homologies of clusterin genes.

Structure and Regulation of Clusterin Genes

7

a useful marker for CNS injury and that their induction may protect the brain from further stress. Kainic acid, hyperthermia, spinal cord trauma, and ischemia can all upregulate heat shock protein production in CNS experimental injury models.55 Clusterin induction has also been observed following heat stress in a variety of cell lines and tissues. Clark and Griswold examined clusterin and hsp70 expression during heat shock in three different cell lines: A431 (human epidermoid carcinoma), MSC1 (mouse sertoli cell line), and primary cultures of rat sertoli cells.56 They observed in all three cells lines that clusterin upregulation during long term heat shock was delayed however, it was sustained in contrast to hsp70 expression. They concluded that clusterin induction during stress was regulated in a cell specific manner, as Sertoli-derived clusterin expression was not upregulated until 12-24 hours of heat shock (41°C). In contrast, clusterin expression in A431 cells was significantly increased by 4 hours of heat shock. Clark and Griswold also showed, using actinomycin D, that increases of clusterin mRNA in MSC1 cells during heat shock was caused by increased transcription rather than increased mRNA stability. The regulatory circuitry responsible for the heat shock induction of clusterin has been partially dissected by Michel et al.57 They identified a cis-acting element contained in the clusterin promoter that interacted with the heat shock transcription factor, HSF-1, in gel shift experiments. The 14 bp cis-acting element, CLE (clusterin element 5'GGTT CCAGAAAGCTCCC3'), is entirely conserved from the quail to the human clusterin genes. They showed that the CLE in multiple arrays conferred heat shock inducibility in transient expression assays, and confirmed the significance of the CLE by mutating the site in a quail promoter reporter construct. Mutation of the CLE site completely eliminated the heat shock response of the reporter gene. Clusterin is also induced in aortic fatty streaks of mice fed an atherogenic diet, and by oxidized LDL.58,59 The recent association of diet-induced atherosclerosis in mice with oxidative stress60 may have relevance for the regulation of clusterin. Susceptibility to diet-induced aortic lesions in mice is genetically linked to the induction of inflammatory genes by oxidized lipids that accumulate in the tissues. Thus, the activation of NF-κB transcription factors by oxidized lipids may account for the activation of NF-κB on responsive genes,61 including clusterin. Other factors have been reported to be oxidation sensitive or inducible, including AP-1 and p53. Interestingly, Jin and Howe showed that the TGF-beta induction was inhibited by a protein kinase C inhibitor and that an AP-1 site in a clusterin promoter reporter gene could confer responsiveness to TGF-beta. Other potential regulatory factors active on the clusterin gene are immediate early factors that mediate a variety of extracellular signaling pathways via the serum response element (SRE) bound by the serum response factor (SRF).62 Extracellular signals that utilize the SRE include both mitogenic stimuli (e.g., epidermal growth factor) and stress stimuli (e.g., UV light).63 At least two pathways exist to activate via SRF.64 One pathway leads to the generation of a ternary complex composed of the SRE, SRF, and an Ets domain transcription factor. This pathway responds to both mitogenic and stress stimuli. The second pathway acts without the Ets mechanism and is responsive to bioactive serum components such as lysophosphatidic acid and the Rho family of GTPases.65

DNase I Hypersensitivity Analysis of the Clusterin Locus As part of an initial approach to identification of clusterin gene regulatory regions, we have used DNase I hypersensitivity analysis. We have done this initially by comparing hypersensitive sites in the liver, where the gene is expressed at a high level, to the thymus, where the gene is expressed at a low level (Fig. 1.2). While this information is not useful in and of itself, when combined with the sequence and functional analyses, rapid progress can be made toward the identification of the individual sequence motifs that are bound by injury response regulatory proteins.

8

Clusterin in Normal Brain Functions and During Neurodegeneration

Fig. 1.2

Structure and Regulation of Clusterin Genes

9

Transgenic Analysis of Clusterin Gene Regulatory Elements The most direct approach to identifying any gene’s regulatory elements is to functionally demonstrate appropriate regulation upon inclusion of cloned segments of the gene into reporter gene constructions. Transgenic mouse technology offers a powerful initial approach. Whereas important clues can sometimes be obtained using DNA sequence analysis, DNase I hypersensitivity or reporter gene transfection into cell lines, these approaches are often very misleading. Transgenic mouse results are also capable of simultaneously distinguishing elements responsible for regulatory control of expression in multiple tissues constitutively, as well as under induced and xenobiotic injured circumstances. Our first reporter construction transgenes have included large clusterin gene segments. This is a good starting point for identifying control regions. We constructed four transgenes that contain a series of segments of the mouse clusterin locus and used these to derive multiple independent transgenic mouse lines (Fig. 1.3). The constructs incorporate 0.7 to 2.0 kb of 5'-flanking and first exon DNA from the mouse clusterin gene linked to the bacterial gene for chloramphenicol acetyl transferase (CAT). Also, two constructs include the clusterin first intron and the initial portion of the second exon; one construct, apoJCAT-2, includes much of the 3' portion of the clusterin locus. The ATG initiator codon located in the second exon of the mouse clusterin gene is placed in-frame with the coding sequences for the CAT gene. In apoJCAT-1 and CAT-2, normal promoter utilization is likely because there is no perturbation of the first exon transcriptional initiation site or the first intron splicing signals.

Transgene Expression in ApojCAT-1 Transgenic Mice We have generated a series of independent transgenic mouse lines from a series of constructions including those above. Individual lines contained multiple unrearranged copies of these constructions and we determined the specific activity of CAT enzyme in extracts from a variety of tissues from F1 animals (Table 1.1). Specific activity of CAT enzyme in each extract is normalized for transgene copy number. We previously presented the CAT activity present in F0 mice (Aronow et al Clusterin: Role in Vertebrate Development, Function, and Adaptation. Landes 1995), and we are now presenting CAT expression in F1 mice.

Clusterin Promoter and First Intron These results show that the transgene construction apoJCAT-1, which contains promoter, clusterin first exon and intron, and second exon fused in frame to CAT coding and polyA sequences is highly functional in all tissues analyzed. Interestingly, there is much more consistent expression of the construct among independent lines in the F1 mice than was previously observed. In situ hybridization analysis (see below) of apoJCAT-1 reveals expression in a similar, but not identical pattern as the endogenous clusterin gene (Fig. 1.4). Comparison of endogenous and transgene expression in the cerebellum reveals that there are strong similarities, yet interesting differences. There is strong expression of the endogenous gene in the cells immediately surrounding the Purkinje cells, but lower expression in the highly cellular granular layer. In contrast, the apoJCAT-1 transgene exhibits high level

Fig. 1.2. (opposite) DNase I hypersensitive site at the clusterin gene promoter. There is a prominent DNAse I hypersensitive site present at -0.1 relative to the promoter start site in the strongly expressing mouse liver, but not in the inactive thymus. Nuclei from each tissue were subjected to increasing concentrations of DNAse I, DNA was purified, digested with BamH1, and Southern blot analyzed with the indicated probe. Note also that the parent band has overall insensitivity to DNAse digestion in the thymus, suggesting a closed chromatin configuration in the inactive tissue.

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Clusterin in Normal Brain Functions and During Neurodegeneration

Fig. 1.3. The organization of the mouse clusterin gene and the construction of transgenic reporter genes to evaluate its regulatory regions. (Top) The mouse clusterin gene contains nine exons. The initiator codon is in exon 2, and the stop codon is located in exon 9. (Lower) Structure of the ApoJCAT-1, -2, -3, -4 reporter genes used to make transgenic mice. A portion of the mouse clusterin gene is linked to a bacterial CAT reporter gene. The clusterin segment of ApoJCAT-1 includes 2 kb of 5' flanking DNA, the first exon, intron, and the first portion of the second exon. The ATG initiator codon located in the second exon of the mouse clusterin gene is placed inframe with the coding sequences for the CAT gene. The CAT gene contains the coding sequences for chloramphenicol acetyl transferase, and splicing and polyadenylating sequences from the SV40 T-antigen gene.

expression in both the peri-Purkinje and granular region cells. Thus, apoJCAT-1 is missing some or many regulatory elements responsible for restricting its expression to the correct cells.

Transgenic Analysis of Injury Response Elements in the Clusterin Locus In the developing kidney, most tubular epithelial cells express clusterin. However, in the adult, only the distal convoluted tubules of the cortex express clusterin.3 As reviewed above, a defining feature of clusterin is its marked induction following renal and other organ injury. To begin an identification of clusterin gene regulatory elements involved in the response to kidney injury, we have subjected apoJCAT-1 mice to toxic amounts of folic acid. Acute renal tubular injury, induced by the intraperitoneal injection of folic acid (250 mg/kg), results in an increase in clusterin mRNA by 31.5 ± 10.2-fold at 24 h, but no change in CAT mRNA or activity (Table 1.2).

Structure and Regulation of Clusterin Genes

11

Table 1.1. Tissue specific reporter gene expression in ApoJCAT transgenic mice. CAT1 LINE Copy#

skin

musc

CAT Activity(cpm/µg/copy) WBC atrium ventr lung liver

24 28 77 62

85 661 830 190

72 2500 3300 860

497 3600 2390 1040

2 15 5 14

589 686 950 400

53 250 330 170

1007 2970 2870 920

1282 8740 20491 2320

kidney

brain

75 300 159 75

388 3000 2044 480

Detailed Brain Dissection LINE 28 CAT Activity (cpm/µg/copy) Olfactory Bulb 2,270 Cortex- anterior 1,850 Cortex- medial 3,000 Cortex- lateral 2,940 Hypothalamus 3,454 Pituitary 190 Midbrain 3,000 Cerebellum 8,200 Medulla 8,000 Spinal Cord 6,400 Independent mouse lines (F1 established lines) were sacrificed to determine the specific activity of CAT enzyme in tissue extracts and DNA was purified for transgene copy number. WBC represents the combined cells present in buffy coat from a 1000xg 15 min centrifugation over Ficoll-Hypaque. CAT activities were determined with minimal amounts of extract (1-100 µg protein) for minimal periods of time (5-15 min) so as to obtain initial rates of enzyme activity. Gene copy number was determined by quantitative Southern blot hybridization with comparison of transgene fragment intensity to that of the endogenous mouse clusterin gene.

Table 1.2. Folic acid nephropathy in ApoJCAT-1 transgenic mice.

Untreated (n = 8) 6 hours (n = 9) 24 hours (n = 9)

Creatinine (mg/dl)

Clusterin mRNA (OD Units)

CAT mRNA (OD Units)

CAT Activity (cpm/h/µg)

0.21 ± 0.02 0.69 ± 0.07* 0.55 ± 0.23*

1.0 7.2 ± 1.4* 31.5 ± 10.2*Ü

1.0 1.0 ± 0.1 1.0 ± 0.1

1350 ± 680 1010 ± 318 1802 ± 651

Results are mean ± SEM; * p<0.01 vs. control, Ü p<0.05 vs. 6 h value.

These results indicate that the apoJCAT-1 transgene lacks response elements that mediate an inductive response to acute renal tubular injury. Thus, despite high level expression of the apoJCAT-1 in the kidney, the first intron and promoter lack genetic elements essential for regulation of clusterin in response to injury.

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Clusterin in Normal Brain Functions and During Neurodegeneration

Fig. 1.4

Structure and Regulation of Clusterin Genes

13

Summary Regulation of clusterin is likely to be highly complex, given the complexity of its expression patterns and inducibility. The constitutive expression of clusterin varies in different organs and even within different cells in a given organ. In addition, specific developmental regulation occurs in many tissues including the heart, lung, brain and kidney. Finally, marked induction of clusterin is seen in a number of pathophysiologic conditions including apoptosis, acute cell injury, and chronic degenerative conditions. Although the role of clusterin in these conditions awaits elucidation, the understanding of its genetic regulation has the potential to provide important insight into the regulation of gene expression in injury states, and in particular at the interface between cell death and cell survival and adaptation.

Acknowledgments We are grateful to Kathy Saalfeld and Pamela Groen for outstanding histological analyses, for the support of the Cincinnati Children’s Hospital Research Foundation, and NIH grants RO1 ES08822 (BJA) and R29DK43075 (M.E.R).

References 1. Burkey BF, Stuart WD, Harmony JA. Hepatic apolipoprotein J is secreted as a lipoprotein. J Lipid Res 1992; 33:1517-1526. 2. Burkey BF, deSilva HV, Harmony JA. Intracellular processing of apolipoprotein J precursor to the mature heterodimer. J Lipid Res 1991; 32:1039-1048. 3. Aronow BJ, Lund SD, Brown TL, Harmony JA, Witte DP. Apolipoprotein J expression at fluid-tissue interfaces: Potential role in barrier cytoprotection. Proc Natl Acad Sci USA 1993; 90:725-729. 4. French LE et al. Murine clusterin: Molecular cloning and mRNA localization of a gene associated with epithelial differentiation processes during embryogenesis. J Cell Biol 1993; 122:1119-1130. 5. Tenniswood MP et al. Active cell death in hormone-dependent tissues. Cancer Metastasis Rev 1992; 11:197-220. 6. Rosenberg ME, Silkensen J. Clusterin: Physiologic and pathophysiologic considerations. Int J Biochem Cell Biol 1995; 27:633-645. 7. Jenne DE, Tschopp J. Clusterin: The intriguing guises of a widely expressed glycoprotein. Trends Biochem Sci 1992; 17:154-159. 8. Collard MW, Griswold MD. Biosynthesis and molecular cloning of sulfated glycoprotein 2 secreted by rat Sertoli cells. Biochemistry 1987; 26:3297-3303. 9. May PC, Finch CE. Sulfated glycoprotein 2: New relationships of this multifunctional protein to neurodegeneration. Trends Neurosci 1992; 15:391-396.

Fig. 1.4. (opposite) In situ hybridization detection of ApoJCAT transgene expression. Sections through the cerebellum of an adult ApoJCAT-1 transgenic mouse (line 28) were subjected to in situ hybridization for endogenous clusterin mRNA and CAT mRNA localization. In Panels (A, C, E, G) the section was hybridized to the clusterin cDNA riboprobe. In Panels (B, D, F, H) the section was hybridized to the CAT mRNA probe. The endogenous clusterin mRNA is strongly expressed in the cells immediately surrounding Purkinje cells, with lower expression in the inner Granular Layer. The CAT mRNA distribution generated by the ApoJCAT-1 transgene is uniformly high throughout the Granular Layer, but is not quite as high as the endogenous clusterin gene within the cells that are immediately adjacent to the Purkinje cells. Panels (A, B, E, F) brightfield illumination, silver grains appear as small black particles. Panels (C, D, G, H) darkfield illumination, silver grains appear as bright white particles. Panels (A-D) (Final magnification x40). Panels (E-H) (Final magnification x400)

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Clusterin in Normal Brain Functions and During Neurodegeneration

10. Pankhurst GJ, Bennett CA, Easterbrook-Smith SB. Characterization of the heparin-binding properties of human clusterin. Biochemistry 1998; 37:4823-4830. 11. Jordan-Starck TC et al. Mouse apolipoprotein J: Characterization of a gene implicated in atherosclerosis. J Lipid Res 1994; 35:194-210. 12. de Silva HV, Harmony JA, Stuart WD, Gil CM, Robbins J. apolipoprotein J: Structure and tissue distribution. Biochemistry 1990; 29:5380-5389. 13. Jin G, Howe PH. Regulation of clusterin gene expression by transforming growth factor beta. J Biol Chem 1997; 272:26620-26626. 14. Witte DP et al. Platelet activation releases megakaryocyte-synthesized apolipoprotein J, a highly abundant protein in atheromatous lesions. Am J Pathol 1993; 143:763-773. 15. Akakura K et al. Effects of intermittent androgen suppression on the stem cell composition and the expression of the TRPM-2 (clusterin) gene in the Shionogi carcinoma. J Steroid Biochem Mol Biol 1996; 59:501-511. 16. Brown TL, Moulton BC, Baker VV, Mira J, Harmony JA. Expression of apolipoprotein J in the uterus is associated with tissue remodeling. Biol Reprod 1995; 52:1038-1049. 17. French LE, Soriano JV, Montesano R, Pepper MS. Modulation of clusterin gene expression in the rat mammary gland during pregnancy, lactation, and involution. Biol Reprod 1996; 55:1213-1220. 18. Bandyk MG, Sawczuk IS, Olsson CA, Katz AE, Buttyan R. Characterization of the products of a gene expressed during androgen-programmed cell death and their potential use as a marker of urogenital injury. J Urol 1990; 143:407-413. 19. Bettuzzi S, Hiipakka RA, Gilna P, Liao ST. Identification of an androgen-repressed mRNA in rat ventral prostate as coding for sulphated glycoprotein 2 by cDNA cloning and sequence analysis. Biochem J 1989; 257:293-296. 20. Guenette RS, Daehlin L, Mooibroek M, Wong K, Tenniswood M. Thanatogen expression during involution of the rat ventral prostate after castration. J Androl 1994; 15:200-211. 21. Morales CR, Griswold MD. Variations in the level of transferrin and SGP-2 mRNAs in Sertoli cells of vitamin A-deficient rats. Cell Tissue Res 1991; 263:125-130. 22. Correa-Rotter R, Hostetter TH, Nath KA, Manivel JC, Rosenberg ME. Interaction of complement and clusterin in renal injury. J Am Soc Nephrol 1992; 3:1172-1179. 23. Schumer M et al. Morphologic, biochemical, and molecular evidence of apoptosis during the reperfusion phase after brief periods of renal ischemia. Am J Pathol 1992; 140:831-838. 24. Nath KA et al. Induction of clusterin in acute and chronic oxidative renal disease in the rat and its dissociation from cell injury. Lab Invest 1994; 71:209-218. 25. Correa-Rotter R et al. Induction of clusterin in tubules of nephrotic rats. J Am Soc Nephrol 1998; 9:33-37. 26. May PC et al. Dynamics of gene expression for a hippocampal glycoprotein elevated in Alzheimer’s disease and in response to experimental lesions in rat. Neuron 1990; 5:831-839. 27. Michel D, Chabot JG, Moyse E, Danik M, Quirion R. Possible functions of a new genetic marker in central nervous system: the sulfated glycoprotein-2 (SGP-2). Synapse 1992; 11:105-111. 28. O’Bryan MK, Cheema SS, Bartlett PF, Murphy BF, Pearse MJ. Clusterin levels increase during neuronal development. J Neurobiol 1993; 24:421-432. 29. Aldskogius H, Kozlova EN. Central neuron-glial and glial-glial interactions following axon injury. Prog Neurobiol 1998; 55:1-26. 30. Scaglia L, Smith FE, Bonner-Weir S. Apoptosis contributes to the involution of beta cell mass in the post partum rat pancreas. Endocrinology 1995; 136:5461-5468. 31. French LE, Sappino AP, Tschopp J, Schifferli JA. Distinct sites of production and deposition of the putative cell death marker clusterin in the human thymus. J Clin Invest 1992; 90:1919-1925. 32. French LE, Sappino AP, Tschopp J, Schifferli JA. Clusterin gene expression in the rat thymus is not modulated by dexamethasone treatment. Immunology 1994; 82:328-331. 33. Chevalier RL, Chung KH, Smith CD, Ficenec M, Gomez RA. Renal apoptosis and clusterin following ureteral obstruction: The role of maturation [see comments]. J Urol 1996; 156:1474-1479.

Structure and Regulation of Clusterin Genes

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34. Chevalier RL. Effects of ureteral obstruction on renal growth. Semin Nephrol 1995; 15:353-360. 35. Correa-Rotter R, Hostetter TH, Manivel JC, Eddy AA, Rosenberg ME. Intrarenal distribution of clusterin following reduction of renal mass. Kidney Int 1992; 41:938-950. 36. D’Mello SR, Galli C. SGP2, ubiquitin, 14K lectin and RP8 mRNAs are not induced in neuronal apoptosis. Neuroreport 1993; 4:355-358. 37. Zoli M, Ferraguti F, Zini I, Bettuzzi S, Agnati LF. Increases in sulphated glycoprotein-2 mRNA levels in the rat brain after transient forebrain ischemia or partial mesodiencephalic hemitransection. Brain Res Mol Brain Res 1993; 18:163-177. 38. Rozovsky I et al. Selective expression of clusterin (SGP-2) and complement C1qB and C4 during responses to neurotoxins in vivo and in vitro. Neuroscience 1994; 62:741-758. 39. Rosenberg ME, Paller MS. Differential gene expression in the recovery from ischemic renal injury. Kidney Int 1991; 39:1156-1161. 40. Swertfeger DK, Witte DP, Stuart WD, Rockman HA, Harmony JA. Apolipoprotein J/ clusterin induction in myocarditis: A localized response gene to myocardial injury. Am J Pathol 1996; 148:1971-1983. 41. Eti S, Cheng CY, Marshall A, Reidenberg MM. Urinary clusterin in chronic nephrotoxicity in the rat. Proc Soc Exp Biol Med 1993; 202:487-490. 42. Silkensen JR, Agarwal A, Nath KA, Manivel JC, Rosenberg ME. Temporal induction of clusterin in cisplatin nephrotoxicity. J Am Soc Nephrol 1997; 8:302-305. 43. Sawczuk IS, Hoke G, Olsson CA, Connor J, Buttyan R. Gene expression in response to acute unilateral ureteral obstruction. Kidney Int 1989; 35:1315-1319. 44. Wong P et al. Genomic organization and expression of the rat TRPM-2 (clusterin) gene, a gene implicated in apoptosis. J Biol Chem 1993; 268:5021-5031. 45. Wong P et al. Molecular characterization of human TRPM-2/clusterin, a gene associated with sperm maturation, apoptosis and neurodegeneration. Eur J Biochem 1994; 221:917-925. 46. Michel D, Chatelain G, Herault Y, Brun G. The expression of the avian clusterin gene can be driven by two alternative promoters with distinct regulatory elements. Eur J Biochem 1995; 229:215-223. 47. Buttyan R et al. Induction of the TRPM-2 gene in cells undergoing programmed death. Mol Cell Biol 1989; 9:3473-3481. 48. Griswold MD, Roberts K, Bishop P. Purification and characterization of a sulfated glycoprotein secreted by Sertoli cells. Biochemistry 1986; 25:7265-7270. 49. Grima J, Pineau C, Bardin CW, Cheng CY. Rat Sertoli cell clusterin, alpha 2-macroglobulin, and testins: Biosynthesis and differential regulation by germ cells. Mol Cell Endocrinol 1992; 89:127-140. 50. Roberts KP, Santulli R, Seiden J, Zirkin BR. The effect of testosterone withdrawal and subsequent germ cell depletion on transferrin and sulfated glycoprotein-2 messenger ribonucleic acid levels in the adult rat testis. Biol Reprod 1992; 47:92-96. 51. Kondoh G et al. Coexpression of multiple Sertoli cell and Leydig cell marker genes in the spontaneous testicular tumor of F344 rat: Evidence for phenotypical bifurcation of the interstitial cell tumor. Jpn J Cancer Res 1997; 88:839-845. 52. Zwain IH, Grima J, Cheng CY. Regulation of clusterin secretion and mRNA expression in astrocytes by cytokines. Mol Cell Neurosci 1994; 5:229-237. 53. Bhat NR, Brunngraber EG. Synthesis of sulfated glycoproteins by glial precursor cells. Neurochem Res 1986; 11:1193-1201. 54. Morgan TE et al. Clusterin expression by astrocytes is influenced by transforming growth factor beta 1 and heterotypic cell interactions. J Neuroimmunol 1995; 58:101-110. 55. Marcuccilli CJ, Miller RJ. CNS stress response: Too hot to handle? Trends Neurosci 1994; 17:135-138. 56. Clark AM, Griswold MD. Expression of clusterin/sulfated glycoprotein-2 under conditions of heat stress in rat Sertoli cells and a mouse Sertoli cell line. J Androl 1997; 18:257-263. 57. Michel D, Chatelain G, North S, Brun G. Stress-induced transcription of the clusterin/ apoJ gene. Biochem J 1997; 328:45-50.

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58. Mackness B, Hunt R, Durrington PN, Mackness MI. Increased immunolocalization of paraoxonase, clusterin, and apolipoprotein A-I in the human artery wall with the progression of atherosclerosis. Arterioscler Thromb Vasc Biol 1997; 17:1233-1238. 59. Navab M et al. Mildly oxidized LDL induces an increased apolipoprotein J/paraoxonase ratio [published erratum appears in J Clin Invest 1997 Jun 15;99(12):3043]. J Clin Invest 1997; 99:2005-2019. 60. Tribble DL et al. Fatty streak formation in fat-fed mice expressing human copper-zinc superoxide dismutase [published erratum appears in Arterioscler Thromb Vasc Biol 1997 Nov;17(11):3363]. Arterioscler Thromb Vasc Biol 1997; 17:1734-1740. 61. Liao F et al. Genetic evidence for a common pathway mediating oxidative stress, inflammatory gene induction, and aortic fatty streak formation in mice. J Clin Invest 1994; 94:877-884. 62. Treisman R. Journey to the surface of the cell: Fos regulation and the SRE. Embo J 1995; 14:4905-4913. 63. Cahill MA, Janknecht R, Nordheim A. Signaling pathways: Jack of all cascades. Curr Biol 1996; 6:16-19. 64. Johansen FE, Prywes R. Two pathways for serum regulation of the c-fos serum response element require specific sequence elements and a minimal domain of serum response factor. Mol Cell Biol 1994; 14:5920-5928. 65. Hill CS, Wynne J, Treisman R. The Rho family GTPases RhoA, Rac1, and CDC42Hs regulate transcriptional activation by SRF. Cell 1995; 81:1159-1170.

CHAPTER 2

Clusterin and Apolipoprotein E Gene Expression in the Adult Brain Marc Danik, Jean-Guy Chabot, Denis Michel and Rémi Quirion

Introduction Clusterin in the Central Nervous System: Historical Overview.

C

lusterin was originally described as a major glycoprotein synthesized in the male reproductive systems of the ram and rat.1,2 Since then it has been identified in a wide range of biological fluids and tissues in many species.1,3-9 Identification of rat clusterin messenger ribonucleic acid (mRNA) to TRPM-2 (testosterone repressed prostatic message 2),10-12 a transcript found to be prevalent in vivo in involuting tissues whether induced in experimental models or naturally during development of the embryo, raised the question of a possible involvement of clusterin in programmed cell death.13,14 Reports by several independent groups of researchers on the upregulation of the clusterin gene in brains of hamsters infected with the scrapie agent15 and of humans afflicted with Alzheimer’s disease (AD),16 epilepsy,17 or gliomas,17 as well as in the degenerating human retina18 gave support to the apoptosis hypothesis and generated strong interest in the role of clusterin in the central nervous system (CNS). It soon became clear that clusterin overexpression occurred in all types of insults to the CNS, ranging from degenerative conditions like AD,16,19 Pick’s disease15 and multiple sclerosis,20 to other neuropathies such as gliomas and epilepsy,17 to retroviral infection (e.g., human immunodeficiency virus and Rous sarcoma virus20,21) and experimental injury such as deafferentation,16,22-24 neurotoxic lesions,16,25-27 ischemia,28-30 and induced status epilepticus.31 A direct role for clusterin in programmed cell death in the rat CNS was subsequently disclosed in a study by Garden and coworkers32 using in situ hybridization. The authors examined the pattern of clusterin mRNA expression in the developing rat embryo and found it not to correlate with regions undergoing developmental cell death. Rather, they found increasing clusterin message to be associated with neuronal differentiation. The fact that nearly every neuron in the adult forebrain was positive for clusterin mRNA made it unlikely that clusterin correlated with programmed cell death in the majority of cells expressing it. Consistent with these data and supportive of a role for clusterin in the maturation of the nervous tissue are the observations made by O’Bryan et al33 during neuronal development in the mouse. In the latter study, virtually all neurons were shown to be clusterin-positive by immunohistochemistry. Staining was observed in the earliest neurons, and the intensity increased in an age-dependent manner. Clusterin in Normal Brain Functions and During Neurodegeneration, edited by Caleb E. Finch. ©1999 R.G. Landes Company.

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Clusterin in Normal Brain Functions and During Neurodegeneration

Clusterin Expression in the Adult Brain of Mammals The normal adult brain had been shown by Northern blot hybridization to be a major site of clusterin mRNA synthesis in several mammalian species, including humans.5,15,34-37 In order to identify the cells responsible for this synthesis and to search for clues to the role of clusterin in the normal CNS, independent research teams have used in situ hybridization or immunohistochemistry techniques to map clusterin transcripts or protein in the unlesioned brains of young and aged adult rats, as well as in a few human cases.

Clusterin mRNA Distribution in the Rodent and Human Brains We, as well as two other groups, have reported on the distribution of clusterin mRNA in the CNS of young adult rats.26,32,38 Transcripts for clusterin were found to be distributed throughout the CNS, although regional differences in their prevalence were readily observed (Fig. 2.1). A strikingly high level of expression was observed in the ependymal lining of the ventricles and the choroid plexus (Figs. 2.1 and 2.2). In keeping with clusterin as a secretion product in other tissues, these results suggest that the protein is secreted locally into the cerebrospinal fluid (CSF), where the demand or turnover rate may be high. Several neuron-rich cell layers and nuclei contained high levels of clusterin mRNA. These included the pyramidal and granule cell layers of the hippocampal formation, the habenular complex, the hypothalamus, several brainstem nuclei, and some motor neurons in the ventral horns of the spinal cord (Figs. 2.1-2.3). Within most of these areas, much heterogeneity in the hybridization signals was observed over individual neuronal cell bodies. It must be pointed out that the apparent labeling of the dentate gyrus granule cell layer was mostly due to heavily labeled cells located at the edge of the layer, which are thought to represent astrocytes.26,38 A rather homogenous pattern of hybridization was observed in the cerebral cortex, where neurons as well as astrocytes were identified as positive for clusterin mRNA.26,32,38 Other major brain regions like the cerebellum, the basal ganglia, the thalamus, and most of the olfactory bulb displayed an overall low to moderate labeling (Figs. 2.1 and 2.2D,E). Nevertheless, these regions contained some astrocytic and/or neuronal cell bodies showing a strong hybridization signal.26,38 Cell type specificity of clusterin mRNA was determined by in situ hybridization in combination with immunohistochemistry.38 Colocalization of clusterin mRNA with neuron-specific enolase or tyrosine hydroxylase immunoreactivities confirmed neuronal expression of clusterin transcripts in the hippocampal pyramidal layer and hilar region, in subsets of neurons of the substantia nigra, as well as in the red nucleus, the trigeminal motor, facial, somatosensory mesencephalic, and other nuclei.38 In general, white matter gave low intensity signals due to a majority of cell bodies not being labeled.26,32 Moderate to heavy clusterin mRNA concentrations over nonidentified

Fig. 2.1. (opposite) Photomicrographs showing the distribution and levels of clusterin mRNA in selected coronal sections of the adult rat brain. Sections were hybridized with a [35S]-labeled (antisense) riboprobe (for details see Danik et al26). Positive hybridization signals with different intensities are observed in various brain structures. 4V, fourth ventricle; 3, oculomotor nucleus; 7, facial nucleus; CG, central gray; CH, choroid plexus; D3V, dorsal third ventricle; DM, dorsomedial hypothalamic nucleus; gr, granular layer of the cerebellar cortex; GrDG, granular layer of the dentate gyrus; HiF, hippocampal fissure; LHb, lateral habenular nucelus, LS, lateral septum, LV, lateral ventricle; MHb, medial habenular nucleus; Me5, mesencephalic trigeminal nucleus; Mo5, motor trigeminal nucleus; mol, molecular layer of the cerebellar cortex; Pa, paraventricular hypothalamus nucleus; Py, pyramidal cell layer of the hippocampus; R, red nucleus; SFO, subfornical organ; SNC, substantia nigra pars compacta; SO, supraoptic nucleus; Tz, nucleus of trapezoid body; VMH, ventromedial hypothalamic nucleus.

Fig. 2.1.

Clusterin and Apolipoprotein E Gene Expression in the Adult Brain

19

Fig. 2.2. Cellular distribution of clusterin mRNA at the level of the choroid plexus (A,C), the ventricular ependyma (A,B), the medial habenular nucleus (A,B) and the cerebellar cortex (D,E). (A) Darkfield photomicrograph at the level of the dorsal third ventricle. The choroid plexus shows the strongest signal. Note the robust labeling over the ependymal cell lining of the ventricle. Numerous clusters of clusterin mRNA positive cells are observed in the medial habenular nucleus. High-power brightfield photomicrographs showing hybridization signals at the level of the medial habenular nucleus (B) and the choroid plexus (C). A moderate labeling (arrowheads) is observed over individual cells in the medial habenular nucleus (B). A very dense labeling is clearly seen over all epithelial cells of the choroid plexus (C). Dark (D) and brightfield (E) photomicrographs illustrating the distribution of clusterin mRNA in the cerebellar cortex. Note the presence of clusters of silver grains over the Purkinje cells (arrows). The large arrows in D and E indicate the same cell. CH, choroid plexus; E, ependyma; gr cerebellar granular layer; MHb, medial habenular nucleus. Scale bars = 100 µm for A and D; 20 µm for B, C and E. (Modified from ref. 26)

20 Clusterin in Normal Brain Functions and During Neurodegeneration

Clusterin and Apolipoprotein E Gene Expression in the Adult Brain

21

Fig. 2.3. Expression of clusterin mRNA in the spinal cord. (A) Autoradiogram showing the distribution of clusterin mRNA in the spinal cord at the level of the fourth lumbar segment. The strongest labeling was clearly seen in the ventral horn, particularly in α-motoneurons of lamina IX and in their vicinity. (B) High-power brightfield photomicrograph showing the high expression of clusterin transcripts over α-motoneurons in lamina IX of the fourth lumbar segment of the spinal cord. Note the lack of significant hybridization signal in these cells after sense strand hybridization (C). IX, lamina IX of the spinal cord. Scale bars = 20 µm for (B) and (C). (Modified from ref. 26)

22

Clusterin in Normal Brain Functions and During Neurodegeneration

scattered glial cells were noted, however, in various white matter areas including the corpus callosum, the anterior commissure, the fimbria, the stria medullaris of the thalamus, the optic tract, and between cerebellar lobules,26 as well as over radially oriented glia in the spinal cord.32 Strongly positive clusterin mRNA-containing glial cells, presumably astrocytes, were located near the glial limitans and blood vessels.26 Together with its presence in ependymocytes, these localizations are suggestive of a role for clusterin at the CNS-periphery interface. To be noted is the absence of labeling over endothelial cells.26 Most brain regions are suspected to contain clusterin-expressing astrocytes. The striatum is the only region where colocalization of clusterin mRNA and glial fibrillary acidic protein (GFAP) immunoreactivity, a marker for fibrous astrocytes, was clearly demonstrated.38 Normal state astrocytic labeling is much weaker than what has been observed for reactive astrocytes in specific brain regions, following an experimental injury.16,22-30 (The reader is referred to Danik et al,26 and Pasinetti et al,38 for a more detailed description of the regions and the different cell types that express clusterin transcripts in the rat brain.) There is only limited data available on the distribution of clusterin mRNA in the human brain. We have previously reported on the relatively homogenous distribution of transcripts in the temporal cortex of an 81 year old patient without any neurological symptoms who died of a myocardial infarction.17 The distribution pattern was similar to what was seen in the rat cortex except for the presence of larger grain clusters in the former. The significance of this clustering is unknown but may be related to aging. More recently, Pasinetti et al38 have examined brains, used as controls for Alzheimer’s disease, in the age range of 76 to 85 years and found similar results for the temporal cortex. These authors have also examined the hippocampal formation of a non-AD case with multi-infarct dementia. As for the rat hippocampus, clusterin mRNA-positive pyramidal neurons were found in all hippocampal fields. In contrast to the rat, clusterin transcripts were more abundant in granule neurons of the human dentate gyrus, although they were less prevalent than in the pyramidal cell layers, and there was no intense labeling of astrocytes along the hilar edge of the layer. Positive astrocytes in the hippocampal molecular layer were also noted. It remains unknown if the differences observed between rat and human are related to factors such as species, age, or the cell response associated with multi-infarction. The above studies demonstrated that many neuronal as well as glial cell populations constitutively express clusterin transcripts in the normal brain. Basal levels of expression vary considerably, being relatively high in ependymal cells, motoneurons of the spinal cord, and several hypothalamic and brainstem neurons. Although in situ hybridization for clusterin mRNA combined with immunocytochemistry (ICC) for specific glial markers did not show colocalization to oligodendrocytes or microglia in the striatum,39 other brain regions should be examined to confirm the incapacity of these cells to synthesize clusterin transcripts. The variable concentrations of clusterin mRNA at the cellular level, the diversity of expressor cell types and the distribution pattern of these cells in the CNS together indicate that the role carried out by clusterin in this tissue must be general.

