Astrocytes in Brain Aging and Neurodegeneration (Neuroscience Intelligence Unit 3)

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

3

Hyman M. Schipper

Astrocytes in Brain Aging and Neurodegeneration

R.G. LANDES C OM PA N Y

NEUROSCIENCE INTELLIGENCE UNIT

Astrocytes in Brain Aging and Neurodegeneration Hyman M. Schipper Department of Neurology and Neurosurgery Department of Medicine (Geriatrics) and Centre for Studies in Aging McGill University and Bloomfield Centre for Research in Aging Lady Davis Institute for Medical Research Sir Mortimer B. Davis-Jewish General Hospital Montreal, Quebec, Canada

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

NEUROSCIENCE INTELLIGENCE UNIT Astrocytes in Brain Aging and Neurodegeneration R.G. LANDES COMPANY Austin, Texas, U.S.A. Copyright © 1998 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: 1-57059-489-9

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

Astrocytes in brain aging and neurodegeneration / [edited by] Hyman M. Schipper. p. cm. -- (Neuroscience intelligence unit) ISBN 1-57059-489-9 (alk. paper) 1. Nervous system--Degeneration. 2. Nervous system--Aging. 3. Astrocytes. I. Schipper, Hyman M., 1954- . II. Series. [DNLM: 1. Neurodegenerative Diseases--physiopathology. 2. Astrocytes-physiology. 3. Brain Diseases--physiopathology. 4. Brain--physiology. 5. Aging-physiology. WL 300A859 1998] RC365.A88 1998 616.8'047--dc21 DNLM/DLC 98-26335 for Library of Congress CIP

NEUROSCIENCE INTELLIGENCE UNIT PUBLISHER’S NOTE

Astrocytes in Brain Aging and Neurodegeneration

Landes Bioscience 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 Department our books are of published within 90Neurosurgery to 120 days of receipt of Neurology and the manuscript. We would like to thank our readers for their Department of Medicine (Geriatrics) continuing interest and welcome any comments or suggestions they and Centre for Studies in Aging may have for future books. McGill University

Hyman M. Schipper

and Judith Kemper Bloomfield Centre for Research in Aging Lady Davis Institute for MedicalProduction Research Manager R.G. Company Sir Mortimer B. Davis-Jewish GeneralLandes Hospital Montreal, Quebec, Canada

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

DEDICATION To my parents, Freda and Mendel, for their unflagging devotion.

CONTENTS Part I: Biology of Astrocytes 1. Astrocyte Ontogenesis and Classification ................................................ 3 James E. Goldman Genesis of Radial Glia and Their Transformation into Astrocytes ....... 4 Genesis of Astrocytes from SVZ Cells .................................................... 5 Control of Astrocyte Differentiation ...................................................... 6 Genesis of Astrocyte Heterogeneity ........................................................ 7 Generation of Astrocytes in the Adult CNS ........................................... 8 2. Functions of Astrocytes........................................................................... 15 Harold K. Kimelberg and Michael Aschner Introduction ........................................................................................... 15 Functions of Astrocytes ......................................................................... 16 Homeostasis of the Extracellular Space ................................................ 17 Transmitter Uptake Systems ................................................................. 21 Receptors for Transmitters ................................................................... 22 Astrocytes and the Blood-Brain Barrier (BBB) .................................... 26 Astrocytes and Immune and Inflammatory Responses in the CNS ... 28 3. Astrocyte Pathophysiology in Disorders of the Central Nervous System ............................................................... 41 Michael D. Norenberg Introduction ........................................................................................... 41 Normal Functions ................................................................................. 41 General Response to Injury ................................................................... 42 Injury to Astrocytes in CNS Disorders (Passive Role) ........................ 43 Active Role of Astrocytes in CNS Disorders ........................................ 44 Clinical Considerations ......................................................................... 47 Perspectives and Conclusions ............................................................... 53 Part II: Astrocytes in Human Brain Senescence and Neurodegenerative Disorders 4. Glial Responses to Injury, Disease, and Aging ...................................... 71 Lawrence F. Eng and Yuen Ling Lee Introduction ........................................................................................... 71 Astrocyte Intermediate Filament, Glial Fibrillary Acidic Protein ....... 71 Astrocytes in Experimental Gliosis ....................................................... 73 Astrocytes in Disease ............................................................................. 73 Astrocyte Activation of GFAP in Astrogliosis ...................................... 74 Microglial Activation ............................................................................. 74 Monocyte/Macrophage Activation ....................................................... 75 Endothelial Cell Activation ................................................................... 75 Astrocytes in Normal Aging .................................................................. 75 Astrocyte Inclusions in Normal Aging ................................................. 77 Astrocyte Inclusions in Disease ............................................................. 78

5. Astrocyte Pathology in Alzheimer Disease ............................................ 91 Jerzy Wegiel and Henryk M. Wisniewski Neuropathological Changes in Alzheimer Disease .............................. 91 Relationships Between Amyloid-β, Neurons, and Glial Cells in AD ......................................................................... 91 Astrogliosis in Aging and AD ................................................................ 93 Astrocyte Degeneration in AD .............................................................. 99 6. Parkinson’s Disease ............................................................................... 111 Donato A. Di Monte Introduction ......................................................................................... 111 Idiopathic Parkinson’s Disease ........................................................... 111 MPTP-Induced Parkinsonism ............................................................ 113 Neuronal-Astrocyte Interactions in Nigrostriatal Degeneration ...... 115 Conclusion ........................................................................................... 121 7. Astrocytes in Transmissible Spongiform Encephalopathies (Prion Diseases) ..................................................................................... 127 Pawel P. Liberski, Radzislaw Kordek, Paul Brown and D. Carleton Gajdusek Introduction ......................................................................................... 127 KURU ................................................................................................... 130 Creutzfeldt-Jakob Disease (CJD) and Gerstmann-Straussler-Scheinker Disease (GSS) .................... 130 GSS ....................................................................................................... 135 The Involvement of Astrocytes in Formation of Amyloid Plaques ......................................................................... 137 Scrapie, Bovine Spongiform Encephalopathy (BSE), and Chronic Wasting Disease (CWD) ........................................... 137 BSE and CWD ...................................................................................... 143 Interaction Between Astrocytes and Oligodendrocytes ..................... 143 A Particular Form of Astrocytic Reaction in TSES ............................ 145 Expression of Glial Fibrillary Acidic Protein (GFAP) and Its mRNA .................................................................................. 145 Astrocytes and the Expression of Cytokines ...................................... 149 Conclusions ......................................................................................... 153 8. Astrocytes in Other Neurodegenerative Diseases ............................... 165 Dennis W. Dickson Introduction ......................................................................................... 165 Neurofibrillary Tangles as an Archetype of Cytoskeletal Inclusions ............................................................... 167 Neurodegenerative Disorders with Filamentous Glial Inclusion Bodies .............................................................................. 169 Progressive Supranuclear Palsy (PSP) ................................................ 171 Pick’s Disease ....................................................................................... 175

Corticobasal Degeneration (CBD) ..................................................... Argyrophilic Grain Dementia (AGD) ................................................ Familial Frontotemporal Dementia and Parkinsonism Linked to Chromosome 17 (FTDP-17) ......................................... Multiple System Atrophy (MSA) ........................................................ Familial Amyotrophic Lateral Sclerosis (FALS) .................................

176 179 180 180 181

Part III: Experimental Models of Astrocyte Senescence: Implications for Neurodegenerative Disease 9. The Peroxidase-Positive Subcortical Glial System .............................. 191 Marc B. Mydlarski, James R. Brawer and Hyman M. Schipper Introduction ......................................................................................... 191 Tinctorial and Histochemical Features .............................................. 191 Topography of the Peroxidase-Positive Astroglia ............................. 192 Modulation of the Peroxidase-Positive Glial System ........................ 193 Peroxidase-Positive Astrocytes in Primary Culture ........................... 196 Subcellular Precursors of Peroxidase-Positive Astroglial Inclusions ........................................................................ 197 Summary and Conclusions ................................................................. 202 10. Astrocyte Granulogenesis and the Cellular Stress Response .............. 207 Marc B. Mydlarski and Hyman M. Schipper HSP Expression in Acutely-stressed Neural Tissues: Effects of Aging ................................................................................ 208 Stress Protein Expression in the Aging and Degenerating Human Brain .................................................... 209 A Cellular Stress Model for the Biogenesis of Astroglial Inclusions ................................................................... 210 Astrocyte Senescence and the Origin of Corpora Amylacea ............. 221 11. Glial Iron Sequestration and Neurodegeneration ............................... 235 Hyman M. Schipper The Free Radical Hypothesis of Parkinson’s Disease ........................ 235 The Redox Neurobiology of Alzheimer’s Disease .............................. 235 Iron Deposition and Neurodegenerative Disease .............................. 236 Iron Sequestration in Aging Astroglia ................................................ 237 The Role of HO-1 in Brain Iron Deposition ...................................... 239 Pro-toxin Bioactivation by Astrocytes in Primary Culture ............... 242 Pathological Glial-Neuronal Interaction in Parkinson’s Disease ..... 243 Conclusion ........................................................................................... 246 Index ................................................................................................................ 253

