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
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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; * p94%. 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
Neurovascular Interactions of Alzheimer's Amyloid β Peptide with apoJ and apoE
75
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-
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Clusterin in Normal Brain Functions and During Neurodegeneration
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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