Clusterin Immunoreactivity and Its Colocalization with Clusterin mRNA In two studies where investigators examined the brains of young adult rats (3-4 months old), only a few clusterin-immunoreactive structures were observed.38,40 The immunoreactivity consisted mostly of neuronal cellular labeling. These observations contrast with the high levels and almost ubiquitous distribution of the clusterin transcript in the rodent brain. They also indicate that translation of the clusterin mRNA into protein is either rather poor in most brain areas, or that the protein has a high turnover rate, and/or it is constitutively secreted into the interstitium, where it does not accumulate. Extracellular deposits of clusterin

Clusterin and Apolipoprotein E Gene Expression in the Adult Brain

23

in the nervous tissue have been reported, although only in brains of aged rats or following experimental lesioning.22,40,41 In young adult rats, intense clusterin immunoreactivity was found, mainly in the walls of the ventricles and over the choroid plexus,38,40 which is consistent with the very high concentrations of clusterin mRNA observed in the ependymal epithelia that lines these structures. Thus, the strong staining could be the consequence of intense clusterin protein synthesis or, alternatively, of substantial cellular uptake of the protein from the CSF, or both. Indeed, clusterin is known to be present in human CSF 4 and to be able to bind to the gp330/ megalin receptor with high affinity.42 Furthermore, there is evidence that this receptor is present on the ependymal lining of the ventricles.43 Noteworthy is the absence of glial labeling for clusterin throughout the brain, with the notable exception of the ependymocytes as just mentioned.38,40 Other major brain areas such as the hippocampal formation and the striatum were also immunonegative. A few discrepancies were apparent between the above two studies on clusterin immunoreactivity in the rat brain. In one study, cortical neurons were immunonegative for clusterin, whereas some neurons in the red nucleus, motor trigeminal nucleus, and the Purkinje cell layer in the cerebellum showed strong immunoreactivity.38 In the other study by Senut et al,40 layer VI of the cingulate cortex and the hypothalamus contained the highest densities of clusterin-positive cells in the telencephalon and the diencephalon, respectively. Positive cell bodies were reminiscent of neurons in both cases. Other areas within the latter main regions, as well as in the mesencephalon and metencephalon, contained occasional labeled cells, when not all unlabeled cells. The reasons for the discrepancies remain unexplained, but could be related to the different antiserum or to the different strain of rats used by the two groups of investigators. In the latter study, clusterin immunoreactivity in the adult rat brain did appear in the red nucleus, the motor trigeminal nucleus, and the cerebellum (extracellularly), as well as in several other areas, but only at an older age. A marked increase in the density of immunoreactive cells and in the number of immunopositive structures occurred in rats that were older than one year.40 In contrast to aged rats, either light staining of some temporal cortex neurons and the neuropil or no clusterin immunoreactivity at all was observed in the hippocampus, as well as in several cortical regions of the brain, in elderly humans, using a monoclonal antibody against human clusterin.19,44,45 It should be specified that in these studies, the antibody strongly stained brain tissues of Alzheimer’s disease patients. Unfortunately, data regarding clusterin’s distribution in other regions of the normal human brain are lacking and therefore comparison with the rat brain is not possible.

In vitro Secretion of Clusterin by Cells of the CNS The fact that clusterin immunoreactivity was observed in some neurons and glial cells does not prove that the protein is being synthesized by the positively-stained cells. The immunostaining could represent cell surface binding or uptake of clusterin. In order to identify the cell types that make and secrete clusterin in the brain, rat primary cultures from embryos or neonatal pups were used for investigation. By this means, both cortical and hippocampal astrocytes were shown to synthesize clusterin mRNA and to secrete the protein into the culture medium.38,46 In contrast to the in vivo situation in the adult rat brain, cortical astrocytes displayed strong clusterin immunoreactivity throughout their cytoplasm and processes.46 It remains to be determined if this strong synthesis is mediated by the in vitro environment or related to the young age of the astrocytes. When hippocampal microglia cultures were examined, no immunoreactive material was found in the media, nor was clusterin mRNA detected by Northern blot.38 Thus, monotypic cultures of microglia do not seem to synthesize clusterin or otherwise they do so at

24

Clusterin in Normal Brain Functions and During Neurodegeneration

levels that are below the detection limit. The possibilities that the protein is stored intracellularly or detectable only when cells are reactive have not been addressed. Furthermore, cocultures of peritubular cells and Sertoli cells have been shown to increase the synthesis of clusterin in the latter cell type via a paracrine factor.47 Thus, cocultures involving microglia and neurons, or other CNS-derived cell types, should be analyzed before ruling out the incapacity of microglial cells to synthesize and secrete clusterin. It should be pointed out, however, that clusterin mRNA was not detected in microglia or oligodendrocytes from mixed glial cultures.39 No detectable clusterin immunoreactivity was found in the media of cultured immature (E16-E18) hippocampal neurons, although these cells contained the corresponding mRNA.38 Hippocampal neurons were not examined for the absence of intracellular clusterin. A negative result would be consistent with the previous observations that tissue sections of the hippocampal formation from adult rats do not stain for clusterin.38,40 However, the possibility that these neurons store small quantities of clusterin inside secretory organelles, which are secreted via a regulated pathway instead of constitutively, cannot be eliminated. This has been demonstrated to be the case for clusterin found in chromaffin granules of adrenal medullary cells.48 In these cells as well as in those of the anterior pituitary, which contain small granules, immunoelectron microscopy revealed that clusterin was confined to, and weakly labeled, the latter.49 It is interesting to note that the staining was significantly higher in cells with larger granules, for example in neurons of the posterior pituitary which are rich in large dense-core vesicles (LDCV).49 These vesicles store and secrete neuropeptides and can be considered as the neuronal counterpart of secretory granules of endocrine cells that store peptide hormones. No attempts have been made, however, to colocalize clusterin with any specific neuropeptide in the CNS or to correlate levels of clusterin expression with LDCV cellular content or solicitation.

Clusterin in the Cerebrospinal Fluid Human CSF contains relatively low concentrations of clusterin with an average value of 2.4 ± 1.2 µg/ml4,44 which is equivalent to approximately 2.5-7% of the concentration found in serum.3,50,51 The presence of clusterin in the CSF had been proposed as early as 1988 and the protein was suspected to be associated with HDL particles.52 The association of clusterin with CSF lipoprotein particles has been confirmed recently. Using different isolation procedures, two groups have demonstrated that clusterin is associated with particles in the size-range of small plasma low-density lipoproteins and also larger complexes,53,54 whereas another group has reported its presence on lipoproteins of higher density as well as in a free state.55 The CSF concentrations for clusterin are too high for what would be expected for a filtered high molecular weight plasma protein.56 We have previously postulated that clusterin is secreted by ependymocytes and perhaps also by CSF-contacting neurons and astrocytes.26 It remains to be determined if CSF clusterin is directed outside the CNS or recaptured by the nervous tissue via the gp330/megalin receptor or other receptors yet to be identified. Clusterin might be secreted into the CSF in order to scavenge and transport lipids or other molecules, like the β-amyloid peptide, for their utilization or their disposal. Indeed, the CSF was shown to normally contain soluble β-amyloid noncovalently complexed to clusterin.57 The possibility that clusterin plays a role in lipid transport in the CNS has been proposed by several investigators. In fact, clusterin was previously identified as an apolipoprotein (apoJ) that shares several features with other apolipoproteins and circulates in plasma as a high-density lipoprotein component carrying cholesterol and other lipids.51,58-60 Furthermore, the human hepatoma-derived cell line HepG2 has been shown to secrete clusterin as a triglyceride-rich lipoprotein particle of very high density into its culture medium.61 Re-

Clusterin and Apolipoprotein E Gene Expression in the Adult Brain

25

cently, it was demonstrated that primary cultures of rat astrocytes secrete discoidal partic that contain clusterin together with little core lipid.54 The size and composition of plasma and CSF apoJ/clusterin-containing lipoproteins, compared to those of secreted nascent lipoproteins from the cultured cells, suggest that the latter particles maturate on their way to the vascular and ventricular compartments, respectively, and thus would participate in lipid clearance. If this is the case, it may explain in part the relative difficulty of staining clusterin in the CNS tissue. This does not preclude the possibility that clusterin-lipid particles are also being taken up and metabolized locally.

Apolipoprotein E Expression in the Adult Brain of Mammals The brain is also a major site of apolipoprotein E (apoE) mRNA expression in humans, marmosets, rats and mice.62 Early data from animal lesion paradigms such as sciatic nerve crush 63,64 and entorhinal cortex lesioning 65,66 suggested that apoE plays a role in the coordinated storage and redistribution of cholesterol and phospholipids among cells within the remodeling area. Apolipoprotein E is now believed to play an important role not only in reactive synaptogenesis, by delivering lipids to remodeling and sprouting neurons in response to tissue injury, but also in physiological ongoing synaptic plasticity and maintenance of neuronal integrity, as well as in cholinergic activity. 67-69 In the vascular compartment, apoE is found in association with several classes of lipoproteins including chylomicrons, the very low-density lipoprotein (VLDL), and a subclass of the HDL.70 These apoE-containing lipoprotein complexes carry lipids such as cholesterol, cholesterol esters, phospholipids, and triglycerides. Apolipoprotein E serves as a ligand for several cell surface receptors that mediate lipoprotein uptake, and thus apoE participates in lipoprotein metabolism and lipid homeostasis. All known receptors for apoE have been shown to be present in the mammalian brain on one or several cell types, including neurons (for a review, see 71). These receptors are members of a single family and include the low-density lipoprotein (LDL) receptor, the very low-density (VLDL) receptor, the apoER2 receptor, the LDL receptor-related protein (LRP), and the gp330/megalin receptor. Interest in the importance of apoE in the CNS grew substantially since the first published reports linking one of the three common human alleles, namely the apoE ε4 allele, to both familial and sporadic late-onset Alzheimer’s disease.72-75 Accordingly, the ε4 allele frequency was shown to increase significantly (~3-fold, i.e., from 14% up to 40%-50%) in the Alzheimer population.73,75,76 Furthermore, a gene dosage effect was observed which translates into the fact that inheritance of one or two apoE ε4 alleles is associated with a doserelated higher risk and younger age of onset distribution of AD.72,75-77

ApoE mRNA Distribution in the Adult Brain Transcripts for apoE have been shown to be distributed throughout all regions of the brain by RNA dot blot hybridization,62 and have been localized by in situ hybridization to glial cells (astrocytes, ependymocytes and microglia) but not in neurons.65,69,78,79 In the adult mouse brain, there was abundant expression of apoE mRNA in the choroid plexus and ependyma in one study79 but apparently absence of labeling in another (data was not shown).78 High amounts of apoE mRNA were also detected in the medial and lateral habenular nuclei, in several thalamic nuclei as well as in the entire hypothalamus, with somewhat higher grain densities in the latter.79 As described above, these structures also contain high levels of clusterin mRNA in the adult rat. With the obvious exception of the ependymal epithelia, it is not known whether the two transcripts colocalize in the same cell populations. ApoE transcripts were distributed over the mouse hippocampus and dentate gyrus but, unlike clusterin mRNA, not in their pyramidal and granular layers, respectively, with

26

Clusterin in Normal Brain Functions and During Neurodegeneration

the highest levels of apoE mRNA seen in the molecular layer of the dentate gyrus.79 These results are consistent with those of two other studies in rats using in situ hybridization in combination with ICC for GFAP and either ED1 or OX42, two different markers for microglia. These latter studies identified astrocytes as the major apoE message-containing cells in the hippocampal formation, but some positive microglial cells could be identified in the CA1 region.65,69 Microglia in the arcuate nucleus of the hypothalamus was also shown to contain apoE transcripts.69 In the cerebral cortex, the apoE message was detected mainly in the outer layers, presumably in astrocytes,79 thereby contrasting with the more ubiquitous expression noted for clusterin transcripts. apoE mRNA-positive cells were also observed in white matter areas in rodent brains, as it was the case for clusterin mRNA.26,65,79 For most of the brain regions, identification of the cell types expressing apoE mRNA would have required ICC for specific glial markers. ApoE transcripts were also mapped in the cortex of aged humans by in situ hybridization and shown to be present in astrocytes only.78 The distribution and cell type specificity of apoE expression in other regions of the human brain have not been reported.

Apolipoprotein E Immunolocalization The distribution of apoE immunoreactivity in the brain of mammals was shown to vary from one species to the other. In addition, cell type labeling within a given species has also been found to vary between studies. This variation has been attributed to differences in the protocols used, mainly with regard to tissue fixation procedures and reactivity of the apoE antiserum.80 In general, neurons stain rather poorly while astrocytes represent the glial cell type most consistently immunopositive. Astrocytes were labeled in the perinuclear region, as well as their processes that terminated on blood vessels and the pial surface.78,81 In rats and mice, apoE immunoreactivity has been shown in a variety of cell types including some fibrous and protoplasmic astrocytes, microglia, ependymocytes, and in the epithelia lining the choroid plexus.78,81,82 With regard to specialized astrocytic cells such as Bergmann glia, one study showed positive staining for apoE (as well as in tanycytes)81 while another did not.82 In most cases, no apoE immunoreactivity was observed in oligodendrocytes identified by morphological and topographic criteria. However, in a study that focused on the normal mature optic nerve both astrocytes and oligodendrocytes were apoEpositive, using specific glial markers.83 Rodent neurons are usually not immunoreactive, but faint staining has been observed in occasional neurons in adult rats and mice.78,82 In primates, apoE immunoreactivity was observed in astrocytes (in both white and gray matter), ependymocytes, microglia (in older specimens), as well as in neurons, where it is common even in young animals.82 Most neuronal staining was faint and perinuclear, but intense labeling of both distal dendrites and axonal processes was often seen in cortical and, especially, in hippocampal CA1-2 pyramidal neurons.82 In contrast, granule neurons in the dentate gyrus were mostly unstained. In prosimians, the density of apoE-immunoreactive neurons generally increased with age.82 Intraneuronal localization of apoE has also been reported in several studies that examined human brain specimens. The occurrence of apoE in cortical and hippocampal neurons without neurofibrillary changes was demonstrated in brains of nondemented elderly individuals.84,85 As seen in primates, numerous apoE-immunoreactive neurons were found in the CA1-2 subfields of the hippocampal formation, whereas the granule cell layer did not stain.84 ApoE ICC also revealed staining of hippocampal glial cells (astrocytes and ependymal cells), although the intensity varied widely between cases.84 In the frontal cortex, apoE immunoreactivity was intense in some neuronal cell bodies and their processes.85 Most of these cells have been identified as small pyramidal neurons in layer III and some as large

Clusterin and Apolipoprotein E Gene Expression in the Adult Brain

27

pyramidal cells in layer V, using double staining with a second antibody to the neuronal marker microtubule associated protein 2. In this brain region, only a few cells with astrocytic morphology were apoE-positive.85 In contrast, in a single case study from a 34 year old individual who died of pneumonia, apoE was found in all major brain subdivisions exclusively in GFAP-positive astrocytes.86 Together these results indicate that neuronal labeling in humans appears with increasing age. Alternatively, optimal sensitivity of the ICC method used in the last study may not have been reached, thereby not allowing for the detection of lower amounts of apoE in neurons. In this regard, it is of note that in a different study, apoE immunoreactivity in temporal cortex neurons from young patients with epilepsy (21-55 years) was found to be less intense than in astrocytes.80

ApoE Secretion Consistent with the localization of its mRNA in vivo and its presence in extracellular sites,81 apoE was shown to be synthesized and secreted in vitro by glial cells, but not by neurons. Astrocytic secretion of apoE was demonstrated in both perinatal rat and fetal human primary cultures.87,88 Confirmation that rat astrocytes synthesize apoE came from electron microscopic immunocytochemical studies which demonstrated the presence of apoE in the Golgi apparatus.81 The same techniques, when applied to the rat optic nerve, also localized apoE in the Golgi apparatus of oligodendrocytes, indicating synthesis by these cells.83 Although apoE secretion by microglia has not been demonstrated, the fact that the corresponding transcripts could be detected in cultured rat microglia suggests that this cell type may represent a possible source of apoE.69,89 Since neurons have not been shown to either secrete apoE or transcribe its corresponding gene, intraneuronal apoE is presumed to reflect cellular uptake, as demonstrated using hippocampal neuronal cell primary cultures.90 The presence in vivo of apoE in CNS neurons was shown by immunoelectron microscopy to be confined to the cytoplasm of cell bodies and proximal dendrites in association with the external membrane surface of some organelles.80 This observation suggests that apoE may affect neuronal metabolism in additional ways not related to cholesterol homeostasis or, alternatively, be involved in the intracellular transport of lipids.

Cerebrospinal Fluid Apolipoprotein E

ApoE is one of the major apolipoproteins found in human CSF.53,55 It is present at slightly higher concentrations (3-6 µg/ml) than clusterin, which correspond to ~4-20% of normal plasma levels.56,91-93 Similar (4-5 µg/ml) or higher (8-12 µg/ml) concentrations of apoE were found in rat and dog CSF, respectively.91,94 CSF to serum ratios are lower in rats (1-2%) and higher in dogs (38-63%) compared to humans. ApoE was shown to be present on particles the size of HDL in all three species,55,91,94 but also on discoidal partic in dogs91 and lipoproteins of lower density in humans.53,95 The presence of apoE in the CSF might be explained by local synthesis in ependymocytes and/or from brain tissue secretion and drainage of the perivascular space.91 In agreement with this, apoE from CSF, brain tissue and astrocytes in culture, was shown to be more highly sialylated than plasma apoE.91

Concluding Remarks In the adult mammalian brain, clusterin sites of expression are more widespread than those of apolipoprotein E (Table 2.1). Both proteins are produced by glial cells, predominantly by astrocytes and ependymal cells. In contrast, only clusterin is synthesized by neurons, although not by all of them, whereas only apoE mRNA can be detected in what appears to be a restricted set of microglial cells. Since both proteins are secreted, the sites of synthesis are not necessarily identical to the sites of final action. However, by analogy to the

28

Clusterin in Normal Brain Functions and During Neurodegeneration

Table 2.1. Clusterin and apolipoprotein E gene and protein expression in the central nervous systems of various species.

Neurons

Astrocytes

Oligodendrocytes

Microglia

Ependymocytes

Clusterin mRNA Protein

Apolipoprotein E mRNA Protein

Human Primates Rat Mouse

+ 16,17,38,

+ 19,44,45

– 78

+ 26,32,38

+ 40 / –38 + 33

– 65,69 – 79

Human Primates Rat Mouse

+ 38

+ 20

+ 78

+ 26,32,38,46

+ 38,46

+ 65,69 + 78,79

– 20

– 78

Human Rat Human Primates Rat Mouse Human Primates Rat Mouse

–

39

– 38,39

– 38

+ 38,40

+ 78,80,84,86,88 + 82 + 81,82,83,87 + 78,82 – 86 + 83/ – 81

– 78 + 82 + 69,89 / – 65 – 81,82 + 82 – 78

+ 26,32

+ 80,84,85 / – 86 + 82 + 82 / – 81 + 78,82

+ 79

+ 84 / – 86 + 82 + 82 / – 81 + 82

(+) denotes that protein or mRNA has been observed [or not (–)] in the corresponding cell type, on brain sections or cultured cells from the indicated species.

recycled membrane-bound form of clusterin in adrenal chromaffin granules,48,96 it remains possible that neuronal clusterin is only partly secreted from LDCV. It was postulated that clusterin may be important in the sorting of glycoproteins along with lipid components to secretory granules.97 In view of its colocalization with neurophysin and chromogranins in LDCV of neurons in the posterior pituitary,49 clusterin may thus play a role in neuropeptide and/or neurotransmitter packaging and processing. The target molecule(s) and site(s) of action for clusterin that is released from neurons and glia remain to be determined. No receptor for clusterin has yet been identified in the nervous tissue, except for the gp330/megalin receptor on the ependymal lining of the ventricles.43 This receptor is a member of the LDL receptor family, all of which are known to bind apoE and to be differentially expressed by several cell types in the CNS. Clusterincontaining lipoprotein particles might be directed towards the CSF for clearance via the perivascular space.91 This does not preclude the possibility that other proteins, apoE for example, may be present on the same particles and serve as a ligand for local uptake in the CNS parenchyma. In this regard, it is interesting to note that a subpopulation of human CSF lipoproteins were found to contain both apoE and clusterin.53 Although additional functions unrelated to lipid transport have been postulated for apoE,70,98-100 its role in cholesterol homeostasis and lipid metabolism has been firmly estab-

Clusterin and Apolipoprotein E Gene Expression in the Adult Brain

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lished.70 In contrast, the role of clusterin in the CNS, or elsewhere in the organism, remains poorly understood. In addition to its putative role in secretory vesicles, it was postulated that clusterin may be involved in cellular defense mechanisms by inhibiting plasma membrane insertion of the membrane attack complex (MAC); thereby, it would protect innocent bystander cells against terminal complement lysis.50 Although complement components are synthesized locally in the CNS, their basal levels are relatively low compared to those of clusterin in non-neurologic cases.19,101 It also remains to be demonstrated that this protective effect, as assayed in vitro on red blood cells, can occur in vivo on nucleated cells capable of response. Furthermore, no association between clusterin and terminal complement complex deposition could be demonstrated in inflammatory CNS lesions of varied etiology.20 We have previously proposed that basally-expressed clusterin mRNA in the brain is linked to natural ongoing synapse turnover.26 This hypothesis was based on clusterin’s putative role as a lipid carrier in biological fluids and in remodeling tissues.14,51,102 In all brain lesion models examined to date, neuronal losses or deafferentation were accompanied by increases in clusterin expression.16,23-26,28,30 Interestingly, both genes for clusterin and apoE were shown to be upregulated in the deafferented zone of the molecular layer of the dentate gyrus following experimental entorhinal cortex lesioning (ECL).16,65 In this model, loss of synaptic input to the granule cells induces synaptic replacement that is essentially completed after 2 months.103 The two genes presented a very similar expression profile after lesioning. Maximum concentrations of both apoE and clusterin mRNA occurred around one week post-ECL, when reactive synaptogenesis and terminal proliferation are in their early phases.16,65 The levels of these transcripts increased at day 2 postlesion, the earliest time point analyzed, and were back to baseline at 30 days, when terminal proliferation is completed and the rate of synaptogenesis is declining.104 The coregulation and parallel time courses in the expression of the two genes support the hypothesis of complementary trophic and coordinated roles between the respective proteins. It was postulated that these roles are related to the scavenging and storage of released lipids during terminal breakdown and their subsequent redistribution to sprouting neurons and neurons undergoing dendritic reorganization.26,67 The brain is a major site of expression for both clusterin and apolipoprotein E in mammals. The importance of this is underscored by the lack of synthesis in the nervous tissue of major plasma apolipoproteins, namely apoB and apoAI, and the presence of the bloodbrain barrier that limits completely or partially, respectively, their access to the CNS. The mammalian CNS has the capability of synthesizing many of the other components found in the periphery that are necessary for lipid transport and utilization. Local synthesis of these components may ensure rapid responses to high demands in lipids for cellular activity, maintenance, and plasticity, particularly following injury to the CNS.

References 1. Blaschuk O, Burdzy K, Fritz IB. Purification and characterization of a cell-aggregating factor (clusterin), the major glycoprotein in ram rete testis fluid. J Biol Chem 1983; 258:7714-20. 2. Sylvester S, Skinner MK, Griswold MD. A sulfated glycoprotein synthesized by Sertoli cells and by epididymal cells is a component of the sperm membrane. Biol Reprod 1984; 31:1087-101. 3. Murphy BF, Kirszbaum L, Walker ID et al. Sp-40,40, a newly identified normal human serum protein found in the sc5b-9 complex of complement and in the immune deposits in glomerulonephritis. J Clin Invest 1988; 81:1858-64. 4. Choi-Miura NH, Ihara Y, Fukuchi K et al. SP-40,40 is a constituent of Alzheimer’s amyloid. Acta Neuropathol 1992; 83:260-4.

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5. de Silva HV, Harmony JA, Stuart WD et al. Apolipoprotein J: Structure and tissue distribution. Biochemistry 1990; 29:5380-9. 6. Sylvester SR, Morales C, Oko R et al. Localization of sulfated glycoprotein-2 (clusterin) on spermatozoa and in the reproductive tract of the male rat. Biol Reprod 1991; 45:195-207. 7. Hartmann K, Rauch J, Urban J et al. Molecular cloning of gp 80, a glycoprotein complex secreted by kidney cells in vitro and in vivo. A link to the reproductive system and to the complement cascade. J Biol Chem 1991; 266:9924-31. 8. Tschopp J, Jenne DE, Hertig S et al. Human megakaryocytes express clusterin and package it without apolipoprotein A-1 into alpha-granules. Blood 1993; 82:118-25. 9. Aronow BJ, Lund SD, Brown TL et al. Apolipoprotein J expression at fluid-tissue interfaces: Potential role in barrier cytoprotection. Proc Natl Acad Sci USA 1993; 90:725-9. 10. Montpetit ML, Lawless KR, Tenniswood M. Androgen-repressed messages in the rat ventral prostate. Prostate 1986; 8:25-36. 11. Cheng CY, Chen CL, Feng ZM et al. Rat clusterin isolated from primary sertoli cell-enriched culture medium is sulfated glycoprotein-2 (SGP-2). Biochem Biophys Res Commun 1988; 155:398-404. 12. Bandyk MG, Sawczuk IS, Olsson CA et al. Characterization of the products of a gene expressed during androgen-programmed cell death and their potential use as a marker of urogenital injury. J Urol 1990; 143:407-13. 13. Leger JG, Montpetit ML, Tenniswood MP. Characterization and cloning of androgen-repressed mRNAs from rat ventral prostate. Biochem Biophys Res Commun 1987; 147:196-203. 14. Buttyan R, Olsson CA, Pintar J et al. Induction of the trpm-2 gene in cells undergoing programmed death. Mol Cell Biol 1989; 9:3473-81. 15. Duguid JR, Bohmont CW, Liu NG et al. Changes in brain gene expression shared by scrapie and Alzheimer disease. Proc Natl Acad Sci USA 1989; 86:7260-4. 16. May PC, Lampert-Etchells M, Johnson SA et al. Dynamics of gene expression for a hippocampal glycoprotein elevated in Alzheimer’s disease and in response to experimental lesions in rat. Neuron 1990; 5:831-9. 17. Danik M, Chabot JG, Mercier C et al. Human gliomas and epileptic foci express high levels of a mRNA related to rat testicular sulfated glycoprotein 2, a purported marker of cell death. Proc Natl Acad Sci USA 1991; 88:8577-81. 18. Jones SE, Meerabux JM, Yeats DA et al. Analysis of differentially expressed genes in retinitis pigmentosa retinas. Altered expression of clusterin mRNA. FEBS Lett 1992; 300:279-82. 19. McGeer PL, Kawamata T, Walker DG. Distribution of clusterin in Alzheimer brain tissue. Brain Res 1992; 579:337-41. 20. Wu E, Brosnan CF, Raine CS. SP-40,40 immunoreactivity in inflammatory CNS lesions displaying astrocyte/oligodendrocyte interactions. J Neuropathol Exp Neurol 1993; 52:129-34. 21. Michel D, Gillet G, Volovitch M et al. Expression of a novel gene encoding a 51.5 kD precursor protein is induced by different retroviral oncogenes in quail neuroretinals cells. Oncogene Res 1989; 4:127-36. 22. Lampert-Etchells M, McNeill TH, Laping NJ et al. Sulfated glycoprotein-2 is increased in rat hippocampus following entorhinal cortex lesioning. Brain Res 1991; 563:101-6. 23. Pasinetti GM, Cheng HW, Morgan DG et al. Astrocytic messenger RNA responses to striatal deafferentation in male rat. Neuroscience 1993; 53:199-211. 24. Zoli M, Ferraguti F, Zini I et al. Increases in sulphated glycoprotein-2 mRNA levels in the rat brain after transient forebrain ischemia or partial mesodiencephalic hemitransection. Mol Brain Res 1993; 18:163-77. 25. Pasinetti GM, Finch CE. Sulfated glycoprotein-2 (SGP-2) mRNA is expressed in rat striatal astrocytes following ibotenic acid lesions. Neurosci Lett 1991; 130:1-4. 26. Danik M, Chabot JG, Hassan-Gonzalez D et al. Localization of sulfated glycoprotein-2/ clusterin mRNA in the rat brain by in situ hybridization. J Comp Neurol 1993; 334:209-27.

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27. Schreiber SS, Tocco G, Najm I et al. Seizure activity causes a rapid increase in sulfated glycoprotein-2 messenger RNA in the adult but not the neonatal rat brain. Neurosci Lett 1993; 153:17-20. 28. May PC, Robison P, Fuson K et al. Sulfated glycoprotein-2 expression increases in rodent brain after transient global ischemia. Mol Brain Res 1992; 15:33-9. 29. Wießner C, Back T, Bonnekoh P et al. Sulfated glycoprotein-2 mRNA in the rat brain following transient forebrain ischemia. Mol Brain Res 1993; 20:345-52. 30. Walton M, Young D, Sirimanne E et al. Induction of clusterin in the immature brain following a hypoxic-ischemic injury. Mol Brain Res 1996; 39:137-52. 31. Dragunow M, Preston K, Dodd J et al. Clusterin accumulates in dying neurons following status epilepticus. Mol Brain Res 1995; 32:279-90. 32. Garden GA, Bothwell M, Rubel EW. Lack of correspondence between mRNA expression for a putative cell death molecule (SGP-2) and neuronal cell death in the central nervous system. J Neurobiol 1991; 22:590-604. 33. O’Bryan MK, Cheema SS, Bartlett PF et al. Clusterin levels increase during neuronal development. J Neurobiol 1993; 24:421-32. 34. Collard MW, Griswold MD. Biosynthesis and molecular cloning of sulfated glycoprotein 2 secreted by rat Sertoli cells. Biochemistry 1987; 26:3297-303. 35. Bettuzzi S, Hiipakka RA, Gilna P et al. Identification of an androgen-repressed mRNA in rat ventral prostate as coding for sulphated glycoprotein 2 by cDNA cloning and sequence analysis. Biochem J 1989; 257:293-6. 36. Palmer DJ, Christie DL. The primary structure of glycoprotein III from bovine adrenal medullary chromaffin granules. J Biol Chem 1990; 265:6617-23. 37. Diemer V, Hoyle M, Baglioni C et al. Expression of porcine complement cytolysis inhibitor mRNA in cultured aortic smooth muscle cells. Changes during differentiation in vitro. J Biol Chem 1992; 267:5257-64. 38. Pasinetti GM, Johnson SA, Oda T et al. Clusterin (SGP-2): A multifunctional glycoprotein with regional expression in astrocytes and neurons of the adult rat brain. J Comp Neurol 1994; 339:387-400. 39. Morgan TE, Laping NJ, Rozovsky I et al. Clusterin expression by astrocytes is influenced by transforming growth factor beta 1 and heterotypic cell interactions. J Neuroimmunol 1995; 58:101-10. 40. Senut MC, Jazat F, Choi NH et al. Protein SP40,40-like immunoreactivity in the rat brain: Progressive increase with age. Eur J Neurosci 1992; 4:917-28. 41. Kida E, Pluta R, Lossinsky AS et al. Complete cerebral ischemia with short-term survival in rat induced by cardiac arrest. II. Extracellular and intracellular accumulation of apolipoproteins E and J in the brain. Brain Res 1995; 674:341-6. 42. Kounnas MZ, Loukinova EB, Stefansson S et al. Identification of glycoprotein 330 as an endocytic receptor for apolipoprotein J/clusterin. J Biol Chem 1995; 270:13070-5. 43. Zheng G, Bachinsky DR, Stamenkovic I et al. Organ distribution in rats of two members of the low-density lipoprotein receptor gene family, gp330 and LRP/alpa 2MR, and the receptor-associated protein (RAP). J Histochem Cytochem 1994; 42:531-42. 44. Harr SD, Uint L, Hollister R et al. Brain expression of apolipoproteins E, J, and A-I in Alzheimer’s disease. J Neurochem 1996; 66:2429-35. 45. Giannakopoulos P, Kovari E, French LE et al. Possible neuroprotective role of clusterin in Alzheimer’s disease: A quantitative immunocytochemical study. Acta Neuropathol (Berl) 1998; 95:387-94. 46. Zwain IH, Grima J, Cheng CY. Regulation of clusterin secretion and mRNA expression in astrocytes by cytokines. Mol Cell Neurosci 1994; 5:229-37. 47. Zwain IH, Grima J, Stahler MS et al. Regulation of Sertoli cell alpha 2-macroglobulin and clusterin (SGP-2) secretion by peritubular myoid cells. Biol Reprod 1993; 48:180-7. 48. Fischer-Colbrie R, Zangerle R, Frischenschlager I et al. Isolation and immunological characterization of a glycoprotein from adrenal chromaffin granules. J Neurochem 1984; 42:1008-16.

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49. Laslop A, Steiner HJ, Egger C et al. Glycoprotein III (clusterin, sulfated glycoprotein 2) in endocrine, nervous, and other tissues: Immunochemical characterization, subcellular localization, and regulation of biosynthesis. J Neurochem 1993; 61:1498-505. 50. Jenne DE, Tschopp J. Molecular structure and functional characterization of a human complement cytolysis inhibitor found in blood and seminal plasma: Identity to sulfated glycoprotein 2, a constituent of rat testis fluid. Proc Natl Acad Sci USA 1989; 86:7123-7. 51. de Silva HV, Stuart WD, Duvic CR et al. A 70-KDa apolipoprotein designated apoJ is a marker for subclasses of human plasma high density lipoproteins. J Biol Chem 1990; 265:13240-7. 52. Hochstrasser AC, James RW, Martin BM et al. HDL particle associated proteins in plasma and cerebrospinal fluid: Identification and partial sequencing. Appl Theor Electrophor 1988; 1:73-6. 53. Borghini I, Barja F, Pometta D et al. Characterization of subpopulations of lipoprotein particles isolated from human cerebrospinal fluid. Biochim Biophys Acta 1995; 1255:192-200. 54. LaDu MJ, Gilligan SM, Lukens JR et al. Nascent astrocyte particles differ from lipoproteins in CSF. J Neurochem 1998; 70:2070-81. 55. Koudinov AR, Koudinova NV, Kumar A et al. Biochemical characterization of Alzheimer’s soluble amyloid beta protein in human cerebrospinal fluid: Association with high density lipoproteins. Biochem Biophys Res Commun 1996; 223:592-7. 56. Roheim PS, Carey M, Forte T et al. Apolipoproteins in human cerebrospinal fluid. Proc Natl Acad Sci USA 1979; 76:4646-9. 57. Ghiso J, Matsubara E, Koudinov A et al. The cerebrospinal-fluid soluble form of Alzheimer’s amyloid beta is complexed to sp-40,40 (apolipoprotein J), an inhibitor of the complement membrane-attack complex. Biochem J 1993; 293:27-30. 58. Jenne DE, Lowin B, Peitsch MC et al. Clusterin (complement lysis inhibitor) forms a high density lipoprotein complex with apolipoprotein A-I in human plasma. J Biol Chem 1991; 266:11030-6. 59. James RW, Hochstrasser AC, Borghini I et al. Characterization of a human high density lipoprotein-associated protein, NA1/NA2. Identity with SP-40,40, an inhibitor of complement-mediated cytolysis. Arterioscl Thromb 1991; 11:645-52. 60. Segrest JP, Jackson RL, Morrisett JD et al. A molecular theory of lipid-protein interactions in the plasma lipoproteins. FEBS Lett 1974; 38:247-58. 61. Burkey BF, Stuart WD, Harmony JA. Hepatic apolipoprotein J is secreted as a lipoprotein. J Lipid Res 1992; 33:1517-26. 62. Elshourbagy NA, Liao WS, Mahley RW et al. Apolipoprotein E mRNA is abundant in the brain and adrenals, as well as in the liver, and is present in other peripheral tissues of rats and marmosets. Proc Natl Acad Sci U S A 1985; 82:203-7. 63. Boyles JK, Zoellner CD, Anderson LJ et al. A role for apolipoprotein E, apolipoprotein A-I, and low density lipoprotein receptors in cholesterol transport during regeneration and remyelination of the rat sciatic nerve. J Clin Invest 1989; 83:1015-31. 64. Goodrum JF. Cholesterol from degenerating nerve myelin becomes associated with lipoproteins containing apolipoprotein E. J Neurochem 1991; 56:2082-6. 65. Poirier J, Hess M, May PC et al. Astrocytic apolipoprotein E mRNA and GFAP mRNA in hippocampus after entorhinal cortex lesioning. Mol Brain Res 1991; 11:97-106. 66. Poirier J, Baccichet A, Dea D et al. Cholesterol synthesis and lipoprotein reuptake during synaptic remodeling in hippocampus in adult rats. Neuroscience 1993; 55:81-90. 67. Poirier J. Apolipoprotein E in animal models of CNS injury and in Alzheimer’s disease. Trends Neurosci 1994; 17:525-30. 68. Masliah E, Mallory M, Ge N et al. Neurodegeneration in the central nervous system of apoE-deficient mice. Exp Neurol 1995; 136:107-22. 69. Stone DJ, Rozovsky I, Morgan TE et al. Astrocytes and microglia respond to estrogen with increased apoE mRNA in vivo and in vitro. Exp Neurol 1997; 143:313-8. 70. Mahley RW. Apolipoprotein E: Cholesterol transport protein with expanding role in cell biology. Science 1988; 240:622-30.