EDITOR Hyman M. Schipper Department of Neurology and Neurosurgery Department of Medicine (Geriatrics) and Centre for Studies in Aging McGill University and Bloomfield Centre for Research in Aging Lady Davis Institute for Medical Research Sir Mortimer B. Davis-Jewish General Hospital, Montreal, Quebec, Canada Chapters 9, 10, 11

CONTRIBUTORS Michael Aschner Department of Physiology and Pharmacology Bowman Gray School of Medicine Winston-Salem, North Carolina, U.S.A. Chapter 2 James R. Brawer Department of Anatomy and Cell Biology McGill University Montreal, Quebec, Canada Chapter 9 Paul Brown Laboratory of Central Nervous System Studies, National Institute of Neurological Disorders and Stroke National Institutes of Health Bethesda, Maryland, U.S.A. Chapter 7 Donato A. Di Monte The Parkinson’s Institute Sunnyvale, California, U.S.A. Chapter 6 Dennis W. Dickson Research Department Mayo Clinic Jacksonville Jacksonville, Florida, U.S.A. Chapter 8

Lawrence F. Eng Pathology Research VAPA Health Care System Palo Alto, California and Stanford University School of Medicine Stanford, California, U.S.A. Chapter 4 James E. Goldman Department of Pathology and The Center for Neurobiology and Behavior Columbia University College of P&S New York, New York, U.S.A. Chapter 1 D. Carleton Gajdusek Laboratory of Central Nervous System Studies, National Institute of Neurological Disorders and Stroke National Institutes of Health Bethesda, Maryland, U.S.A. Chapter 7 Harold K. Kimelberg Department of Pharmacology and Neuroscience Division of Neurosurgery Albany Medical College Albany, New York, U.S.A. Chapter 2

Radzislaw Kordek Laboratory of Central Nervous System Studies National Institute of Neurological Disorders and Stroke National Institutes of Health Bethesda, Maryland, U.S.A. and Laboratories of Tumor Biology Laboratory of Electron Microscopy and Neuropathology Medical Academy Lodz Lodz, Poland Chapter 7 Yuen Ling Lee Pathology Research VAPA Health Care System Palo Alto, California and Stanford University School of Medicine Stanford, California, U.S.A. Chapter 4 Pawel P. Liberski Laboratory of Central Nervous System Studies National Institute of Neurological Disorders and Stroke National Institutes of Health Bethesda, Maryland, U.S.A. and Laboratories of Tumor Biology Laboratory of Electron Microscopy and Neuropathology Medical Academy Lodz and Laboratory of Electron Microscopy Department of Pathology Polish Mother Memorial Hospital Lodz, Poland Chapter 7

Marc B. Mydlarski Department of Neurology and Neurosurgery McGill University and Bloomfield Centre for Research in Aging Lady Davis Institute for Medical Research Sir Mortimer B. Davis-Jewish General Hospital Montreal, Quebec, Canada Chapters 9, 10 Michael D. Norenberg Laboratory of Neuropathology Veterans Administration Medical Center and Departments of Pathology, and Biochemistry and Molecular Biology University of Miami School of Medicine Miami, Florida, U.S.A. Chapter 3 Jerzy Wegiel Department of Pathological Neurobiology New York State Institute for Basic Research in Developmental Disabilities Staten Island, New York, U.S.A. Chapter 5 Henryk M. Wisniewski Department of Pathological Neurobiology New York State Institute for Basic Research in Developmental Disabilities Staten Island, New York, U.S.A. Chapter 5

PREFACE

T

he last decade or so has witnessed a remarkable proliferation of original scientific papers, review articles and books devoted to the neuroglia and their involvement in health and disease. In the prefaces to the many excellent compendia currently available on this topic, the editors almost invariably take pains to point out that for almost 150 years the study of neuroglia in general, and astrocytes in particular, has been largely eclipsed by the effort to decipher the properties of what has traditionally been regarded as the “business” end of the nervous system, the neurons and their connections. To be sure, no one would deny the paramount importance of neurons to the workings of the brain and its ailments. Yet, there is a rapidly-growing awareness, fueled by a biotechnological prowess permitting exquisitely refined analyses of cellular behavior, that the astroglia engage in intimate, mutually-dependent interactions with virtually all neural cell types, including neurons, and subserve a multitude of adaptive functions vital to the maintenance of normal brain structure and activity. To cite but a few examples, astrocytes are known to assume pivotal roles in the establishment of the blood-brain barrier and the regulation of ion homeostasis, the elaboration of a scaffolding for neuronal migration during embryogenesis, the sequestration and metabolism of various neurotransmitters and other neuroactive substances, and the production of immunomodulatory and proinflammatory cytokines and neuropeptides. In this regard, it should come as no surprise that astrocyte dysfunction resulting from injury or disease may mediate a host of dystrophic effects within the CNS and thereby contribute to a decline in neurological status. The formation of epileptogenic scar tissue in response to CNS trauma, the release of excitotoxic amino acids following tissue hypoxia, metal exposure or oxidative stress, neoplastic transformation and malignant behavior, and the bioactivation of pro-toxins (such as MPTP) to potent neurotoxins (MPP+) are illustrative of some clinically-relevant pathophysiologic processes which directly implicate the astroglial compartment. Astrocyte hypertrophy and hyperplasia, the biosynthesis of GFAP-associated intermediate filaments (reactive gliosis) and the accumulation of discrete cytoplasmic inclusions are characteristic pathological features of the major aging-related neurodegenerative disorders, including Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis. Gliosis and inclusion body formation also figure prominently in the relatively uncommon human neurodegenerative conditions, such as Pick’s disease and corticobasal ganglionic degeneration, and occur to a lesser extent in the course of normal brain aging. The raison d’être of this monograph was to consolidate information concerning the established and putative roles of astroglia in brain aging and neurodegeneration gleaned from vast and often disparate literatures on the biology and pathology of these cells. To achieve this objective, I invited the participation of respected investigators from a mix of basic and clinical departments whose interests in the neuroglia are diverse and long-standing. In addition to providing thorough reviews of their respective fields, each team of

contributors was requested to speculate freely on the question “In the condition under consideration, do the astrocytic changes actively contribute to the degenerative process or do they merely represent passive responses to primary neuronal injury?” Given the divergence of opinion on this question, a certain degree of overlap of material covered by the authors (e.g., the role of astroglia in Alzheimer’s disease) was not only tolerated but encouraged. The chapters in this monograph are grouped in three sections: I. Biology of Astrocytes. Collectively, the chapters in this section constitute a comprehensive discussion of the origin and known functions of astroglia in the mammalian CNS and the roles these cells may play in the pathophysiology of neurological disorders. II. Astrocytes in Human Brain Senescence and Neurodegenerative Disorders. In this section, detailed accounts of the pathology of astrocytes and their involvement in human brain aging and various neurodegenerative conditions are presented. III. Experimental Models of Astrocyte Senescence: Implications for Neurodegenerative Disease. In this final part, experimental approaches to the delineation of the role of astroglia in brain aging and degeneration are described. We hope that this compendium will appeal to basic neuroscientists interested in various aspects of neuroglial biology, as well as to clinically-oriented investigators concerned with the pathogenesis of the major human neurodegenerative disorders. I am deeply grateful to the many mentors, colleagues and students at home and abroad who have helped shape my interest and refine my knowledge of the neuroglia and their place in clinical medicine. Hyman M. Schipper