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71. Beffert U, Danik M, Krzywkowski P et al. The neurobiology of apolipoproteins and their receptors in the CNS and Alzheimer’s disease. Brain Research reviews 1998; 27:119-42. 72. Corder EH, Saunders AM, Strittmatter WJ et al. Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer’s disease in late onset families. Science 1993; 261:921-3. 73. Strittmatter WJ, Saunders AM, Schmechel D et al. Apolipoprotein E: High-avidity binding to beta-amyloid and increased frequency of type 4 allele in late-onset familial Alzheimer disease. Proc Natl Acad Sci USA 1993; 90:1977-81. 74. Saunders AM, Strittmatter WJ, Schmechel D et al. Association of apolipoprotein E allele epsilon 4 with late-onset familial and sporadic Alzheimer’s disease. Neurology 1993; 43:1467-72. 75. Poirier J, Davignon J, Bouthillier D et al. Apolipoprotein E polymorphism and Alzheimer’s disease. Lancet 1993; 342:697-9. 76. Farrer LA, Cupples LA, Haines JL et al. Effects of age, sex, and ethnicity on the association between apolipoprotein E genotype and Alzheimer disease. A meta-analysis. apoE and Alzheimer Disease Meta Analysis Consortium. JAMA 1997; 278:1349-56. 77. Nalbantoglu J, Gilfix BM, Bertrand P et al. Predictive value of apolipoprotein E genotyping in Alzheimer’s disease: Results of an autopsy series and an analysis of several combined studies. Ann Neurol 1994; 36:889-95. 78. Diedrich JF, Minnigan H, Carp RI et al. Neuropathological changes in scrapie and Alzheimer’s disease are associated with increased expression of apolipoprotein E and cathepsin D in astrocytes. J Virol 1991; 65:4759-68. 79. Lorent K, Overbergh L, Moechars D et al. Expression in mouse embryos and in adult mouse brain of three members of the amyloid precursor protein family, of the alpha-2-macroglobulin receptor/low density lipoprotein receptor-related protein and of its ligands apolipoprotein E, lipoprotein lipase, alpha-2-macroglobulin and the 40,000 molecular weight receptor-associated protein. Neuroscience 1995; 65:1009-25. 80. Han SH, Einstein G, Weisgraber KH et al. Apolipoprotein E is localized to the cytoplasm of human cortical neurons: A light and electron microscopic study. J Neuropathol Exp Neurol 1994; 53:535-44. 81. Boyles JK, Pitas RE, Wilson E et al. Apolipoprotein E associated with astrocytic glia of the central nervous system and with nonmyelinating glia of the peripheral nervous system. J Clin Invest 1985; 76:1501-13. 82. Schmechel DE, Tiller MO, Tong P, McSwain M, Han SH, Ange R, Burkhart DS, Izard MK. Pattern of apolipoprotein E immunoreactivity during brain aging. In: Roses AD, Weisgraber KH, Christen Y eds, Apolipoprotein E and Alzheimer’s Disease. New York:Springer-Verlag Berlin Heidelberg. 1996:29-48. 83. Stoll G, Meuller HW, Trapp BD et al. Oligodendrocytes but not astrocytes express apolipoprotein E after injury of rat optic nerve. GLIA 1989; 2:170-6. 84. Han SH, Hulette C, Saunders AM et al. Apolipoprotein E is present in hippocampal neurons without neurofibrillary tangles in Alzheimer’s disease and in age-matched controls. Exp Neurol 1994; 128:13-26. 85. Metzger RE, LaDu MJ, Pan JB et al. Neurons of the human frontal cortex display apolipoprotein E immunoreactivity: Implications for Alzheimer’s disease. J Neuropathol Exp Neurol 1996; 55:372-80. 86. Murakami M, Ushio Y, Morino Y et al. Immunohistochemical localization of apolipoprotein E in human glial neoplasms. J Clin Invest 1988; 82:177-88. 87. Pitas RE, Boyles JK, Lee SH et al. Astrocytes synthesize apolipoprotein E and metabolize apolipoprotein E-containing lipoproteins. Biochim Biophys Acta 1987; 917:148-61. 88. Baskin F, Smith GM, Fosmire JA et al. Altered apolipoprotein E secretion in cytokine treated human astrocyte cultures. J Neurol Sci 1997; 148:15-8. 89. Nakai M, Kawamata T, Taniguchi T et al. Expression of apolipoprotein E mRNA in rat microglia. Neurosci Lett 1996; 211:41-4. 90. Beffert U, Aumont N, Dea D et al. Beta-amyloid peptides increase the binding and internalization of apolipoprotein E to hippocampal neurons. J Neurochem 1998; 70:1458-66.

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91. Pitas RE, Boyles JK, Lee SH et al. Lipoproteins and their receptors in the central nervous system. Characterization of the lipoproteins in cerebrospinal fluid and identification of apolipoprotein B,E(LDL) receptors in the brain. J Biol Chem 1987; 262:14352-60. 92. Lehtimaki T, Pirttila T, Mehta PD et al. Apolipoprotein E (apoE) polymorphism and its influence on ApoE concentrations in the cerebrospinal fluid in Finnish patients with Alzheimer’s disease. Hum Genet 1995; 95:39-42. 93. Rall SC, Jr., Weisgraber KH, Mahley RW. Isolation and characterization of apolipoprotein E. Methods Enzymol 1986; 128:273-87:273-87. 94. Chiba H, Mitamura T, Fujisawa S et al. Apolipoproteins in rat cerebrospinal fluid: A comparison with plasma lipoprotein metabolism and effect of aging. Neurosci Lett 1991; 133:207-10. 95. Guyton JR, Miller SE, Martin ME et al. Novel large apolipoprotein E-containing lipoproteins of density 1.006-1.060 g/ml in human cerebrospinal fluid. J Neurochem 1998; 70:1235-40. 96. Patzak A, Winkler H. Exocytotic exposure and recycling of membrane antigens of chromaffin granules: ultrastructural evaluation after immunolabeling. J Cell Biol 1986; 102:510-5. 97. Palmer DJ, Christie DL. Identification of molecular aggregates containing glycoproteins III, J, K (carboxypeptidase H), and H (Kex2-related proteases) in the soluble and membrane fractions of adrenal medullary chromaffin granules. J Biol Chem 1992; 267:19806-12. 98. Weisgraber KH, Roses AD, Strittmatter WJ. The role of apolipoprotein E in the nervous system. Curr Opin Lipidol 1994; 5:110-6. 99. Vogel T, Guo NH, Guy R et al. Apolipoprotein E: A potent inhibitor of endothelial and tumor cell proliferation. J Cell Biochem 1994; 54:299-308. 100. Riddell DR, Graham A, Owen JS. Apolipoprotein E inhibits platelet aggregation through the L-arginine:nitric oxide pathway. Implications for vascular disease. J Biol Chem 1997; 272:89-95. 101. Walker DG, McGeer PL. Complement gene expression in human brain: Comparison between normal and Alzheimer disease cases. Mol Brain Res 1992; 14:109-16. 102. Jenne DE, Tschopp J. Clusterin: The intriguing guises of a widely expressed glycoprotein. TIBS 1992; 17:154-9. 103. Cotman CW, Nieto-Sampedro M. Cell biology of synaptic plasticity. Science 1984; 225:1287-94. 104. Steward O, Vinsant SL, Davis L. The process of reinnervation in the dentate gyrus of adult rats: An ultrastructural study of changes in presynaptic terminals as a result of sprouting. J Comp Neurol 1988; 267:203-10.

CHAPTER 3

Regulation of apoJ and apoE by Ovarian Steroids in the Brain David J. Stone and Irina Rozovsky

Introduction

T

he steroidal regulation of apolipoproteins in the brain is of interest in light of recent research in Alzheimer’s Disease (AD). Both an estrogen-deficient state1-2 and the apoE ε4 allele3-4 increase the risk of late-onset AD, although neither is a necessary condition for the disease. There are indications that the apoE ε4 allele is a greater risk factor in women than in men4-5 with risk in heterozygous women being twice that in heterozygous men (OR = 2.11; p<0.03).5 This suggests a link between the two most common risk factors for AD, apoE ε4 and estrogen deficits. We note that, while estrogen replacement therapy (ERT) may lower the risk of AD, the evidence is still emergent and mainly based on post hoc studies. Regulation of lipoproteins by steroids is well established in the periphery6-7 suggesting that the reason for the increased penetrance of the apoE ε4 allele in women may be estrogenic control of lipoprotein metabolism or production in the CNS. Here we discuss evidence for the steroidal control of apolipoproteins E and J in the CNS and periphery, and the implications of this control for AD.

Estrogen and Apolipoproteins E and J in the Periphery A considerable amount of data exists on estrogenic control of the blood-lipid profile and apoE levels. Estrogen replacement therapy (ERT) after natural or surgically induced menopause prevents elevations of total cholesterol and LDL-cholesterol, while maintaining high density lipoprotein (HDL)-cholesterol.6-10 Although ERT initially increases production of both HDL11 and LDL-cholesterol, ERT also increases the clearance of LDL6,12 and VLDL.13 One mechanism of faster clearance of postprandial lipids is an increase in hepatic LDL-receptor activity.14 However, HDL clearance does not appear to be affected by estrogen.6 Thus ERT shifts blood cholesterol and lipoproteins to a less atherogenic profile with a lower LDL/HDL ratio. The risk of cardiovascular disease is correlated with earlier age of menopause, with the greatest risk if menopause occurs before 39 years; the risk of cardiovascular disease is reduced by 2% for each year that menopause is delayed.15 The effects of estrogen on apoE in the periphery are less clear, and appear to be species/ genotype-specific. Both C57BL and C57L inbred strains of mice respond to estrogen with increased serum apoE, and a shift in apoE distribution from LDL and HDL fractions to LDL and IDL.16 However, CH3 and BALBc mice in the same study showed no serum apoE increase and a shift in apoE lipoprotein distribution from LDL to HDL particles. Estrogen Clusterin in Normal Brain Functions and During Neurodegeneration, edited by Caleb E. Finch. ©1999 R.G. Landes Company.

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treatment decreases plasma apoE, however, in humans9,17 and baboons10 and a shift in apoE distribution from HDL to VLDL.9 Estrogenic control of apoJ is evident during normal development and tissue reorganization. Apolopoprotein J is found in various areas of the developing rat brain as early as embryonic day 17.18 In addition to being widely expressed in the developing epithelia in skin, teeth, duodenum, lung, and kidney tissue19 apoJ is found in the uterus wall during pregnancy.20 During the estrous cycle, changes in apoJ levels are associated with uterine cell turnover and involution. In the mouse, apoJ increases in the lumenal epithelial cells during uterine involution through estrus and metestrus I. After ovariectomy (OVX), during the most intense periods of uterine atrophy, apoJ protein accumulates in the lumenal epithelial cells. Patterns are similar in humans with apoJ levels fluctuating throughout the menstrual cycle, reaching a peak in the mid-late secretory phase.21

Steroids and Lipoproteins in the CNS Apolipoprotein E production in the adult rodent brain is also influenced by estrogen. Again, this effect is dependent upon genotype and varies between brain regions. Of four mouse strains studied,22 brain apoE mRNA was affected by estrogen in only the C3H mouse strain, which showed increased brain apoE mRNA levels in response to 17β-estradiol (E2) for 5 days. Changes in apoE mRNA are also apparent in various brain regions of the F344 rat during the normal estrous cycle. In the arcuate nucleus, synaptic remodeling occurs during the normal rat estrous cycle, with synapse number decreasing on the afternoon of proestrus when estrogen levels are highest (Fig. 3.1). This decrease occurs prior to the luteinizing hormone (LH) surge which cases ovulation.23 This decrease is transient, and caused by estrogen.24 We found that during this estrogen-induced synaptic remodeling, apoE mRNA levels are also transiently increased (Fig. 3.2A). Likewise, the hippocampal CA1 also undergoes transient estrogen-induced synaptic remodeling.25 In the stratium radiatum of the CA1 both dendritic spine and synaptic density are increased on proestrus, and apoE mRNA levels also appear to be under estrogenic control in this region. On proestrus, when circulating estrogen levels are highest, apoE mRNA levels were found to be 30 to 70% higher than on other cycle days.26 In the same study, however, apoE mRNA levels were highest on diestrus in the CA3, when circulating estrogen levels are considerably lower. These results are not surprising as specialized subpopulations of astrocytes occur with different frequencies in the CA1 and CA3.27 This would suggest differential regulation of apoE mRNA levels in different astrocyte subpopulations/different brain regions. These findings, however, do not constitute direct evidence that estrogen influences apoE mRNA in vivo, as levels of several other hormones also differ between cycle days (Fig. 3.1).28 Estrogenic control of apoE mRNA is directly shown in vitro. In mixed glial cultures (65-75% astrocytes, 15-20% microglia, 10-15% oligodendrocytes; originated from cerebral cortex from 1-5 day old rat pups), the levels of apoE mRNA are increased 2-fold by the addition of 17β-estradiol at a concentration of 0.1nM (approximately blood levels at late proestrus or pregnancy). This apoE induction occurs in both astrocytes and microglia. However, no induction is caused by corticosterone or dihydrotestosterone (Fig. 3.3). This upregulation is dependent upon cell-cell interactions, as neither astrocytes or microglia respond to estrogen with increased apoE mRNA when grown in monotypic culture.26 Cellcell contact is also necessary for this increase,29 as separation of astrocytes and microglia with a 0.45 µm pore-size filter removed the estrogen effect. Astrocytic apoJ mRNA is also under estrogen control in vitro: We demonstrated 2-fold increase of apoJ mRNA in both monotypic astrocyte and mixed glial cultures (Rozovsky et al, unpublished data).

Regulation of apoJ and apoE by Ovarian Steroids in the Brain

Fig. 3.1. Concentration of progesterone, prolactin, estradiol, LH, and FSH in peripheral plasma obtained at 2 h intervals throughout the 4 d rat estrous cycle. Each point represents mean (± SE) of 5–6 rats. Black bars represent dark interval (1800-0600); numbers below represent time of day. Redrawn from ref. 28.

37

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Fig. 3.2. Apolopoprotein E mRNA levels are increased with estrogen in vivo. (A) In the rat arcuate nucleus, which undergoes transient synaptic remodeling during the estrous cycle, apoE mRNA levels peak on proestrus, when estrogen levels are highest.26 (B) In EC–lesioned mice, apoE mRNA levels at the wound site and in the deafferented dentate gyrus followed estrogen–dependent trends at 14 days post lesion which were not significant. Redrawn from ref. 26.

In vivo, gonadal steroids also appear to have an effect on apoJ mRNA, and this effect is again dependent upon the species. Castration of male rats for 3 weeks increases the level of apoJ mRNA in both the normal and deafferented hippocampus.30 In a different study we manipulated estrogen levels in mice through OVX and estrogen replacement, after which we lesioned the entorhinal cortex to examine reactive synaptogenesis and apolipoprotein production.31 This study was carried out on both wild type mice (WT) and transgenics lacking the apoE gene (apoE KO). While OVX did not decrease apoJ mRNA production, the OVXed mice with E2 replacement showed a 1.7-fold increase in apoJ mRNA

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Fig. 3.3. Glial cells in vitro respond to E2 17β-estradiol treatment with increased apoE mRNA. (A) Dose response curve: Mixed glial cells show a 2-fold increase in apoE mRNA at 0.1 nM, approximating high proestrous levels. (B) Secreted protein shows a small, nonsignificant increase, while cellular protein shows a highly significant 4-fold increase (p<0.001). This increase in cellular protein may signify an increase in uptake, which could be mediated by either increased receptor expression or affinity. (C) Analysis by in situ hybridization demonstrates that both astrocytes and microglia respond to estradiol, but not to other steroids. Redrawn from ref. 26.

levels in the lesioned entorhinal cortex (Fig. 3.4). Thus there may be an interaction between estrogen and progesterone (or some other ovarian hormone), such that E2 only causes an apoJ increase in the absence of progesterone. Apolopoprotein E-KO mice also showed a complex hormonal effect, with apoJ mRNA increasing in the deafferented dentate gyrus after OVX, with or without E2 replacement (Fig. 3.4B). Experiments with rats also suggest some form of interactions between apoJ mRNA and ovarian steroids other than estrogen. After entorhinal cortex lesioning (ECL), rats which were also OVXed have 20% higher apoJ mRNA levels in the deafferented dentate gyrus (p<0.025) over intact controls (Stone and Rozovsky, unpublished data). Estrogen replacement

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Fig. 3.4. Both WT and apoE-KO mice show a possible progesterone effect on apoJ mRNA levels. (A) In WT mice, OVXed mice with E2 replacement show increased levels of apoJ mRNA in the entorhinal cortex after EC–lesioning, suggesting estrogen/progesterone interactions. (B) In apoE KO mice, removal of the ovaries increases apoJ mRNA levels in the deafferented dentate gyrus with or without E2 replacement, suggesting a progesterone effect. These results also suggest some form of coordination in apoE and apoJ production, as the apoE–null state changes the apoJ response to ovarian hormones. Lesioning/OVX experiments with progesterone replacement will be necessary to elucidate the interaction of estrogen and progesterone on cholesterol transport. Redrawn from ref. 31.

did not reverse this effect, implicating progesterone or some other ovarian hormone as the cause of this increase. Progesterone was not directly manipulated in these studies.

Estrogen, AD, and Possible Mechanisms of Estrogen-Induced Neuroprotection Recent evidence indicates that ERT both reduces the risk of AD in women and slows cognitive decline.1-2 The duration of ERT also seems to be important, because women with

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41

long-term use of ERT have the lowest risk.2 Gender differences were suggested as a possible explanation for the higher incidence of the familial AD in women that is also linked to the apoE-associated risk factor.5 In addition, Phillips and Sherwin32 showed that exogenous E2 maintains short-term memory in surgically-induced menopausal young women. Several levels of evidence demonstrated multiple sites of estrogen actions in the brain. The specific mechanism/s by which estrogen reduces dementia are unclear, and they might be combined in order to be beneficial in improvement of clinical symptoms. Estrogen and several other estrogenic steroids which are contained in Premarin (the most common ERT drug) were also indicated as potential neurotrophins that increased survival and growth of hippocampal and cortical neurons in vitro.33 Direct actions of E2 and other estrogenic steroids on neurons occurred rapidly, suggesting involvement of membrane receptor(s) that mediate estrogen-induced responses.34 Rapid membrane-mediated E2-induced responses are also consistent with E2 induction of cAMP, which was observed in breast cancer cells.35 The colocalization of estrogen receptors in several brain regions (hippocampus, frontal cortex, etc.) with nerve growth factor receptors suggests a possible role of growth factors in mediated estrogen beneficial effects.36

Estrogen and Synaptic Sprouting: The Role of Apolopoprotein E Entorhinal cortex lesioning (ECL) is used as a model of the deafferenting aspects of AD. The outer 2/3 of the molecular layer of the dentate gyrus receive the majority of their input from the stellate neurons from layer 2 of the entorhinal cortex, which make up the majority of the perforant path.37 In ECL, the outer and middle molecular layers of the dentate gyrus are deafferented by transection or ablation of the perforant path. In the response to ECL, the molecular layer of the dentate gyrus is reinnervated by sprouting from multiple pathways. The inner molecular layer receives an increase in commissural/ associational (C/ A) afferents, while the outer one-third receives acetylcholine esterase (AChE) positive afferents from the septo-hippocampal pathways, contralateral entorhinal cortex, and local interneurons.38-39 Similarly, one aspect of AD is the death of entorhinal cortex neurons and the loss of input to the dentate gyrus. As in ECL, the AD brain responds to the deafferentation with an increase in the C/A pathway, and AChE-positive fibers in the outer molecular layer.40 Ovariectomy (OVX) reduces and E2 replacement reinstates compensatory sprouting41 and synapse formation31 by C/A neurons to the inner molecular layer. If the increased synaptic sprouting in response to estrogen is mediated by the upregulation of apoE described above, differences in the effect of estrogen on synaptic sprouting would be expected between WT and apoE-KO mice. We examined reactive synaptogenesis in the above described experiment two weeks after EC-lesioning by synaptophysin (SYN, a presynaptic vesicle protein) (Fig. 3. 5). In the WT mice, SYN immunoreactivity in the inner molecular layer (an estimate of C/A sprouting) was decreased by OVX, and reinstated by E2 replacement (Fig. 3.5A-C; Fig. 3.6A). Apolopoprotein E-KO mice, however, did not show this trend (Fig. 3.5D-F; Fig. 3.6A). The outer molecular layer also showed increases in thickness with E2 treatment (Fig. 3.5C). Sprouting to the outer molecular layer is believed to originate in the septum,39-40 although recently it has been suggested that these fibers may be from local interneurons.42 Whichever the origin, SYN immunoreactivity showed estrogen-dependent trends after ECL. In WT mice, OVX did not affect outer molecular layer width, but estrogen treatment increased the width of the SYN immunoreactive band (Fig. 3.5A-C; Fig. 3.6B). Apolopoprotein E-KO mice did no show any response to estrogen in outer molecular layer sprouting (Fig. 3.5D-F, Fig. 3.6B).

42

Clusterin in Normal Brain Functions and During Neurodegeneration

Fig. 3.5. Synaptophysin immunoreactivity after entorhinal cortex lesioning shows estrogen– dependent trends in WT but not apoE KO mice. After ECL, both WT (A) and apoE KO (D) mice show a "banded" pattern of synaptphysin immunoreactivity in the molecular layer of the dentate gyrus. In WT mice, OVX decreases (B) and E2 replacement reinstates (C) inner and outer molecular layer width and inner molecular layer optical density. In apoE KO mice, neither OVX (E) or E2 replacement (F) affected the SYN immunoreactivity in the outer or inner layers. From ref. 31.

Regulation of apoJ and apoE by Ovarian Steroids in the Brain

43

Fig.3.6. Estimates of compensatory synaptogenesis to inner and outer molecular layer show estrogen–dependent trends in WT but not apoE–KO mice. (A) Synaptophysin immunoreactivity in the inner molecular layer (an estimate of C/A sprouting) is decreased by OVX; levels are reinstated by E2 replacement. *significantly different from control, p<0.05. (B) AChE histochemical staining in the outer molecular layer (relative to the inner molecular layer) is decreased by OVX and reinstated by E2 replacement in WT mice. In apoE–KO mice, OVX increases staining in the outer layer, regardless of whether E2 was replaced, suggesting a progesterone effect. Optical density is given as a percent of that in the inner layer. *significantly greater than inner molecular staining p<0.05; **p<0.0131

44

Clusterin in Normal Brain Functions and During Neurodegeneration

These results suggest that estrogen exhibits its enhancement of neuronal sprouting in part through the upregulation of the cholesterol transport system. This may be accomplished through increased production of apolipoproteins. Another action of estrogen, however, is to increase the level of cellular protein in comparison to secreted protein in vitro (Fig. 3.3B). This would suggest changes in number or affinity of one or more apoE receptors. Another possibility, however, is that the removal of the apoE gene compromises the ability to sprout new synapses so severely that no neurotrophic agents can exhibit their effects.

Possible Compensatory Role of Apolopoprotein J Apolopoprotein E is strongly linked to the ethiology of AD. The presence of the apoE4 allele increases risk of AD and decreases the age of AD onset.3-4 Bertrand et al43 showed reduced apoE protein in the hippocampus and cortex of apoE4 AD carriers compared with apoE3/3 controls. This decreased levels in brain apoE protein is consistent with the reduced plasma apoE in young living apoE4/4 homozygotes44 and markedly reduced apoE in CSF of AD subjects when compared with age-matched controls.45 Poirier46 suggested that correlation between the apoE4 allele and decreased apoE might have a direct impact on impaired synaptogenesis in AD. Like apoE, apoJ mRNA is increased in experimental lesions and AD brain47 and is also under steroidal control.29-30 Bertrand et al43 examined the relationship between apoE and apoJ proteins as a function of apoE genotype in hippocampus and cortex of AD. In these studies, apoE and apoJ were found in inverse correlation in AD brain: apoJ protein showed 60% higher levels in apoE4 AD subjects vs. controls, while apoE protein levels were 20% lower in AD vs. controls43 (Fig. 3.7, 3.8). These results suggest a possible compensatory increase of apoJ in the brain of apoE4 AD subjects which showed low levels of apoE. Our recent studies with apoE KO mice also demonstrated possible compensatory trend: In unlesioned hippocampus apoJ mRNA levels showed a nonsignificant 1.4-fold increase in apoE-KO mice over wild type.31

Summary Apolipoproteins J and E in the brain have assumed major importance in many brain functions. Recent findings demonstrated that production of apoJ and apoE in the CNS is closely coregulated. In the optic tract cholesterol transport involves lipoprotein particles with both apoE and apoJ components.48 Primary astrocytes in culture produce nascent lipoprotein particles that contain both apoE and apoJ.49 Our recent studies with apoE-KO mice described above demonstrated that both the E2-induced increase in apoJ mRNA in lesion site and the lesion-induced apoJ mRNA increase in the deafferented dentate gyrus appear to be apoE-dependent. In a view of the association of risk for AD with apoE ε4 alleles, it is of great interest that in AD brain hippocampal apoE content showed a partial inverse correlation with the apoE ε4 allele dose.43 One of the working hypotheses of AD is that the compromised phospholipid and cholesterol transport which would result from lower brain apoE could contribute to impaired compensatory synaptogenesis seen in AD.46 Moreover, we suggest that in AD an E2-induced increase in apoE or apoJ level could counteract this impairment and be a mechanism by which estrogens are neuroprotective.

Regulation of apoJ and apoE by Ovarian Steroids in the Brain

45 Fig. 3.7. Apolopoprotein J levels in the hippocampus of control and Alzheimer's disease (AD) subjects. (A) Apolopoprotein J protein levels by Western blot analysis (***p<0.001). (B) Genotypic variations in apoJ of AD subjects with known apoE genotypes. Correlational analyses were performed using multiple general linear model package from Systat Corporation. R.O.D.= relative optical density. Redrawn from ref. 43.

Fig. 3.8. ApoE protein levels in the hippocampal and cortical areas in AD and control subjects. Left graph: Relative apoE levels in AD vs. controls (**p<0.02). Right graph: The impact of apoE genotype on brain apoE levels in hippocampus and cortex of AD subjects. R.O.D.= relative optical density. Redrawn from ref. 43.

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Clusterin in Normal Brain Functions and During Neurodegeneration

References 1. Schneider LS, Finch CE. Can estrogen prevent neurodegeneration? Drugs and Aging 1997; 11:87-95. 2. Tang MX, Jacobs D, Stern Y et al. Effect of oestrogen during menopause on risk and age at onset of Alzheimer’s disease. Lancet 1996; 388:429-432. 3. Strittmatter WJ, Saunder AM, Schmechel D et al. Apolipoprotein E: High avidity binding to β-amyloid and increased frequency of type 4 allele in late-onset familial Alzheimer disease. Proc Nat Acad Sci 1993; 90:1977-1981. 4. Poirier J, Davignon J, Bouthillier D et al. Apolipoprotein E polymorphism and Alzheimer’s disease. Lancet 1993; 342:697-699. 5. Payami H, Zareparsi S, Montee KR et al. Gender differences in apolipoprotein E-associated risk for familial Alzheimer-disease: A possible clue to the higher incidence of Alzheimer disease in women. Amer J Hum Gen 1996; 58:803-811. 6. Walsh BW, Schiff I, Riosner B et al. Effects of postmenopausal estrogen replacement on the concentrations and metabolism of plasma lipoproteins. New England J Med 1991; 325:1196-1204. 7. Sacks FM, Walsh BW. Sex hormones and lipoprotein metabolism. Current Opinion Lipidology 1994; 5:236-240. 8. Schram JHN, Boerrigter PJ, The TY. Influences of two hormone replacement therapy regimens, oral oestradiol valerate and cyproterone acetate versus transdermal oestradiol and oral dydrogesterone, on lipid metabolism. Maturitas, 1995; 22:121-130. 9. Muesing RA, Miller VT, LaRosa JC et al. Effects of unapposed conjugated equine estrogen on lipoprotein composition and apolipoprotein-E distribution. J Clin Endocrinol Metab 1992; 75:1250-1254. 10. Kushwaha RS, Foster DM, Barrett PH et al. Metabolic regulation of plasma apolipoprotein E by estrogen and progesterone in the baboon (Papio sp). Metabolism 1991; 40:93-100. 11. Walsh BW, Sacks FM. Estrogen treatment raises plasma HDL concentrations by increasing HDL production. Circulation Supp II 1994; 84:0557. 12. Eriksson M, Berglund L, Rudling P, Henriksson P, Angelin B. Effects of estrogen on low density lipoprotein metabolism in males. J Clin Invest 1989; 84:802-810. 13. Floren CH, Kushwaha RS, Hazzard WR et al. Estrogen-induced uptake of cholesterol-rich very low density lipoproteins in perfused rabbit liver. Metabol 1981; 30:367-375. 14. Angelin B, Olivecrona H, Reiher E et al. Hepatic cholesterol metabolism in estrogen-treated men. Gastroenterol 1992; 103:1657-1663. 15. van der Schouw YT, van der Graaf Y, Steyerberg EW et al. Age at menopause as a risk factor for cardiovascular mortality. Lancet 1996; 347:714-718. 16. Srivastava RAK, Srivastava N, Averna M et al. Estrogen up-regulates apolipoprotein E (apoE) gene expression by increasing apoE mRNA in the translating pool via the estrogen receptor a-mediated pathway. J Biol Chem 1997; 272:33360-33366. 17. Applebaum-Bowden D, McLean P, Steinmetz A et al. Lipoprotein, apolipoprotein, and lypolytic enzyme changes following estrogen administration in postmenopausal women. J Lipid Res 1989; 30:1895-1906. 18. O’Bryan MKO, Cheema SS, Bartlett PF et al. Clusterin levels increase during neural development. J Neurobiol 1992; 24:421-432. 19. French LE, Chonn A, Ducrest D et al. Murine clusterin: Molecular cloning and mRNA localization of a gene associated with epithelial differentiation processes during embryogenesis. J Cell Biol. 1993; 122:1119-1130. 20. Ahuja HS, Tenniswood M, Lockshin R et al. Expression of clusterin in cell differentiation and cell death. Biochem Cell Biol 1994; 72:523-530. 21. Brown TL, Moulton BC, Baker et al. Expression of apolipoprotein J in the uterus is associated with tissue remodeling. Biol Reproduc 1995; 52:1038-1049. 22. Srivastava RA, Bhasin N, Srivastava N Apolipoprotein E gene expression in various tissues of mouse and regulation by estrogen. Biochem Mol Biol Internat 1996; 38:91-101.

Regulation of apoJ and apoE by Ovarian Steroids in the Brain

47

23. Olmos G, Naftolin F, Perez J et al. Synaptic remodeling in the rat arcuate nucleus during the estrous cycle. Neurosci 1989; 32:663-667 24. Naftolin F, Mor G, Horvath TL et al. Synaptic remodeling in the arcuate nucleus during the estrous cycle is induced by estrogen and precedes the preovulatory gonadotropin surge. Endocrinol 1996; 137:5576-5580 25. Woolley CS, McEwen BS. Roles of estradiol and progesterone in regulation of hippocampal dendritic spine density during the estrous cycle in the rat. J Comp Neurol 1993; 336:293-306. 26. Stone DJ, Rozovsky I, Morgan TE et al. Astrocytes and microglia respond to estrogen with increased apoE mRNA in vivo and in vitro. Exp Neurol 1997; 143:313-318. 27. D’Ambrosio R, Wenzel J, Schwartkrion PA et al. Functional specialization and topographic segregation of hippocampal astrocytes. J Neurosci 1998; 18:4425-4438. 28. Smith MS, Freeman ME, Neill JD. The control of progesterone secretion during the estrous cycle and early pseudopregnancy in the rat: Prolactin, gonadotropin and steroid levels associated with rescue of the corpus luteum. Endocrinol 1975; 96:219-226. 29. Rozovsky I, Stone DJ, Morgan TE et al. Microglial apoE is regulated by estrogen and TGFβ-1: Role of cell-cell interactions. Soc Neurosci 1997; 641.19. 30. Day JR, Laping NJ, McNeill TH et al. Castration enhances expression of glial fibrillary acidic protein and sulfated glycoprotein-2 in the intact and lesion-altered hippocampus of the adult male rat. Mol Endocrinol 1990; 4:1995-2002. 31. Stone DJ, Rozovsky I, Morgan TE et al. Increased synaptic sprouting in response to estrogen via an apolipoprotein E-dependent mechanism: Implications for Alzheimer’s disease. J Neurosci 1998; 18:3180-3185. 32. Phillips SM, Sherwin B. Effects of estrogen on memory function in surgically menopausal women. Psychneuroendocrinol 1992; 17:485-495. 33. Brinton RD. 17β-estradiol induction of filopodial growth in cultured hippocampal neurons within minutes of exposure. Mol Cell Neurosci 1993; 4:36-46. 34. Watters JJ, Campbell JS, Cunningham MJ et al. Rapid membrane effects of steroids in neuroblastoma cells: Effects of estrogen on mitogen activated protein kinase signaling cascade and c-fos immediate early gene transcription. Endocrinol 1997; 138:4030-4033. 35. Aronica SM, Kraus WL, Katzenellenbogen BS. Estrogen action via the cAMP signaling pathway: Stimulation of adenylate cyclase and cAMP-regulated gene transcription. Proc Nat Acad Sci 1994; 91:8517-8521. 36. Torres-Aleman I, Rejas MT, Pons S et al. Estradiol promotes cell shape changes and GFAP redistribution in hypothalamic astrocytes in vitro: A neuronal-mediated effect. Glia 1992; 6:180-187. 37. Amaral DG, Witter MP. Hippocampal formation. In: Paxinos G, ed. The rat nervous system. San Diego: Academic Press, Inc., 1995:443-493. 38. Steward O, Loesche J. Quantitative autoradiographic analysis of the time course of proliferation of contralateral entorhinal effeferents in the dentate gyrus denervated by ipsilateral entorhinal lesions. Brain Res 1977; 125:11-21. 39. Scheff SW. Synaptic reorganization after injury: The hippocampus as a model system. In: Neural Regeneration and Transplantation. Alan R. Liss, Inc., 1989:137-156. 40. Geddes JW, Monaghan DT, Cotman CW et al. Plasticity of hippocampal circuitry in Alzheimer’s disease. Science 1985; 230:1179-1181. 41. Morse JK, Scheff SW, DeKosky ST. Gonadal steroids influence axon sprouting in the hippocampal dentate gyrus: A sexually dimorphic response. Exp Neurol 1986; 94:649-658. 42. Aubert I, Poirier J, Gauthier S et al. Multiple cholinergic markers are unexpectedly not altered in the rat dentate gyrus following entorhinal cortex lesions. J Neurosci 1994; 14:2476-2484. 43. Bertrand P, Poirier J, Oda T et al. Association of apolipoprotein E genotype with brain levels of apolipoprotein E and apolipoprotein J (clusterin) in Alzheimer’s disease. Mol Brain Res 1995; 33:174-178. 44. Gregg RE, Zecg LA, Schaefer EJ et al. Abnormal in vivo metabolism of apolipoprotein ε4 in humans. J Clin Invest 1986; 78:815-821.

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45. Blennow K, Hesse C, Fredman P. Cerebrospinal fluid apolipoprotein E is reduced in Alzheimer’s disease. Neuroreport 1994; 5:2534-2536. 46. Poirier J. Apolipoprotein E in the brain and its role in Alzheimer’s disease. J Psychi Neurosci 1996; 21:128-134. 47. May PC, Lampert-Etchells M, Johnston SA et al. Dynamics of gene expression for a hippocampal glycoprotein elevated in Alzheimer’s disease and in response to experimental lesions in rat. Neuron 1990; 5:831-839. 48. Shanmugaratnam J, Berg E, Kimmer L et al. Retinal Muller glia secrete apolipoprotein E and J which are efficiently assembled into lipoprotein particles. Mol Brain Res 1997; 50:113-120. 49. LaDu MJ et al. Nascent astrocyte particles differ from lipoprotein particles in CSF. J. Neurochem 1998; 70:2070-2081.

CHAPTER 4

Lipoprotein Receptors in Brain G. William Rebeck and Bradley T. Hyman

I

nterest in the biology of apoE in the CNS has increased with the recognition that apoE is a genetic risk factor for AD1 and is involved in the nervous system’s response to lesion,2 synaptic stability,3 and neurite outgrowth.4 Apolipoprotein E is present in the CNS on lipoproteins, and its metabolism is mediated via lipoprotein receptors.5 In this chapter, we will focus on the structure and functions of CNS lipoproteins containing exchangeable apolipoproteins (apoE, apoJ, apoA-I), the expression of lipoprotein receptors (the LDL receptor family, scavenger receptors) in the CNS, and the potential roles of these molecules in the pathophysiology of Alzheimer’s disease.

Lipoproteins in the CNS—Source and Structure There are at least three main populations of lipoproteins in the CSF. Apolipoprotein E is associated with high density lipoproteins (HDL) which are primarily spherical, and 14 to 20 nm in size.5-8 A second class of high density lipoproteins contains primarily apoA-I;5,6 these particles are slightly denser than the apoE-lipoproteins.7,8 A third class of lipoprotein is larger (32 nm), and contains neither apoE or apoA-I.6 Apolipoprotein J is found associated with each of these classes of lipoproteins, although loosely; if these lipoproteins are isolated by density centrifugation, much of the apoJ is dissociated.7 There is further complexity associated with these three populations of lipoproteins, with many other apolipoproteins present in the CSF, including apoA-II, apoA-IV, apoC-I, apoC-II, apoC-III, and apoD.5,6,9,10 Apolipoprotein E is expressed by astrocytes2,11,12 and is released in association with disc-like, high density lipoproteins.7 Apolipoprotein E is also synthesized in microglia,13,14 and released on similar particles (Rebeck et al, unpublished data). The particles presumably are the precursors of the apoE-lipoproteins found in the CSF, which are larger due to a higher percentage of esterified cholesterol.7 Studies of apoE isoforms in CSF of individuals after liver transplant indicated that nearly all of the apoE present in the CNS is synthesized locally.15 Apolipoprotein J and apoD are also made in the CNS.16,17 Apolipoprotein J is synthesized and secreted from astrocytes as part of high density lipoproteins.7 Apolipoprotein J message is also found in ependymal cells, the choroid plexus, and neurons;16,18 whether this apoJ is also secreted with lipoproteins from these cells is unknown.

Lipoprotein Receptors in the CNS In fibroblasts and hepatocytes, the initial binding of apoE-containing lipoproteins occurs via heparan sulfate proteoglycans.19 Apolipoprotein E secreted from cells remains associated with cell surface proteoglycans,20 and this apoE aids in the binding of exogenous lipoproteins.21 It is likely that a similar situation occurs in the CNS with secretion of apoE by astrocytes. After binding to the cell surface, the apoE-containing lipoprotein is then transClusterin in Normal Brain Functions and During Neurodegeneration, edited by Caleb E. Finch. ©1999 R.G. Landes Company.

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Clusterin in Normal Brain Functions and During Neurodegeneration

ferred to a second binding site, and the receptor-ligand complex is internalized and directed to the endosomal/lysosomal pathway.22 There are two families of receptors that may act to clear lipoproteins in the CNS. They are the LDL receptor family and the scavenger receptors.

The LDL Receptor Family So far, five members of the LDL receptor family have been identified in humans: the LDL receptor, the VLDL receptor, apoER2, LRP (also referred to as the α2-macroglobulin receptor), and gp330 (also referred to as megalin or LRP-2) (Figure 4.1). LRP and gp330 are considerably larger than the other receptors (each is over 500 kDa) and thus could be considered as their own subfamily. These receptors share a number of features. They each have a short cytoplasmic C-terminus and a single transmembrane region. Extracellularly, they contain a variable number of EGF repeats, ligand binding repeats, and linker regions. The ligand binding repeats each contain six cysteines, which form three sets of disulfide bonds. The EGF repeats are involved in the dissociation of ligand from receptor in the acidic environment of the early endosomes.

Expression Patterns Each member of the LDL receptor family is expressed in a subpopulation of cells in the CNS. The LDL receptor is expressed at low levels in both neurons and glia, as shown by immunostaining5,23 and in situ hybridization18,24 (Fig. 4.2A). There is occasionally stronger expression in astrocytes, suggesting that under some conditions there may be up-regulation of LDLR. Increased LDL binding in the brain was observed after acute lesions,25 suggesting that LDLR expression is increased during neuronal remodeling as a way for neurons to increase their lipid content.26 The VLDL receptor is also expressed in the CNS. Immunohistochemistry localized strong expression in both resting and activated microglia27 and to a subset of neurons,27,28 including granule cells of the dentate gyrus, pyramidal neurons of CA4, CA3, presubiculum, subiculum, entorhinal cortex, and layers III and IV of temporal lobe neocortex. Interneurons were also occasionally VLDLR-positive. In situ hybridization analysis showed only weak VLDLR expression in brain, mainly localized to neurons (Fig. 4.2B).18 Apolipoprotein E receptor 2, in contrast, seems limited to neurons throughout the brain, both by in situ hybridization29 and immunohistochemistry.30 Apolipoprotein ER2 was observed in granule cells and CA pyramidal neurons of the hippocampus, Purkinje cells of the cerebellum, and cortical neurons. No glial staining was observed.30 LRP is strongly expressed in the CNS. LRP is present on most neurons throughout the cortex,23,31 with particularly strong expression in the hippocampus. It is also present in the granular cells of the cerebellum.32 Activated astrocytes, but not resting astrocytes, are LRP-positive,23,33 but microglia have not been identified with LRP antibodies. In situ hybridization showed strong expression in neurons, with lower level expression also in astrocytes and oligodendrocytes (Fig. 4.2C).18 There was no up-regulation of LRP mRNA after lesion.18 gp330, as a receptor for apoJ,34 is discussed in detail elsewhere in this volume. Expression has been reported in ependymal cells in the CNS.35 We have not observed expression in neurons or glia, under normal conditions or for three to eleven days after lesioning.18 Each of the apoE receptors binds the 39 kDa receptor associated protein (RAP).36-40 RAP is an endoplasmic reticulum resident protein,41 where it acts to prevent LRP from binding to endogenous ligands in the endoplasmic reticulum.42 The expression pattern of RAP is very similar to that seen for LRP (Figure 4.2C and D).43 RAP may also help receptors to fold properly.44 In vitro, RAP is used as a pharmacologic agent to block interactions at the

Lipoprotein Receptors in Brain

51 Fig. 4.1. Diagram of LDL receptor family members. The extracellular domains of the receptor family members are above the double line; transmembrane domains within the double line; and intracellular domains below the double line. Full-length products are shown, but there are additional splice forms and soluble forms of the receptors.

Fig. 4.2. In situ hybridization analysis of LDL receptor and related molecules in mouse brain. (A) LDL receptor; (B) VLDL receptor; (C) LRP; (D) RAP.

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Clusterin in Normal Brain Functions and During Neurodegeneration

Table 4.1. Receptor ligands

Lipoprotein-associated ligands apoE apoB apoJ lipoprotein lipase hepatic lipase Proteinase/inhibitors activated α2-macroglobulin α1-antitrypsin tissue factor pathway inhibitor 1 protease nexin II tissue plasminogen activator urokinase plasminogen activator plasminogen activator inhibitor 1 aprotinin Other lactoferrin thrombospondin receptor associated protein

LDLR

VLDLR

apoER2

LRP

gp330

+ +

+

+

+

+ + + +

+

+

+ +

+ + +

+ + + + + + +

+

+ + +

+

+ + + + + +

The “+” sign indicates an interaction of the ligand with the receptor; the individual studies are referenced in the text.

cell surface between members of the LDL receptor family and their ligands.39,45 Thus, RAP has become an important reagent for identifying new ligands for these receptors. Each member of the LDL receptor family binds more than one ligand (see Table 4.1). The ligand binding regions of the apoE receptors have been dissected using monoclonal blocking antibodies and receptor fragments. LRP and gp330 are both quite large, containing 4 groups of ligand binding repeats, totaling 31 and 36 ligand binding repeats, respectively. Analysis of the second group of ligand binding repeats of LRP and gp330, using monoclonal antibodies and receptor fragments, have implicated this region in binding of most ligands, including RAP, plasminogen activators and inhibitor, α2-macroglobulin, apoE, lactoferrin and lipoprotein lipase.46-49 The LDL receptor, VLDL receptor, and apoER2 are much smaller, each containing a single group of 7-8 ligand binding repeats. Despite the smaller size, these receptors share a number of ligands with LRP and gp330. The VLDLR still binds plasminogen activators and inhibitor50 and lipoprotein lipase.51 Furthermore, each of the smaller receptors bind apoE; the fifth ligand binding repeat of the LDL receptor is necessary for binding apoE,52 although the homologous repeat in apoER2 (the sixth) is not necessary for apoE binding.53 Together, these observations indicate that the numerous ligand binding repeats in the members of the LDL receptor family are probably each multifunctional, and binding of many ligands can probably occur at several sites.

Splice Variants and Soluble Forms of apoE Receptors Several of the receptors are found in alternatively spliced versions in the CNS. VLDLR exists in several forms, with two exons alternatively spliced: exon 4, containing sequence for

Lipoprotein Receptors in Brain

53

one of the ligand binding repeats; and exon 16, containing sequence for the O-linked glycosylation region.27 Apolipoprotein ER2 is even more complex, with at least four exons alternatively spliced:30,53,54 exon 5, containing three ligand binding repeats; exon 8, containing one EGF repeat; exon 15, containing the O-linked glycosylation region; and exon 18, containing a cytoplasmic domain that is not homologous to sequence in other members of the family. No splicing has been reported for LDLR, LRP, or gp330. We have examined brain cDNA for alternate splicing of LRP, but, despite having 89 exons, we have observed no alternative splicing (unpublished data). Soluble forms of several of the receptors have also been detected. The N-terminal 28 kDa of LDLR was identified in plasma, and suggested to protect cells against infection by vesicular stomatitis virus.55 Similarly, an extremely large fragment of LRP was found in plasma, and shown to be competent to bind several ligands;56 we have observed this sLRP in CSF.57 A similar form of gp330 was found shed from proximal tubule cells in Heymann nephritis.58 There is also a suggestion that some splice forms of apoER2 may contain an extracellular cleavage site to generate a soluble form of this receptor.54 These shed versions of cell surface receptors may bind various ligands and sequester them, without mediating their cellular uptake and degradation.

The Scavenger Receptor Family The second group of multifunctional lipoprotein receptors is the scavenger receptor family. This family consists of three major classes, SR-A SR-B, and SR-C.59 Unlike the LDL receptor family, the scavenger receptors do not have an NPxY sequence for internalization; nonetheless, they mediate uptake of various ligands.60 All receptors in these classes bind acetylated lipoproteins, and most bind oxidized lipoproteins. Scavenger receptor B-I binds HDL without apoE.61 There is a wide variety of other ligands which include primarily polyanionic molecules.60 We have detected scavenger receptor A-I on microglia in the CNS.62

Functions of Lipoproteins and Their Receptors in the CNS There are two main functions of lipoproteins: removal of lipids from loaded cells, and delivery of lipids to starved cells. In the plasma, HDL are used for the removal of lipids from cells. Plasma HDL contain primarily apoA-I and apoA-II; a small fraction of plasma HDL contains only apoE, and is termed γ-LpE.63 Both of these types of HDL remove cholesterol from cells. CSF lipoproteins also remove cholesterol from loaded cells in culture,64 perhaps via both the apoA-I- and the apoE-containing lipoproteins. The transfer of lipids to lipoproteins presumably changes the composition of the particles, leading to a larger, less dense lipoprotein. Delivery of lipids to cells occurs primarily through the interactions of apoE and apoB with cell surface receptors. Since there is no apoB in the CSF, uptake of lipoproteins in the CNS is presumably primarily mediated by apoE. CSF lipoproteins interact with the LDL receptor in vitro.5,64 When they are enriched with exogenous apoE, these particles can interact with LRP.65,66 Thus, under normal conditions, CSF lipoproteins may be endocytosed via the low level of LDL receptors or scavenger receptors present in the brain. However, when damage occurs and glia produce high levels of apoE, particles may be directed to the more abundant LRP. These two functions of apoE-containing lipoproteins may explain the up-regulation of apoE seen in models of peripheral nerve damage67-69 and CNS damage.2,18 Apolipoprotein E is synthesized by non-neuronal cells around the site of injury, and delivers cholesterol to the damaged axons, a process necessary for regeneration. Apolipoprotein A-I is also found

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in increased levels around the inury site, perhaps as a mechanism for removal of damaged membranes.69 However, nerve regeneration can occur in the absence of apoE and apoA-I, and other apolipoproteins may be able to substitute;70 for example, apoD69 and apoJ18 are increased around sites of damage. Surprisingly, there is no up-regulation of LDL receptor or LRP mRNA after brain injury,18 although presumably the need for lipid clearance increases. It is possible that distribution of lipoprotein receptors from intracellular vesicles to the cell surface accounts for observed increases in lipoprotein binding after lesioning.25 Other potential mechanisms to regulate the rate of clearance have not been explored, but include removal of CSF lipoproteins to the plasma via the arachnoid granulations. Many other ligands are present in the CNS, including tissue plasminogen activator,71 α2-macroglobulin,72 lactoferrin,73 and, of course, apoJ.74 Ligand-receptor interactions may allow clearance of molecules that have lost their usefulness, such as proteinase-proteinase inhibitor complexes. Ligands may also direct cell changes through interactions with receptors. For example, α2-macroglobulin72 and apoE-lipoproteins4 stimulate neurite outgrowth via interactions with LRP.65,75,76

Relationship to Alzheimer’s Disease Alzheimer’s disease is neuropathologically defined by the presence in the brain of two features, amyloid deposits of the Aβ peptide and neurofibrillary tangles (NFT) of the tau protein. The risk of AD is increased by inheritance of the ε4 allele of apoE, and decreased by inheritance of the ε2 allele of apoE.1 Apolipoprotein E ε4 is associated with increased amyloid,23,77,78 but not increased NFTs.78 Thus, it has been postulated that apoE4 is involved either in increased deposition of Aβ, or decreased clearance of Aβ. The putative apoE-Aβ interactions have been supported by several different studies. Apolipoprotein E binds Aβ in vitro, whether apoE is delipidated79 or bound to lipoproteins.80 Apolipoprotein E can also promote Aβ aggregation in vitro;81-83 this model is supported in vivo in mouse models of amyloidogenesis showing that apoE knockout mice have considerably delayed Aβ deposition.84 Aβ clearance can be promoted by interactions with the lipoprotein receptors LRP,85,86 gp330,87,88 and scavenger receptor A-I.89,90 The apoE isoforms may differentially affect these various clearance mechanisms. Apolipoprotein E may also affect Aβ levels by altering the interaction between the amyloid precursor protein and LRP.91,92 The studies of apoE as a genetic risk factor in AD promoted analysis of apoE receptor genes as candidate risk factors for AD. No consistent linkage was found between AD and polymorphisms in the LDL receptor gene,93 the VLDL receptor gene,28,93-95 and a polymorphism in the 3' untranslated region of LRP.93,96,97 However, a polymorphism in exon 3 of LRP has been linked to AD in several studies.98-101 While this polymorphism does not alter the amino acid sequence of LRP, it has sparked further interest in analysis of other regions of LRP and other ligands of LRP as potential genetic risk factors. In summary, recent investigations have revealed a complex family of exchangeable apolipoproteins which can be synthesized by resident CNS cells and contribute to the classes of lipoproteins found in the brain. These lipoproteins form a unique class of particles, distinct from that seen in the periphery, which appears to be capable of mediating both lipid removal and delivery. The CNS lipoproteins have available a wide array of at least five potential receptors, each with a unique cellular and regional distribution and with multiple splice forms and soluble forms. This much diversity no doubt underlies a similar diversity in cellular function which we are only beginning to understand. Efforts in this direction are progressing rapidly, however, due in large part to the identification of members of this family of ligands and receptors as candidate genes in Alzheimer’s disease.

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References 1. Strittmatter W, Roses A. Apolipoprotein E and Alzheimer’s disease. Ann Rev Neurosci 1996; 19:53-77. 2. Poirier J, Hess M, May PC et al. Astrocytic apolipoprotein E mRNA and GFAP mRNA in hippocampus after entorhinal cortex lesioning. Mol Brain Res 1991; 11:97-106. 3. Masliah E, Mallory M, Ge N et al. Neurodegeneration in the central nervous system of apoE-deficient mice. Exp Neurol 1995; 136:107-122. 4. Handelmann GE, Boyles JK, Weisgraber KH et al. Effects of apolipoprotein E, β-very low density lipoproteins, and cholesterol on the extensions of neurites by rabbit dorsal root ganglion neurons in vitro. J Lipid Res 1992; 33:1677-1688. 5. Pitas RE, Boyles JK, Lee SH et al. Lipoproteins and their receptors in the central nervous system. J Biol Chem 1987; 262:14352-14360. 6. Borghini I, Barja F, Pometta D et al. Characterization of subpopulations of lipoprotein particles isolated from human cerebrospinal fluid. Biochim Biophys Acta 1995; 1255:192-200. 7. LaDu MJ, Gilligan SM, Lukens JR et al. Nascent astrocyte particles differ from lipoproteins in CSF. J Neurochem 1998; 70:2070-2081. 8. Guyton JR, Miller SE, Martin ME et al. Novel large apolipoprotein E-containing lipoproteins of density 1.006-1.060 g/ml in human cerebrospinal fluid. J Neurochem 1998; 70:1235-1240. 9. Koudinov AR, Koudinova NV, Kumar A et al. Biochemical characterization of Alzheimer’s soluble amyloid beta protein in human cerebrospinal fluid: Association with high density lipoproteins. Biochem Biophys Res Commun 1996; 223:592-597. 10. Roheim PS, Carey M, Forte T et al. Apolipoproteins in human cerebrospinal fluid. Proc Natl Acad Sci USA 1979; 76:4646-4649. 11. Boyles JK, Pitas RE, Wilson E et al. Apolipoprotein E associated with astrocytic glia of the central nervous system and with nonmyelinating glia of the peripheral nervous system. J Clin Invest 1985; 76:1501-1513. 12. Pitas RE, Boyles JK, Lee SH et al. Astrocytes synthesize apolipoprotein E and metabolize apolipoprotein E-containing lipoproteins. Biochim Biophys Acta 1987; 917:148-161. 13. Nakai M, Kawamata T, Taniguchi T et al. Expression of apolipoprotein E mRNA in rat microglia. Neurosci Lett 1996; 211:41-44. 14. Stone DJ, Rozovsky I, Morgan TE et al. Astrocytes and microglia respond to estrogen with increased apoE mRNA in vivo and in vitro. Exp Neurol 1997; 143:313-318. 15. Linton MF, Gish R, Hubl ST et al. Phenotypes of apolipoprotein B and apolipoprotein E after liver transplantation. J Clin Invest 1991; 88:270-281. 16. Aronow BJ, Lund SD, Brown TL et al. Apolipoprotein J expression at fluid-tissue interfaces: Potential role in barrier cytoprotection. Proc Natl Acad Sci USA 1993; 90:725-729. 17. Provost PR, Villeneuve L, Weech PK et al. Localization of the major sites of rabbit apolipoprotein D gene transcription by in situ hybridization. J Lipid Res 1991; 32:1959-1970. 18. Page K, Hollister RD, Hyman BT. Dissociation of apolipoprotein and apolipoprotein receptor response to lesion in the rat brain: An in situ hybridization study. Neurosci 1998; 85:1161-1171. 19. Ji Z-S, Brecht WJ, Miranda RD et al. Role of heparan sulfate proteoglycans in the binding and uptake of apolipoprotein E-enriched remnant lipoproteins by cultured cells. J Biol Chem 1993; 268:10160-10167. 20. Lilly-Stauderman M, Brown TL, Balasubramaniam A et al. Heparin releases newly synthesized cell surface associated apolipoprotein E from HepG2 cells. J Lipid Res 1993; 34:190-200. 21. Ji Z-S, Fazio S, Lee Y-L et al. Secretion-capture role for apolipoprotein E in remnant lipoprotein metabolism involving cell surface heparan sulfate proteoglycans. J Biol Chem 1994; 269:2764-2772. 22. Weisgraber KH. Apolipoprotein E: Structure-function relationships. Adv Prot Chem 1994; 45:249-302. 23. Rebeck GW, Reiter JS, Strickland DK et al. Apolipoprotein E in sporadic Alzheimer’s disease: Allelic variation and receptor interactions. Neuron 1993; 11:575-580.

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24. Swanson LW, Simmons DM, Hofmann SL et al. Localization of mRNA for low density lipoprotein receptor and a cholesterol synthetic enzyme in rabbit nervous system by in situ hybridization. Proc Natl Acad Sci USA 1988; 85:9821-9825. 25. Poirier J, Baccichet A, Dea D et al. Cholesterol synthesis and lipoprotein reuptake during synaptic remodeling in hippocampus in adult rats. Neurosci. 1993; 55:81-90. 26. Poirier J. Apolipoprotein E in animal models of CNS injury and in Alzheimer’s disease. TINS 1994; 17:525-530. 27. Christie RH, Chung H, Rebeck GW et al. Expression of the very low density lipoprotein receptor (VLDL-r), an apolipoprotein E receptor, in the central nervous system and in Alzheimer disease. J Neuropath Exp Neurol 1996; 55:491-498. 28. Okuizumi K, Onodera O, Namba Y et al. Genetic association of the very low density lipoprotein (VLDL) receptor with sporadic Alzheimer’s disease. Nature Genet 1995; 11:207-209. 29. Kim D-H, Iijima H, Goto K et al. Human apolipoprotein E receptor 2. J Biol Chem 1996; 271:8373-8380. 30. Clatworthy AE, Stockinger W, Christie RH et al. Expression and alternate splicing of apoE receptor 2 in brain. Neurosci 1998; in press. 31. Wolf BB, Lopes MBS, VandenBerg SR et al. Characterization and immunohistochemical localization of α2-macroglobulin receptor (low-density lipoprotein receptor-related protein) in human brain. Am J Path 1992; 141:37-42. 32. Moestrup SK, Gliemann J, Pallensen G. Distribution of the α2-macroglobulin receptor/low density lipoprotein receptor-related protein in human tissues. Cell Tissue Res 1992; 269:375-382. 33. Lopes MBS, Bogaev CA, Gonias SL et al. Expression of α2-macroglobulin receptor/low density lipoprotein receptor-related protein is increased in reactive and neoplastic glial cells. FEBS Lett 1994; 338:301-305. 34. Kounnas MZ, Loukinova EB, Stefansson S et al. Identification of glycoprotein 330 as an endocytic receptor for apolipoprotein J/clusterin. submitted 1995. 35. Zheng G, Bachinsky DR, Stamenkovic I et al. Organ distribution in rats of two members of the low-density lipoprotein receptor gene family, gp330 and LRP/α2MR, and the receptor-associated protein (RAP). J Histochem Cytochem 1994; 42:531-542. 36. Medh JD, Fry GL, Bowen SL et al. The 39-kDa receptor-associated protein modulates lipoprotein metabolism by binding to LDL receptors. J Biol Chem 1995; 270:536-540. 37. Battey FD, Gafvels ME, FitzGerald DJ et al. The 39 kDa receptor-associated protein regulates ligand binding by the very low density lipoprotein receptor. J Biol Chem 1994; 269:23268-23273. 38. Novak S, Hiesberger T, Schneider WJ et al. A new low density lipoprotein receptor homologue with 8 ligand binding repeats in brain of chicken and mouse. J Biol Chem 1996; 271:11732-11736. 39. Herz J, Goldstein JL, Strickland DK et al. 39-kDa protein modulates binding of ligands to low density lipoprotein receptor-related protein/α2-macroglobulin receptor. J Biol Chem 1991; 266:21232-21238. 40. Kounnas MZ, Argraves WS, Strickland DK. The 39-kDa receptor-associated protein interacts with two members of the low density lipoprotein receptor family, α2-macroglobulin receptor and glucoprotein 330. J Biol Chem 1992; 267:21162-21166. 41. Bu G, Geuze HJ, Strous GJ et al. 39 kDa receptor-associated protein is an ER resident protein and molecular chaperone for LDL receptor-related protein. EMBO J 1995; 14:2269-2280. 42. Willnow TE, Rohlmann A, Horton J et al. RAP, a specialized chaperone, prevents ligandinduced ER retention and degradation of LDL receptor-related endocytic receptors. EMBO J 1996; 15:3632-3639. 43. Rebeck GW, Harr SD, Strickland DK et al. Multiple, diverse senile plaque-associated proteins are ligands of an apolipoprotein E receptor, the α2-macroglobulin receptor/low-density-lipoprotein receptor-related protein. Ann Neurol 1995; 37:211-217.

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44. Bu G, Rennke S. Receptor-associated protein is a folding chaperone for low density lipoprotein receptor-related protein. J Biol Chem 1996; 271:22218-22224. 45. Williams SE, Ashcom JD, Argraves WS et al. A novel mechanism for controlling the activity of α2-macroglobulin receptor/low density lipoprotein receptor-related protein. J Biol Chem 1992; 267:9035-9040. 46. Moestrup SK, Holtet TL, Etzerodt M et al. α2-macroglobulin-proteinase complexes, plasminogen activator inhibitor type-1-plasminogen activator complexes, and receptor associated protein bind to a region of the α2-macroglobulin receptor containing a cluster of eight complement-type repeats. J Biol Chem 1993; 268:13691-13696. 47. Willnow TE, Orth K, Herz J. Molecular dissection of ligand binding sites on the low density lipoprotein receptor-related protein. J Biol Chem 1994; 269:15827-15832. 48. Horn IR, van der Berg BMM, van der Meijden PZ et al. Molecular analysis of ligand binding to the second cluster of complement-type repeats of the low density lipoprotein receptorrelated protein. J Biol Chem 1997; 272:13608-13613. 49. Orlando RA, Exner M, Czekay R-P et al. Identification of the second cluster of ligandbinding repeats in megalin as a site for receptor-ligand interactions. Proc Natl Acad Sci USA 1997; 94:2368-2373. 50. Heegaard CW, Simonsen ACW, Oka K et al. Very low density lipoprotein receptor binds and mediates endocytosis of urokinase-type plasminogen activator-type-1 plasminogen activator inhibitor complex. J Biol Chem 1995; 270:20855-20861. 51. Takahashi S, Suzuki J, Kohno M et al. Enhancement of the binding of triglyceride-rich lipoproteins to the very low density lipoprotein receptor by apolipoprotein E and lipoprotein lipase. J Biol Chem 1995; 270:15747-15754. 52. Russell DW, Brown MS, Goldstein JL. Different combinations of cysteine-rich repeats mediate binding of low density lipoprotein receptor to two different proteins. J Biol Chem 1989; 264:21682-21688. 53. Kim D, Magoori K, Inoue TR et al. Exon/intron organization, chromosome localization, alternative splicing, and transcription units of the human apolipoprotein E receptor 2 gene. J Biol Chem 1997; 272:8498-8504. 54. Brandes C, Novak S, Stockinger W et al. Avian and murine LR8B and human apolipoprotein E receptor 2: Differentially spliced products from corresponding genes. Genomics 1997; 42:185-191. 55. Fischer DG, Tal N, Novick D et al. An antiviral soluble form of the LDL receptor induced by interferon. Science 1993; 262:250-253. 56. Quinn KA, Grimsley PG, Dai Y-P et al. Soluble low density lipoprotein receptor-related protein (LRP) circulates in human plasma. J Biol Chem 1997; 272:23946-23951. 57. Rebeck GW, Harr S, West HL et al. Apolipoprotein E, LRP, and Alzheimer disease: New observations of shed receptors and isoform specific complex formation in CSF. Soc Neurosci (Abstract) 1994; 20:1076. 58. Bachinsky DR, Zheng G, Niles JL et al. Detection of two forms of gp330. Am J Path 1993; 143:598-611. 59. Freeman MW. Scavenger receptors in atherosclerosis. Current Opin Hematol 1997; 4:41-47. 60. Krieger M, Herz J. Structures and functions of multiligand lipoprotein receptors: Macrophage scavenger receptors and LDL receptor-related protein (LRP). Annu Rev Biochem 1994; 63:601-37. 61. Acton S, Rigotti A, Landschulz KT et al. Identification of scavenger receptor SR-BI as a high density lipoprotein receptor. Science 1996; 271:518-520. 62. Christie RH, Freeman M, Hyman BT. Expression of the macrophage scavenger receptor, a multifunctional lipoprotein receptor, in microglia associated with senile plaques in Alzheimer’s disease. Am J Path 1996; 148:399-403. 63. Huang Y, von Eckardstein A, Wu S et al. A plasma lipoprotein containing only apolipoprotein E and with γ mobility on electrophoresis releases cholesterol from cells. Proc Natl Acad Sci USA 1994; 91:1834-1838.

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64. Rebeck GW, Alonzo NC, Berezovska O et al. Structure and functions of human cerebrospinal fluid lipoproteins from individuals of different apoE genotypes. Exp Neurol 1998; 149:175-182. 65. Bellosta S, Nathan BP, Orth M et al. Stable expression and secretion of apolipoproteins E3 and E4 in mouse neuroblastoma cells produces differential effects on neurite outgrowth. J Biol Chem 1995; 270:27063-27071. 66. Fagan AM, Bu G, Sun Y et al. Apolipoprotein E-containing high density lipoprotein promotes neurite outgrowth and is a ligand for the low density lipoprotein receptor-related protein. J Biol Chem 1996; 271:30121-30125. 67. Snipes GJ, McGuire CB, Norden JJ et al. Nerve injury stimulates the secretion of apolipoprotein E by non-neuronal cells. Proc Natl Acad Sci USA 1986; 83:1130-1134. 68. Ignatius MJ, Gebicke-Haerter PJ, Skene JHP et al. Expression of apolipoprotein E during nerve degeneration and regeneration. Proc Natl Acad Sci USA 1986; 83:1125-1129. 69. Boyles JK, Notterpek LM, Anderson LJ. Accumulation of apolipoproteins in the regenerating and remyelinating mammalian peripheral nerve. J Biol Chem 1990; 265:17805-17815. 70. Goodrum JF, Bouldin TW, Zhang SH et al. Nerve regeneration and cholesterol reutilization occur in the absence of apolipoproteins E and A-I in mice. J Neurochem 1995; 64:408-416. 71. Qian Z, Gilbert ME, Colicos MA et al. Tissue plasminogen activator is induced as an immediate-early gene during seizure and long term potentiation. Nature 1993; 361:453-457. 72. Mori T, Iijima N, Kitabatake K et al. α2-Macroglobulin is an astroglia-derived neuritepromoting factor for cultured neurons from rat central nervous system. Brain Res 1990; 527:55-61. 73. Kawamata T, Tooyama I, Yamada T et al. Lactotransferrin immunocytochemistry in Alzheimer and normal human brain. Am J Path 1993; 142:1574-1585. 74. de Silva HV, Harmony JAK, Stuart WD et al. Apolipoprotein J: Structure and tissue and distribution. Biochem 1990; 29:5380-5389. 75. Holtzman DM, Pitas RE, Kilbridge J et al. Low density lipoprotein receptor-related protein mediates apolipoprotein E-dependent neurite outgrowth in a central nervous system-derived neuronal cell line. Proc Natl Acad Sci USA 1995; 92:9480-9484. 76. Ishii M, Osada T, Gliemann J et al. Neurite-promoting effect of a2-macroglobulin in rat cerebral cortex is mainly associated with α2-macroglobulin receptor. Brain Res 1996; 737:269-274. 77. Schmechel DE, Saunders AM, Strittmatter WJ et al. Increased amyloid β-peptide deposition in cerebral cortex as a consequence of apolipoprotein E genotype in late-onset Alzheimer’s disease. Proc Natl Acad Sci 1993; 90:9649-9653. 78. Gomez-Isla T, West HL, Rebeck GW et al. Clinical and pathological correlates of apolipoprotein E e4 in Alzheimer disease. Ann Neurol 1996; 39:62-70. 79. Strittmatter WJ, Weisgraber KH, Huang D et al. Binding of human apolipoprotein E to βA4 peptide: Isoform specific effects and implications for late onset Alzheimer disease. Proc Natl Acad Sci USA 1993; 90:8098-8102. 80. LaDu MJ, Pederson TM, Frail DE et al. Purification of apolipoprotein E attenuates isoformspecific binding to β-amyloid. J Biol Chem 1995; 270:9039-9042. 81. Ma J, Yee A, Brewer Jr AYH et al. Amyloid-associated proteins α1-antichymotrypsin and apolipoprotein E promote assembly of Alzheimer β-protein into filaments. Nature 1994; 372:92-94. 82. Sanan DA, Weisgraber KH, Russell SJ et al. Apolipoprotein E associates with β amyloid peptide of Alzheimer’s disease to form novel monofibrils. J Clin Invest 1994; 94:860-869. 83. Wisniewski T, Castano EM, Golabek A et al. Acceleration of Alzheimer’s disease fibril formation by apolipoprotein E in vitro. Am J Path 1994; 145:1030-1035. 84. Bales KR, Verina T, Dodel RC et al. Lack of apolipoprotein E dramatically reduces amyloid b-peptide deposition. Nature Genet 1997; 17:263-264. 85. Jordan J, Galindo MF, Miller RJ et al. Isoform-specific effect of apolipoprotein E on cell survival and β-amyloid-induced toxicity in rat hippocampal pyramidal neuronal cultures. J Neurosci 1998; 18:195-204.

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86. Urmoneit B, Prikulis I, Wihl G et al. Cerebrovascular smooth muscle cells internalize Alzheimer amyloid beta protein via a lipoprotein pathway: Implications for cerebral amyloid angiopathy. Lab Invest 1997; 77:157-166. 87. Zlokovic BV, Martel CL, Matsubara E et al. Glycoprotein 330/megalin: Probable role in receptor-mediated transport of apolipoprotein J alone and in a complex with Alzheimer disease amyloid β at the blood-brain and blood-cerebrospinal fluid barriers. Proc Natl Acad Sci USA 1996; 93:4229-4234. 88. Hammand SM, Ranganathan S, Loukinova E et al. Interaction of apolipoprotein J-amyloid β peptide complex with low density lipoprotein receptor-related protein-2/megalin. J Biol Chem 1997; 272:18644-18649. 89. El Khoury J, Hickman SE, Thomas CA et al. Scavenger receptor-mediated adhesion of microglia to β-amyloid fibrils. Nature 1996; 382:716-719. 90. Paresce DM, Ghosh RN, Maxfield FR. Microglial cells internalize aggregates of the Alzheimer’s disease amyloid beta-protein via a scavenger receptor. Neuron 1996; 17:553-565. 91. Kounnas MZ, Moir RD, Rebeck GW et al. LDL receptor-related protein, a multifunctional apoE receptor, binds secreted β-amyloid precursor protein and mediates its degradation. Cell 1995; 82:331-340. 92. Knauer MF, Orlando RA, Glabe CG. Cell surface APP751 forms complexes with protease nexin 2 ligands and is internalized via the low density lipoprotein receptor-related protein (LRP). Brain Res 1996; 740:6-14. 93. Lendon CL, Talbot CJ, Craddock NJ et al. Genetic association studies between dementia of the Alzheimer’s type and three receptors for apolipoprotein E in a Caucasian population. Neurosci Lett 1997; 222:187-190. 94. Chung H, Roberts CT, S G et al. Lack of association of trinucleotide repeat polymorphisms in the very-low-density lipoprotein receptor gene with Alzheimer’s disease. Ann Neurol 1996; 39:800-803. 95. Pritchard ML, Saunders AM, Gaskell PC et al. No association between very low density lipoprotein receptor (VLDL-R) and Alzheimer disease in American Caucasians. Neurosci Lett 1996; 209:105-108. 96. Wavrant-DeVrieze F, Perez-Tur J, Lambert J-C et al. Association between the low density lipoprotein receptor-related protein (LRP) and Alzheimer’s disease. Neurosci Lett 1997; 227:68-70. 97. Clatworthy AE, Gomez-Isla T, Rebeck GW et al. Lack of association of a polymorphism in the low density lipoprotein receptor-related protein gene with Alzheimer disease. Arch Neurol 1997; 54:1289-1292. 98. Kang DE, Saitoh T, Chen X et al. Genetic association of the low-density lipoprotein receptor-related protein gene (LRP), an apolipoprotein E receptor, with late-onset Alzheimer’s disease. Neurol 1997; 49:56-61. 99. Hollenbach E, Ackermann S, Hyman BT et al. Confirmation of an association between a polymorphism in exon 3 of the low density lipoprotein receptor-related protein gene and Alzheimer’s disease. Neurol 1998; 50:1905-1907. 100. Kamboh MI, Ferrell RE, DeKosky ST. Genetic association studies between Alzheimer’s disease and two polymorphisms in the low density lipoprotein receptor-related protein gene. Neurosci Lett 1998; 244:65-68. 101. Baum L, Chen L, Ng H-K et al. Low density lipoprotein receptor related protein gene exon 3 polymorphism association with Alzheimer disease in Chinese. Neurosci Lett 1998; 247:33-36.

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CHAPTER 5

Clusterin-apoE Lipoprotein Particles David M. Holtzman, Mary Jo LaDu and Anne M. Fagan

T

he intercellular transport of lipids through the aqueous circulatory system as well as within tissues and other body fluids requires the packaging of these hydrophobic molecules into water soluble carriers (lipoproteins) and their regulated targeting to appropriate tissues by receptor-mediated endocytic pathways as well as scavenger receptormediated pathways.1 Lipoproteins have been classified into several major groups on the basis of the density at which they float by ultracentrifugation. In the plasma, chylomicrons and very low density lipoproteins (VLDL) are large particles that have a high lipid to protein ratio and are the major carriers of triglycerides. Intermediate density lipoproteins (IDL) and low density lipoproteins (LDL) are intermediate sized particles that are high in cholesterol and cholesteryl esters; in humans, LDL are the principal cholesterol transporting lipoproteins in the plasma. High density lipoproteins (HDL) are the smallest particles and contain the highest protein to lipid ratio. Certain kinds of HDL particles are involved in the process of “reverse cholesterol transport,” a pathway whereby these particles acquire cholesterol from peripheral tissues and transport it to the liver for excretion.2 In addition to the plasma, lipoproteins are also present in other body fluids such as the cerebrospinal fluid (CSF). In contrast to plasma, the majority of CSF lipoproteins are HDL-like in both density and size (see below).3,4

Clusterin/apoJ: An Apolipoprotein in Plasma and CSF Lipoproteins have different protein (called apolipoprotein) and lipid compositions, and varying physiological activities as a result of this compositional heterogeneity. Apolipoproteins not only facilitate the binding of lipoprotein particles to specific cell surface receptors, but are also important in maintaining the structural integrity of lipoproteins and in modulating enzymatic reactions important in lipid/cholesterol transport. One of the more recently identified apolipoproteins is apolipoprotein J (apoJ) also known as clusterin. In regard to its potential role as an apolipoprotein, apoJ was first identified as a component of a very specific class of plasma HDL in the 1980s. Two proteins termed PLS:29 and PLS:305 and then later termed NA1 and NA26 were shown by immunoaffinity chromatography to be components of at least a portion of apoA-I-containing HDL particles in both plasma and CSF.7 Hochstraffer et al7 showed that NA1 and NA2 were HDL-associated glycoproteins and provided partial N-terminal sequence data. Studies published in 1990 by Harmony and coworkers convincingly demonstrated that the proteins initially termed NA1 and NA2 were two subunits of apoJ. They showed that apoJ was a plasma apolipoprotein consisting of two disulfide-linked subunits of 34-36 kDa (apoJα) and 36-39 kDa (apoJβ).8,9 Utilizing immunoaffinity chromatography, it was shown that apoJ is a component of a specific plasma HDL subclass which also contains apoA-I and cholesteryl ester transfer protein (CETP) activity and was present in the HDL3 and VHDL density ranges.8,9 Jenne et al also Clusterin in Normal Brain Functions and During Neurodegeneration, edited by Caleb E. Finch. ©1999 R.G. Landes Company.