Part I Biology of Astrocytes

CHAPTER 1

Astrocyte Ontogenesis and Classification James E. Goldman

A

strocytes, first named for their star-shaped appearance as visualized with heavy metal impregnations,1 in fact display a extensive variety of morphologies. All are united in their astrocyte nature, however, by common features, including multiple, thin processes, close interactions with both the neuronal and mesenchymal elements of the CNS, the presence of intermediate filaments of several types (vimentin, GFAP, nestin), and the expression of a variety of other molecules, such as S-100β and glutamine synthetase. Besides their complex, multiprocess shapes the other salient histological characteristic of astrocytes is their interactions with specific sets of other cells. First, the basal lamina that surrounds blood vessels in the brain and that lines the pial surface of the brain is covered with astrocyte end feet (the ends of astrocyte processes).2 This is indeed a very large surface area, and thus requires an exceedingly large number of astrocyte processes. Second, astrocytes intimately associate with neurons, wrapping neuronal perikarya and dendrites, contacting neurons in zones between synaptic contacts.2-4 Thus, astrocytes serve to isolate individual synapses or groups of synapses, perhaps those that share functional connections or characteristics. Such isolation of synapses makes sense in view of the astrocytes’ abilities to take up neurotransmitters with high affinities and to buffer potassium (see chapter 2). These interactions may well serve to condition and maintain astrocyte shape (see below). Astrocytes are not distributed randomly in the brain, but rather lie in separate domains with some peripheral overlap. For example, the “domain” of a neocortical astrocyte is roughly spherical with a diameter of about 100 microns.5 Similarly, “domains” of retinal astrocytes are spatially separate at 100-150 microns, with a modest degree of overlap in the peripheral processes.6 Subpial astrocytes are not spherical, but look like truncated spheres or columns.7 Thus, astrocyte development must somehow produce a matrix of astrocyte spheres which intersect only at their peripheries. It is at the periphery, incidentally, that astrocytes are connected by gap junctions, allowing movement of ions and small molecules through an astrocyte “syncytium” over many hundreds of cubic microns.8 How this regular spacing is accomplished is not known. Since glia continue to divide as they migrate through the brain (see below), sibling astrocytes begin life next to each other after a mitotic division. Do they migrate away from each other, or does the growth of the brain continue to separate related glial cells? Astrocytes display a wonderful variety of sizes and shapes. In most gray matter regions, where astrocytes have been traditionally termed “protoplasmic,” the cell body and domains of all processes roughly describe a sphere or ellipsoid. Processes branch into ever-finer twigs, more like the boughs of a tree than the rays of a star, eventually reaching tremendous numbers Astrocytes in Brain Aging and Neurodegeneration, edited by Hyman M. Schipper. ©1998 R.G. Landes Company.

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Astrocytes in Brain Aging and Neurodegeneration

and microscopic size.3 In the cerebellar granule cell layer, “velate” astrocytes wrap thin sheetlike extensions about the mossy fiber glomeruli.9,10 Astrocytes in white matter (classically termed “fibrous”) display fewer processes and a less complex branching pattern than their gray matter relatives. Processes separate fascicles of axons, a characteristic particularly easily observed in spinal cord tracts and optic nerve.11,12 Astrocyte processes also contact nodes of Ranvier,13 where they may play a role in spatial ion buffering. Some astrocytes display processes oriented “radially,” perpendicular to the pial surface. These include the Bergmann glia of the cerebellar molecular layer, which retain their radially-oriented nature first established for granule cell migration.9,14 Muller glia of the retina are also first established as radially oriented cells, coursing through all retinal layers, and remain so for life. Radial type glia in periventricular regions, particularly around the third and fourth ventricles and aqueduct of Sylvius, display long processes beginning at the ventricular surface and extending for hundreds of microns into the parenchyma of the hypothalamus and brain stem.15-17

Genesis of Radial Glia and their Transformation into Astrocytes The term “radial glia” is used to describe elongated, bipolar glial cells that arise during early histogenesis of the CNS. Heavy metal impregnations and more recently, immunocytochemistry, have produced a detailed view of the radial glial scaffolding in the developing brain.18-20 Oriented “radially” (perpendicular to the pial surface), these cells extend from the ventricular zone to the pia and develop concurrently with the first sets of neurons.21-23 Not only are radial glia generated contemporaneously with some neuronal populations, but also they share a lineage with neurons, since early progenitors give rise to both radial glia and to the neurons that migrate along them.22-24 Thus, there is a glial-neuronal fate decision for a subpopulation of cells in the early ventricular zone, although how this decision is accomplished is not known. Radial glia have long been considered a form of astrocyte, based upon the expression of the intermediate filaments vimentin and nestin, and in primates, GFAP, as well as the storage of glycogen and the interactions with the pial surface, all characteristics of astrocytes. Furthermore, radial glia have been considered the source of many of the astrocytes in the mature CNS. This transformation of radial glia into astrocytes has been inferred from several observations. Radial glia disappear in cortex concurrently with the emergence of the multiprocess forms of mature astrocytes. During this time several studies have noted forms “transitional” between radial glia and astrocytes: cells with both long, radially-oriented processes and smaller branches emerging from the cell body.18,19,25,26 While undoubtedly some of these “transitional” forms reflect passing stages from radial glia to astrocytes, similar forms are produced by subventricular zone (SVZ) cells that migrate into the cortex after neurogenesis.27 Cells cultured from embryonic rodent CNS and expressing antigenic markers for radial glia begin to express GFAP in culture and assume the morphologies of cultured astrocytes.28 One dynamic study provides direct evidence for such a transformation, however.29 In work with postnatal ferret brain, the application of the lipophilic fluorescent tracer dye, diI, to the cortex initially labeled radial glia. After maturation of the brain and the disappearance of radial glia, the dye was found in astrocytes. What controls this transformation of radial glia to astrocytes and why does such transformation apparently take place in some regions (cortex, for example), but not, or to a lesser extent, in others (periventricular zones in diencephalon and brain stem)? Studies in cell culture suggest a role of extrinsic factors in promoting the change in shape from elongated to branched with many processes. Such a transformation takes place in primary cultures from embryonic forebrain,28 and can be reversibly promoted by soluble signals from the

Astrocyte Ontogenesis and Classification

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embryonic CNS.30 Cerebellar astrocytes cocultured with granule neurons assume elongated shapes, suggesting that interactions with immature neurons helps determine astrocyte shape.31 A critical, and necessary, change in the transformation of radial glia is the loss of subpial connections. This process, which has not been examined, requires a loss of adhesion between the end of the glial process and the pial surface. Breaking such adhesion in turn may require local extracellular protease activity or redistribution of surface adhesion molecules such as integrins that may interact with mesenchymal tissue matrix, or contraction of the microfilament network in the process. Loss of adhesion to the pia does not represent a lack of adhesive properties of the cell in general, since radial glia that transform into astrocytes presumably contact blood vessels as they are detaching from the pia, or shortly thereafter.

Genesis of Astrocytes from SVZ Cells In addition to the generation of astrocytes from radial glia, astrocytes are also derived from immature cells of the subventricular zone, without apparently going through a radial intermediate stage. The genesis of astrocytes from immature cells in the forebrain SVZ was originally suggested from thymidine labeling in the postnatal rodent brain.32-34 These classic studies showed that the SVZ population is a highly proliferative one and that the thymidine label could be “chased” into mature glial cells in white matter and gray matter. More recent antigen expression studies35 and Golgi impregnations of the developing CNS7 have also supported a nonradial glial derivation of some astrocytes. Through the use of recombinant retroviruses, a direct demonstration of SVZ cell migration and differentiation into mature glia has illuminated many of the details of this developmental process.5,36-39 In these experiments, immature, cycling cells of the postnatal SVZ were labeled in vivo by stereotactic injection of retroviruses directly into the SVZ. The fates of labeled cells and their routes of migration into the striatum, overlying white matter, and neocortex could then be traced. How do astrocytes derived from the SVZ colonize the CNS? Glial colonization, to produce the distributions described above, is not a random process, but takes place in definable spatial and temporal patterns. Migration of progenitors from SVZ into white matter and cortex occurs in a coronal plane.36 Perhaps the migratory pathways are defined in part by the radial glial scaffolding. The idea that SVZ cells migrate along radial glia is supported by several observations. First, radial glia persist in the rodent neocortex through the first 1-2 postnatal weeks.20,25,26 During this period, SVZ cells distribute into white matter and cortex.5,37 By postnatal day 14 (P14), however, progenitors that migrate out of the SVZ remain in white matter and do not enter cortex.36 Thus, a restriction in migration coincides exactly with the loss of the cortical radial glial tracks. Second, we have observed progenitors from the SVZ aligning along radial glia in the cortex during early postnatal development.38 Third, progenitors from the SVZ migrate along “radial glial”-like cables in culture (Newman et al, in preparation). In contrast to the laminar colonization of neurons of the neocortex, however, astrocytes do not differentiate in a layered pattern. In fact, astrocytes derived from SVZ cells appear to differentiate at all depths of the cortex, from the pial surface to deep layers, at the same time. It is common to see radially oriented clusters of young astrocytes derived from SVZ cells, clusters we believe are clonal. At present we favor a model in which progenitors migrate into cortex, and continue to divide therein. Some of the progeny cease migration, while others continue toward the pial surface, thus leaving progeny behind at a number of cortical levels. What induces a particular progenitor to stop migrating and begin to differentiate into an astrocyte will be considered below.

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Astrocytes in Brain Aging and Neurodegeneration

In other regions of the CNS that do not have an SVZ, the genesis of astrocytes may be different. For example, in the optic nerve, astrocytes arise prenatally, while those progenitors that migrate into and along the nerve in postnatal life do not differentiate into astrocytes, but only into oligodendrocytes.40,41 Thus, there appear to be separate lineages for astrocytes and oligodendrodcytes in this tract. Astrocytes in optic nerve likely arise from radial glial cells that formed during earlier telencephalic development and were carried into the nerve when the optic outpouching occurred. And the oligodendrocytic fate of progenitors that migrate into the nerve postnatally may be analogous to the oligodendrocytic fate of forebrain SVZ cells that settle in subcortical white matter. In culture, the postnatal progenitors (O-2A cells) can differentiate into oligodendrocytes or into astrocytes,42 showing their bipotential nature, but there is apparently an oligodendrocyte fate restriction in vivo. Astrocyte development in the spinal cord may be similar, with many of the astrocytes developing from radial glia, as suggested from antigen and morphology studies.43,44 In the cord, oligodendrocytes arise from proliferating, immature cells in the centro-ventral region,45,46 but whether astrocytes also arise from this proliferative population is not known.

Control of Astrocyte Differentiation Much recent work has utilized cell culture systems to examine the control of astrocyte differentiation, and has led to the general conclusion that cell-extrinsic factors contribute substantially to the determination of astrocyte cell fate. Oligodendrocyte progenitors isolated from the optic nerve are induced to express astrocyte genes and cease oligodendrocyte development by exposure to serum,42 serum fractions,47 and ciliary neurotrophic factor (CNTF).48 Although the nature of the serum stimulus(i) is not known, extracellular molecules isolated from endothelial and meningeal cells will also induce astrocyte genes,48,49 in combination with CNTF. This induction by matrix may well be an in vitro counterpart to astrocyte induction by cues from blood vessels and pia in vivo (see below). More recent studies identify CNTF as an attractive candidate for an important inducer of astrocytic differentiation in immature CNS cells.50-52 CNTF induces GFAP expression and a flat, astrocytic morphology in immature cortical cells via a JAK-STAT signaling pathway52 and also upregulates GFAP transcription in the CG-4 glial cell line.53 The 5' upstream region of the GFAP gene contains a consensus STAT binding site,52,54 which in transfection assays appears to be essential for the CNTF regulation of GFAP expression.52 The GFAP gene also contains consensus sequences for CREB, AP-2, and AP-1 binding,54,55 the former two possibly used for cyclic-AMP increases in GFAP transcription,54,56 the latter possibly utilized in stress-regulated increases in GFAP. Another candidate class of signaling molecules that can induce astrocyte differentiation are members of the transforming growth factor-β (TGF-β) family, in particular, the bone morphogenic proteins (BMP) 2 and 7, which cause astrocytic development and suppress oligodendrocytic development in bipotential progenitors from neonatal rat forebrain and immature cells expanded from embryonic CNS by epidermal growth factor (EGF).57,58 Notably, serum and BMPs can induce GFAP expression in progenitors that have already begun to express the early oligodendrocyte marker, O4, giving rise to a hybrid glial cell type. It is not known whether glial progenitors begin to express O4 and then become astrocytes in vivo, during normal glial development, but the possibility seems unlikely. However, under pathological conditions (such as the development of brain tumors composed of progenitor-like cells) the acquisition of astrocyte gene expression in oligodendrocyte lineage cells might occur. In contrast to BMPs, such growth factors as EGF, platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), thyroid hormone and insulin-like growth factor 1 (IGF1) do not promote astrocyte differentiation of O-2A progenitors. Rather, they either

Astrocyte Ontogenesis and Classification

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promote division of progenitors without differentiation, as in the case of PDGF and bFGF,59 or are permissive for oligodendrocyte differentiation and/or survival.60,61 In most of the experiments cited above, astrocytic “differentiation” was measured by the induction of GFAP expression. While this intermediate filament protein is characteristic of astrocytes and therefore denotes the acquisition of at least one astrocyte feature, it is not clear whether there is a group of genes expressed coordinately during astrocyte development and whether in vitro systems fully capture that differentiated state. That other genes are both necessary and sufficient for astrocytic differentiation is clear from the several GFAP knockout transgenic mice, in which astrocytes do develop.62-64 In the future, control of specific receptors, transporters, and astrocyte enzymes will be required to characterize the developmental pathway. As discussed below, perhaps a progenitor makes several decisions during astrocyte development—the first to differentiate into an astrocyte and the second to acquire specific characteristics required for specific functions in the local CNS environment. Determinants of astrocyte fate in vivo has not been examined in as much detail as determinants in culture. Many of the factors suggested from the culture studies to play a role in fate determination exist in the developing brain. However, when a given progenitor becomes responsive to those signals and even whether such signals play a role in vivo is not yet known. Clues as to the nature of developmental signals may come from considering the anatomic changes that take place during astrocyte development. The peak period of astrocyte genesis coincides with the rapid growth of blood vessels65-67 and pial surface, the elaboration of dendritic arbors, and the establishment of synapses (both from cortical afferents and from intracortical circuits). For example, in the rat forebrain, thalamocortical afferents enter the cortex around P2-4 and cortico-cortical fibers around P6-8.68,69 Thus, the differentiation of astrocytes takes place during the establishment of synaptic connections and of the vascular supply. How is the development of glia coordinated with vascular and synaptic growth to assure the appropriate glial-vascular and glial-neuronal interactions? Furthermore, does the development of astrocytes and/or oligodendrocytes play a role in vascular growth or synapse formation? There is evidence for mutual interactions between astrocytes and endothelial cells. Astrocytes may participate in the formation of endothelial tight junctions, the anatomic substrate of the blood-brain barrier, and in inducing specific endothelial cell properties, such as polarization of transporters, increases in γ-glutamyl transaminase.70-72 Furthermore, the presence of astrocytes in the mammalian retina correlates with the presence of blood vessels.73 In examining the fates of progenitors from the SVZ after migration into the cortex, we have noted a close concordance between the early stages of astrocyte differentiation, as judged by an increase in intermediate filament expression and the beginnings of a complex, multiprocess cell shape and contact with blood vessels or the pial surface.39 These observations do not prove a causal relationship between astrocyte differentiation and vessel contact, but the model suggests a way in which astrocyte development can be coordinated with the tremendous growth of blood vessels and the pial surface in late gestational and postnatal CNS development.

Genesis of Astrocyte Heterogeneity Astrocytes vary both in morphology and in the expression of certain antigens from region to region. One example is the well known morphological distinctions between the “fibrous” astrocytes of white matter and the “protoplasmic” astrocytes of gray matter, the former expressing a much higher level of GFAP than the latter.74 A number of studies have clearly shown functional heterogeneity among astrocytes, although most of these experiments have been performed in vitro. Thus, astrocytes cultured from different regions of the

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Astrocytes in Brain Aging and Neurodegeneration

CNS differ in their abilities to support process growth of neurons, in their responses to neurotransmitters, and in their expressions of proteoglycans.75-77 Astrocytes from one region appear to be matched functionally to support neurons of the same region; mesencephalic neurons grow better on mesencephalic astrocytes than on astrocytes from other regions, for example.77 In cultures from neonatal forebrain, which includes all cortical areas and white matter and some subcortical gray matter nuclei, there is a heterogeneity in the uptake of and responses to neurotransmitters within the astrocyte population.78,79 Whether this heterogeneity was determined in vivo before the cultures were established or in vitro is not clear, but the observations dramatically illustrate that astrocytes are able to acquire important functional differences. In another study, the clonal progeny of single spinal cord astrocytes in culture were examined, and both homogeneous and heterogeneous clones were observed,80 showing clearly that an individual proliferating astrocyte, or an individual progenitor, is able to generate a mixture of astrocytic forms. Less is known about heterogeneity in vivo, however, but techniques exist to study astrocyte physiology in slices, where responses to transmitters or uptake mechanisms could be studied in real time. In vivo retroviral labeling studies suggest (although do not yet prove) that different astrocyte forms can arise from a single progenitor. For example, the proximity of retrovirally labeled Bergmann glia and velate astrocytes in the cerebellar cortex suggests a clonal heterogeneity (Fig. 1.1).81,82 And, as noted above, the astrocytic progeny of a single progenitor in the neocortex probably span the entire cortical depth, and would therefore be exposed to different microenvironments. How is the heterogeneity of astrocytes determined? One model would suggest that progenitors first are induced to differentiate into astrocytes and then signals peculiar to the local environment dictate specific morphological and functional patterns. This model makes sense if an astrocyte’s functional properties must match those of the neurons in the immediate proximity. Thus, the heterogeneity of astrocytes may not be lineage related, in the sense that such heterogeneity has little to do with the astrocyte fate decision. Astrocytes can change morphology and expression of many molecules, including surface gangliosides, intermediate filaments, enzymes, and stress proteins, in response to pathological conditions (see for examples refs. 83, 84). So, even in the mature CNS, astrocytes maintain a remarkable malleability.