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showed that a population of HDL contains apoJ and that these particles are lipid-poor, containing ~22% (w/w) lipids.10 It appears that some but not all of the apoJ-containing plasma HDL particles also contain apoA-I.11 The primary structure of apoJ was deduced by protein sequencing and cDNA cloning and sequencing.12 It was shown that the two apoJ subunits were encoded by a single gene in both mouse and humans and that apoJ was identical to the protein clusterin originally cloned from other organs.13,14 Both apoJ subunits were shown to be post-translationally cleaved between Arg-205 and Ser-206, after which the subunits associate through disulfide bonds. The primary structure predicted the presence of amphiphilic helices, which likely account for association of apoJ with lipoproteins, as well as the presence of heparin-binding motifs.12 Expression of apoJ was shown to be present in most organs, with high levels in brain, testis, ovary, and liver.12 It is interesting to note that compared to other apolipoproteins, apoJ has several similarities to apoE. First, the expression pattern of apoJ is similar to apoE12,15 with high expression in liver and brain. Second, the concentration of both proteins in plasma is similar (~50 µg/ml).12,15,16 The fact that both apoE and apoJ are expressed within organs such as brain, which is isolated from the circulation by the blood-brain barrier, suggested that apoJ may play a role in not only plasma lipid transport but also lipid transport among cells within organs. This may be an important role for apoE and apoJ within the CNS (see below). While there is a clear requirement for apoE in normal plasma cholesterol homeostasis15 a specific role for apoJ in plasma lipoprotein metabolism has not yet been defined. It has been shown that HepG2 cells, a hepatocellular carcinoma cell line, secrete nascent apoJ-containing dense lipoproteins. In contrast to plasma apoJ-HDL, these lipoproteins are rich in triglyceride and do not contain apoA-I.17 This suggests that apoJ-containing plasma lipoproteins undergo remodeling which results in triglyceride depletion and association with apoA-I. A recent study has demonstrated that purified apoJ can facilitate cholesterol and phospholipid efflux from foam cells in vitro.18 While this study suggests a potential role for apoJ in reverse cholesterol transport, further studies are required to define its role in plasma lipid homeostasis in vivo.

apoE and apoJ: Presence and Potential Roles in CNS Despite the fact that cholesterol is one of the most abundant molecules in the brain, accounting for approximately 10% of dry brain weight in contrast to less than 1% found in most other organs, surprisingly little is known about cholesterol and lipid transport and metabolism in the CNS. In addition to being a membrane component in the CNS, cholesterol is also found associated with lipoprotein particles in the CSF. Analysis of the constituents of human CSF provided the first information that lipoprotein metabolism in the CNS was distinct from that in the periphery.3 CSF is produced by the choroid plexus and also contains nonresorbed products derived from the interstitial space of the brain and, to a lesser degree, the plasma. Several apolipoproteins are present in human CSF, the two most abundant being apoE and apoA-I; each are found at concentrations between 2.5-5 µg/ml.3,4,19,20 Both proteins appear to reside in distinct lipoprotein particles which are predominantly HDL-like in both density and size.4,19,20 Recently, it has been shown that a small percentage of larger, more buoyant lipoprotein particles containing apoE are present which are similar in density and size to plasma HDL1 and LDL.20,21 Interestingly, apoE present within the CSF is produced within the BBB. In patients who have undergone liver transplantation, the plasma apoE phenotype changes to that of the donor, while apoE protein within the CSF remains the phenotype of the recipient.22 In addition to apoE and apoA-I, apoA-II and apoJ are also present in CSF lipoproteins. There is heterogeneity within CSF lipoproteins with respect to the size distribution of these apolipoproteins.20 ApoE is in larger particles which range in size from HDL1 to HDL3, with

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Fig. 5.1. Lipid distribution in human CSF (A,B) and serum-free conditioned media from primary rat astrocytes (C,D) fractionated by size (A,C) and density (B,D). 50 mls of either CSF (A,B) or astrocyte conditioned media (C,D) were concentrated to 1 ml and then fractionated by gel filtration chromatography using tandem Superose 6 columns (A,C) or single-spin equilibrium ultracentrifugation using 3-20% NaBr gradients (B,D). The resulting fractions were analyzed for lipid, which is expressed as µg/fraction. Reprinted with permission from J Neurochem 1998; 70:2070-2081.

apoA-I and apoA-II being present in progressively smaller particles (HDL2-VHDL). ApoJ is distributed fairly evenly across the HDL-like particle size range. Not surprisingly, as has been described for plasma lipoproteins,9,23 significant amounts of both apoE and apoJ are disrupted from CSF lipoproteins during the process of density ultracentrifugation and are then observed in free protein fractions.20 This is not the case when techniques such as size exclusion or immunoaffinity chromatography are utilized. This suggests that functional analysis of native CNS-produced lipoproteins will require isolation by these techniques if physiological properties of native particles are sought. Lipid analysis of CSF lipoproteins has revealed that like plasma HDL, there is a high protein/lipid ratio. The lipids are composed of esterified and unesterified cholesterol and phospholipid with little or no triglyceride (Fig. 5.1).3,4,20,24 Several studies utilizing electron microscopy have shown that these lipoproteins range in size from 7-15 nM and are spherical in shape (Fig. 5.2).3,4,19-21 Some CSF lipoproteins, especially those containing apoA-I, appear to be predominantly derived from plasma. It is likely, however, that both apoE and apoJ-containing CSF lipoproteins are derived from within the CNS. While apoE in the CNS is synthesized predominantly by astrocytes,25,26 apoE mRNA is also in microglia.27,28 ApoJ mRNA is also present in astroctyes, ependymal cells lining the ventricles, and in neurons.29,30 Since a significant

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Fig. 5.2. Negative stained electron micrographs of lipoproteins from human CSF and rat astrocyte conditioned media. Lipoproteins were isolated by gel filtration chromatography and a concentrated aliquot (0.5 mg protein/ml) from fractions 37/38 (A) and 43/44 (B) of CSF and 37/38 (C) of astrocyte media was placed on a carbon coated EM grid and negatively stained with 2% phosphotungstic acid. Size bar is 25 nm. Arrows indicate a large spherical particle from human CSF (A), a small spherical particle from human CSF (B) and a stack of discoidal particles from rat astrocyte conditioned media (C). Reprinted with permission from J Neurochem 1998; 70:2070-2081.

amount of CSF lipoproteins containing apoE and apoJ are likely to be derived from brain parenchyma, our group has characterized lipoproteins secreted by astrocytes, cells likely to be secreting apoE and apoJ-containing lipoproteins within brain tissue. There were similarities and differences between astrocyte-secreted vs. CSF lipoproteins. We found that primary cultured rat astrocytes produce nascent lipoprotein particles which are HDL-like in size, contain little core lipid, are primarily discoidal in shape, and contain apoE and apoJ.20 In preliminary experiments using coimmunoprecipitation of astrocyte-secreted lipoproteins under nondenaturing conditions, we have found that most apoJ is in distinct nonapoE-containing particles. Whether there are some apoE-containing particles which also contain apoJ remains to be determined. In recent studies we have also studied astrocytederived lipoprotein particles from wild type mice and apoE knockout (-/-) mice. Like rat astrocytes, cultured wild type mouse astrocytes also secrete nascent lipoprotein particles containing cholesterol, apoE and apoJ. In contrast, we have found that while apoE–/– astrocytes continue to secrete apoJ, following gel filtration chromatography of astrocyte-conditioned media, there is no longer easily detectable cholesterol or phospholipid across the lipoprotein size range (LaDu MJ, Fagan AM, Holtzman DM, unpublished observations). This suggests that apoE is required for the secretion of normal levels of HDL-like lipoproteins by these cells. Though size exclusion chromatography reveals that the apoJ secreted by apoE–/– astrocytes continues to elute in the small HDL size range, further experiments will be required to determine whether the apoJ is in an aggregated lipid-free form or is present in very lipid-poor particles. This may be an important issue in regard to the interaction of apoJ with receptors as well as other disease-related proteins such as the amyloid β protein (Aβ) (see next section). Because spherical CSF lipoproteins are in part derived from the brain parenchyma, it seems likely that nascent astrocyte lipoproteins secreted as discs may be able to convert to

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spheres by acquiring cholesteryl esters before reaching the CSF. For example, small discoidal plasma HDL have been shown to be efficient acceptors of cholesterol.31 Further, CSF particles isolated via density ultracentrifugation have been shown to accept cholesterol.24 Thus, it seems likely that nascent astrocyte-secreted lipoproteins will also participate in the efflux of cholesterol from neural cells. This may be of particular importance following neural injury (see next section). Further studies will be required to determine if astrocyte-secreted lipoproteins can promote “reverse cholesterol transport”, and the role of apoE and apoJ in this process. It is of note that enzymes that participate in cholesterol esterification (lecithin: cholesterol acyltransferase) and in cholesteryl ester transfer (cholesteryl ester transfer protein) are present in the brain32-34 and could theoretically promote the acquisition of cholesteryl esters by HDL-like particles in brain parenchyma. What are some of the other potential functions of apoE/apoJ-containing lipoproteins produced in the brain? In addition to cholesterol removal, astrocyte-secreted particles are likely to interact with a variety of cellular receptors and deliver their constituents to cells. There is direct evidence that astrocyte secreted apoE-containing particles can directly interact with the LDL-receptor related protein (LRP) on neurons to facilitate processes such as neurite outgrowth in vitro.35,36 Effects by these particles on neural process outgrowth may be related to the delivery and utilization of lipoprotein constituents by neurons. The role of CNS lipoprotein particles in such processes in vivo remains to be defined further. In addition to LRP, other cell surface receptors which may interact with astrocyte-secreted lipoprotein particles includes gp330 or megalin, a receptor for apoJ expressed on ependymal cells.37 The described apoE receptors known to be expressed in the brain include the following: LRP (neurons and glia), VLDLR (neurons), apoER2 (neurons), and LDLR (astrocytes and low levels by neurons).38-42 The cellular distribution and level of expression of these receptors will likely affect their relative contribution to receptor-mediated endocytosis of apoE-containing lipoproteins produced in the brain and is discussed in greater detail in the chapter by Rebeck and Hyman.

Potential Roles for apoE and apoJ in CNS Disease Further studies to understand the structure and function of apoE and apoJ-containing lipoproteins produced by cells within the brain is likely to provide important insights into the role of these proteins in neurodegenerative and other diseases of the CNS. This issue will be considered in regard to Alzheimer’s disease (AD) and brain repair following CNS injury. Genetic epidemiological studies have shown that the ε4 allele of apoE is a major risk factor for AD.43 In addition, recent data suggests that apoE4 is also a risk factor for poor outcome after head trauma,44,45 cerebral hemorrhage,46 cardiac bypass,47 and possibly stroke.48 ApoE4 also appears to influence the age of onset of Parkinson’s disease.49 In regard to AD, one hypothesis is that the association between apoE4 and AD is due to the ability of apoE to interact with the Aβ protein. Aβ deposition in the AD brain appears to be an early and important pathogenetic event in AD.50 A recent in vivo study highlights the potential importance of both apoE and apoJ in Aβ deposition. Mice have recently been developed in which overexpression of a mutant form of the amyloid precursor protein (APP) results in age-dependent Aβ deposition in plaque-like structures resembling those found in AD.51 Bales et al52 found that there was a marked decrease in Aβ deposition and thioflavin-Spositive amyloid deposits when the mutant APP transgenic mice were bred onto a mouse apoE–/– background. This finding suggests that the presence of mouse apoE promotes Aβ deposition through increasing Aβ fibrillogenesis or inhibiting its clearance. Interestingly, the other major apolipoprotein produced in brain outside of apoE is apoJ, and apoJ like apoE has been shown to bind to Aβ both in vivo and in vitro.53-55 Since apoE and apoJ are both present in lipoprotein particles in brain and CSF, this suggests that

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important interactions influencing Aβ metabolism may occur between Aβ, apoE, and apoJ in CNS lipoprotein particles. In fact, recently published data suggests that: 1) Aβ in CSF is found in HDL-like lipoproteins56 and 2) apoE and apoJ associate with Aβ in CSF.53-55 It will be important in future studies to characterize the interaction between apoE, apoJ, and Ab in lipoprotein particles likely to be found in brain, such as those derived from astrocytes. This is highlighted by studies which have shown that while lipid free apoE isoforms can interact with Aβ,57,58 cell secreted apoE isoforms containing lipid-interact with Ab in a different isoform-specific fashion.59,60 Studies examining apoE, apoJ, and Aβ interactions under physiological conditions may give insight into mechanisms underlying Aβ deposition. ApoE and apoJ-containing CNS lipoproteins may also play a role in modulation of CNS lipid metabolism and impact on recovery after CNS injury. Several in vitro studies suggest that apoE3-containing lipoproteins can enhance neurite outgrowth, whereas apoE4 either has no effect or decreases neurite outgrowth.61,62 Similar findings have also been made using astrocyte-derived apoE-containing lipoproteins and primary hippocampal neurons.35,36 While apoJ is also present in these astrocyte-secreted particles, it is unclear if it plays a role in neurite outgrowth. Some studies have suggested that the effects of apoE-containing lipoproteins on neurite outgrowth require neuronal LRP.36,62-64 This implies that uptake of lipoprotein particles containing cholesterol and phospholipids may be required for this effect. While the mechanism of this LRP-mediated effect is unclear, these in vitro findings suggest the hypothesis that following CNS injury or in a CNS disease, apoE-containing lipoproteins may facilitate cholesterol/lipid delivery and utilization in regenerating neuronal processes. For example, it was reported that reactive synaptogenesis following a dernervating hippocampal lesion is enhanced by estrogen in ovariectomized wild type mice. This enhancement by estrogen was not observed in apoE–/– mice.65 In addition to the potential role of apoE/apoJ in cholesterol lipid delivery in the CNS, another important role they may play is in “reverse cholesterol transport”. This is suggested by our recent data showing that nascent rat astrocyte-secreted lipoproteins are cholesteryl esterpoor lipoproteins shaped like discs. Our recent in vivo study also supports this potential role of apoE in CNS following injury. We showed that following a hippocampal denervating lesion, that apoE–/– mice cleared lipid-laden axonal debris much more slowly than wild type mice.66 If there are isoform, specific differences in the ability of apoE to clear cholesterol and lipid following CNS injury, this could impact on neurologic outcome. Studying the physiological form and functions of apoE/apoJ lipoproteins produced in CNS will be critical in further characterizing the role of these proteins in lipid metabolism following CNS injury.

Summary ApoE and apoJ are apolipoproteins found in plasma in distinct subpopulations of lipoproteins. While an important role for apoE has been found in plasma cholesterol metabolism, a role of apoJ in plasma cholesterol metabolism remains to be defined. Both apoE and apoJ appear to be the principal apolipoproteins produced in brain. Characterization of lipoprotein particles produced by cells intrinsic to the brain is underway. Both genetic, biochemical, and cell biological studies suggest potential roles for apoE and apoJ in AD and other CNS diseases. Further studies to characterize the role of apoE/apoJ-containing lipoproteins produced in brain on lipid/cholesterol metabolism as well as interactions with receptors and disease-related proteins such as Aβ are likely to give important insights into the role of apoE/apoJ in the injured brain.

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References 1. Krieger M, Herz J. Structures and functions of multiligand lipoprotein receptors: Macrophage scavenger receptors and LDL receptor-related protein (LRP). Annu Rev Biochem 1994; 63:601-637. 2. Fielding CJ, Fielding PE. Molecular physiology of reverse cholesterol transport. J Lipid Res 1995; 36:211-228. 3. Roheim PS et al. Apolipoproteins in human cerebrospinal fluid. Proc Natl Acad Sci USA 1979; 76:4646-4649. 4. Pitas RE et al. Lipoproteins and their receptors in the central nervous system. J Biol Chem 1987; 262:14352-14360. 5. Anderson L, Anderson NG. Some perspectives on two-dimensional protein mapping. Clin Chem 1984; 30:1898-1905. 6. James RW et al. Protein heterogeneity of lipoprotein particles containing apolipoprotein A-I without apolipoprotein A-II and apolipoprotein A-I with apolipoprotein A-II isolated from human plasma. J Lipid Res 1988; 29:1557-1571. 7. Hochstrasser A-C et al. HDL particle associated proteins in plasma and cerebrospinal fluid: Identification and partial sequencing. Appl Theoret Electroph 1988; 1:73-76. 8. de Silva HV et al. A 70-kDa apolipoprotein designated apoJ is a marker for subclasses of human plasma high density lipoproteins. J Biol Chem 1990; 265:13240-13247. 9. de Silva HV et al. Purification and characterization of apolipoprotein J. J Biol Chem 1990; 265:14292-14297. 10. Jenne DE et al. Clusterin (complement lysis inhibitor) forms a high density lipoprotein complex with apolipoprotein A-I in human plasma. J Biol Chem 1991; 266:11030-11036. 11. Stuart WD et al. Structure and stability of apolipoprotein J-containing high-density lipoproteins. Biochemistry 1992; 31:8552-8559. 12. de Silva HV et al. Apolipoprotein J: Structure and tissue distribution. Biochemistry 1990; 29:5380-5389. 13. Cheng CY, Mathur PP, Grima J. Structural analysis of clusterin and its subunits in ram rete testis fluid. Biochemistry 1988; 27:2079-4088. 14. Collard MW, Griswold MD. Biosynthesis and molecular cloning of sulfated glycoprotein 1 secreted by rat Sertoli cells: sequence similarity with the 70-kilodalton precursor to sulfatide/ GM1 activator. Biochemistry 1987; 26:3297-3303. 15. Mahley RW. Apolipoprotein E: Cholesterol transport protein with expanding role in cell biology. Science 1988; 240:622-630. 16. Jenkins SH et al. Quantitation of plasma apolipoprotein J. Methods Enzymol 1996; 263:309-316. 17. Burkey BF, Stuart WD, Harmony JA. Hepatic apolipoprotein J is secreted as a lipoprotein. J Lipid Res 1992; 33:1517-1526. 18. Gelissen IC et al. Apolipoprotein J (clusterin) induces cholesterol export from macrophage-foam cells: A potential anti-atherogenic function? Biochem J 1998; 331:231-237. 19. Borghini I et al. Characterization of subpopulations of lipoprotein particles isolated from human cerebrospinal fluid. Biochem Biophys Acta 1995; 1255:192-200. 20. LaDu MJ et al. Nascent astroctye particles differ from lipoproteins in CSF. J Neurochem 1998; 70:2070-2081. 21. Guyton JR et al. Novel large apolipoprotein E-containing lipoproteins of density o1.006-1.060 g/ml in human cerebrospinal fluid. J Neurochem 1998; 70:1235-1240. 22. Linton MF et al. Phenotypes of apolipoprotein B and apolipoprotein E after liver transplantation. J Clin Invest 1991; 88:270-281. 23. Castro GR, Fielding CJ. Evidence for the distribution of apolipoprotein E between lipoprotein classes in human normocholesterolemic plasma and for the origin of unassociated apolipoprotein E (Lp-E). J Lipid Res 1984; 25:58-67. 24. Rebeck GW et al. Structure and functions of human cerebrospinal fluid lipoproteins from individuals of different APOE genotypes. Exp Neurol 1998; 149:175-182.

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25. Boyles JK et al. Apolipoprotein E associated with astrocytic glia of the central nervous system and with nonmyelinating glia of the peripheral nervous system. J Clin Invest 1985; 76:1501-1513. 26. Pitas RE et al. Astrocytes synthesize apolipoprotein E and metabolize apolipoprotein E-containing lipoproteins. Biochim Biophys Acta 1987; 917:148-161. 27. Nakai M et al. Expression of apoE mRNA in rat microglia. Neurosci Lett 1996; 211:41-44. 28. Stone DJ et al. Astrocytes and microglia respond to estrogen with increased apoE mRNA in vivo and in vitro. Exp Neurol 1997; 143:313-318. 29. May PC, Finch CE. Sulfated glycoprotein 2: New relationships of this multifunctional protein to neurodegeneration. Trends Neurol Sci 1992; 15:391-396. 30. Aronow BJ et al. Apolipoprotein J expression at fluid-tissue interfaces: Potential role in barrier cytoprotection. Proc Natl Acad Sci USA 1993; 90:725-729. 31. Forte TM, Nordhausen RW. Electron microscopy of negatively stained lipoproteins. Methods Enzymol 1986; 128:442-457. 32. Warden CH et al. Tissue-specific expression, developmental regulation and chromosomal mapping of the lecithin: Cholesterol acyltransferase gene. J Biol Chem 1989; 264:21573-21581. 33. Smith KM, Lawn RM, Wilcox JN. Cellular localization of apolipoprotein D and lecithin: cholesterol transferase mRNA in rhesus monkey tissues by in-situ hybridization. J Lipid Res 1990; 31:995-1004. 34. Albers JJ et al. Cholesteryl ester transfer protein in human brain. Int J Clin Lab Res 1992; 21:264-266. 35. Narita M et al. The low density lipoprotein receptor-related protein (LRP), a multifunctional apoE receptor, modulates hippocampal neurite development. J Neurochem 1997; 68:587-595. 36. Sun Y et al. GFAP-apoE transgenic mice: Astrocyte specific expression and differing biological effects of astrocyte-secreted apoE3 and apoE4 lipoproteins. J Neurosci 1998; 18:3261-3272. 37. Kounnas MZ et al. Immunological localization of glycoprotein 330, low density lipoprotein receptor related protein and 39 kDa receptor associated protein in embryonic mouse tissues. In Vivo 1994; 8:343-351. 38. Moestrup SK, Gliemann J, Pallesen G. Distribution of the α2-macroglobulin receptor/low density lipoprotein receptor-related protein in human tissues. Cell Tiss Res 1992; 269:375-382. 39. Rebeck GW et al. Apolipoprotein E in sporadic Alzheimer’s disease: Allelic variation and receptor interactions. Neuron 1993; 11:575-580. 40. Christie RH et al. Expression of the very low-density lipoprotein receptor (VLDL-r), an apolipoprotein-E receptor, in the central nervous system and in Alzheimer’s disease. J Neuropath Exp Neurol 1996; 55:491-498. 41. Kim DH et al. Human apolipoprotein E receptor 2. A novel lipoprotein receptor of the low density lipoprotein receptor family predominantly expressed in brain. J Biol Chem 1996; 271:8373-8380. 42. Poirer J. Apolipoprotein E in animal models of CNS injury and Alzheimer’s disease. Trends Neurol Sci 1994; 17:525-530. 43. Strittmatter WJ, Roses AD. Apolipoprotein E and Alzheimer’s disease. Annu Rev Neurosci 1996; 19:53-77. 44. Nicoll JAR, Roberts GW, Graham DI. ApoE E4 allele is associated with deposition of amyloid beta-protein following head injury. Nature Med 1995; 1:135-137. 45. Teasdale GM et al. Association of apolipoprotein E polymorphism with outcome after head injury. Lancet 1997; 350:1069-1071. 46. Alberts MJ et al. ApoE genotype and survival from intracerebral hemorrhage. Lancet 1995; 346:575. 47. Tardiff BE et al. Preliminary report of a genetic basis for cognitive decline after cardiac operations. The neurologic Outcome Research Group of the Duke Heart Center. Ann Thorac Surg 1997; 64:715-720.

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48. Slooter AJC, Tang MX, Vanduijn CM. Apolipoprotein E epsilon-4 and the risk of dementia with stroke-a population based investigation. J Am Med Assoc 1997; 277:818-821. 49. Zarepesi S et al. Modulation of the age at onset of Parkinson’s disease by apolipoprotein E genotype. Ann Neurol 1997; 42:655-658. 50. Selkoe DJ. Normal and abnormal biology of the beta-amyloid precursor protein. Ann Rev Neurosci 1994; 17:489-517. 51. Games D et al. Alzheimer-type neuropathology in transgenic mice overexpressing V717F β-amyloid precursor protein. Nature 1995; 373:523-527. 52. Bales KR et al. Lack of apolipoprotein E dramatically reduces amyloid β-peptide deposition. Nature Genet 1997; 17:263-264. 53. Choi-Miura NH et al. SP-40,40 is a constituent of Alzheimer’s amyloid. Acta Neuropath 1992; 83:260-264. 54. Ghiso J et al. The cerebrospinal-fluid form of Alzheimer’s amyloid beta is complexed to SP-40,40 (apolipoprotein J), an inhibitor of the complement membrane-attack complex. Biochem J 1993; 293:27-30. 55. Matsubara E, Frangione B, Ghiso J. Characterization of apolipoprotein J-Alzheimer’s Aβ interaction. J Biol Chem 1995; 270:7563-7567. 56. Koudinov AR et al. Biochemical characterization of Alzheimer’s soluble beta protein in human cerebrospinal fluid-association with high density lipoprotein. Biochem Biophys Res Commun 1996; 223:592-597. 57. Strittmatter WJ et al. Apolipoprotein E: High avidity binding to β-amyloid and increased frequency of type 4 allele in late-onset familial Alzheimer disease. Proc Natl Acad Sci USA 1993; 90:1977-1981. 58. Strittmatter WJ et al. Binding of human apolipoprotein E to synthetic amyloid β peptide: Isoform specific-effects and implications for late-onset Alzheimer disease. Proc Natl Acad Sci USA 1993; 90:8098-8102. 59. LaDu MJ et al. Isoform-specific binding of apolipoprotein E to β-amyloid. J Biol Chem 1994; 269:23404-23406. 60. LaDu MJ et al. Purification of apolipoprotein E attenuates isoform-specific binding to β-amyloid. J Biol Chem 1995; 270:9030-9042. 61. Nathan BP et al. Differential effects of apolipoproteins E3 and E4 on neuronal growth in vitro. Science 1994; 264:850-852. 62. Holtzman DM et al. LRP mediates apolipoprotein E-dependent neurite outgrowth in a CNS-derived neuronal cell line. Proc Natl Acad Sci USA 1995; 92:9480-9484. 63. Bellosta S et al. Stable expression and secretion of apolipoproteins E3 and E4 in mouse neuroblastoma cells produces differential effects on neurite outgrowth. J Biol Chem 1995; 270:27063-27071. 64. Fagan AM et al. Apolipoprotein E-containing high density lipoprotein promotes neurite outgrowth and is a ligand for the low density lipoprotein receptor-related protein. J Biol Chem 1996; 271:30121-30125. 65. Stone DJ et al. Increased synaptic sprouting in response to estrogen via an apolipoprotein E-dependent mechanism: Implications for Alzheimer’s disease. J Neurosci 1998; 18:3180-3185. 66. Fagan AM et al. Evidence for normal aging of the septo-hippocampal cholinergic system in apoE (-/-) mice but impaired clearance of axonal degeneration products following injury. Exp Neurol 1998; 151:314-325.

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CHAPTER 6

Neurovascular Interactions of Alzheimer’s Amyloid β Peptide with Apolipoproteins J and E Berislav V. Zlokovic, Blas Frangione and Jorge Ghiso

D

eposition of amyloid β peptide (Aβ) in the central nervous system (CNS) and cerebral vessels occurs during normal aging and is accelerated by Alzheimer’s disease (AD).1,2 AD is the most common form of human amyloidosis and the major cause of dementia, affecting >5% of the population over the age of 65 years. Neuropathologically, AD is characterized by: 1. Intraneuronal deposits of neurofibrillary tangles (NFT); 2. Parenchymal amyloid deposits—neuritic plaques; 3. Vascular amyloidosis; and 4. Synaptic loss. Aβ is considered to be implicated in neuropathogenesis and the development of cerebrovascular pathology in AD and related disorders.1-8 Recent studies from our and other laboratories suggest a major role for the blood-brain barrier (BBB) and cerebrospinal fluid (CSF) clearance in regulating the concentrations of Aβ in brain and cerebral vasculature.9-20 The process of Aβ amyloid formation is highly tissue-specific, and normally with aging occurs in brain extracellular fluid (ECF) space, in the walls of cortical and leptomeningeal vessels and in the choroid plexus (CP) in humans, nonhuman primates and some mammalian species.1,21 Specific predisposing genetic factors (e.g., apolipoprotein E4 (apoE4) genotype, mutations in amyloid-β-precursor protein (βPP), presenilin 1 and 2 genes, α2-macroglobulin), age-dependent mechanisms (e.g., expression of specific receptors, binding proteins) and/or provocative stimuli (e.g., cytokines) may increase the aggregation and/or enhance the cytotoxicity of Aβ, and are important for the development of AD and Aβ-related pathology.1 Apolipoproteins J and E (apoJ, apoE) may critically influence fibrillogenesis, cytotoxicity, transport across biological membranes and cell-specific uptake of Aβ within the CNS.

Amyloid β Aβ is produced by most somatic cells as a soluble peptide that circulates in blood and CSF22-24 and is present in brain.25,26 It is not known exactly how much of the Aβ in the circulation, CSF and other extracellular body fluids is free, and how much is transported on apoJ27,28 and apoE isoforms,29 transthyretin (TTR),30 lipoproteins, and/or albumin.31 More recent studies in 36 AD cases, 28 age-matched controls and more than 60 young controls indicate that >90% of Aβ in the plasma circulates in association with lipoprotein particles Clusterin in Normal Brain Functions and During Neurodegeneration, edited by Caleb E. Finch. ©1999 R.G. Landes Company.

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(Matsubara et al, personal communication). Aβ is generated through still unclear proteolytic mechanisms by peripheral and CNS cells, as an internal degradation product (39-44 residue) of a larger precursor, βPP, encoded by a gene on chromosome 21.32-35 The Aβ extracted from senile plaques of AD patients contains mainly 42/44 amino acids.36 The vascular amyloid is three residues shorter,37 but 1-42 sequences are also found.38-40 A major form of secreted sAβ is Aβ1-40, while Aβ1-42 accounts for about 10% of sAβ.41 An increased amount of sAβ has been detected in brains in AD and Down’s syndrome (DS) patients.25,42,43 This increase importantly precedes amyloid plaque formation44 and correlates with the development of vascular changes.43

Origin of Aβ The “neuronal” theory suggests that sAβ secreted by brain becomes Aβ deposits. The fact that neuronal cells in culture can produce sAβ45 supports this view. It has been suggested that clearance of sAβ and/or its binding apolipoproteins, apoE and apoJ, and/or other amyloid-associated proteins from the CSF may be altered in Aβ-related disorders predisposing to parenchymal and vascular deposition of Aβ.1,19,20,46 On the other hand, the discovery of sAβ in the circulation and CSF suggests that the role of amyloid in AD may be similar to systemic amyloidoses.47 The “vascular” theory is supported by pathological changes in patients with hereditary cerebral hemorrhage with amyloidosis—Dutch type (HCHWA-D), a genetic form of cerebral amyloid angiopathy (CAA).48,49 Recent studies on Aβ transport across vascular barriers of the CNS support the theory that BBB and bloodCSF barrier may play a critical role in the development of AD and amyloidrelated pathology.9-20

Amyloidogenesis The Aβ peptide is the principal component of brain parenchymal and cerebrovascular amyloid, and has unique physicochemical properties.1 This includes characteristic fibrillar, unbranched appearance on electron microscopy, being 8 to 10 nm thick and up to 100 nm long. Next, special tinctorial properties are widely employed for histopathological analysis, such as apple-green birefringence under polarized light and yellow-green fluorescence after Congo red and thioflavin S staining. Predominantly β-pleated sheet secondary structure has been demonstrated by X-ray diffraction analysis. It is generally believed that a high degree of insolubility may preclude complete proteolytic degradation of fibrils in vivo. The exact mechanisms of amyloid fibril formation and deposition, as well as their tissue-specific localization, are poorly understood.1 Spontaneous fibril formation has been demonstrated in vitro with synthetic Aβ peptides.47 The early onset familial forms of AD in certain Dutch and Swedish pedigrees indicated that the primary structure and concentration of Aβ may be critical in influencing fibril formation.50,51 A mutation at codon 693 (βPP770) of Gln substituting Glu at residue 22 in HCHWA-D 52 converts Aβ1-40 into a highly amyloidogenic and pathologic form, AβQ.22,53,54 Post-translational modification of amino acids (e.g., oxidation, phosphorylation, glycosylation, methylation) may be important.47 According to the ‘amyloid hypothesis’ Aβ is first deposited in preamyloid lesions55 associated with few or no dystrophic neurites. In DS patients with three copies of the βPP genes, preamyloid lesions can appear as early as the age of 12.56-58 These lesions become compacted over a period of many years, and by the end of the third decade acquire the characteristics of amyloid plaques associated with neuronal damage and NFT.55-58 Clinically normal aged individuals can develop extensive preamyloid deposits which may herald the later development of AD.59 The amyloid-associated proteins including apoE and apoJ, as discussed below, as well as amyloid P-component, proteoglycans, a1-antichymotrypsin (ACT),

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vitronectin, TTR, may act to either inhibit amyloid formation, or as pathological chaperones, stabilizing a β-pleated sheet structure.47,59

Cytotoxicity of Aβ

Aβ accumulation is considered a central part in the pathogenesis of AD.1-8 The pathogenic role of Aβ is supported by the demonstration that Aβ peptides have neurotoxic properties in vitro 61-63 and in vivo.64-66 An endoplasmic reticulum-associated binding protein was shown to be important for Aβ cellular toxicity.67 Aβ induces neuronal oxidant stress directly and indirectly by activating microglia.68,69 Activated microglia may play a central role in the inflammatory process associated with amyloid plaques.70 The cells of macrophage/monocyte lineage are also associated with CAA71 and found along vessel walls in HCHWA-D.72 Vascular damage, leukocyte activation and migration across the vessel wall in response to Aβ were shown in vivo.73 Aβ may produce endothelial injury by generating superoxide radicals.74 Some forms of Aβ such as 25-35 may alter the BBB permeability and produce apoptosis in endothelial cells.75 Aβ cerebrovascular deposition is associated with degeneration and eventual disappearance of smooth muscle cells (SMC) 76-78 and degeneration of the CP epithelium.2 The length, form (wild or mutant) and conformational state (e.g., aggregated or soluble) importantly influence the cytotoxic effects of Aβ. Whether or not amyloid is directly causative in the pathogenesis of AD, the accumulation of Aβ seems to be the starting point for the development of several cellular pathologies. The interaction of Aβ or sAβ with apolipoproteins may critically influence its cytotoxic effects,1,79 as discussed below.

Aβ-Receptors Several putative receptors may be involved in interactions with free or bound forms of Aβ. RAGE69 and SR80,81 have been recently identified as receptors for free Aβ. RAGE is a member of the immunoglobulin superfamily of cell-surface molecules expressed in a variety of cells, including vascular endothelial cells, SMC, CP epithelium and phagocytes.82 It has been suggested that RAGE mediates pathophysiological responses in the vasculature when occupied with glycated ligands or Aβ.17,18,69,82 SR is a homotrimeric cell surface molecule that contains α-helical coiled-coil, collagen-like and cysteine-rich domains.83 It is expressed on macrophages, microglia and vascular endothelium and promotes endocytosis and degradation of modified oxidized LDL and glycated ligands. The SR type I and II receptors are both expressed in cerebral capillaries.84 Both RAGE and SR are up-regulated in different cells in AD brain, including vascular SMC and endothelium,69,85 and are involved in signaling microglia to accumulate at sites of Aβ deposition.69,80,85 RAGE mediates Aβ toxicity on vasculature by producing oxidant stress.69 Both RAGE and SR are mediators of endocytosis and transcytosis of macromolecules82,83 and can bind and/or transport AGE and Aβ across vascular endothelium including the BBB.17,18,86

Transport of Free/Unbound Aβ Across the BBB Recent studies from our and other laboratories have indicated that circulating free Aβ1-40 may increase the level of Aβ in the vessel wall and brain parenchyma in rodents9-12 and nonhuman primates.13,14 Specific receptor-mediated BBB transport mechanism(s) responsible for microvascular accumulation, deposition and brain transfer of blood-borne unbound Aβ1-40, AβQ22 and Aβ1-42 have been suggested.9-14,17,18,87 In squirrel monkey, brain transport of circulating Aβ1-40 is detectable by SPECT.88 The microvascular sequestration and BBB permeability to circulating Aβ1-40 were increased by aging in squirrel monkey, and several age-related systemic changes may further enhance these effects.14 In addition, the intra-arterial infusion of Aβ1-40 and Aβ1-42 in aged squirrel and Rhesus monkey resulted in

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colocalization of radiolabeled peptide onto vascular and parenchymal amyloid lesions, respectively.13,89 Brain metabolism11,87,90 and rapid clearance from the CSF may serve to prevent Aβ accumulation in vascular and parenchymal tissues.20 It has been suggested based on in vitro studies and CSF clearance studies that transport of Aβ from abluminal-to-luminal side and/ or from brain-to-blood, in normal CNS is negligible.28,91 Although these studies may indicate that the BBB does not have a capability to reduce the level of Aβ in normal brain by transporting Aβ peptides out of the CNS, the definitive evidence from direct brain clearance measurements is still not available. Transport of Aβ by a bulk flow of the brain extracellular fluid (ECF) into CSF, and from CSF transport across the arachnoid villi and choroid plexus back into cerebral venous blood and drainage into the cervical lymphatic system may play important functions in normal Aβ homeostasis.92 Whether a similar situation exists in the aging brain and/or AD, how Aβ clearance from brain is influenced by the presence of CAA and parenchymal amyloid deposits, and how altered expression of Aβ vascular and CNS receptors affects its transport out of brain, is not known. The role of Aβ endothelial receptors in binding and transcytosis of a synthetic peptide homologous to human sAβ1-40 has been recently characterized using an in vitro model of human BBB.18 125I-sAβ1-40 binding to brain microvascular endothelial cell monolayer was time-dependent, polarized to the apical side, and saturable with a high and low affinity dissociation constants of 7.8 ± 1.2 and 52.8 ± 6.2 nM, respectively. Binding of 125I-sAβ1-40 was inhibited by anti-RAGE antibody (63%) and by acetylated low density lipoproteins (33%). Consistent with these data, transfected cultured cells overexpressing RAGE or macrophage SR type A displayed binding and internalization of 125I-sAβ1-40. The internalized peptide remains intact >94%. Transcytosis of 125I-sAβ1-40 was time and temperature dependent, asymmetrical from apical-to- basolateral side, saturable with a Michaelis constant of 45 ± 9 nM, and partially sensitive to RAGE blockade (36%) but not to SR blockade. The findings indicate that RAGE and SR mediate binding of sAβ1-40 at the apical side of the human blood-brain barrier, while RAGE is also involved in transcytosis of free sAβ1-40.

Apolipoprotein J Recent findings indicated several possible relationships between apoJ or clusterin and AD. ApolipoproteinJ is a component of the senile plaques in AD and DS.93,94 In a rodent model of neurodegeneration, apoJ expression is increased in astrocytes.95,96 Apolipoprotein J has been shown to be the major carrier protein of sAβ in the CSF and plasma.27 In vitro studies have demonstrated that the sAβ1-40 -apoJ complex cannot be dissociated by apoE2, apoE3, apoE4, ACT, TTR and vitronectin at their physiological plasma concentrations.28 Plasma sAβ-apoJ complexes are normally incorporated into HDL3 particles.97 In its native HDL form, apoJ is fully active to interact with Aβ and protects sAβ from proteolytic degradation.98 Other studies indicated that in addition to forming soluble heterodimers, lipidfree apoJ may also form highly aggregated complexes with Aβ,79 while lipidated apoJ prevents aggregation and polymerization of synthetic Aβ in vitro.98 Recent studies indicate that apoJ at physiologic concentrations prevents the cytotoxic effect of Aβ1-42 on rat primary hippocampal neurons (Pappolla et al, unpublished observations). It has been shown that apoE ε4 allele dose correlates positively with apoJ load and inversely with apoE load in AD brains.99

Glycoprotein 330/Megalin A receptor for apoJ has been identified, the gp330/megalin or low density lipoprotein receptor (LDLR) related protein 2, LRP-2, that is capable of mediating its endocytosis with subsequent lysosomal degradation,100 or transcytosis in certain tissues,15 similarly to LDLR.101

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gp330/megalin is expressed in vascular CNS tissues including the CP, the BBB endothelium and ependyma.15,102 A recent study indicated that gp330/megalin mRNA and protein are expressed in brain capillaries and choroid plexus in smaller amounts than in kidney, yet sufficient to carry important functions of the receptor in the CNS apoJ/Aβ homeostasis.103 In brain lesion models, gp330 immunostaining is enhanced both in neurons and glia (Pasinetti et al, unpublished observations). It has been suggested that expression of gp330 at sites of neuronal injury may mediate the endocytosis of apoJ, perhaps complexed with membrane debris from synaptic remodeling. It has been reported that astrocytes secrete a novel apoE-apoJ lipoprotein particle.104 In AD brains, the cells containing significant nuclear DNA fragmentation express the highest level of cell surface gp330-like immunoepitopes.105 Although the relationship of gp330 to apoJ has not been studied in AD, it has been shown that dying neurons in AD brain accumulate apoE (also ligand for gp330), that correlates with the detection of intracellular Aβ immunoreactivity and expression of gp330. It has been suggested that uptake of lipids may stabilize Aβ protein within the cell, and that neuronal cell death likely precedes the extracellular deposition of Aβ in AD brains.105 Brain ISF Aβ deposits are only detected upon neuronal death, initially as halos of Aβ immunoreactivity around individual dying neurons, and subsequently as Aβ plaques containing numerous neuronal cell ghosts.