Generation of Astrocytes in the Adult CNS Thymidine labeling studies in the adult mammalian CNS show a low level of cell division in the mature CNS85-87 and several investigators have inferred a slow turnover of astrocytes. Genesis must be balanced by cell death, since numbers of astrocytes in the cortex do not appear to increase during adult life.88 The nature of the dividing cells is not clear; that is, astrocytes might be generated from dividing astrocytes or from dividing, immature cells that then differentiate into astrocytes. Under pathological conditions, such as trauma, astrocytes in the region of the lesion divide, although the capacity for proliferation appears limited.84 Whether new astrocytes are generated from immature cells in pathological circumstances is not known. Cycling cells in adult rat white matter, labeled with recombinant retroviruses, do not differentiate into astrocytes, either under normal conditions, demyelination, or trauma (refs. 89, 90 and our unpublished observations). This finding contrasts with studies that find a population of immature cells isolated from adult optic nerve, cord, or forebrain that can differentiate into either oligodendrocytes or astrocytes in culture (“adult O-2A progenitors”91,92). Again, there may be fate restrictions in vivo, or perhaps appropriate pathological conditions have yet to be found in vivo to induce astrocyte differentiation in cycling immature cells.

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9

Fig. 1.1. Morphological transformations in the development of astrocytes as revealed by a Lac-Z encoding retrovirus. Newborn rat pups were injected into the forebrain SVZ or cerebellar white matter as described.5,82 Labeled cells were visualized by X-gal staining. (a) two unipolar cells in the SVZ, 1 day after injection. (b) a bipolar cell oriented radially in the cortex, 3 days after injection; such cells do not express astrocyte markers and presumably represent progenitors. (c) an early astrocyte in the cortex, 3 days after injection; one process has wrapped around a blood vessel (arrowhead); cells at this stage are expressing intermediate filament proteins. (d) two velate astrocytes in the cerebellar granule cell layer, 2 weeks after injection, displaying mature forms. (e) a Bergmann glial cell (top), with a cell body in the Purkinje cell layer and processes extending into the molecular layer, adjacent to a velate astrocyte (bottom) in the granule cell layer, 2 weeks after injection.

Acknowledgments The work from the author’s lab has been supported by NIH grant NS-17125. Many thanks to Bernetta Abramson, Cathy Chuang, JoAnn Gensert, Steven Levison, Sharon Newman, Marielba Zerlin, and Lei Zhang for all of their many major contributions to our studies.

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References 1. Andriezen WL. The neuroglia elements in the human brain. Br J Med 2:227-230. 2. Peters A, Palay SL, Webster H deF. The Fine Structure of the Nervous System, 3rd Ed. Oxford: Oxford University Press, 1991. 3. Hama K, Arii T, Kosaka T. Three-dimensional organization of neuronal and glial processes: high voltage electron microscopy. Microsc Res Tech 1993; 29:357-367. 4. Kosaka T, Hama K. Three-dimensional structure of astrocytes in the rat dentate gyrus. J Comp Neurol 1986; 249:242-260. 5. Levison SW, Goldman JE. Both oligodendrocytes and astrocytes develop from progenitors in the subventricular zone of postnatal rat forebrain. Neuron 1993; 10:201-212. 6. Chan-Ling T, Stone J. Factors determining the morphology and distribution of astrocytes in the cat retina: a ‘contact-spacing’ model of astrocyte interaction. J Comp Neurol 1991; 303:387-399. 7. Marin-Padilla M. Prenatal development of fibrous (white matter), protoplasmic (gray matter), and layer I astrocytes in the human cerebral cortex: a Golgi study. J Comp Neurol 1995; 357:554-572. 8. Dani JW, Chernjavsky A, Smith SJ. Neuronal activity triggers calcium waves in hippocampal astrocyte networks. Neuron 1992; 8:429-440. 9. Palay SL, Chan-Palay V. Cerebellar Cortex, Cytology, and Organization. New York: SpringerVerlag, 1974. 10. Chan-Palay V, Palay SL. The form of velate astrocytes in the cerebellar cortex of monkey and rat: high voltage electron microscopy of rapid Golgi preparations. Z Anat Entwicklungsgesch 1972; 138:1-19. 11. Bovolenta P, Liem RHK, Mason CA. Glial filament protein expression in astroglia in the mouse visual pathway. Brain Res 1987; 430:113-126. 12. Butt AM, Ransom BR. Visualization of oligodendrocytes and astrocytes in the intact rat optic nerve by intracellular injection of Lucifer yellow and horseradish peroxidase. Glia 1989; 2:470-475. 13. Sims TJ, Gilmore SA, Waxman SG. Radial glia give rise to perinodal processes. Brain Res 1991; 549:25-35. 14. Bovolenta P, Liem RKH, Mason CA. Development of cerebellar astroglia: transitions in form and cytoskeletal content. Dev Biol 1984; 102:248-259. 15. Seress L. Development and structure of the radial glia in the postnatal rat brain. Anat Embryol (Berl) 1980; 160:213-226. 16. Mori K, Ikeda J, Hayaishi O. Monoclonal antibody R2D5 reveals midsagittal radial glial system in postnatally developing and adult brainstem. Proc Natl Acad Sci USA 1990; 87:5489-5493. 17. Edwards MA, Yamamoto M, Caviness VS Jr. Organization of radial glia and related cells in the developing murine CNS. An analysis based upon a new monoclonal antibody marker. Neuroscience 1990; 36:121-144. 18. Choi BH, Lapham LW. Radial glia in the human fetal cerebrum: A combined Golgi, immunofluorescent, and electron microscopic study. Brain Res 1978; 148:295-311. 19. Schmechel DE, Rakic P. A Golgi study of radial glial cells in developing monkey telencephalon: morphogenesis and transformation into astrocytes. Anat Embryol (Berl.) 1979; 156:115-152. 20. Misson J-P, Edwards ME, Yamamoto M et al. Identification of radial glial cells within the developing murine central nervous system: studies based upon a new immunohistochemical marker. Dev Brain Res 1988; 44:95-108. 21. Levitt P, Cooper ML, Rakic P. Coexistence of neuronal and glial precursor cells in the cerebral ventricular zone of the fetal monkey: An ultrastructural immunoperoxidase analysis. J Neurosci 1981; 1:27-39. 22. Halliday AL, Cepko CL. Generation and migration of cells in the developing striatum. 1992; Neuron 9:384-396. 23. Gray G, Sanes J. Lineage of radial glia in the chicken optic tectum. Development 1992; 114:271-283.

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24. Galileo DS, Gray GC, Owens G et al. Neurons and glia arise from a common progenitor in chicken optic tectum: demonstration with two retroviruses and cell-type-specific antibodies. Proc Natl Acad Sci USA 1990; 87:458-462. 25. Misson J-P, Takahashi T, Caviness VS Jr. Ontogeny of radial and other astroglial cells in murine cerebral cortex. Glia 1991; 4:138-148. 26. LeVine SM, Goldman JE. Embryonic divergence of oligodendrocyte and astrocyte lineages in developing rat cerebrum. J Neurosci 1988; 8:3992-4006. 27. Zerlin M, Levison SW, Goldman JE. Early patterns of migration, morphogenesis, and intermediate filament expression of subventricular zone cells in the postnatal rat forebrain. J Neuroscience 1995; 15:7238-7249. 28. Culican SM, Baumrind NL, Yamamoto M et al. Cortical radial glia: Identification in tissue culture and evidence for their transformation to astrocytes. J Neurosci 1990; 10:684-692. 29. Voigt T. Development of glial cells in the cerebral wall of ferrets: Direct tracing of their transformation from radial glia into astrocytes. J Comp Neurol 1989; 289:74-88. 30. Hunter KE, Hatten ME. Radial glial cell transformation to astrocytes is bidirectional: regulation by a diffusible factor in embryonic forebrain. Proc Natl Acad Sci USA 1995; 92:2061-2065. 31. Hatten ME, Liem RKH, Mason CA. Two forms of cerebellar glial cells interact differently with neurons in vitro. J Cell Biol 1984; 98:193-204. 32. Altman J. Proliferation and migration of undifferentiated precursor cells in the rat during postnatal gliogenesis. Exp Neurol 1966; 16:263-278. 33. Imamoto K, Paterson JA, Leblond CP. Radioautographic investigation of gliogenesis in the corpus callosum of young rats. I. Sequential changes in oligodendrocytes. J Comp Neurol 1978; 180:115-138. 34. Paterson JA, Privat A, Ling EA et al. Investigation of glial cells in semithin sections III. Transformation of subependymal cells into glial cells as shown by radioautography after 3H-thymidine injection into the lateral ventricle of the brain of young rats. J Comp Neurol 1973; 149:83-102. 35. Gressens P, Richelme C, Kadhim HJ et al. The germinative zone produces the most cortical astrocytes after neuronal migration in the developing mammalian brain. Biol Neonate 1992; 61:4-24. 36. Levison, SW, Chuang C, Abramson B et al. The migrational patterns and developmental fates of glial precursors in the rat subventricular zone are temporally regulated. Development 1993; 119:611-622. 37. Luskin MB, McDermott K. Divergent lineages for oliogodendrocytes and astrocytes originating in the neonatal forebrain subventricular zone. Glia 1994; 11:211-226. 38. Zerlin M, Levison SW, Goldman JE. Early patterns of migration, morphogenesis, and intermediate filament expression of subventricular zone cells in the postnatal rat forebrain. J Neurosci 1995; 15:7238-7249. 39. Zerlin M, Goldman JE. Interactions between glial progenitors and blood vessels during early postnatal corticogenesis: blood vessel contact represents an early stage of astrocyte differentiation. J Comp Neurol 1997; 387:537-546. 40. Skoff RP. Gliogenesis in rat optic nerve: Astrocytes are generated in a single wave before oligodendrocytes. Dev Biol 1990; 139:149-168. 41. Fulton BP, Burne JF, Raff MC. Visualization of O-2A progenitor cells in developing and adult rat optic nerve by quisqualate-stimulated cobalt uptake. J Neurosci 1995; 12:4816-4833. 42. Raff MC, Miller RH, Noble M. A glial progenitor cell that develops in vitro into an astrocyte or an oligodendrocyte depending on culture medium. Nature 1983; 303:390-396. 43. Hirano M, Goldman JE. Gliogenesis in rat spinal cord: Evidence for origin of astrocytes and oligodendrocytes from radial precursors. J Neurosci Res 1988; 21:155-167. 44. Maier CE, Miller RH Development of glial architecture in the frog spinal cord. Dev Neurosci 1995; 178:149-159. 45. Warf BC, Fok-Seang J, Miller RH. Evidence for the ventral origin of oligodendrocyte precursors in the rat spinal cord. J Neurosci 1991; 11:2477-2488.

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46. Richardson WD, Pringle NP, Yu W-P et al. Origins and early development of oligodendrocytes. In: Jessen KR, Richardson WD, eds. Glial Cell Development, Basic Principles and Clinical Relevance. Oxford, UK: Bios Scientific Publishers, 1996:53-70. 47. Levison SW, McCarthy KD. Characterization and partial purification of AIM: a plasma protein that induces rat cerebral type 2 astroglia from bipotential glial progenitors. J Neurochem 1991; 57:782-794. 48. Hughes SM, Lillien LE, Raff MC et al. Ciliary neurotrophic factor induces type-2 astrocyte differentiation in culture. Nature 1988; 335:70-72. 49. Lillien LE, Sendtner M, Raff MC. Extracellular matrix-associated molecules collaborate with ciliary neurotrophic factor to induce type-2 astrocyte development. J Cell Biol 1990; 111:635-644. 50. Gard AL, Williams WC, Burrell MR. Oligodendroblasts distinguished from O-2A glial progenitors by surface phenotype (O4+/GalC-) and response to cytokines using signal transducer LIFRβ. Dev Biol 1995; 167:596-608. 51. Johe KK, Hazel TG, Muller T et al. Single factors direct the differentiation of stem cells from fetal and adult central nervous system Genes Dev 1996; 10:3129-3140. 52. Bonni A, Sun Y, Nadal-Vicens M et al. Regulation of gliogenesis in the central nervous system by the JAK-STAT signaling pathway. Science 1997; 278:477-483. 53. Kahn MA, Huang CJ, Caruso A et al. Ciliary neurotrophic factor activates JAK/Stat signal transduction cascade and induces transcriptional expression of glial fibrillary acidic protein in glial cells. J Neurochem 1997; 68:1413-1423. 54. Besnard F, Brenner M, Nakatani Y et al. Multiple interacting sites regulate astrocyte-specific transcription of the human gene for tglial fibrillary acidic protein. J Biol Chem 1991; 266:18877-18883. 55. Masood K, Besnard F, Su Y et al. Analysis of a segment of the human glial fibrillary acidic protein gene that directs astrocyte-specific transcription. J Neurochem 1993; 61:160-166. 56. Shafit-Zagardo B, Iwaki AK, Goldman, JE. Astrocytes regulate GFAP mRNA levels by cAMP and protein kinase C dependent mechanisms. Glia 1988; 1:346-354 57. Gross RE, Mehler MF, Mabie PC et al. Bone morphogenetic proteins promote astroglial lineage commitment by mammalian subventricular zone progenitor cells. Neuron 1996; 17:595-606. 58. Mabie P, Mehler MF, Marmur R et al. Bone morphogenetic proteins induce astroglial differentiation of oligodendroglial-astroglial progenitor cells. J Neurosci 1997; 117:4112-4120. 59. Noble M, Murray K, Stroobant P et al. Platelet-derived growth factor promotes division and inhibits premature differentiation of the oligodendrocyte/type 2 astrocyte progenitor cell. Nature 1988; 333:560-562. 60. Behar T, McMorris FA, Novotny EA, Barker JL, Dubois-Dalcq M. Growth and differentiation properties of O-2A progenitors purified from rat cerebral hemispheres. J Neurosci Res 1988; 21:168-180. 61. Barres BA, Raff M. Axonal control of oligodendrocyte development. In: Jessen KR, Richardson WD, eds. Glial Cell Development, Basic Principles and Clinical Relevance. Oxford, UK: Bios Scientific Publishers, 1996:71-83. 62. Pekny M, Leveen P, Pekna M et al. Mice lacking glial fibrillary acidic protein display astrocytes devoid of intermediate filaments but develop and reproduce normally. EMBO J 1995; 14:1590-1598. 63. Shibuki K, Gomi H, Chen L et al. Deficient cerebellar long term depression, impaired eyeblink conditioning, and normal motor coordination in glial fibrillary acidic protein mutant mice. Neuron 1996; 16:587-599. 64. Liedtke W, Edelmann W, Bieri PL et al. GFAP is necessary for the integrity of CNS white matter architecture and long-term maintenance of myelination. Neuron 1996; 17:607-615. 65. Caley DW, Maxwell DS. Development of the blood vessels and extracellular spaces during postnatal maturation of rat cerebral cortex. J Comp Neurol 1970; 138:31-48. 66. Phelps CH. The development of glio-vascular relationships in the rat spinal cord. Z Zellforsch 1972; 128:555-563.

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67. Robertson PL, Du Bois M, Bowman PD et al. Angiogenesis in developing rat brain: an in vivo and in vitro study. Dev Brain Res 1985; 23:219-223. 68. Wise SP, Jones ED. Organization and postnatal development of the commissural projection of the rat somatic sensory cortex. J Comp Neurol 1976; 168:313-343. 69. Wise SP, Jones ED. Developmental studies of thalamocortical and commissural connections in the rat somatic sensory cortex. J Comp Neurol 1978; 178:187-208. 70. DeBault LE, Cancilla PA. g-Glutamyl transpeptidase in isolated brain endothelial cells: induction by glial cells in vitro. Science 1980; 207:653-655. 71. Janzer, RC, Raff MC. Astrocytes induce blood-brain barrier properties in endothelial cells. Nature 1987; 325:253-257. 72. Beck DW, Roberts RL, Olson JJ. Glial cells influence membrane-associated enzyme activity at the blood-brain barrier. Brain Res 1986; 381:131-137. 73. Schnitzer J. Astrocytes in the guinea pig, horse, and monkey retina: their occurrence coincides with the presence of blood vessels. Glia 1988; 1:74-89. 74. Kitamura T, Nakanishi K, Watanabe S et al. GFA-protein gene expression on the astroglia in cow and rat brains. Brain Res 1987; 423:189-195. 75. Wilkin GP, Marriott DR, Cholewinski AJ. Astrocyte heterogeneity. TINS 1990; 13:43-46. 76. Garcia-Abreu J, Neto VM, Carvelho SL et al. Regionally specific properties of midbrain glia: I. Interactions with midbrain neurons. J Neurosci Res 1995; 40:471-477. 77. Denis-Donini S, Glowinski J, Prochaintz A. Glial heterogeneity may define the three-dimensional shape of mouse mesencephalic dopaminergic neurons. Nature 1984; 307:641-643. 78. Amundson RH, Goderie SK, Kimelberg HK. Uptake of [3H] serotonin and [3H] glutamate by primary astrocyte cultures. II. Differences in cultures prepared from different brain regions. Glia 1992; 6:9-18. 79. McCarthy KD, Salm AK. Pharmacologically distinct subsets of astroglia can be identified by their calcium response to neuroligands. Neuroscience 1991; 41:325-333. 80. Miller RH, Szigeti V. Clonal analysis of astrocyte diversity in neonatal rat spinal cord cultures. Development 1991; 113:353-362. 81. Miyake T, Fujiwara T, Fukunaga T et al. Glial cell lineage in vivo in the mouse cerebellum. Develop Growth Differ 1995; 37:273-285. 82. Zhang L, Goldman JE. Developmental fates and migratory pathways of dividing progenitors in the postnatal rat cerebellum. J Comp Neurol 1996; 370:536-550. 83. Eddleston M, Mucke L. Molecular profile of reactive astrocytes—implications for their role in neurologic disease. Neuroscience 1993; 54:15-36. 84. Norton WT, Aquino DA, Hozumi I et al. Quantitative aspects of reactive gliosis: a review. Neurochem Res 1992; 17:877-885. 85. Altman J Autoradiographic investigation of cell proliferation in the brains of rats and cats. Anat Rec 1963; 145:573-591. 86. Kaplan MS, Hinds JW. Gliogenesis of astrocytes and oligodendrocytes in the neocortical gray and white matter of the adult rat: electron microscopic analysis of light radioautographs. J Comp Neurol 1980; 193:711-727. 87. McCarthy GF, Leblond CP. Radoautographic evidence for slow astrocyte turnover and modest oligodendrocyte production in the corpus callosum of adult mice perfused with 3H-thymidine. J Comp Neurol 1988; 2761:589-603. 88. Ling EA, Leblond CP. Investigation of glial cells in semithin sections. II. Variation with age in the numbers of the various glial cell types in rat cortex and corpus callosum. J Comp Neurol 1973; 149:73-82. 89. Gensert JM, Goldman JE. In vivo characterization of proliferating cells in adult rat subcortical white matter. Glia 1996; 17:39-51. 90. Gensert JM, Goldman JE. Remyelination by endogenous progenitors in the adult rat CNS. Neuron 1997; 19:197-203. 91. ffrench-Constant C, Raff MC. Proliferating bipotential glial progenitor cells in adult optic nerve. Nature 1986; 319:499-502. 92. Wren D, Wolswijk G, Noble M. In vitro analysis of origin and maintenance of O-2A adult progenitor cells. J Cell Biol 1992; 116:167-176.