Transport of apoJ and Aβ-apoJ Complexes Across the BBB Our recent study has demonstrated remarkable cerebrovascular permeability to circulating apoJ and apoJ-Aβ1-40 complexes, and significant uptake of these ligands by the choroid plexus.15 The cerebrovascular permeability expressed as the permeability coefficient (P) x capillary surface area (S) product (PS) for Aβ1-40-apoJ was 9 to 14-fold higher than for insulin, a peptide considered to have significant BBB permeability mediated by endothelial insulin receptor,106 which also transports anti-insulin receptor monoclonal antibody.107 In comparison to transferrin and nerve growth factor, the PS value for the complex was 45 to 99-fold higher.106 The PS value of OX26 antibody to transferrin BBB receptor, which mediates BBB transport of OX26-Aβ1-40 complex,108 was 17-fold lower than apoJ-Aβ1-40 complex. When compared to vascular markers albumin and dextran (Mr, 70,000), the PS value for the complex was 1350- to 3600-fold higher.106 High affinity transport systems with a Km of 0.2 and 0.5 nM were demonstrated for apoJ at the BBB and choroid epithelium in vivo, suggesting a specific receptor-mediated mechanism. The apoJ-Aβ1-40 complex shared the same transport mechanism and exhibited 2.4 to 10.2-fold higher affinity than apoJ itself. Binding to microvessels, transport into brain parenchyma, and choroidal uptake of both apoJ and apoJ-Aβ1-40 complexes (Fig. 6.1) were markedly inhibited (74-99%) in the presence of monoclonal antibody to gp330/megalin and were virtually abolished by perfusion with the receptor-associated protein (RAP), which blocks all known ligands to gp330. RAP is a 39-44 kDa protein that forms a complex with gp330 shortly after biosynthesis and remains largely confined to the rough endoplasmic reticulum.109 The findings suggests an important role of gp330/megalin in mediating cellular uptake and transport of apoJ and Aβ1-40-apoJ complex at the cerebral vascular endothelium and choroid epithelium.

Apolipoprotein E

Apolipoprotein E, a 34.2 kDa protein110,111 is important in the lipoprotein transport system, and resides in VLDL, IDL and HDL particles and chylomicron remnants. Apolipoprotein E is synthesized by the liver and majority of peripheral tissues,112 including astrocytes,113,114 SMC115 and epithelial cells.116 Apolipoprotein E-containing lipoproteins in the CSF are both spherical and discoidal in shape and can bind efficiently to the LDL

40 1-

β

J 33 0 sA po + β 1J RA 40 P + -a po ap o J J + sA J

po

-a

sA

β

1-

40

40

1-

β

-a

po -a

+

40 1-

J

β

sA

po -a 40

1-

β

sA

sA

gp

40 1-

β

J 33 0 po + β 1J RA 40 P + -a po ap o J J + sA J

po

40

1-

β

sA

-a

-a

40

1-

β

sA

sA

po -a

+

40 1-

J

β

sA

po -a 40

1-

β

sA

sA

β

1-

40

-a

po

-a

40

1-

β

gp

1-

β

J 33 0 J sA po + β 1J RA 40 P + -a po ap o J J + sA

po

gp

-a

+

40 1-

J

β

sA

po -a 40

1-

β

sA

sA

40

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76

Fig. 6.1. Compartmental brain distribution of radioiodinated sAβ1-40-apoJ complex in the presence of unlabeled anti-gp330 antibodies (5.4 nM), RAP (20 nM), apoJ (9 nM), and sAβ1-40 (9 nM) in capillary-depleted brain (A), microvessels (B), and choroid plexus (C). Volumes of tracer distribution, VD, are means ± SE; n = 5-8. [VD(test) - VD(sucrose)] denotes that VD values for test tracers were corrected by VD values for sucrose. * P<0.001 for radiolabeled sAβ1-40-apoJ in the presence and absence of either anti-gp330 antibodies, RAP, or apoJ, by ANOVA. ns, Nonsignificant for radiolabeled sAβ1-40-apoJ in the presence and absence of sAβ1-40, by ANOVA. VD, volume of distribution.

Table 6.1. Distribution of LDL family receptors in the CNS parenchymal and vascular tissues.

Brain parenchyma Brain capillaries Choroid Plexus Ependyma

LDLR

LRP-1

LRP-2 (gp330/megalin)

VLDLR

+ + ? ?

+ + +

-/+ + + +

+ + + ?

Adapted from refs. 101-103, 105, 123, 125-128

receptors present in neuronal cells.117 Newly synthesized apoE is secreted as a lipid-free apoprotein or as a lipoprotein particle.118,119 There is a common genetic polymorphism of apoE in humans which is the result of the three common apoE alleles, ε4, ε3 and ε2, at a single genetic locus.120 These three alleles give a rise to three homozygous (E4/4, E3/3 and E2/2) and three heterozygous (E4/3, E3/2, E4/2) phenotypes. The average frequency of apoE alleles in Caucasians is ε4 = 0.15, ε3 = 0.77, ε4 = 0.08. The three common apoE variants result from amino acid substitutions at residues 112 and/or 158. The E4 isoform contains Arg-112, Arg-158, the E3 contains Cys-112, Arg-158, and the E2 contains Cys-112, Cys-158. The ε4 allele is associated with higher and the ε2 allele with lower total and LDL cholesterol levels as compared to the ε3 allele.121

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Lipoprotein Receptors Apolipoprotein E bound to different lipoprotein particles in the brain as well as lipidfree apoE may have different physiological functions relevant to AD. Apolipoprotein E is the ligand that promotes the recognition and catabolism of apoE-containing lipoproteins such as chylomicron remnants, βVLDL, hypertriglyceridemic VLDL, IDL and HDL containing apoE by the LDL receptor (LDLR),122 the E/α2M macroglobulin receptor LRP-1,123 gp330/ megalin or LRP2 receptor,105,124 and possibly the VLDL receptor.125 Brain cells contain all four types of receptors.101-103,105,123,125-128 These receptors are also distributed in vascular CNS tissues including the microvascular endothelium,15,101,103,127 i.e., the site of the BBB; the CP epithelium,15,102,103 i.e., the site of the blood-CSF barrier; and the ependymal wall lining the CNS ventricles102 that separates CSF from brain ISF (Table 6.1). The role of these receptors in lipid transport and cholesterol homeostasis in the CNS is poorly understood, as well as their potential roles in the uptake and transport of apoE-Aβ complexes between blood and CSF, the clearance of these complexes from the CSF-CNS system and exchanges between CSF and brain ISF.

apoE in AD Epidemiological and genetic data have linked apoE to AD: 1. AD maps on chromosome 19 and is linked to apoE;129 2. apoE is associated with the senile plaques and congophilic angiopathy of AD;130,131 3. The ε4 allele is a risk factor for familial late onset AD;130,132-134 4. The frequency of the ε4 allele is approximately three times higher in patients with AD than in non-AD subjects, and there is a dosage effect of the ε4 allele on lifetime risk for AD and the age and onset of the disease.132,134 Older persons with one or more apoE4 alleles, in addition to being predisposed to AD have also greater vascular Ab load, CAA and amyloid deposition in the CP.2,135,136 The amount of deposited Ab1-40 is increased in AD brains according to the number of copies of E4 alleles.137 Major ethnic differences with no effect of apoE e4 allele on the development of AD among African Americans and Hispanics have also been reported.138 Biochemical and functional data have revealed isoform-specific properties of apoE on binding to Aβ,29,46,139-144 binding to tau,145-148 cholinergic deficit in the frontal cortex and the hippocampus,149,150 effects on neuronal morphology and cytoskeletal structure in vitro,150-153 effects on neuronal degeneration and dendritic remodeling in vivo,154 and binding to soluble βPP that enhances protection against excitotoxicity in rat hippocampal neurons155 and inhibits activation of microglia.156 Four main theories have been formulated to explain the role of apoE isoforms in the pathogenesis of AD. The first theory is focused on interactions of apoE with Aβ and discussed in detail below. The second theory implies the lack of apoE4 interaction with the microtubule-associated protein tau.145,147,148 Apolipoprotein E3 binds well to tau, whereas apoE4 does not. Binding of tau to apoE3 protects it from phosphorylation, while the inability of apoE4 to bind to tau leads to tau hyperphosphorylation and formation of NFTs inside the neurons.148 The third theory suggests that neuronal degeneration in AD may result from impairments in lipid transport caused by apoE4 in contrast to apoE3.151 The fourth theory implicates the ε4 allele in the dysfunction of the lipidtransport system, which may affect phospholipid homeostasis and cholinergic function, and the efficiency of synpathogenesis and neuronal cell remodeling during compensatory reinnervation following neuronal cell loss.149,157

apoE-Aβ Interactions There have been conflicting reports as to whether there is greater or similar binding of apoE3 and apoE4 to Aβ,29,158 and whether or not the apoE enhances or inhibits Aβ fibril

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Table 6.2. Kd values (in nM) for the interaction between apolipoproteins E3 and E4 and Aβ peptides in the presence and absence of lipids. Peptide

apoE3 apoE4

Aβ1-40 + lipids 1.6 3.7

Aβ1-42 - lipids 11.8 12.9

+ lipids 1.0 2.7

- lipids 9.3 10.3

Fig. 6.2. Compartmental brain distribution of radioiodinated sAβ1-40-apoE2, sAβ1-40-apoE3, and sAβ1-40-apoE4 and of the latter in the presence of antibodies to gp330, after 10 min of vascular brain perfusion in the guinea pig. The complexes were either iodinated on sAβ1-40 or on the apolipoprotein component, as indicated below. (A) Transport of test tracers into capillary-depleted brain. (B) Sequestration of test tracers by cerebral microvessels. (C) Sequestration of test tracers by choroid plexus. [VD(test) - VD(sucrose)] denotes that VD (volume of distribution) values for test tracers were corrected by VD values for sucrose. Values are expressed as mean ± SE values; n = 5-7. Uptake of sAβ1-40-apoE3 was not significantly greater than zero in any compartment. 1: 125I-sAβ1-40-apoE2; 2: 125I-sAβ1-40-apoE3; 3: sAβ1-40-125I-apoE3; 4: 125I-sAβ1-40-apoE4; 5: sAβ1-40-125I-apoE4; 6: 125I-sAβ1-40-apoE4, in the presence of antibody to gp330. ap < 0.005 to 0.05 for sAβ1-40-apoE2 and sAβ1-40-apoE3 vs. sAβ1-40-apoE4. nsNot significantly different from uptake in the absence of anti-gp330 antibodies.

formation and seeding in vitro in isoform-dependent fashion.159-161 Most experimental studies with apoE have not used native lipoprotein complexes. Our data indicate that human recombinant lipid-free apoE2, apoE3 and apoE4 have similar binding affinities for Aβ1-40.16 It has been suggested140,141 that the isoform-specific binding of apoE varies depending on whether apoE is lipidated or not, with lipidation enhancing the binding of sAβ to apoE3 relative to apoE4.142 As indicated in Table 6.2, incorporation of apoE3 and apoE4 into reconstituted HDL particles not only increased several fold their affinity for Aβ peptides but also clearly revealed differences between the apoE isoforms. Under our experimental conditions, apoE3 exhibited higher affinity than apoE4 for both Aβ1-40 and Aβ1-42 only in the presence of lipids. Newly synthesized apoE2 and apoE3 bind strongly to Aβ,162 which may prevent its polymerization and inhibit fibrillogenesis providing that Aβ-apoE complexes are efficiently removed from the CNS.163 On the other hand, nascent apoE4 binds more weakly to Aβ, which may favor polymerization and aggregation of free Aβ1-40 and Aβ1-42 in ε4 homozygous patients.162 These hypotheses were, however, all based on the assumption

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that apoE-Aβ complexes are removed from the CNS and cleared from the CSF more efficiently than the free forms of peptides.163 The actual cellular mechanisms responsible for the removal of either complexes or free Aβ remained unidentified, and movements of Aβ/ apolipoproteins along brain ECF routes have not been studied. The initial observations indicating that apoE from CSF binds with high affinity to Aβ,131,143 and that apoE may be a pathological “chaperone” stabilizing Aβ β-pleated conformation may be correct, in particularly if there is a failure in CSF clearance mechanisms and the CP cleansing function resulting in the CNS and vascular accumulation of apoE-Aβ complexes.164 A recent study confirmed that lack of apoE dramatically reduces Aβ peptide deposition,165 supporting the “pathological” chaperone theory for apoE131,143 by an in vivo model.

apoE and apoE-Aβ Complexes at the BBB We have recently demonstrated an absence of brain uptake of circulating apoE isoforms, with low but detectable capillary sequestration and significant uptake by the choroid plexus.16,163 Complexing of Aβ to E2 and E3 isoforms almost abolished the uptake of the peptide by cerebral microvessels, choroid plexus and/or brain parenchyma. In contrast, cerebrovascular sequestration and BBB permeability to Aβ1-40-apoE4 were significant (Fig. 6.2). After 10 min, 85% of Aβ1-40-apoE4 taken up at the BBB remained as intact complex, whereas Aβ1-40 was 51% degraded. The mechanism for BBB uptake of Aβ1-40-apoE4 is unknown. The difference between apoE4 and its complex with Aβ1-40 may be explained by a conformational transformation induced by apoE4 binding to Aβ that allows uptake at the BBB. One apoE binding receptor, gp330, has been ruled out as a likely candidate on the basis that gp330 antibodies (in contrast to apoJ) failed to inhibit transport and binding of apoE4-Aβ complexes at the BBB. Other possible candidates may include the BBB LDLR,101 or RAGE and SR that take part in endothelial internalization and BBB trancytosis of Aβ,18 or perhaps adsorptive-mediated transport and/or nonspecific diffusion. Apolipoprotein E4 by preventing Aβ degradation may increase the probability of Aβ-induced cytotoxicity and possibly formation of amyloid fibrils. If apoE4 does indeed increase the rate of fibrillogenesis as suggested,159 this could also serve as a mechanism by which the vascular sequestration and brain entry of the Aβ1-40-apoE4 could contribute directly to amyloid formation.

Conclusions Recent work from our laboratories has suggested important functions of Aβ putative receptors such as RAGE and SR, and the lipoprotein receptors such as gp330/megalin, and possibly some other yet nonidentified LDL receptor family members in controlling the levels of free Aβ and apoJ and apoE4-bound Aβ in brain ECF, CSF and the vascular wall. Studies have indicated that complexing of Aβ to apoJ and apoE isoforms in body fluids has differential effects on the levels within the CNS. Thus, in a complex with apoJ, the rate of Aβ transport from the circulation across the vascular CNS barriers is significantly enhanced, while the rate of metabolism is remarkably slowed down. Whether this event may keep Aβ predominantly in a soluble form, and what is the long-term effect of Aβ-apoJ complexing on the clearance of Aβ from the CNS and CSF, and the accumulation of this peptide by leptomenigeal vessels and uptake by the choroid plexus, remains presently unknown. In contrast to apoJ, the formation of Aβ complexes with apoE2 and apoE3 isoforms completely prevents peptide transport into the CNS. The isoform-specific apoE effect is seen at its best with apoE4, which in contrast to apoE2 and apoE3 carries Aβ across the BBB, and protects the peptide (similar to apoJ) from its normal metabolic degradation in brain parenchyma. It is still unknown whether altered Aβ CNS and vascular homeostasis under pathological conditions as in AD, is due to changes in the number and cell-specific redistribution of Aβ and lipoprotein vascular and CNS receptors. Since neurovascular receptor-mediated

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interactions of Aβ peptides with apoJ and apoE isoforms significantly affect, in addition to the CNS accumulation, also the fibrillogenic potential and cell-specific cytotoxicity of Aβ, these events are likely to be of major importance in the development of AD pathology and formation of senile plaques and vascular amyloid.

Acknowledgments This study was supported by the National Institutes of Health grants NS34467 and AG05891.

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66. Kowall NW, McKee AC, Yankner BA et al. In vivo neurotoxicity of beta-amyloid [β (1-40)] and the β (25-35) fragment. Neurobiol Aging 1992; 13:537-42. 67. Yan SD, Fu J, Soto C et al. ERAB: A novel intracellular amyloid-beta peptide binding protein which mediates neurotoxicity in Alzheimer’s disease. Nature 1997; 389:689-95. 68. Smith MA, Sayre LM, Monnier VM et al. Radical aging in Alzheimer’s disease. Trends Neurosci 1995; 18:172-6. 69. Yan SD, Chen X, Fu J et al. RAGE and amyloid-β peptide neurotoxicity in Alzheimer’s disease. Nature 1996; 382:685-91. 70. McGeer PL, McGeer EG. The inflammatory response system of brain: Implications for therapy of Alzheimer and other neurodegenerative diseases. Brain Res Rev 1995; 21:195-218. 71. Yamada M, Itoh Y, Shintaku M et al. Immune reactions associated with cerebral amyloid angiopathy. Stroke 1996; 27:1155-62. 72. Maat-Schieman MLC, van Duinen SG, Rozemuller AJM et al. Association of vascular amyloid β and cells of the mononuclear phagocyte system in hereditary cerebral hemorrhage with amyloidosis (Dutch) and Alzheimer disease. J Neuropathol 1997; 56(3):273-84. 73. Thomas T, Sutton ET, Bryant MW et al. In vivo vascular damage, leukocyte activation and inflammatory response induced by β -amyloid. J Submicrosc Cytol Pathol 1997; 29(3):293-304. 74. Thomas T, Thomas G, McLendo C et al. β-Amyloid-mediated vasoactivity and vascular endothelial damage. Nature 1996; 380:115-8. 75. Blanc EM, Toboreck M, Mark RJ et al. Amyloid β-peptide induces cell monolayer albumin permeability, impairs glucose transport, and induces apoptosis in vascular endothelial cells. J Neurochem 1997; 68(5):1870-81. 76. Coria F, Larrondo-Lillo M, Frangione B. Degeneration of smooth muscle cells in β-amyloid angiopathies. J Neuropathol Exper Neurol 1989; 48:368-75. 77. Kawai M, Kalaria RN, Cras P et al. Degeneration of vascular muscle cells in cerebral amyloid angiopathy of Alzheimer disease. Brain Res 1993; 623:142-46. 78. Vinters HV, Secor DL, Read SL et al. Microvasculature in brain biopsy specimens from patients with Alzheimer’s disease: An immunohistochemical and ultrastructural study. Ultrastruct Path 1994; 18:333-48. 79. Oda T, Wals P, Osterburg HH et al. Clusterin (apoJ) alters the aggregation of amyloid beta-peptide (Aβ1-42) and forms slowly sedimenting Aβ complexes that cause oxidative stress. Exp Neurol 1995; 136:22-31. 80. El Khoury J, Hickman SE, Thomas CA et al. Scavenger receptor-mediated adhesion of microglia to β-amyloid fibrils. Nature 1996; 382:716-9. 81. Christie RH, Freeman M, Hyman BT. Expression of the macrophage scavenger receptor, a multifunctional lipoprotein receptor, in microglia associated with senile plaques in Alzheimer’s disease. Am J Pathol 1996; 148:399-403. 82. Brett J, Schmidt AM, Yan SD et al. Survey of the distribution of a newly characterized receptor for advanced glycation end products in tissues. Am J Pathol 1993; 143:1699-1712 83. Krieger M, Herz J. Structures and functions of multiligand lipoprotein receptors: Macrophage scavenger receptors and LDL receptor-related protein (LRP). Annu Rev Biochem 1994; 63:601-637. 84. Lucarelli M, Gennarelli M, Cardeli R et al. Expression of receptors for native and chemically modified low-density lipoproteins in brain microvessels. FEBS Lett 1997; 401:53-8. 85. Mattson MP, Rydel RE. Amyloid ox-tox transducers. Nature 1996; 382:674-5. 86. Schmidt AM, Hasu M, Popov D et al. Receptor for advanced glycation end products (AGE) has a central role in vessel wall interactions and gene activation in response to circulating AGE proteins. Proc Natl Acad Sci USA 1994; 91:8807-11. 87. Martel CL, Mackic JB, McComb JG et al. Blood-brain barrier uptake of the 40 and 42 amino acid sequences of circulating Alzheimer’s amyloid β in guinea-pigs. Neurosci Lett 1996; 206:157-60. 88. Bading JR, Kirkman E, Mackic JB et al. SPECT imaging of Alzheimer’s amyloid β peptide kinetics in brain of the squirrel monkey. Cereb Vasc Biol Conf March 26-28, Portland 1998.

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89. Zlokovic BV, Wisniewski T, Walker LC et al. On Alzheimer’s amyloid β peptide transport at the blood-brain barrier. J Clin Invest 1997; submitted. 90. Saito Y, Buciak J, Yang J et al. Vector-mediated delivery of 125I-labeled β-amyloid peptide Aβ1-40 through the blood-brain barrier and binding to Alzheimer’s disease amyloid to the Aβ1-40 /vector complex. Proc Natl Acad Sci USA 1995; 92:10227-31. 91. Shayo M, Maness LM, Banks WA et al. Passage of amyloid-beta (1-40) from brain to blood is not significantly different from that of albumin. Soci Neurosci Abstr 1997; 23:539. 92. McComb JG, Zlokovic BV. Cerebrospinal fluid and the blood-brain interface. In: Check WR, Marlin AE, McLone DG et al. Pediatric Neurosurgery: Surgery of the Developing Nervous System. Pennsylvania: W.B. Saunders Company, 1994; 167-84. 93. McGeer PL, Kawamata T, Walker DG. Distribution of clusterin in Alzheimer brain tissue. Brain Res 1992; 579:337-41. 94. Choi-Miura NH, Ihara Y, Kukuchi K et al. SP-40,40 is a constituent of Alzheimer’s amyloid Acta Neuropath 1992; 83:260-4. 95. May PC, Finch CE. Sulfated glycoprotein-2: New relationships of this multifunctional protein to neurodegeneration. TINS 1992; 15:391-6. 96. Pasinetti GM, Cheng HW, Morgan DG et al. Astrocytic responses to striatal deafferentation in male rat. Neuroscience 1993; 53:199-211. 97. Koudinov A, Matsubara E, Frangione B et al. The soluble form of Alzheimer’s amyloid β protein is complexed to high density lipoprotein 3 and very high density lipoprotein in normal human plasma. Biochem Biophys Res Commun 1994; 205:1164-71. 98. Matsubara E, Soto C, Governale S et al. Apolipoprotein J and Alzheimer’s amyloid β solubility. Biochem J 1996; 316:671-9. 99. Bertrand P, Poirier J, Oda T et al. Association of apolipoprotein E genotype with brain levels of apolipoprotein E and apolipoprotein J (clusterin) in Alzheimer disease. Mol Brain Res 1995; 33:174-8. 100. Kounnas MZ, Loukinova EB, Stefansson S et al. Identification of glycoprotein 330 as an endocytic receptor for apolipoprotein J/clusterin. J Biol Chem 1995; 270:13070-5. 101. Dehouck B, Fenart L, Dehouck MP et al. A new function for the LDL receptor: Transcytosis of LDL across the blood-brain barrier. J Cell Biol 1997; 138:877-89. 102. Zheng G, Bachinsky DR, Stamenkovic I et al. Organ distribution in rats of two members of the low-density lipoprotein receptor gene family, gp330 and LRP/alpha 2MR, and the receptor-associated protein (RAP). J Histochem Cytochem 1994; 42:531-42. 103. Chun JT, Wang L, Pasinetti et al. Glycoprotein 330/megalin: Low to moderate mRNA and protein expression in brain microvessels and choroid plexus relative to kidney. submitted. 104. LaDu MJ, Gilligan SM, Lukens JR et al. Nascent astrocyte particles differ from lipoproteins in CSF. J Neurochem 1998; 70(5):2070-81. 105. LaFeria FM, Troncoso JC, Strickland DK et al. Neuronal cell death in Alzheimer’s disease correlates with apoE uptake and intracellular A β stabilization. J Clin Invest 1997; 100(2):310-20. 106. Poduslo JF, Curran GL, Berg C. Macromolecular permeability across the blood-nerve and blood-brain barriers. Proc Natl Acad Sci USA 1994; 91:5705-9. 107. Pardridge WM, Kang YS, Buciak JL et al. Human insulin receptor monoclonal antibody undergoes high affinity binding to human brain capillaries in vitro and rapid transcytosis through the blood-brain barrier in vivo in the primate. Pharm Res 1995; 12(6):807-16. 108. Zlokovic BV. Cerebrovascular permeability to peptides: Manipulations of transport systems at the blood-brain barrier. Pharm Res 1995; 12(10):1395-1406. 109. Farquhar MG, Saito A, Kerjaschki D et al. The Heymann nephritis antigenic complex: Megalin (gp330) and RAP. J Am Soc Nephrol 1995; 6:35-47. 110. Rall SC, Weisgraber KH, Mahley RW. Human apolipoprotein E: The complete amino acid sequence. J Biol Chem 1981; 257:4171-8. 111. Paik YK, Chang DJ, Reardon CA et al. Nucleotide sequence and structure of the human apolipoprotein E gene. Proc Natl Acad Sci USA 1985; 82:3445-51. 112. Newman TC, Dawson PA, Rudel LL et al. Quantitation of apolipoprotein E mRNA in the liver and peripheral tissues of nonhuman primates. J Biol Chem 1985; 260:2452-7.

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113. Poirier J, Hess M, May PC et al. Astrocytic apolipoprotein E mRNA and GFAP mRNA hippocampus after entorhinal cortex lesioning. Mol Brain Res 1991; 11:97-106. 114. Pitas RE, Boyles JK, Lee SH et al. Astrocytes synthesize apolipoprotein E and metabolize apolipoprotein E-containing lipoproteins. Biochim Biophys Acta 1987; 917:148-161. 115. Hussain MM, Bucher NLR, Faris B et al. Tissue-specific post-translation modification of rat apoE synthesis of sialated apoE forms by neonatal rat smooth muscle cells. J Lipid Res 1988; 29:915-23. 116. Driscoll DM, Getz GS. Extrahepatic synthesis of apolipoprotein E. J Lipid Res 1984; 25:1368-74. 117. Pitas RE, Boyles JK, Lee SH et al. Lipoproteins and their receptors in the central nervous system. Characterization of the lipoproteins in cerebrospinal fluid and identification of apolipoprotein B,E (LDL) receptors in the brain. J Biol Chem 1987; 262:14352-60. 118. Hussain MM, Zanni EE, Kelly M, et al. Synthesis, modification and flotation properties of rat hepatocyte apolipoproteins. Biochim Biophys Acta 1989; 101:90-101. 119. Hussain MM, Roghani A, Cladaras C et al. Secretion of lipid poor nascent human apoA-I, apoCIII and apoE by cell clones expressing the corresponding genes. Electrophoresis 1991; 12:273-83. 120. Zannis VI, Just PW, Breslow JL. Human apolipoprotein E isoprotein subclasses are genetically determined. Am J Hum Genet 1981; 33:11-24. 121. Davignon J, Gregg RE, Sing CF. Apolipoprotein E polymorphism and atherosclerosis. Arteriosclerosis 1988; 8:1-21. 122. Handelmann GE, Boyles JK, Weisgraber KH et al. Effects of apolipoprotein E, β-very low density lipoproteins, and cholesterol on the extension of neurites by rabbit dorsal root ganglion neurons in vitro. J Lipid Res 1992; 33:1677-88. 123. Wolf BB, Lopes MB, VandenBerg SR et al. Characterization and immunohistochemical localization of α-2-microglobulin receptor (low density lipoprotein receptor-related protein) in human brain. Am J Pathol 1992; 141:37-42. 124. Kounnas MZ, Loukinova EB, Stefansson S et al. Identification of glycoprotein 330 as an endocytic receptor for apolipoprotein J/clusterin. J Biol Chem 1995; 270:13070-5. 125. Takahashi S, Kawarabayasi Y, Nakai T et al. Rabbit very low density lipoprotein receptor: A low density lipoprotein receptor-like protein with distinct ligand specificity. Proc Natl Acad Sci USA 1992; 89:9252-6. 126. Okuizumi K, Onodera O, Namba Y et al. Genetic association of the very low density lipoprotein (VLDL) receptor gene with sporadic Alzheimer’s disease. Nature Genet 1995; 11:207-9. 127. Wyne KI, Pathak K, Seabra MC et al. Expression of the VLDL receptor in endothelial cells. Arterioscler Thromb Vasc Biol 1996; 16:407-15. 128. Christie RH, Chung H, Rebeck GW et al. Expression of the very low-density lippoprotein receptor in the central nervous system and in Alzheimer’s disease. J Neuropath & Exper Neurol 1996; 55:491-8. 129. Pericak-Vance MA, Bebout JL, Gaskell Jr PC et al. Linkage studies in familial Alzheimer disease: Evidence for chromosome 19 linkage. Am J Hum Genet 1991; 48:1034-50. 130. Strittmatter W, Saunders A, Schmecher D et al. Apolipoprotein E: High-avidity binding to β-amyloid and increased frequency of type 4 allele in late-onset familial Alzheimer’s disease Proc Natl Acad Sci USA 1993; 90:1977-81. 131. Wisniewski, Frangione B. Apolipoprotein E: A pathologic chaperone protein in patients with cerebral and systemic amyloid. Neurosci Lett 1992; 135:235-8. 132. Corder EH, Saunders AM, Risch NJ et al. Protective effect of apolipoprotein E type 2 allele for late-onset Alzheimer disease Nature Genetics 1994; 7:180-4. 133. Saunders AM, Strittmatter WJ, Schmechel D et al. Association of apolipoprotein E allele E4 with late-onset familial and sporadic Alzheimer’s disease. Neurology 1994; 43(8):1467-72. 134. Corder EH, Saunders AM, Strittmatter WJ et al. Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer’s disease in late onset families. Science 1993; 261:921-3.

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135. Premkumar DRD, Cohen DL, Hedera P et al. Apolipoprotein ε-E4 alleles in cerebral amyloid angiopathy and cerebrovascular pathology associated with Alzheimer’s disease. Am J Path 1996; 148:2083-95. 136. Greenberg SM, Rebeck GW, Vonsattel JPG et al. Apolipoprotein E(epsilon)4 and cerebral hemorrhage associated with amyloid angiopathy. Ann Neurol 1995; 38:254-9. 137. Mann DM, Iwatsubo T, Pickering-Brown SM et al. Preferential deposition of amyloid β protein (Aβ) in the form Aβ40 in Alzheimer’s disease is associated with a gene dosage effect of the apolipoprotein E,E4 allele. Neurosci Let 1997; 221:81-4. 138. Tang MX, Stern Y, Marder K et al. The apoE-epsilon4 allele and the risk of Alzheimer disease among African Americans, whites, and Hispanics. JAMA 1998; 279(10):751-5. 139. Naslund J, Thyberg J, Tjemberg LO et al. Characterization of stable complexes involving apoplipoprotein E and the amyloid β peptide in Alzheimer’s disease brain. Neuron 1995;15:219-28. 140. Zhou Z, Smith JD, Greengard P et al. Alzheimer amyloid-β peptide forms denaturantresistant complex with type E3 but not type E4 isoform of native apolipoprotein E. Molecul Med 1996; 2:175-80. 141. LaDu MJ, Falduto MT, Manelli AM et al. Isoform-specific binding of apolipoprotein E to β-amyloid. J Biol Chem 1994; 269:23403-6. 142. LaDu MJ, Pederson TM, Frail DE et al. Purification of apolipoprotein E attenuates isoformspecific binding to β-amyloid. J Biol Chem 1995; 9039-9042 143. Wisniewski T, Golabek A, Matsubara E et al. Apolipoprotein E: Binding to soluble Alzheimer’s β-amyloid. Biochem Biophys Res Commun 1993; 192:359-65. 144. Sanan DA, Weisgraber KH, Russel SJ et al. Apolipoprotein E associates more efficiently than apoE3. J Clin Invest 1994; 94:860-9. 145. Strittmatter WJ, Saunders AM, Goedert M et al. Isoform-specific interactions of apolipoprotein E with microtubule-associated protein tau: Implications for Alzheimer’s disease. Proc Natl Acad Sci 1994; 94:11183-6. 146. Strittmatter W, Roses AD. Apolipoprotein E and Alzheimer disease. Proc Natl Acad Sci USA 1995; 92:4725-7. 147. Huang DY, Goedert M, Jakes R et al. Isoform-specific interactions of apolipoprotein E with the microtubule-associated protein MAP2C: Implications for Alzheimer’s disease. Neurosci Lett 1994; 182:55-8. 148. Strittmatter WJ, Weisgraber KH, Godert M et al. Hypothesis: Microtubule instability and paired helical filament formation in the Alzheimer disease brain are related to apolipoprotein E genotype. Exp Neurol 1994; 125:163-71. 149. Poirier J, Delisle MC, Quirion R et al. Apolipoprotein E4 allele as a predictor of cholinergic deficits and treatment outcome in Alzheimer disease. Proc Natl Acad Sci USA 1995; 92:12260-4. 150. Weisgraber KH, Mahley RE. Human apolipoprotein E: The Alzheimer’s disease connection. FASEB J 1996; 10:1485-94. 151. Nathan BP, Bellosta S, Sanan DA et al. Differential effects of apolipoproteins E3 and E4 on neuronal growth in vitro. Science 1994; 264:850-2. 152. Nathan BP, Chang KC, Bellosta S et al. The inhibitory effect of apolipoproteins E3 and E4 on neuronal growth in vitro. Science 1994; 264:850-2. 153. Bellosta S, Nathan BP, Orth M et al. Stable expression and secretion of apolipoproteins E3 and E4 in mouse neuroblastoma cells produces differential effects on neurite outgrowth. J Biol Chem 1995; 270:27063-71. 154. Arendt T, Schindler C, Bruckner MK et al. Plastic neuronal remodeling is impaired in patients with Alzheimer’s disease carrying apolipoprotein E4 allele. J Neurosci 1997; 17:516-29. 155. Barger SW, Mattson MP. Isoform-specific modulation by apolipoprotein E of the activities of secreted β-amyloid precursor protein. J Neurochem 1997; 69:60-7. 156. Barger SW, Harmon AD. Microglial activation by Alzheimer amyloid precursor protein and modulation by apolipoprotein E. Neurosci Lett 1997; 388:878-81.

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157. Poirier J. Apolipoprotein E in animal models of CNS injury and in Alzheimer’s disease. Trends Neurosci 1994; 17:525-30. 158. Chan W, Fornwald J, Brawner M et al. Native complex formation between apolipoprotein E isoforms and the Alzheimer’s disease peptide Aβ. Biochemistry 1996; 35:7123-30. 159. Castano EM, Prelli F, Golabek R et al. Fibrillogenesis in Alzheimer’s disease of amyloid β peptides and apolipoprotein E. Biochem J 1995; 306:599-604. 160. Evans KC, Berger EP, Cho CG et al. Apolipoprotein E is a kinetic but not a thermodynamic inhibitor of amyloid formation: Implications for the pathogenesis and treatment of Alzheimer’s disease. Proc Natl Acad Sci USA 1995; 92:763-7. 161. Wood SJ, Chan W, Wetzel R. Seeding of Aβ fibril formation is inhibited by all three isotypes of apolipoprotein E. Biochemistry 1996; 35:12623-8. 162. Harper JD, Wong SS, Lieber CM et al. Observation of metastable Aβ amyloid produced by atomic force microscopy. Chemistry & Biology 1997; 4:119-25. 163. Martel CL, Ghiso J, Frangione B, et al. Transport of apolipoproteins across the bloodbrain barrier: Relevance to Alzheimer’s Disease. STP Pharma Sci 1997; 7(1):28-36. 164. Urmoneit B, Prikulis I, Wihl G et al. Cerebrovascular smooth muscle cells internalize Alzheimer amyloid beta protein via a lipoprotein pathway: Implications for cerebral amyloid angiopathy. Lab Invest 1997; 77:157-66. 165. Bales KR, Verina T, Dodel RC et al. Lack of apolipoprotein E dramatically reduces amyloid β-peptide deposition. Nat Genet 1997; 17:263-4.

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CHAPTER 7

Apolipoprotein E and Apolipoprotein J (Clusterin) in the Brain in Alzheimer’s disease Edith G. McGeer, Claudia Schwab and Patrick L. McGeer

Introduction

T

he apolipoproteins are a class of proteins which bind to insoluble fats so as to solubilize them in body fluids. Two members of this class, apolipoprotein E (apoE) and apolipoprotein J (clusterin, SP-40,40, complement lysis inhibitor, TRPM-2, SGP-2) have been shown to be associated with the lesions in the brain in Alzheimer's disease (AD). Additional reasons for interest in these two materials in regard to AD exist. Inheritance of one particular form of apoE, apoE4, is a risk factor for AD. The many names given to clusterin suggest its multiple functions. One of these is to bind the membrane attack complex (the MAC), the terminal complex of the complement cascade, and thus protect host tissue against bystander lysis. The appearance of clusterin in AD brain may, therefore, be partly a protective response against the chronic inflammation which exists in such a brain.1,2 In this chapter, we will review the literature on the appearance of these two apolipoproteins in AD brain.

Association with Lesions in Alzheimer Brains Both apoE and clusterin bind avidly to amyloid, so it is not surprising that both are found in association with amyloid deposits in AD brains. Indeed, some other apolipoproteins, namely apolipoprotein A-I3 and apolipoprotein B,4 have also been reported to be associated with the senile plaques. Apolipoprotein B, like apoE and clusterin, is also reported to be associated with some neurofibrillary tangles (NFTs) in AD brain.4 More detailed descriptions of the localizations reported for clusterin and apoE in AD brain are given in the following sections.