CHAPTER 2

Functions of Astrocytes Harold K. Kimelberg and Michael Aschner

Introduction

I

n the previous chapter, Goldman covered the structure and development of astrocytes, and that chapter should be read before reading this chapter to better understand the functional properties we will discuss. Thus, an appreciation of the complex morphologies of all types of astrocytes, their interrelationships with other cells and brain structures such as blood vessels, and the complexity of astrocyte development must surely reasonably lead, based on the principle that form reflects function, to the conclusion that astrocytes are likely to have many complex properties closely associated with many aspects of brain function. Indeed, there has been no dearth of hypotheses regarding astrocyte function emerging simply from contemplation of the complexities of astrocyte morphology and interrelationships dating from the work of Golgi, Cajal and others,1 which first showed their structures in precise detail in the late nineteenth century. For example, per Lugaro2 in 1907; “the neuronal articulation* would be the center of the chemical exchange, and this would comprise therefore in all the most proximal, vacant interstitial spaces, a region for infiltration of the protoplasmic prolongations or feathery extensions of the neuroglia, perhaps with the purpose of collecting and instantly processing the smallest amount of waste product.” Golgi and Cajal among others speculated that the roles of glia included neuronal nutrition, structural and metabolic support and involvement involved in nervous system development.1 However, these and other hypotheses could not then be tested. Experimental studies on glial function began with the work of Kuffler and his colleagues in the mid 1960s. They focused on the electrical and ion transport properties of glia in simple invertebrate nervous systems and the relatively simple preparation of the amphibian optic nerve.3 Beginning in the 1970s primary astrocyte cultures from neonatal rodent CNS began to be used extensively to study the properties of astroglia.4,5 The primary cultures prepared from neonatal rodents consist predominately of GFAPpositive astrocytes and provide preparations of cells in sufficient numbers to allow for a variety of biochemical, electrophysiological, molecular and general cell biological studies. It is still unclear as to why all the cells in these astrocyte cultures, which consist primarily of flat cells which have been proposed to be analogous to protoplasmic astrocytes, stain for GFAP whereas protoplasmic astrocytes in situ in many regions, such as the cerebral cortex, stain variably for GFAP.6 This has led to the view that the cultures may consist predominantly of reactive astrocytes, which in situ are characterized by prominent GFAP staining.7 * synapse Astrocytes in Brain Aging and Neurodegeneration, edited by Hyman M. Schipper. ©1998 R.G. Landes Company.

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It needs to be emphasized that the bulk of the current information on the properties of mammalian astrocytes has come from these preparations, as they are relatively easily studied. However, their properties are often imprecisely referred to as astrocytic properties, without any qualifications. Studies on primary astrocyte cultures have always had the implicit caveat that one is uncertain as to how their properties are altered by growth in vitro.8 In our view, such cultures have had two major and critical advantages. One, they led to a major expansion of studies on astrocytes, albeit mainly in these culture systems, which otherwise would probably not have been done in any other system. Second, the results of such studies suggested to neuroscientists that astrocytes and other glial cells could have a number of properties such as receptors and uptake systems for transmitters and gated and rectifying channels which, based on a few negative studies on glial cells in situ had, rather prematurely, been considered specific to neurons in the CNS. The primary cultures have now provided a long list of putative functions which need to be tested in systems more representative of the in vivo state, for the numerous differences that have now been reported between the properties of the primary astrocyte cultures and the properties of astrocytes expressed in preparations closer to the in situ state, such as brain slices, makes it impossible to use primary cultures by themselves to define astrocyte function. Thus there is currently less emphasis on such cultures and a reemphasis on in situ preparations that appear to more closely correspond to in vivo situations. The relative paucity of reliable information is presumably why astrocytes so rarely figure in discussions of brain function. In contrast, the characteristic properties of neurons have been studied in great detail and were found to lend themselves relatively easily to hypotheses of information processing by the postulation of electrically active loops and networks. Also, experimental interference with neuronal function led to clear effects on neural function, so that discussions of how the nervous system functions at the most complex levels are currently almost exclusively based on the properties of neurons.9 Thus a certain circularity of reasoning is apparent that can only be broken by sufficient rigorous study of the properties that astrocytes and other glia have in the CNS. In many respects we are still searching for experimental systems in which hypotheses advanced at the turn of this century can be rigorously tested.

Functions of Astrocytes We will arrange this section based first on the properties of astrocytes established both in cultured and acutely isolated cells or in slices of the intact brain, taking care to note the experimental systems in which they were obtained. We will also discuss potential functions that these properties indicate, and then mention the very limited number of cases in which functions have actually been demonstrated. It must be born in mind, however, that the properties studied are not only limited by the experimental systems, but have been conceptually restricted, notably by concepts based on what has been developed for neurons which have led to success in understanding nervous function. Thus, it is not surprising that many studies of astrocytes have involved investigations of their membrane electrical properties and ion channels, even though this approach has led a well-respected worker in the field of glial electrophysiology to conclude that “generation of glial electric signals is not among their (i.e., astrocytes) functions.”10 This then begs the question: What is (are) the role(s) of the predominant K+ channels seen in astrocytes and what is (are) the role(s) of the -70 to -80 mV membrane potentials that are a consequence of them?