Clusterin

Levels of the mRNA for clusterin5-7 or of the protein itself 8 are increased in extracts from AD hippocampus or cortex as compared with controls. One group9 reported the increases in clusterin levels in the hippocampus and frontal cortex to be proportional to the

Clusterin in Normal Brain Functions and During Neurodegeneration, edited by Caleb E. Finch. ©1999 R.G. Landes Company.

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number of apoE4 alleles, while another3 found a significant decrease in the amount of clusterin to be associated with the apoE4 allele. The reason for this discrepancy is not clear. All immunohistochemical studies with anti-clusterin antibodies on AD cortex and hippocampal tissue have reported positive staining of senile plaques and vascular amyloid.10-13 No reaction was seen in control tissue except for faint staining of residual serum in vessels.10,12 The diffuse deposits of amyloid seen in the striatum and cerebellum of both AD and controls did not stain for clusterin,12 although some diffuse deposits of amyloid in AD cortex were positive.10 We10 found that the staining of dystrophic neurites in senile plaques, neuropil threads and a few intraneuronal NFTs was often stronger than that of the senile plaques (Fig. 7.1). Occasional normal appearing pyramidal neurons and fibers showed punctate staining. Others2 have reported that clusterin immunoreactivity was seen in an unusually high percentage of NFT-free neurons in cortical areas affected in AD but rarely in NFT-containing neurons, supporting a protective role. The staining of dystrophic neurites, neuropil threads and the occasional normal appearing fibers may correspond to the “fibrous background structures” which Takamaru12 reported to be positive for clusterin in AD brain. The staining of dystrophic neurites and neuropil threads in AD brain with the antibody to clusterin is similar to the staining of those structures with antibodies to vitronectin, protectin and the membrane attack complex (MAC) of complement proteins.10 Vitronectin and protectin, like clusterin, bind to components of the MAC and inhibit its insertion into cell membranes. Thus, the similarity of the staining of dystrophic neurites and neuropil threads for the three inhibitors with the staining for MAC may reflect their protective action. Further comparative studies with antibodies to these four materials indicated that amyloid deposits were only stained for clusterin and vitronectin, and the clusterin antibody showed the least tendency of the four to stain NFTs.10 The staining of occasional normal appearing pyramidal neurons with the antibody to clusterin is consistent with the detection, by in situ hybridization, of its mRNA in pyramidal cells of both human6 and rat.13 This is somewhat contrary to the indications of cell culture studies which have been reported to show production by glia but not by neuroblastoma cell lines,14 and to studies in injured rat brain where reactive astrocytes appear to be the major producers.15

Apolipoprotein E In contrast to clusterin, levels of apoE have been reported to be reduced in the frontal cortex and hippocampus in AD, with the reduction variously reported as proportional to the apoE4 allele dose9 or independent of apoE type.16 The mRNA for apoE, on the other hand, has been reported to be significantly more abundant in AD than control brain tissue,17,18 with the amount in AD being decreased in relation to the apoE4 gene dose.17 The upregulation of the mRNA occurs in reactive astrocytes.18 And, astrocytes rich in the apoE gene appear to shift in AD hippocampus from the neuropil to regions with densely packed neurons.19 The discrepancies between reports on the levels of the protein and its mRNA may depend upon the cases or regions of brain used, but need to be clarified by further work. Another possibility is that some of the apoE in AD brain is so tightly bound to amyloid that it does not appear as apoE in the separation of brain extracts. It has already been reported that, in attempts to purify amyloid, a complex of apoE (or apoE fragments) and amyloid purifies as such from AD brain.20,21 Complexes of apoE with soluble Aβ peptides have been found in AD brain supernatants.22 Reports of immunohistochemical studies on AD brain with antibodies to apoE emphasize the staining of plaques and NFTs, but differ somewhat in the extent of such staining. The senile plaques found in aged, nondemented individuals are also variably

apoE and apoJ (Clusterin) in the Brain in Alzheimer's disease

91 Fig. 7.1. Clusterin immunostaining in the entorhinal cortex in Alzheimer's disease (A) and the Parkinson dementia complex of Guam (B). (A) The antibody against clusterin stained many dystrophic neurites, NFTs and amyloid plaques in Alzheimer's disease. (B) In the Parkinson's dementia complex, dystrophic neurites and some NFTs are intensely immunoreactive, while most NFTs remain unstained. Both photomicrographs are at the same magnification; bar in (B) = 100 µm.

immunopositive for apoE.23 Astrocytes,24,25 blood vessels and some neurons are also stained as they are in normal brain.24,26,27 Similar staining of neurons and glial cells for apoE has been found in transgenic mice bearing a human apoE gene, and this pattern differs from the staining seen in wild type mice.28 With regard to the senile plaques, most reports suggest that apoE antibodies label most but not all classic senile plaques (Fig. 7.2) and some diffuse amyloid deposits.29,30 All plaques in the hippocampus, and especially in CA1, have been found positive for apoE, while some in cortical regions were not.31 Aβ plaques showing high apoE immunoreactivity have been reported to be localized in layers II, III and V of the neocortex, with those in layer I being generally unlabeled.32 Yamaguchi et al33 reported that senile plaques were consistently labeled with the apoE antiserum even in early stages of plaque formation. In double staining experiments on temporal cortical samples from AD patients, apoE was present in 83-86% of neuritic plaques but in only about 6% of non-neuritic plaques whether they were of the diffuse or “burned out” type.25 In the neocortex in general, apoE-positive plaques were greater

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Fig. 7.2. Comparison of apoE (A, C) and Aβ (B, D) demonstrated with the E50 antibody against Aβ17-31, immunoreactivity in the CA1 region of the hippocampus in nearby sections of a case of Alzheimer’s disease (A, B) and a case of the Parkinson’s dementia complex of Guam (C, D). (A) The apoE antibody in AD showed intense staining of some senile plaques with lighter staining of tangles. (B) With the antibody to Aβ, plaques in AD tissue are intensely stained but the tangles are not reactive. (C) Many extracellular NFTs are positive with the antibody to apoE in the case of Parkinson’s dementia of Guam (PDC). (D) Only a few of the NFTs in the PDC case show immunoreactivity with the antibody to Aβ. No plaques or diffuse deposits are visible in this field. All photomicrographs are at the same magnification; bar in (D) = 200 µm.

in E4/4 AD cases than in those with other genotypes but, overall, were less numerous than Aβ-positive plaques.35 Apolpoprotein E immunoreactivity was found in association with senile plaques in all brain regions examined but there was marked regional variation in its occurrence in diffuse deposits. Staining has been reported as absent27 or strong in many cerebellar (cf. refs. 11,33,34) diffuse plaques but was absent or weak in such deposits in the striatum and thalamus. (cf. refs. 34,35) It is speculated that the difference is due to the reported presence of small amounts of fibrillar amyloid in the diffuse deposits in the cortex and cerebellum.36 The labeling of diffuse deposits for apoE was more common than for clusterin in the cortex of AD (and Down’s syndrome) cases.11 Using confocal laser scan microscopy, Nishiyama et al37 found a clear difference in the distribution and shape of deposits stained with antibodies to apoE and Aβ. Several Aβ deposits of typical and primitive plaques were often included in one diffuse deposit of apoE. Some apoE deposits did not exhibit any Aβ immunoreactivity, and many typical plaques staining for Aβ had little apoE immunoreactivity (Fig. 7.2). Using a number of different antibodies, Aizawa et al38 found that an amino-terminal truncated apoE was the major form associated with senile plaques.

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There is also some confusion as to the extent of apoE labeling of NFTs. Apolpoprotein E was not found in dystrophic neurites in early stages of NFT formation, suggesting it is probably not primarily involved in neurofibrillary pathology.32 Yamaguchi et al33 reported all extracellular NFTs were also positive for apoE—even those not positive for Aβ, while Namba et al39 implied that only those NFTs having amyloid showed such staining. The staining of extracellular NFTs for apoE is somewhat less intense than the staining of senile plaques (Fig. 7.2).30 In normal brain, antibodies to apoE give widespread staining of the cell bodies and proximal dendrites of neurons.26,40 One report41 mentions an increased density in AD of olfactory receptor neurons staining for apoE. Neurons are also strongly stained with antibodies to the apoE receptor known as the low density lipoprotein receptor-related protein (LRP),31,42,43 while astrocytes are lightly stained.43 Neuronal staining for apoE is decreased in the cortex and hippocampus in AD,31 although apoE-immunoreactive neurites are closely associated with Aβ-containing senile plaques.24 Senile plaques are strongly reactive with antibodies to the LRP.43 Another receptor for apoE, the very low density lipoprotein receptor, is normally also found on neurons, as well as on microglia—both resting and activated. But it occurs particularly on the activated microglia near senile plaques in AD.44

Specificity to Alzheimer’s Disease Upregulation of clusterin and abnormal staining of lesions for apoE is not limited to AD. Brain levels of clusterin seem to be elevated in many conditions involving injury or chronic inflammation of the brain. Elevated levels of the mRNA for clusterin are seen in the hippocampus in Pick’s disease as well as in AD.4 Abnormal staining for clusterin has been seen in dystrophic neurites and some NFTs in the Parkinson’s dementia complex of Guam (Fig. 7.1). It has also been seen in humans in ischemic Purkinje cells which showed the shrunken and pyknotic appearance characteristic of irreversible damage.45 Intense staining for clusterin has been seen in hypertrophic astrocytes in cases of multiple sclerosis, stroke and AIDS encephalitis. In these cases, however, the distribution of clusterin did not appear to correlate with that of the MAC,46 a correlation which does appear to occur in AD.9 Clusterin levels in rat brain neurons appear to increase with aging,47 while the increased levels seen after experimental lesions seem to be in both neurons and astrocytes. Perforant path transection led to the appearance of clusterin in scattered hippocampal neurons as well as extracellularly.48 Excitotoxic lesions of the hippocampus led to a marked increase in the number of clusterin-immunopositive pyramidal neurons within the degenerating CA3 and CA4 pyramidal cell layer6,49 and to an overexpression of the clusterin mRNA.15,49 The overexpression of the mRNA appeared to involve reactive astrocytes. Glutamate treatment of primary hippocampal cell cultures49 or cultures of embryonic rat spinal cord50 also induced increases in clusterin mRNA. Abnormally high levels of the mRNA have also been reported in scrapie-infected hamster brain4 but the type of cell involved was not identified. Treatment of animals with the toxic ricin resulted in upregulation of clusterin in reactive astrocytes and in the axotomized motoneurons.51 Complete cerebral ischemia in the rat led to the accumulation of both apoE and clusterin in neurons exhibiting signs of ischemic damage and in multiple extracellular deposits located close to the microvessels.52 Apolpoprotein E staining has been found associated with the amyloid in various types of human cerebral or systemic amyloidosis,53,54 including the kuru plaques in CreutzfeldtJakob disease,39 and with the mainly extracellular NFTs and occasional diffuse Aβ deposit in the Parkinson’s dementia complex of Guam.30,55 As in AD, not all of the tau-positive tangles were also stained for apoE.55 A comparison of staining for Aβ and apoE in AD and the Parkinson’s dementia complex is shown in Figure 7.2.

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Apolpoprotein E immunoreactivity has also been reported in a few percent of the Pick’s bodies in the brains of two patients with Pick’s disease. Apolpoprotein E-positive inclusions were limited to limbic areas such as the dentate gyrus.56 Nor is the colocalization of apoE with amyloid deposits limited to humans. In aged monkeys,57,58 chimpanzees,59 dogs60 and Microcebus murinus61 apoE is found colocalized with amyloid beta protein in senile plaques and blood vessels. In the last named species, astrocytes and some neurons are also immunostained for apoE, suggesting further parallels to the human. Upregulation of apoE is also found in astrocytes in a mouse model of scrapie where the increase occurs soon after the amyloid-forming abnormal form of the prion protein accumulates in astrocytes.18 An increased abundance of apoE mRNA in astrocytes close to neuronal cell bodies, similar to that seen in AD, has been observed in the hippocampus of rats with unilateral ablation of the entorhinal cortex.19

Possible Mechanisms for Changes in Clusterin and Apolipoprotein E in AD The expression of clusterin is clearly upregulated in AD brain, in several other neurodegenerative diseases, and in brain injury of various types. It has been called an extracellular version of a heat shock protein.62 In many of the conditions where clusterin is upregulated it has been shown that there is an immune response with a profusion of activated microglia and reactive astrocytes in the affected brain regions. This is notably true of AD.1 Upregulation of inflammatory cytokines occurs and some have already been shown to stimulate astrocytic production of clusterin (see chapter 6). Since one of the many functions of clusterin is to complex with the MAC of complement, it is tempting to hypothesize that the excessive production in these conditions may be a protective mechanism against complement attack. In the case of AD, this speculation gains some support from the correspondence of much of the immunostaining for clusterin with that for the MAC.9 Whether or not apoE levels are significantly upregulated in AD brain remains to be determined. It seems clear, however, that there is some dislocation of the apoE from a predominantly neuronal and astrocytic localization to one also involving also senile plaques and NFTs. The association of apoE with senile plaques and diffuse amyloid deposits is usually attributed to the tight binding of apoE to amyloid. Another factor that may play a role is the appearance of the LRP in senile plaques. The LRP is also known as the α2-macroglobulin receptor. It is a multifunctional receptor which binds and rapidly internalizes at least 7 ligands, and all of these, including apoE, accumulate on senile plaques in AD.63 The appearance of apoE immunoreactivity on NFTs was originally thought to indicate the presence of some amyloid in such tangles. However, apoE has been shown to bind to tau64 (and to microtubulin associated protein 2c),65 and such binding may account for the appearance of apoE on some tangles. If so, the apoE present may well be the apoE3 form, since this has been shown to bind to both tau and MAP2c more avidly than the apoE4 form.

Acknowledgment Work in the Kinsmen Laboratory on Alzheimer's disease is supported by grants from the Alzheimer Society of B.C. and the Jack Brown and Family A.D. Research Fund, as well as donations from individual British Columbians.

References 1. McGeer PL, McGeer EG. The inflammatory response system of brain: Implications for therapy of Alzheimer and other neurodegenerative diseases. Brain Res Rev 1995; 21:195-218.

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2. Giannakopoulos P, Kovari E, French LE et al. Possible neuroprotective role of clusterin in Alzheimer’s disease: A quantitative immunocytochemical study. Acta Neuropathol 1998; 95:387-94. 3. Harr SD, Uint L, Hollister R et al. Brain expression of apolipoproteins E, J and A-I in Alzheimer’s disease. J Neurochem 1996; 66:2429-35. 4. Namba Y, Tsuchiya H, Ikeda K. Apolipoprotein B immunoreactivity in senile plaque and vascular amyloids and neurofibrillary tangles in the brain of patients with Alzheimer’s disease. Neurosci Lett 1992; 134:264-6. 5. Duguid JR, Bohmont CW, Liu N et al. Changes in gene expression shared by scrapie and Alzheimer’s disease. Proc Natl Acad Sci USA 1989; 86:7260-4. 6. May PC, Lampert-Etchells M, Johnson SA et al. Dynamics of gene expression for a hippocampal glycoprotein elevated in Alzheimer’s disease and in response to experimental lesions in rat. Neuron 1990; 5:831-9. 7. May PC, Johnson SA, Poirier J et al. Altered gene expression in Alzheimer’s disease brain tissue. Can J Neurol Sci 1989; 16:473-6. 8. Oda T, Pasinetti GM, Osterburg HH et al. Purification and characterization of brain clusterin. Biochem Biophys Commun 1994; 204:1131-6. 9. Bertrand P, Poirier J, Oda T et al. Association of apolipoprotein E genotype with brain levels of apolipoprotein E and apolipoprotein J (clusterin) in Alzheimer's disease. Mol Brain Res 1995; 33:174-8. 10. McGeer PL, Kawamata T, Walker DG. Distribution of clusterin in Alzheimer brain tissue. Brain Res 1992; 579:337-41. 11. Kida E, Choi-Miura NH, Wisniewski KE. Deposition of apolipoproteins E and J in senile plaques is topographically determined in both Alzheimer’s disease and Down’s syndrome brain. Brain Res 1995; 685:211-6. 12. Takamaru Y. Analysis of the 80 kDa antigen in senile plaque amyloid. Hokkaido J Med Sci 1994; 69:191-201. 13. Pasinetti GM, Johnson SA, Oda T et al. Clusterin (SGP-2): A multifunctional glycoprotein with regional expression in astrocytes and neurons of the adult rat brain. J Comp Neurol 1994; 339:387-400. 14. Choi-Miura NH, Ihara Y, Fukuchi M et al. SP-40,40 is a constituent of Alzheimer’s amyloid. Acta Neuropathol 1992; 83:260-4. 15. Danik M, Chabot JG, Hassan-Gonzalez D et al. Localization of sulfated glycoprotein-2/ clusterin mRNA in the rat brain by in situ hybridization. J Comp Neurol 1993; 334:209-27. 16. Pirttila T, Soininen H, Heinonen O et al. Apolipoprotein E (apoE) levels in brains from Alzheimer's disease patients and controls. Brain Res 1996; 722:71-7. 17. Yamada T, Kondo A, Takamatsu J et al. Apolipoprotein E mRNA in the brains of patients with Alzheimer’s disease. J Neurol Sci 1995; 129:56-61. 18. Diedrich JF, Minnigan H, Carp RI et al. Neuropathological changes in scrapie and Alzheimer’s disease are associated with increased expression of apolipoprotein E and cathepsin D in astrocytes. J Virol 1991; 65:4759-68. 19. Zarow C, Vitoroff J. Increased apolipoprotein E mRNA in the hippocampus in Alzheimer's disease and in rats after entorhinal cortex lesioning. Exp Neurol 1998; 149:79-86. 20. Naslund J, Thyberg J, Tjernberg LO et al. Characterization of stable complexes involving apolipoprotein E and the amyloid beta peptide in Alzheimer’s disease brain. Neuron 1995; 15:219-28. 21. Wisniewski T, Lalowski M, Golabek A et al. Is Alzheimer’s disease an apolipoprotein E amyloidosis? Lancet 1995; 345:956-8. 22. Permanne B, Perez C, Soto C et al. Detection of apolipoprotein E dimeric soluble amyloid beta complexes in Alzheimer’s disease brain supernatants. Biochem Biophys Res Commun 1997; 240:715-20. 23. Ogeng’o JA, Cohen DL, Sayi JG et al. Cerebral amyloid beta protein deposits and other Alzheimer lesions in nondemented elderly east Africans. Brain Pathol 1996; 6:101-7.

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24. Han S-H, Hulette C, Saunders AM et al. Apolipoprotein E is present in hippocampal neurons without neurofibrillary tangles in Alzheimer’s disease and in age-matched controls. Exp Neurol 1994; 128:13-26. 25. Sheng JG, Mrak RE, Griffin WST. Apolipoprotein E distribution among different plaque types in Alzheimer’s disease: Implications for its role in plaque progression. Neuropath Appl Neurobiol 1996; 22:334-41. 26. Han S-H, Einstein G, Weisgraber KH et al. Apolipoprotein E is localized to the cytoplasm of human cortical neurons: A light and electron microscopic study. J Neuropath Exp Neurol 1994; 53:535-44. 27. Styren SD, Kamboh MI, Dekosky ST. Expression of differential immune factors in temporal cortex and cerebellum: The role of alpha-1-antichymotrypsin, apolipoprotein E, and reactive glia in the progression of Alzheimer’s disease. J Comp Neurol 1998; 396:511-20. 28. Xu PT, Schmechel D, Rothrock-Christian T et al. Human apolipoprotein E2, E3 and E4 isoform-specific transgenic mice: Human-like pattern of glial and neuronal immunoreactivity in central nervous system not observed in wild type mice. Neurobiol Dis 1996; 3:229-45. 29. Uchihara T, Duyckaerts C, Lazarini F et al. Inconstant apolipoprotein E (apoE)-like immunoreactivity in amyloid beta protein deposits: Relationship with APOE genotype in aging brain and Alzheimer’s disease. Acta Neuropathol 1996; 92:180-5. 30. Schwab C, Steele JC, Akiyama H et al. Distinct distribution of apolipoprotein E and β-amyloid immunoreactivity in the hippocampus of Parkinson dementia complex of Guam. Acta Neuropathol 1996; 92:378-85. 31. Thal DR, Glas A, Schneider W et al. Differential pattern of beta-amyloid, amyloid precursor protein and apolipoprotein E expression in cortical senile plaques. Acta Neuropathol 1997; 94:255-65. 32. Dickson TC, Saunders HL, Vickers JC. Relationship between apolipoprotein E and the amyloid deposits and dystrophic neurites of Alzheimer’s disease. Neuropath Appl Neurobiol 1997; 23:483-91. 33. Yamaguchi H, Ishiguro K, Sugihara S et al. Presence of apolipoprotein E on extracellular neurofibrillary tangles and on meningeal blood vessels precedes the Alzheimer β-amyloid deposition. Acta Neuropathol 1994; 88:413-9. 34. Shao YP, Gearing M, Mirra SS. Astrocyte-apolipoprotein E associations in senile plaques in Alzheimer's disease and vascular lesions: A regional immunohistochemical study. J Neuropath Exp Neurol 1997; 56:376-81. 35. Gearing M, Schneider JA, Robbins RS et al. Regional variation in the distribution of apolipoprotein E and Aβ in Alzheimer’s disease. J Neuropath Exp Neurol 1995; 54:833-41. 36. Kida E, Golabek AA, Wisniewski T et al. Regional differences in apolipoprotein E immunoreactivity in diffuse plaques in Alzheimer’s disease brain. Neurosci Lett 1994; 167:73-6. 37. Nishiyama E, Iwamoto N, Ohwada J et al. Distribution of apolipoprotein E in senile plaques in brains with Alzheimer’s disease: Investigation with the confocal laser scan microscopy. Brain Res 1997; 750:20-4. 38. Aizawa Y, Fukatsu R, Takamaru Y et al. Amino-terminus truncated apolipoprotein E is the major species in amyloid deposits in Alzheimer’s disease-affected brains: A possible role for apolipoprotein E in Alzheimer’s disease. Brain Res 1997; 768:208-14. 39. Namba Y, Tomonaga M, Kawasaki H et al. Apolipoprotein E immunoreactivity in cerebral amyloid deposits and neurofibrillary tangles in Alzheimer’s disease and kuru plaque amyloid in Creutzfeldt-Jakob disease. Brain Res 1991; 541:163-6. 40. Metzger RE, LaDu MJ, Pan JB et al. Neurons of the human frontal cortex display apolipoprotein E immunoreactivity: Implications for Alzheimer’s disease. J Neuropath Exp Neurol 1996; 55:372-80. 41. Yamagishi M, Takami S, Getchell ML et al. Increased density of olfactory receptor neurons immunoreactive for apolipoprotein E in patients with Alzheimer’s disease. Ann Otol, Rhinol Laryngol 1998; 107:421-6.

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42. Tooyama I, Kawamata T, Akiyama H et al. Subcellular localization of the low density lipoprotein receptor-related protein (a2-macroglobulin receptor) in human brain. Brain Res 1995; 691:235-8. 43. Rebeck GW, Reiter JS, Strickland DK et al. Apolipoprotein E in sporadic Alzheimer’s disease: Allelic variations and receptor interactions. Neuron 1993; 11:575-80. 44. Christie RH, Chung H, Rebeck GW et al. Expression of the very low-density lipoprotein receptor (VLDL-R), an apolipoprotein E receptor, in the central nervous system and in Alzheimer’s disease. J Neuropath Exp Neurol 1996; 55:491-8. 45. Yasuhara O, Aimi Y, Yamada T et al. Clusterin as a marker for ischaemic Purkinje cells in human brain. Neurodegeneration 1994; 3:325-9. 46. Wu E, Brosnan CF, Raine CS. SP-40,40 immunoreactivity in inflammatory CNS lesions displaying astrocyte/oligodendrocyte interactions. J Neuropath Exp Neurol 1993; 52:129-34. 47. Senut MC, Jazat F, Choi NH et al. Protein SP40, 40-like immunoreactivity in the rat brain: Progressive increase with age. Eur J Neurosci 1992; 4:917-28. 48. Johnson SA, Young-Chan CS, Laping NJ et al. Perforant path transection induces complement C9 deposition in rat hippocampus. Exp Neurol 1996; 138:198-205. 49. Rozovsky I, Morgan TE, Willoughby DA et al. Selective expression of clusterin (SGP-2) and complement C1qB and C4 during responses to neurotoxins in vivo and in vitro. Neuroscience 1994; 62:741-85. 50. Messmer-Joudrier S, Sagot Y, Mattenberger L et al. Injury-induced synthesis and release of apolipoprotein E and clusterin from rat neural cells. Eur J Neurosci 1996; 8:2652-61. 51. Tornqvist E, Liu L, Aldskogius H et al. Complement and clusterin in the injured nervous system. Neurobiol Aging 1996; 17:695-705. 52. Kida E, Pluta R, Lossinsky AS et al. Complete cerebral ischemia with short-term survival in rat induced by cardiac arrest. II. Extracellular and intracellular accumulation of apolipoproteins E and J in the brain. Brain Res 1995; 674:341-6. 53. Wisniewski T, Frangione B. Apolipoprotein E: A pathological chaperone protein in patients with cerebral and systemic amyloid. Neurosci Lett 1992; 135:235-8. 54. Charge SB, Esiri MM, Bethune CA et al. Apolipoprotein E is associated with islet amyloid and other amyloidoses: Implications for Alzheimer’s disease. J Pathol 1996; 179:443-7. 55. Buee L, Perez-Tur J, Leveugle B et al. Apolipoprotein E in Guamanian amyotrophic lateral sclerosis/parkinsonism-dementia complex: Genotype analysis and relationships to neuropathological changes. Acta Neuropathol 1996; 91:247-53. 56. Hayashi S, Eakabayashi K, Iwanaga K et al. Pick’s disease: Selective occurrence of apolipoprotein E-immunoreactive Pick bodies in the limbic system. Acta Neuropathol 1998; 95:1-4. 57. Poduri A, Gearing M, Rebeck GW et al. Apolipoprotein E and beta amyloid in senile plaques and cerebral blood vessels of aged rhesus monkeys. Am J Pathol 1994; 144:1183-7. 58. Hartig W, Bruckner G, Schmidt C et al. Colocalization of beta-amyloid peptides, apolipoprotein E and glial markers in senile plaques in the prefrontal cortex of old rhesus monkeys. Brain Res 1997; 751:315-22. 59. Gearing M, Rebeck GW, Hyman BT et al. Neuropathology and apolipoprotein E profile of aged chimpanzees: Implications of Alzheimer's disease. Proc Natl Acad Sci USA 1994; 91:9382-6. 60. Uchida K, Kuroki K, Yoshino T et al. Immunohistochemical study of constituents other than beta-protein in canine senile plaques and cerebral amyloid angiopathy. Acta Neuropathol 1997; 93:277-84. 61. Calenda A, Jallageas V, Silhol S et al. Identification of a unique apolipoprotein E allele in Microcebus murinus; apoE brain distribution and colocalization with beta-amyloid and tau proteins. Neurobiol Dis 1995; 2:269-76. 62. Michel D, Chatelain G, North S et al. Stress-induced transcription of the clusterin/Apo J gene. Biochem J 1997; 328:45-50. 63. Rebeck GW, Harr SD, Strickland SK et al. Multiple, diverse senile-plaque associated proteins are ligands of an apolipoprotein E receptor, the alpha 2-macroglobulin receptor/low density-lipoprotein receptor-related protein. Ann Neurol 1995; 37:211-7. 64. Strittmatter WJ, Saunders AM, Goedert M et al. Isoform-specific interactions of apolipoprotein E with microtubule-associated protein tau: Implications for Alzheimer's disease. Proc Natl Acad Sci USA 1994; 91:11183-6.

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CHAPTER 8

Clusterin in Models of Central and Peripheral Injury and for Ischemia and Trauma Håkan Aldskogius, Li Liu and Mikael Svensson

A

n increased expression of clusterin and clusterin mRNA in the CNS occurs in a wide range of insults to the nervous system. The cellular localization and temporal characteristics of the increase show great variability. Injury of peripheral motor axons or axons of intrinsic CNS neurons causes rapid and transient upregulation of clusterin and its mRNA in axotomized neuronal perikarya and perineuronal astrocytes. Wallerian degeneration in the CNS causes a delayed and prominent increase in white matter oligodendrocytes in the degeneration zone. Direct lesions (contusion or stab injury) of the CNS induce a multicellular clusterin upregulation, involving affected nerve cell bodies, axon stumps, reactive astrocytes and—with time—oligodendrocytes. Cerebral ischemia represents a condition in which clusterin upregulation appears to occur mainly in astrocytes. This multitude of conditions with different pathogenetic backgrounds suggest that the increased clusterin expression is an indicator of disturbed CNS homeostasis. In addition, the pattern and cellular localization of clusterin expression is compatible with one or more of the properties of this multifunctional protein, such as complement regulation, lipid transport and cell–cell interactions. It is still unclear, however, whether clusterin upregulation in the models discussed here is involved in regenerative or degenerative processes.

Introduction One of the most challenging issues in neurobiology is to understand the mechanisms which underlie the potential for repair after various nervous system insults. These insults range from anatomically discrete conditions, such as transection or crushing of a peripheral nerve, to the massive destruction of brain or spinal cord tissue which may follow upon ischemia or contusion injuries. These different conditions of insult display a wide range of cellular and molecular complexity (Fig. 8.1). Interestingly, they are all associated with a distinct and often very pronounced upregulation of clusterin expression. As will be seen, this expression sometimes also has somewhat bewildering temporal characteristics and cellular localization. These circumstances are undoubtedly compatible with the presence of a more or less unique basis for clusterin upregulation and function in each individual situation. Nevertheless, until otherwise is proven it seems worthwhile to search for possible common regulating factors of clusterin expression and some common elements in the function(s) of clusterin in these conditions. Clusterin in Normal Brain Functions and During Neurodegeneration, edited by Caleb E. Finch. ©1999 R.G. Landes Company.

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Peripheral Nerve Injury Produces an Increased Expression of Clusterin in CNS Neuronal Perikarya The prototypic experimental model for exploring the neuron-glial responses following axon injury is the motoneuron nuclei of cranial or spinal nerves. Shortly after axotomy, the neurons express many features of dedifferentiation, downregulating functions associated with synaptic transmission and upregulating growth-associated processes.1,2 These modifications are believed to enhance regrowth of the injured axons. Motoneurons already express low levels of immunoreactivity for clusterin from birth. These levels tend to increase during early postnatal development (Tao, Aldskogius, unpublished observations).3 One-two weeks after injury of motor axons in adult animals, clusterin immunoreactivity and labeling for its mRNA are increased (Fig. 8.2).2,4 This upregulation does not occur uniformly in the population of axotomized motoneurons but predominate in regionally distinct groups. Under the same lesioning conditions in immature rats immunoreactivity for clusterin appeared to be increased a few days after injury in virtually the entire population of affected motoneurons (Tao, Aldskogius, unpublished observations). These two experimental situations show important differences with respect to neuronal vulnerability. Neonatal injury produces rapid and extensive nerve cell death, but adult injury produces little nerve cell death and that only after a protracted postoperative survival time. Injury of peripheral sensory axons results in similar structural and molecular modifications in the pseudounipolar sensory neurons located in dorsal root ganglia or in sensory ganglia of cranial nerves as in motoneurons. Neurons in sensory ganglia express variable but often relatively high levels of clusterin protein and mRNA (Pfaller, Liu, Aldskogius, unpublished observations). These levels do not seem to change after axon injury. Interestingly, however, a group of corresponding pseudounipolar sensory neurons with the peculiar feature of being located within the CNS, i.e., the mesencephalic trigeminal nucleus, show a distinct upregulation of clusterin protein and clusterin mRNA (Pfaller, Liu, Aldskogius, unpublished observations). From these observations we conclude that peripheral nerve injury produces an increased expression of clusterin in neurons situated in the central, but not in the peripheral nervous system. Furthermore, our findings show that there is no distinct relationship between clusterin upregulation and neuronal death or survival after axotomy.

Peripheral Nerve Injury Produces an Increased Expression of Clusterin in Perineuronal Astrocytes Concomitantly with the response of axotomized motoneurons, astrocytes in their immediate surrounding undergo hypertrophy and express several molecular features of increased activity.1 A structural modification which may be of particular significance for the forthcoming discussion is an increased astrocytic coverage of the axotomized perikarya. This process is probably the consequence of an extensive stripping of presynaptic terminals from the surface of the injured nerve cell bodies, perhaps transiently exposing areas of the neuronal membrane to the extracellular space. Immunohistochemical preparations demonstrate a distinct increase in clusterin staining intensity in astrocytes in the vicinity of axotomized motoneurons2 and mesencephalic trigeminal nucleus neurons (Pfaller, Liu, Aldskogius, unpublished observations). Similarly, in the spinal cord dorsal horn where primary sensory fibers terminate, astrocytes increase their expression of clusterin and clusterin mRNA following peripheral nerve injury.4 In all these situations the increased immunoreactivity appears to reasonably well match the astrocytic hypertrophy following peripheral nerve injury. With in situ hybridization for clusterin mRNA, the neuropil labeling is typically relatively weak compared to that of the

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Fig. 8.1. Cartoon summarizing the localization of clusterin upregulation following various types of injury, brain contusion (arrow), lesion (//) of a pathway in the central nervous system (tractotomy) or axotomy (//)of peripheral motor and sensory axons. The different cell types displaying clusterin upregulation are symbolized in the following way: neurons (), astrocytes (▲), oligodendrocytes (●), axon stumps (✳).

neurons, and a possible increase in perineuronal labeling is difficult to assess. Taken together, these findings are compatible with the conclusion that astrocytic clusterin is upregulated, but not exceeding the general upregulation associated with the axotomy-induced astrocytic response.

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Injury of Central Neural Pathways Also Produces an Increased Expression of Clusterin in Axotomized Neuronal Perikarya and Perineuronal Astrocytes Injury of axons confined to the CNS results in a response by the injured neurons, which in many ways is fundamentally different from that displayed by axotomized motoneurons. Recent experiments in which the descending rubrospinal tract was transected in the cervical spinal cord show that the neurons in the red nucleus, which give rise to this pathway, transiently and markedly increase their expression of clusterin protein and clusterin mRNA (Liu, Svensson, Aldskogius, unpublished observations). Although less prominent, we also observed an upregulation of these molecules in the perineuronal astrocytes. Interestingly, this upregulation occurs in the absence of astrocytic hypertrophy, indicating that this response is uncoupled from the regulation of clusterin expression in the red nucleus.

Wallerian Degeneration of Nerve Fibers in the CNS Is Accompanied by Increased Expression of Clusterin in Astrocytes and Oligodendrocytes In peripheral nerves Wallerian degeneration is associated with proliferation and changes in Schwann cell phenotype as well as recruitment and activation of macrophages. In combination with the extracellular matrix of the peripheral nerve, these reactions create favorable conditions for axon regeneration. We have not observed clusterin expression along the degenerating nerve at any time point after injury. In central pathways, Wallerian degeneration creates distinctly different non-neuronal responses, which involve clusterin in two apparently unrelated ways. Along degenerating CNS pathways and in their termination territories there is proliferation of microglia and hypertrophy of astrocytes.1 Microglia gradually become phagocytes which incorporate and remove nerve fiber debris. Subsequently, tightly packed astrocytic processes replace the lost nerve fibers and develop a glial “scar”. In the area where axon terminals degenerate, astrocytes may even undergo limited proliferation. In the zone of degenerating white matter these responses are accompanied by increased levels of clusterin and clusterin mRNA in astrocytes.5 The regulation of clusterin levels in terminal areas appears to be variable. Degeneration of hippocampal afferents,6,7 primary sensory afferents to the spinal cord5 and cortical afferents to the striatum8-10 is associated with an increase in clusterin, while nigrostriatal degeneration lacks this.10 These upregulations appear to match the general elevation of astrocytic activity. We hypothesize that clusterin plays a similar role in this injury situation to that in the perineuronal astrocytes which respond to a peripheral nerve injury (see below). With the progression of Wallerian degeneration in the white matter, high levels of clusterin and clusterin mRNA are gradually expressed de novo in profiles which we could identify as oligodendrocytes (Fig. 8.3).5 It should be noted that these expressions appear late and increase in intensity with increasing postlesion survival time. During this period, removal of the degenerating nerve fiber debris by microglia is already underway. At some (unknown) stage prior to this removal the connections are interrupted between oligodendrocytes and their myelin internodes. This event may create a critical change in the functional state of the oligodendrocytes. Results from previous studies in other systems or species indicate that a significant fraction of oligodendrocytes in degenerating white matter degenerate (Aldskogius, Arvidsson, 1989).11 This process may be the one triggering the induction of clusterin.

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Fig. 8.2. In situ hybridization with a (32S)-labeled oligonucleotide probe for clusterin mRNA in the L4 spinal cord segment one week following unilateral transection of the sciatic nerve in the adult rat. Note the increased labeling over the ventral and dorsal horns reflecting upregulation in ventral horn motoneurons as well as in astrocytes around the axotomized motoneurons and in the projection territory of the peripherally axotomized primary sensory neurons (right). Bar = 500µm.

Neuronal and Glial Cell Structures at a Lesion Site in the CNS Express High Levels of Clusterin Direct injuries of the brain or spinal cord can be produced, e.g., by dropping a defined weight onto the surface of the CNS in order to mimic a contusion injury or by making a “stab wound”, such as transection of the spinal cord. Both of these lesions create a central area of primary injury and physical breakdown of the blood-brain barrier. Here a core of neurons and glial cells undergo acute degeneration which produces short- and long-term glial responses. Surrounding this area there will be other complex secondary changes involving neurons as well as glial cells, i.e., astrocytes and microglia/macrophages. Complement may play an important role in the development of these secondary changes. Activation of the complement cascade influences microglia to develop into powerful phatocytic cells and leads to the generation of its cytotoxic end product, the terminal complex of complement or membrane attack complex (MAC). There is evidence for complement activation within a few days after the contusion injury in the “penumbra”, i.e., the ischemic zone surrounding the primary lesion area.12 Concomitantly, there is an upregulation of clusterin and clusterin mRNA in astrocytes and some neurons close to the lesion, while other neurons express only low levels of the protein (Fig. 8.4). These variable levels of clusterin expression may reflect differential capability by the affected neurons to cope with complement “attack”.

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Fig. 8.3. Large numbers of clusterin immunoreactive profiles are present in the lumbar dorsal column three months after ipsilateral transection of the L4-L6 dorsal roots in the adult rat (A). Double labeling with a mouse monoclonal antibody to clusterin and a polyclonal antibody specific for oligodendrocytes (transferrin) shows many colabeled profiles (B). Bar = 50 µm.

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Fig. 8.4. Frontal section through a brain contusion injury showing a marked upregulation of clusterin mRNA adjacent to the actual lesion in the cerebral cortex. Bar = 3 µm.

Fig. 8.5. The lesion site in the spinal cord two days after spinal cord hemi– section. Heavily clusterin immunoreactive profiles of different sizes are present in the lateral funiculus. Double labeling experiments (not shown here) revealed that the large, spherical profiles represent swollen axons, while the smaller, irregular clusterin positive profiles were immunoreactive for the astrocytic marker glial fibrillary acidic protein. Bar = 100 µm.

Perhaps the most remarkable injury-associated clusterin expression reported so far occurs at the site of a direct white matter injury. Within a few days after injury of the spinal cord white matter, the stumps of the injured axons become markedly enlarged, probably as a result of accumulation of intra-axonally transported organelles and other cytoplasmic components. The axoplam of these enlargements display homogeneous expression of high levels of clusterin (Fig. 8.5). However, one week after injury this particular expression has already disappeared. Strong clusterin expression in astrocytes in the lesion area persists for months, however (Liu, Svensson, Aldskogius, unpublished observations). The source of the intra-axonal clusterin is not entirely clear. Increased expression for clusterin mRNA has been demonstrated with in situ hybridization but appears to be mainly associated with non-neuronal cells (Liu, Svensson, Aldskogius, unpublished observations). The axonal clusterin therefore seems to have accumulated as a result of intra-axonal transport from distant sites of synthesis. It is intriguing that injury to peripheral motor axons

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does not result in a similar axoplasmic deposition of clusterin (Liu, Svensson, Aldskogius, unpublished observations). Clusterin mRNA has been shown at the site of a peripheral nerve lesion, however, although the cellular source of this induction was not defined.13

Clusterin Expression Following CNS Ischaemia Appears to Be Primarily Astrocyte–Associated A restricted period of cerebral ischemia induced by occlusion of cerebral blood vessels results in a marked increase in the expression of clusterin and its mRNA a few days after the insult.14-17 This increase is found in specific anatomical regions known to be particularly susceptible to ischemic insults, such as the hippocampus and the striatum. Clusterin expression in ischemia appears to be largely or entirely associated with astrocytes which also respond to this injury with a prominent increase in GFAP and its mRNA. Neuronal clusterin expression in degenerating Purkinje cells has been reported in human autopsy material following hypoxia.17 This expression pattern appears to be a consequence of clusterin secretion by astrocytes and subsequent uptake by degenerating neurons.18,19

Possible Functional Implications of Clusterin Expression Following Acute Insults of the Nervous System From the foregoing review it is clear that clusterin is regularly upregulated in astrocytes and frequently in neurons as well in the early phases following a number of acute insults to the nervous system. In addition, a delayed but prominent response is evident in oligodendrocytes under specific conditions. The available data on expression patterns and cellular localizations give very limited clues to understanding the possible functional role of clusterin in the conditions described here. A general problem in these conditions is to define whether clusterin has a role in the ongoing degenerative or regenerative processes, or in both. The discussion below serves to suggest possible roles of clusterin based on its previously demonstrated variety of functional properties.20 While clusterin in many other tissues and in many other CNS disorders appears to be strongly associated to cell degeneration, the diverse pattern of changes in clusterin expression following injury or ischemia suggest a role in less dramatic events. The earliest increase in clusterin expression is observed following traumatic or ischemic injury to the CNS. In trauma the upregulation occurs in astrocytes and neurons. We suggest that this increased expression of clusterin has a role in the early phases of structural rearrangements within affected cells, in their mutual interactions or interactions with other cells, in particular microglia/macrophages. These cells, which rapidly invade the lesion area, are able to secrete a variety of powerful mediators, including complement. One of the roles of clusterin could therefore be to moderate the effect of complement on lesioned but surviving neurons. This effect could be mediated by neurons as well as astrocytes, either as cellbound or secreted clusterin.21 As a result of the injury, there is extensive axonal remodeling, involving breakdown of axon stabilizing elements as well as generating the basis for a sprouting reaction, although this is typically abortive in the CNS. The rapid induction of clusterin in the axons at the site of injury could be important for molecular rearrangements such as lipid transfer in the structural modification of the injured axons. The puzzling role of clusterin in CNS insults is highlighted in ischemia. This condition is associated with a marked astrocytic upregulation of clusterin expression, which is therefore regarded as associated with ischemic neurodegeneration.14 While substantial and general neuroprotection can be obtained by treatment of experimental cerebral ischemia with antioxidants, clusterin upregulation is counteracted only in selected brain regions.15 This discrepancy is additionally intriguing in the light of the downregulation of GFAP expres-

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sion brought about by antioxidant treatment in all brain areas affected by neuronal degeneration. Axotomy at a distance from the injured neuronal perikarya produce a regular pattern of clusterin upregulation in the affected neurons and surrounding astrocytes (Liu, Svensson, Aldskogius, unpublished observations).2 These responses show little correlation to nerve cell death. Besides significant intracellular modifications in both cell types, there is also a marked increase in their interactions, the most striking one being an increased astrocyticneuronal cell membrane apposition.1 Clusterin may be involved in mediating this process.22 Since Wallerian degeneration of CNS fiber tracts activates astrocytes, an early upregulation of astrocytic clusterin is expected. A surprising observation is, however, the late and intense induction of clusterin and its mRNA in oligodendrocytes located in the degenerating zone. Most probably this induction reflects the fact that degenerating CNS axons can not regrow and become remyelinated. This situation may result in a state of permanent “target deprivation” and survival stress for oligodendrocytes which were associated with the lost myelin internodes. Since these cells do not express any clusterin under physiological conditions, CNS Wallerian degeneration could provide a useful in vivo experimental model for elucidating the role of clusterin in cell survival or death in the CNS.23,24 Undoubtedly, clusterin has attracted considerable interest in Alzheimer’s disease and in experimental models relevant for this disease: this review underscores the potential significance of this molecule for a wide variety of pathological conditions in the CNS. From the present descriptive stage, we now need to explore the experimental models discussed here with biological, genetic, molecular and pharmacological probes which specifically interfere with clusterin function.

Acknowledgments The authors’ work was supported by the Swedish Medical Research Council, project 5420, and Alzheimer Föreningen.

References 1. Aldskogius H, Kozlova EN. Central neuron–glial and glial–glial interactions following axon injury. Progr Neurobiol 1998; 55:1-26. 2. Svensson M, Liu L, Mattsson P et al. Evidence for activation of the terminal pathway of complement and upregulation of sulfated glycoprotein (SGP)-2 in the hypoglossal nucleus following peripheral nerve injury. Chem Molec Neuropathol 1995; 24:53-68. 3. O’Bryan MK, Chema SS, Bartlett PF et al. Clusterin levels increase during neuronal development. J Neurobiol 1993; 24:421-32. 4. Liu L, Törnqvist E, Mattsson P, Eriksson NP et al. Complement and clusterin in the spinal cord dorsal horn and gracile nucleus following sciatic nerve injury in the adult rat. Neurosci 1995; 68:167-179. 5. Liu, L, Persson, JKE, Svensson et al. Glial cell responses, complement and clusterin in the central nervous system following dorsal root transection. Glia 1998; 6. Lampert–Ethcells M, McNeill TH, Laping NJ et al. Sulfated glycoprotein-2 is increased in rat hippocampus following entorhinal cortex lesioning. Brain Res 1991; 563:101-6. 7. Johnson SA, Young–Chan CS, Laping NJ et al. Perforant path transection induces complement C9 deposition in hippocampus. Exp Neurol 1996; 138:198-205. 8. Pasinetti GW, Cheng HW, Morgan DG et al. Astrocytic messenger RNA responses to striatal deafferentation in male rat. Neuroscience 1993; 53:199-211. 9. Cheng HW, Jiang T, Brown SA et al. Response of striatal astrocytes to neuronal differentiation: An immunocytochemical and ultrastructural study. Neuroscience 1994; 62:425-39. 10. Schauwecker PE, Cogen JP, Jiang T et al. Differential regulation of astrocyte mRNAs in the rat striatum after lesions of the cortex or substantia nigra. Exp Neurol 1998; 149:87-96.

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11. Franson P. Quantitative electron microscopic observations on the non–neuronal cells and lipid droplets in the posterior funiculus of the cat after dorsal rhizotomy. J Comp Neurol 1985; 231:490-9 12. Bellander BM, von Holst H, Fredman P et al. Activation of the complement cascade and increase of clusterin in the brain following a cortical contusion in the adult rat. J Neurosurg 1996; 85:468-75. 13. Bonnard AS, Chan, P, Fontane M. Expression of clusterin and C4 mRNA during rat peripheral nerve regeneration. Immunopharmacol 1997; 38:81-86. 14. May PC, Robison P, Fuson K et al. Sulfated glycoprotein-2 expression increases in rodent brain after transient global ischemia. Molec Brain Res 1992; 15:33-9. 15. May PC, Clemens JA, Panetta JA et al. Induction of sulfated glycoprotein-2 (clusterin) and glial fibrillary acidic protein (GFAP) RNA expression following transient global ischemia is differentially influenced by LY231617. Molec Brain Res 1996; 42:145-8 16. Weissner C, Back T, Bonnehoh P et al. Sulfated glycoprotein-2 mRNA in the rat brain following transient forebrain ischemia. Molec Brain Res 1993; 20:345-52. 17. Yasuhara O, Aimi Y, Yamada T et al. Clusterin as a marker for ischaemic Purkinje cells in human brain. Neurodegeneration 1994; 3:325-9. 18. Dragunow M, Preston K, Dodd J et al. Clusterin accumulates in dying neurons following status epilepticus. Molec Brain Res 1995; 32:279-90. 19. Walton M. Young D, Strimanne E et al. Induction of clusterin in the immature brain following a hypoxic–ischemic injury. Molec Brain Res 1996; 39:137-52. 20. Rosenberg ME, Silkensen J. Clusterin: Physiologic and pathophysiologic considerations. Int J Biochem Cell Biol 1995; 27:633-645. 21. Messmer–Joudrier S, Sagot Y, Mattenberger L et al. Injury–induced synthesis and release of apolipoprotein E and clusterin from rat neural cells. Eur J Neurosci 1996; 81:2652-61. 22. Fratelli M, Galli G, Minto M et al. Role of clusterin in cell adhesion during early phases of programmed cell death in P19 embryonic carcinoma cells. Biochim Biophys Acta 1996; 1311:71-6. 23. French LE, Wohlwend A, Sappino AP et al. Human clusterin expression is confined to surviving cells during in vitro programmed cell death. J Clin Invest 1994; 93:877-84. 24. Koch–Brandt C, Morgans C. Clusterin: A role in cell survival in the face of apoptosis? Progr Molec Subcellul Biol 1996; 16:130-49.

CHAPTER 9

Clusterin as a Neuroprotectant Todd Morgan and Patrick May

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s in other organ systems the role of clusterin in the central nervous system is not known. In fact, it is still not clear whether clusterin is a unifunctional or multifunctional protein. This chapter explores the potential beneficial role clusterin has in the central nervous system. The potential benefits clusterin may serve in the brain include support of normal and repair synaptic plasticity, protection from toxic agents, and/or neuronal survival. After a brief introduction on clusterin we will present results from current literature supporting the hypothesis that normal and disease-induced clusterin expression is beneficial to the proper functioning of the CNS.

Introduction Clusterin was first identified as a glycoprotein from chromaffin granules of the bovine adrenal medulla,1 and then in ram testes as a protein that promoted the aggregation of primary Sertoli cells (hence the name clusterin).2,3 Subsequently, clusterin was independently identified in other organ systems and found to possess other in vitro activities, suggesting a potential role in lipid transport (apolipoprotein J), inhibition of complementmediated cell lysis (serum protein 40,40; cytolysis inhibitor, CLI), secretion (glycoprotein (gp) III; gp80; secretogranin IV), programmed cell death (testosterone-repressed prostate message 2, TRPM-2), and/or cell-cell interactions (clusterin; sulfated glycoprotein 2, SGP-2). Clusterin is a highly conserved heterodimeric glycoprotein encoded by a single copy gene.4 Translated as a 449 amino acid precursor, clusterin possesses a signal peptide that guides it into the endoplasmic reticulum and is glycosylated and internally cleaved before it is secreted. Recently, a truncated form of clusterin has been identified that lacks the signal peptide and may represent an intracellular form of clusterin.5 Clusterin is constituitively expressed in mammalian body fluids and most tissues. Clusterin expression increases in regressing, involuting, or injured tissues.6

Clusterin Expression and Localization in the Brain

After its identification in bovine neural tissue1 clusterin was identified in the CNS as a mRNA species with increased abundance in AD hippocampus7,8 and scrapie-infected hamster brain.9 Clusterin mRNA has a similar regional distribution in normal rat and human brain, being widely found in astrocytes throughout the brain, with regional selectivity for neurons.10 Immunohistological localization of clusterin reveals similar selectivity for neurons but few clusterin-positive astrocytes in normal embryonic and postnatal rodent brains.11,12 Three different clusterin immunostaining patterns exist: diffuse, cytoplasmic punctate, and granules without visible cell membranes.11 Unlike apoE, clusterin (apoJ) does not appear to be made by microglia.13 In vitro studies using primary neurons or astrocytes Clusterin in Normal Brain Functions and During Neurodegeneration, edited by Caleb E. Finch. ©1999 R.G. Landes Company.

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indicate that astrocytes, but not neurons, secrete clusterin.10,13 Clusterin expression (mRNA and immunoreactivity) in normal adult rat brain is highest in ventricular ependyma and choroid plexus epithelial cells.10,14 Also, clusterin has been identified as a component of the cerebrospinal fluid (CSF).15 Clusterin mRNA and protein levels increase in a variety of human neurologic disorders, including Alzheimer’s disease (AD),7-9,16,17 multiple sclerosis,18 acquired immune deficiency syndrome (AIDS),18 Pick’s disease,9 epilepsy19 and gliomas19 (see chapter 7 for complete review). Clusterin expression is also increased in a variety of experimentally-induced brain injury rodent models representing ischemia,20,21 synaptic repair,22,23 epilepsy,8,24,25 or hormonal alterations26,27 (see chapter 3 for complete review). Human CSF clusterin is increased in patients with evidence for de-myelination, but at normal levels in patients with neurodegenerative and meningeal disease.28,29 Although both neurons and astrocytes contain clusterin mRNA, the increase of clusterin mRNA in the acute-lesioned rodent brain appears to occur primarily within reactive astrocytes.14,21,24 Depending on the lesion paradigm, increased clusterin mRNA is detected in reactive astrocytes within 12 to 48 hours postlesion. Experimentally, astrocytic clusterin mRNA levels can also be manipulated by hormones (corticosterone26; testosterone27) or cytokines (transforming growth factor-β113,30). This increase in astrocytic clusterin mRNA is followed by an increase in clusterin immunoreactivity.22,24,27 Immunohistochemically, clusterin is detected as both cytoplasmic and extracytoplasmic punctate deposits.24 Since cultured astrocytes secrete clusterin,10,13 the increased astrocytic clusterin expression observed after acute trauma may be a source of extracellular clusterin. Increased clusterin immunoreactivity is also associated with neurons after brain lesions in rodents 8,24,25 and humans.17,28,31-33 Additionally, clusterin is found in association with dystrophic neurites and β-amyloid (Aβ) in plaques of AD.28,31,32,34

Elucidation of Clusterin’s Role in the Brain A number of studies implicate a potential beneficial role for clusterin within the CNS. These studies include examination of clusterin’s temporal and spatial expression in relation to known brain activities, biological characterization of secreted or purified clusterin and genetic manipulation of clusterin expression in model systems to ascertain function. A great many of these studies focus on clusterin’s role in AD. These studies are discussed below.

Clues from Localization Studies

As in peripheral systems,35 clusterin is highly expressed in cells lining the fluid compartments of the brain, including ventricular ependyma and choroid plexus epithelial cells.10,14 The only known receptor for clusterin, gp330, is also expressed by ependymal cells that line the ventricles.36 Clusterin binds gp330 with high affinity and is internalized and degraded37 (see chapter 6 for complete review). Because of clusterin’s hydrophobic binding potential, clusterin could bind toxic fatty acids, bile salts or membrane fragments and shuttle them to be degraded, providing protection for brain cells. In the CSF clusterin is the predominant Aβ-binding protein as determined in vitro and in vivo.38 Clusterin is associated with parenchymal and vascular deposits of Aβ (discussed below),28,31 suggesting a possible role for clusterin in transporting Aβ into or out of the brain. Using a well characterized in situ perfused guinea pig brain model, Zlokovic’s group39 demonstrated that clusterin and Aβ-clusterin complexes are transported across the bloodbrain barrier and the blood-cerebrospinal fluid barrier. gp330 is implicated in this process, as the uptake is blocked with antibodies to gp330 or the presence of receptor-associated protein (RAP, blocks binding of all known ligands to gp330).39

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In the prostate, clusterin (TRPM-2) is implicated to play a role in apoptosis, as clusterin mRNA is found to be induced during regression of the rat ventral prostate after castration.40 Since clusterin mRNA is also increased in neurodegenerative diseases (AD, scrapie, epilepsy) where apoptosis may occur, it has been hypothesized that clusterin may be a marker of apoptosis. This hypothesis has been challenged because not all apoptotic neurons contain increased levels of clusterin mRNA41 and many normal neurons contain clusterin mRNA without evidence of apoptosis.10,41 More direct evidence comes from two recent studies using rodent models of neuronal apoptosis.42,43 In the rd mice (model of retinitis pigmentosa, where photoreceptors die by apoptosis) the clusterin mRNA increase is associated with surviving, not apoptotic, motor neuron cells.42 Removal of the olfactory bulb (only synaptic target for sensory neurons of olfactory nasal mucosa) causes massive degeneration of sensory neurons of olfactory nasal mucosa through apoptosis. Clusterin mRNA accumulation coincides with apoptosis but is not induced in dying neurons, but rather in the glial sheath surrounding the axon bundles of the degenerating olfactory neurons.43 These studies suggest that clusterin may play a protective role in apoptotic mechanisms (discussed below). The expression and localization of clusterin in experimentally-lesioned rodent brains provide clues as to its possible function(s). The increase of clusterin mRNA after brain lesion occurs primarily in astrocytes adjacent to degenerating axons. Immunohistochemically, clusterin is found as punctate deposits (apparently extracellular) in the surrounding neuropil. After perforant path transection, clusterin is localized to the outer molecular layer of the dentate gyrus where synaptic reorganization occurs.22,27 Therefore, it could play a role in synaptic remodeling through its cell adhesion properties or lipid transport capacity. After kainic acid-induced neurodegeneration, clusterin mRNA increases in astrocytes within 2 days, whereas clusterin peptide is localized to surviving pyramidal neurons in the degenerating CA3 layer of the hippocampus by 14 days after lesion (Fig. 9.1). Therefore, clusterin may protect neurons from damage through its inhibition of complement-mediated cytolysis, as complement factors are found in the same area.24 Clusterin immunoreactivity is associated with both parenchymal and vascular deposits of Aβ as well as neurofibrillary tangles,17,28,31,32,34,44,45 the pathologic hallmarks of Alzheimer’s disease. Despite these numerous reports demonstrating clusterin immunoreactivity with amyloid deposits, there is no consensus about whether clusterin associates preferentially with one or all of the different plaque types observed in AD. For example, Harr and colleagues observed clusterin immunoreactivity in neuritic Aβ deposits but not in diffuse deposits of Aβ,44 while Kida et al45 observed clusterin immunoreactivity in numerous neuritic plaques as well as diffuse deposits of Aβ (but the latter association with diffuse deposits varied by brain region). This distinction is important ,as the principal neuropathologic features of Alzheimer’s, e.g., neuronal degeneration and glial activation, are associated predominantly with the neuritic Aβ plaques, while diffuse deposits of amyloid appear to be relatively benign.46 Thus, the source of clusterin that decorates neuritic amyloid plaques may be secretions from nearby reactive astrocytes,10,13 perhaps in response to complement activation.47 However, a source of clusterin associating with diffuse deposits of Aβ is less clear, as in general microglia and astrocytes are quiescent, and little, if any complement activation is observed around these diffuse deposits of Aβ. Clearly this issue should be revisited, particularly since many of the early colocalization studies with clusterin preceded our current understanding of the different isoforms of Aβ, (e.g., 1-40, 1-42, 17-42) that are now known to populate various plaque types. The association of clusterin with parenchymal amyloid-β deposits (see above) is intriguing with respect to Aβ plaque pathology in AD. In vitro models have been developed which mimic the apparent conformation-dependent neurodegeneration associated with Aβ. In particular, direct addition of synthetic Aβ to primary neuronal cultures results in neu-

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Fig. 9.1. Hippocampal clusterin immunoreactivity 14 days after i.p. kainic acid injections. (A) Hippocampal pyramidal neurons from control animal do not immunoreact with anti-clusterin antibody. (B) Extended postlesion time (14 days) showed clusterin-immunoreactive pyramidal neurons; boxed areas: (a) CA1 hippocampal pyramidal neurons; (b) CA3 hippocampal pyramidal neurons. x45 magnification in A and B; scale bar = 100µm. x200 magnification in panels (a) and (b) Reprinted with permission from Rozovsky et al, 1994; Neurosci 62:746.

ronal degeneration over the course of the next few days;48,49 however, to demonstrate direct neurotoxicity, the Aβ must be in the appropriate aggregation/conformation state.50,51 Similarly, Aβ-mediated potentiation of cytokine release from a human astrocytoma is affected by the aggregation state of the peptide.52 Thus, clusterin, as well as other plaque associated proteins which bind Aβ, may directly influence amyloid plaque pathology (discussed below). Distinct from its ability to affect Aβ aggregation, clusterin may protect cells from complement-mediated cell lysis, as complement proteins are also found near the Aβ plaque.47 In regard to clusterin’s association with neurofibrillary tangles (NFT), a quantitative immunocytochemical study revealed a significantly higher percentage of NFT-free neurons containing clusterin in AD versus in nondemented cases.17 Since clusterin immunoreactivity was rarely associated with NFT-containing neurons, these authors suggest that increased clusterin expression protects neurons from AD-related neurodegeneration.

Clues from Studies on Secreted and Purified Clusterin As already mentioned, clusterin is secreted from astrocytes in vitro. This extracellular source of clusterin could perform beneficial roles within the CNS. As in the CSF, clusterin is found as a lipoprotein particle in culture medium from astrocytes.53,54 Distinct from the lipoprotein particles in the CSF, astrocytic lipoprotein particles contain little core lipid, are discoidal in shape, and the predominant apolipoproteins are clusterin (apoJ) and apoE. As proposed by LaDu54 apoJ-containing lipoprotein particles secreted by astrocytes may de-

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liver constituents to cells and participate in cholesterol clearance, developing a core of esterified cholesterol before reaching the CSF (see chapter 5 for complete review). Interestingly, clusterin is only one of several presumptive endogenous “chaperones” for Aβ. For example, apoE is another protein demonstrated to bind Aβ in vitro55 and in vivo56 and the epsilon 4 isoform of apo E is a well described risk factor for late-onset AD.57 It is intriguing that both clusterin and apoE are secretory products of astrocytes and transported by homologous receptors (LRP for apoE and LRP-2 (gp330) for clusterin) (see above). One attractive hypothesis is that clusterin and apoE compete for binding to soluble Aβ in brain parenchyma. This competition may be partially driven by the conformational state of the peptide, as Ghiso and colleagues suggest that clusterin preferentially binds Aβ in a random coil conformation, while apoE preferentially binds Aβ in a beta-pleated sheet conformation.58,59 Nonetheless, in this scheme, clusterin binding to soluble Aβ promotes its clearance from the brain parenchyma and into the CSF mediated by LRP-2/gp330 (see above). In contrast, apoE binding promotes fibrillogenesis and deposition of Aβ in the brain parenchyma. Mating APP transgenic mice, in which there are amyloid deposits, to apoE knockout mice (PDAPP x apoE KO) strikingly reduced parenchymal amyloid deposits.60 This model may be an extreme example of clusterin “outcompeting” apoE for Aβ. Perhaps crossing an amyloid-depositing transgenic mouse with a transgenic mouse overexpressing clusterin would result in a similar abrogation of Aβ deposition in brain parenchyma. The recent report of a polymorphism in α2-macroglobulin, whose gene product also binds Aβ in vitro,62 as yet another major risk factor for AD61 suggests that processes driving amyloid deposition are multifactorial. Using purified recombinant clusterin, May and colleagues demonstrated robust protection against in vitro Aβ neurotoxicity in dispersed primary neuronal cultures by coadministration of recombinant clusterin.63 Clusterin neuroprotection against Aβ was dosedependent, with an ED50 of 60 to 100 nM (4-8 µg/ml), i.e., concentrations of clusterin near those found in normal CSF (0.5-3 µg/ml) (Fig. 9.2). The mechanism of neuroprotection is unclear, but may involve a direct association with the Aβ peptide, as clusterin afforded no neuroprotection against either kainate-mediated excitotoxicity or an H2O2-mediated oxidative stress. As already discussed, several groups have demonstrated that clusterin binds Aβ in vitro with high avidity.58,59,64 Given that clusterin binds to monomeric Aβ with a higher affinity that it binds aggregated Aβ,59 one hypothesis for the observed neuroprotection is that clusterin is preventing adoption of the necessary aggregation state of Aβ to achieve a neurotoxic state (but see below). Recently, we extended these results to organotypic hippocampal slice cultures.65 Pretreatment of hippocampal slices for 16 hours with clusterin protects CA pyramidal neurons from the cytotoxic effects of aggregated Aβ1-42 or glutamate. Clusterin pretreatment is necessary as no protection is observed if clusterin is added only at the same time as the toxic agent. Toxicity was determined by measuring the increase in uptake of propidium iodide in CA neurons and lactate dehydrogenase leakage into the culture medium. Clusterin’s neuroprotective actions may be specific for a particular toxicity mechanism, as no protection occurs for oxidative stress (hydrogen peroxide), calcium influx (A23187) or apoptosis (staurosporine). As outlined above, in vitro Aβ toxicity has been attributed to its state of aggregation.50,51 In vitro experiments indicate that clusterin inhibits the aggregation state of Aβ.58,59,64 Therefore, it was believed that clusterin would protect neurons from Aβ by keeping Aβ in an unaggregated state. Interestingly, aggregating Aβ in the presence of clusterin in vitro inhibits the aggregation of Aβ but results in a slowly sedimenting Aβ-clusterin fraction (referred to as Aβ-derived diffusible ligands, ADDLs).64,66 ADDLs cause oxidative damage to primary neurons and PC12 cells,64 kill neurons in organotypic hippocampal slices65,66 and inhibit

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Fig. 9.2. Clusterin protects against Aβ1-40 neurotoxicity. Phase-contrast photomicrographs show primary rat hippocampal cultures (10 days in vitro) treated for 4 days with (A) phosphate-buffered saline (PBS) vehicle, (B) 25 µM Aβ1-40 (lotZL508), (C) 25 µM Aβ and 125 nM clusterin, and (D) 25 µM Aβ and 125 nM bovine serum albumin (BSA). x200. Reprinted with permission from Boggs et al, 1996; J Neurochem 67:1326.

hippocampal long-term potentiation.66 Ongoing studies will determine whether ADDLs occur in the AD brain and ADDLS effects on in vivo brain functions. One caveat that needs to be applied to all of these in vitro studies with exogenously added clusterin is drawn from the recent apoE literature. Similar neuroprotection and Aβ interaction studies have been conducted with recombinant apoE of various isoforms, and its becoming quite clear that the physiologic effects of apoE in a lipid particle are quite different from the rather artificial effects achieved with naked protein.67 Similarly, the studies described above with exogenous clusterin also used either recombinant or purified protein devoid of any associated lipid. Towards that end, LaDu and colleagues54 are characterizing the normal physiologic state of clusterin secreted from astrocytes as well as that found in CSF.

Clues from Genetic Manipulation of Clusterin Expression To date in vitro genetic manipulation of clusterin levels within brain cells has not been reported. A number of important studies utilizing nonbrain cell types suggest a potential

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cytoprotective role for clusterin. For example, overexpression of clusterin has been shown to protect clonal prostate cancer cells from tumor necrosis factor-(TNF-)alpha-induced cytotoxicity.68 Additionally, Sensibar determined that antisense depletion of clusterin caused an increase in cell death. Similarly, Humphreys et al69 showed that clusterin transfected L929 (murine fibrosarcoma) cells are protected from TNF-alpha-mediated cytotoxicity. Overexpressed clusterin did not protect these cells from other death-inducing agents such as colchicine, staurosporine or azide. In this system, clusterin’s protective activity may be an intracellular property as protection was no observed with addition of exogenous clusterin or from secreted proteins.69 An intracellular role for clusterin has also been implicated in other cell systems where a truncated form of clusterin interacts with TGF-β receptors and may target them to the cell nucleus.5,70 Therefore, clusterin may be involved in TGF-β’s neuroprotective activities.71,72 An intracellular activity for clusterin is especially pertinent for the CNS since a specific membrane receptor for clusterin, like gp330, has not been localized to parenchymal brain cells.36 Clusterin knock-out animals have been successfully made in the laboratories of Judith Harmony and Bruce Aronow. Mice without detectable clusterin survive, mature and reproduce and have no gross alterations in brain structures (personal communication Harmony and Aronow). Compensation from other molecules (e.g., apolipoprotein E?) may enable these animals to develop into mature mice under “nonstressed” conditions. However, because clusterin expression is greatly enhanced in response to most brain perturbations it will be informative to examine these knock-out mice in their response to brain lesion. We are currently utilizing two experimentally-induced brain lesions, kainic acid induced neurodegeneration and perforant path transection, to explore these questions.

Concluding Remarks This brief review has focused upon the possible role of clusterin as a neuroprotectant, particularly following neurodegenerative insults or in association with neurodegenerative diseases. Within this context, 3 general levels of “neuroprotection” can be envisioned for clusterin: 1. Direct neuroprotection; 2. Indirect neuroprotection; and 3. Neurotrophic functions.

Direct Neuroprotection A direct neuroprotective function of clusterin seems most tenuous, as under certain conditions exogenous clusterin affords protection against Aβ neurotoxicity in vitro, but not against other insults, e.g., oxidative and apoptotic, which are thought to share similar pathways to Aβ neurotoxicity. Nonetheless, the observation that pretreatment with clusterin is required to block excitotoxicity in hippocampal slices suggests that either clusterin is activating a neuroprotective gene(s), or conversely blocking a neurodegenerative signaling pathway. Future experiments utilizing cDNA chip array/differential display technology or molecular signaling pathway technology would be useful in directly testing the hypothesis that clusterin elicits changes in gene expression and/or signaling cascades that affect neurodegenerative pathways. As indicated, clusterin could provide direct neuroprotection by transporting molecules into the cell’s nucleus, thus participating in neuroprotective signal transduction mechanisms such as those initiated by TGF-β. For these reasons, it would be highly informative to construct clusterin transgenes encoding the truncated, presumably intracellular, form of clusterin.

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Indirect Neuroprotection Indirect neuroprotection by clusterin is a slightly more attractive hypothesis that has some experimental support, albeit in vitro. This is best exemplified by the ability of clusterin to block Aβ aggregation and limit neuronal degeneration in vitro. More speculative is whether clusterin could afford indirect neuroprotection by promoting rapid clearance of Aβ. In addition to experiments described above with a clusterin knock-in mouse, a gp330/LRP-2 knockout mouse might also prove useful in determining the validity/relevance of clusterin in mediating Aβ clearance. The potential for indirect neuroprotection by clusterin is not limited to Aβ-mediated pathways. In particular, the ability of clusterin to interfere with formation of the membrane attack complex of complement could effect delayed neuroprotection against a wide range of neuronal insults and conditions which elicit an inflammatory response in the brain, e.g., stroke, temporal lobe epilepsy, etc.

Neurotrophic Functions Rather than acting as a neuroprotectant per se, it seems likely that clusterin also plays a neurotrophic role; not in the sense of a growth factor like NGF or BDNF, but in promoting general recovery from neuronal injury via lipid transport or membrane recycling. In support of this notion, clusterin expression in brain is markedly induced a few days after a variety of neuronal insults (see above). While this delayed induction of clusterin may serve to limit neurodegeneration developing after the primary insult has dissipated, it also likely plays a prominent role in re-establishing lipid homeostasis in neurons recovering from a sublethal insult. In conclusion, the precise role(s) of clusterin in brain function under normal and neurodegenerative conditions still awaits elucidation. Clearly, the availability of clusterin knockin and knockout animals, as well as primary neuronal/glial cultures derived from these genetically-modified animals, will afford critical in vivo and in vitro models in which to test hypotheses directed at each of the possible “neuroprotectant” roles for clusterin.

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INDEX hippocampal formation 4, 18, 22-24, 26, 36, 51, 92, 112 hippocampus 4 hypothalamus 18, 23, 25, 26 neocortex 91 olfactory 18, 93 perforant path 41 posterior pituitary 24, 28 pyramidal neuron 90, 93 red nucleus 18, 23 sciatic nerve 25 spinal 18, 22, 99, 100, 102, 103, 105 striatum 22, 23 thalamus 18, 22, 23, 25, 26 trigeminal nucleus 100 white matter 18, 22, 26

A α2-macroglobulin 50, 52, 54, 94

Age 65 aged rats 23 aging 22, 28 elderly 23, 26 immature 100 Alzheimer's disease 2, 45, 77, 89, 91, 94 dementia 71 Amyloid Aβ 54 Aβ-derived diffusible ligand (ADDLs) 113, 114 amyloid deposits 89-91, 94 amyloid hypothesis 72 soluble β-amyloid 24 Antioxidant 106, 107 Apolipoprotein E (apoE) alleles 44, 45, 76, 77, 89 Apolipoprotein E (apo E) protein 12, 45, 62, 89, 90 Astrocytes 4, 5, 39

B Baboons 36 Blastocyst 3 Blood-brain barrier 75, 79 Brain region basal ganglia 18 brainstem 18, 22 ciliary body 3 corpus callosum 22 dentate gyrus 4 entorhinal cortex 25, 29 ependyma 3 ependymocytes 22-27 frontal cortex 89, 90 habenular complex 18

C Carcinoma 2, 7 Cell death cytotoxicity 17, 75 apoptosis 1, 3, 11 nephrotoxic 4 Cerebrospinal fluid 18, 23, 27, 64, 74 Chaperone 2, 5 Chimpanzee 94 Cholesterol 24, 25, 27, 28, 61-66 Choroid 3, 71, 74, 75, 79 Clusterin gene alleles 54 clusterin element (CLE) 7 DNase I hypersensitivity 7 Ets 7 intron 5, 9, 11 NF-κB 7 organization 6, 12 serum response element (SRE) 7 Clusterin protein Cys-rich motifs 2 EGF repeats 50

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heparin binding 1 nucleus 2 signal peptide 2, 109 Colchicine 4, 115 Colon 3 Complement 90, 94 membrane attack complex (MAC) 29 terminal complement complex 29 Creutzfeldt-Jakob disease 93 Cytokines 2, 5, 71, 115

D Dementia 71 Dogs 94 Dystrophic neurite 90, 93

E Electron microscopy 24, 27 Endoplasmic reticulum 109 Endosomal 50 Epilepsy 27 Extracellular fluid (ECF) 71, 74, 79

F Foam cells 62

G Guinea pig 110

H Heat shock proteins 5 HSF-1 7 Homeostasis 25, 27, 28, 62 Hormones androgen 2, 4 corticosterone 110 dexamethasone 3 estradiol 36 estrogen 35, 36, 38, 39, 41, 44 estrous cycle 36 testosterone 109, 110 ovariectomy (OVX) 36, 38, 39, 41

I IL-1β 5 IL-2 5 IL-3 5 IL-6 5

K Kainic acid 4, 7 Kidney 3, 4, 10, 11

L Lipid metabolism 28 Lipoproteins apoA 49, 53, 54 apoC 49 apoD 49, 54 apo E 12, 45, 62, 89, 90 discoidal particle 25, 27, 64 gp330/megalin 23-25, 28 HDL particles 24 LDL receptor-related protein (LRP) 25, 50, 52-54 low-density lipoproteins 24 oxidized LDL 7 receptor associated protein (RAP) 50, 52 reverse cholesterol transport 62, 65, 66 VLDL (very low density receptor) 25

M Mammary gland 3, 4 Menopause 35 Microcebus murinus 94 Microglia 22-27 Motoneurons 93, 100, 102 Mouse strains apoE knockout 38, 40, 41, 43, 64, 113 BALBc 35 C57BL 35 CH3 35

Index

clusterin knockout 115 rd 111 Myocarditis 4

N Neurite outgrowth 54 Neuroblastoma 90 Nucleus 2

O Oligodendrocytes 22, 24, 26, 27, 102, 106, 107 Oxidative stress H2O2 113

P Parkinson’s disease 65 Primates 71, 73 prosimians 26

Q Quail 5, 7

R Rat genotype F344 36 Ricin 93

123

S Scavenger receptor 50, 53, 54 Schwann cell 102 Squirrel monkey 73 Stab wound 103 Staurosporine 113, 115 Stroke 65 Synapse 36, 41, 44 Synaptic plasticity 25

T TGF-β 2, 7 Tumor necrosis factor (TNF) 115

U Uterus 3

V Vascular atherosclerotic vascular disease 2 blood brain barrier 71, 74 cardiovascular disease 35 microvascular 73, 74, 77

W Wallerian degeneration 102, 107