Functions of Astrocytes

17

Homeostasis of the Extracellular Space Regulation of Extracellular K+ One answer to the question just posed was proposed by Kuffler et al.3 Namely, that the selective K+ permeability implied a role in control of extracellular potassium levels ([K+]o). The original reason for Kuffler and his colleagues embarking on their pioneering studies was that electron microscopic studies of mammalian CNS had shown, at this time, that astrocytes generally formed enlarged watery compartments seemingly obliterating the extracellular space (ECS). This led to the proposal that astrocytes actually formed the extracellular space of the brain. This would require that they would uniquely be high Na+ cells, and it was to examine this question that Kuffler and his associates studied the easily accessible glial cells in the leech nervous system and the amphibian optic nerve. In both cases, glial cells were found to have a membrane potential of -80 to -90 mV, and showed a close to Nernstian response to varying [K+]o. Thus these glial cells had to have high intracellular K+ rather than Na+ concentrations, and thus could not form the ECS. The finding of an essentially selective K+-dependent membrane potential implied that the cell membranes were operationally impermeable to sodium, and possibly chloride, and led to a mechanism for uptake of K+ released by active neurons.3 The mechanism would be that a localized release of K+ from neurons during excitation would depolarize the astrocyte at this point with a 60 mV depolarization for a 10-fold increase in [K+]o. This would set up a current loop with other nondepolarized parts of the cell and, since the membrane was permeable only to K+, there would be an inward current at the depolarized point carried by extracellular K+ crossing the membrane. Since K+ is the major electrolyte inside the cell, it would also be the major current carrier inside the cell, and the current loop would be connected by efflux of K+ at some distant point. The return part of the loop would be carried by major extracellular ions such as Na+, or Cl– in the opposite direction. This led to the concept of “K+ spatial buffering” in which K+ is transferred from a region of localized release to some distant point, traveling through the astrocyte or the astrocytic syncytium. Work since the studies of Kuffler and his colleagues has concentrated on identifying the types and location of K+ channels in different glial preparations using modern patch clamp methods. K+ channels are the most diverse ionic channel type11 and, as reviewed over the past several years, a wide variety of K+ channels have been found, predominantly using cultured astrocytes.12-15 These K+ channels include an inward rectifying K+ channel (K+in), Ca2+-dependent K+ channels (K+Ca), delayed rectifying channels (K+D) and an inactivating potassium channel (K+a). K+ channels sensitive to ATP have also been found in astrocytes, such as an ATP-regulated, strongly inward rectifying K+ channel that has been observed on Bergmann glia in situ.16 Some of these channels may be related to the K+ spatial buffering phenomenon just discussed. When there is also a significant chloride permeability (see later), net KCl uptake leading to swelling will occur when [K+]o rises. Some of these K+ channels should also be responsible for the large negative K+ diffusion potentials characteristic of astrocytes. The work of Newman17 using acutely isolated astrocytes has indicated inward K+ rectifying channels at very high densities in areas of the astrocyte where it seems to be adapted to K+ spatial buffering, namely at the capillary-facing astrocytic end-feet. If the membrane potential is very close to the K+ equilibrium potential, then the net outward leak of K+ will always be very low, but this may be increased when there is depolarization of the astrocyte caused by other than an increased [K+]o, such as by receptor activation. In this case, there will be an outward flux of potassium which would be later replenished by reuptake on the Na+/K+ pump or repolarization and reestablishment of Em ≅ Ek (see later). If K+ channels are important in astrocyte function, then it is likely that alterations in their functioning would affect astrocyte properties and they are likely to be targets of the

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Astrocytes in Brain Aging and Neurodegeneration

activation of astrocyte receptors. This is currently an active and fruitful area of investigation. Thus, it has been shown that β-receptor activation modulates K+IR currents in cultured rat spinal cord astrocytes,18 as well as altering astrocyte proliferation in vitro.15 AMPA/kainate receptor activation blocks outward K+ currents in cultured stellate mouse cortical astrocytes.19 This was suggested as a mechanism whereby astrocytes do not lose too much K+ when they become depolarized in pathological states. The K+ currents of glial cells in situ in hippocampal brain slices have been studied from different aged animals in both nonexcitable, GFAP-negative “complex cells” from younger animals and in GFAP-positive cells from older animals (>P20). The complex cells exhibited more types of ion currents. They showed a delayed outward K+ rectifier (K+D) and a transient outward A-type K+ current. They also showed a TTX-sensitive Na+ current. In the older cells, the voltage-gated Na+ and K+ outward currents downregulated and were replaced by passive and inward rectifier K+ conductances.20 These changes are consistent with a precursor glial cell with a more complex array of ion channels changing into a mature astrocyte which exhibits K+ channels that have predominantly [K+]o regulating properties. It has also been shown that a variety of K+ channel blockers inhibit cell proliferation in cultured astrocytes.15 Recent work has also shown that application of cesium for >2 min to hippocampal slices blocks long term depression (LTD) and synchronous, interictal-like bursting in the CA1 region.21 Studies using patch-clamp electrophysiology showed this to be due to a direct blockade of the K+IR currents of astrocytes. The increase in [K+]o was considered to block the pyramidal cell activity since there was no change in the pyramidal cell conductance. This experiment is reminiscent of the 30 year old study of Krnjevic and Schwartz22 wherein they attempted to detect transmitter-induced conductance changes in glial cells from the cerebral cortex using sharp electrodes. They found no such changes, possibly due to the insensitivity of their techniques, and concluded that depolarization of the glial cells was due to a rise in [K+]o rather than a transmitter-mediated conductance change in the astrocyte

Na+ Channels This is a more controversial area because if astrocytes are nonexcitable there would appear to be no need for Na+ channels, or at least voltage-sensitive ones. Na+ currents in glia were first described in astrocytes in primary cultures.23,24 Like neuronal channels, these were sensitive to tetrodotoxin (TTX), but it was found that there were both TTX-sensitive and relatively insensitive Na+ channels which had different characteristics in terms of the depolarization required to activate them.25 The depolarizations needed to open these channels were always thought to be greater than would ever be seen in astrocytes “clamped” at a highly negative membrane potential by their large K+ conductances.5 However, recent work by Sontheimer et al26 has found that astrocytes cultured from certain regions of the brain, such as the spinal cord, have a very high density of Na+ channels which would have some open probability at the resting membrane potential of these cells. It was hypothesized that the Na+ channels may function in regulating entry of Na+ in order to activate the Na+/K+ pump when active uptake K+ is required, such as when [K+]o rises from its normal level of 3 mM to 5-10 mM during periods of sustained neuronal activity. This thus represents a self-regulating mechanism for active K+ clearance by astrocytes that does not require any special properties of the Na+/K+ pump, and such special properties have not been clearly shown (see below). The major question in regard to the Na+ channels, as with other astrocytic properties mainly described in astrocytes in culture, is whether, when and in what cells Na+ channels are expressed in situ. The type II sodium channel has been seen in astrocytes in situ in the dorsal and ventral columns of the spinal cord of the adult rat using immunocytochemistry,

Functions of Astrocytes

19

but can only assumed to be functional.27 The function(s) of these channels must at present be purely speculative. Do they provide a voltage-dependent path for Na+ entry? There are, however, several other routes for Na+ entry into astrocytes. There are cotransporters for glutamate and aspartate and other substances which utilize the energy of the Na+ gradient to actively accumulate these substances. Are the channels a source of Na+ channels for the axolemmal membrane of the node, as suggested by Ritchie?28 Do they in some way add to the ability of astrocytes to control the ionic composition of the ECS? However, these channels are only activated when the cell membrane is depolarized to at least -40 mV and the question still remains as to what extent such depolarizations do occur in astrocytes in situ.

Ca2+ Channels Voltage-gated L-type Ca2+ channels were also first identified in primary astrocyte cultures.29 This was again a surprising finding because it was thought that such channels in the CNS were specific to neurons and were responsible for such properties as Ca2+ action potentials, depolarization-induced Ca2+ influx required for exocytosis at nerve terminals and modulation of neuronal firing rates by hyperpolarization of the membrane potential via Ca2+-activated K+ channels. The question raised in regard to voltage-sensitive Na+ channels can also be raised in regard to voltage-sensitive Ca2+ channels; namely, are the astrocytes ever depolarized enough to activate these channels? Thus the function of the Ca2+ channels in astrocytes has not yet been satisfactorily explained, but the occurrence of large changes in [Ca2+]i levels in astrocytes when stimulated by receptors such as glutamate, mechanical stimulation or swelling raise the possibility that part of the intracellular Ca2+ may be entering via such channels.30 Ca2+ channels will also be necessary for the entry of Ca2+ to replenish intracellular Ca2+ stores after their depletion.30 Intracellular Ca2+ is a pleiotropic intracellular second messenger affecting processes from gene regulation to ion channel activities. The reader is referred to several reviews on this topic in astrocytes.30-35 In this context, the changes in [Ca2+]i levels can be viewed as a ubiquitous intracellular signaling mechanism present in astrocytes, as in other cells, rather than subserving a specific CNS function such as regulated release of neurotransmitters at CNS synapses.

Anion Channels As in other cells, anion channels have been less studied in astrocytes than cation channels, but it is now becoming clear that anion channels do have fundamental functions in many, if not all, cells. A number of anion channels have now been identified in cultured astrocytes, including small conductance chloride channels (5-25 pS) and a high conductance chloride channel (250-400 pS).14,15 These channels also transport Cl– and HCO3–, but the high conductance channel in astrocytes may also transport organic anions such as amino acids,35 as may some of the other channels.36 The roles of anion channels may also include the uptake of HCO3– or chloride to accompany uptake of K+; a mechanism additional to K+ spatial buffering (see above) to control [K+]o. However, this will lead to cell swelling, and many pathological states involve exaggerated astrocyte swelling.37 Cell swelling causes activation of a number of ion channels and Strange et al38 and Okada39 have recently reviewed the different anion channels that may be involved in anion or amino acid efflux in swollen cells, including astrocytes. It is important to define the types of different anion channels normally present on astrocytes, and which ones are activated during swelling or are responsible for K+-dependent Donnan swelling, because, if release of excitatory amino acids in ischemia and other pathological states occurs through a particular type of anion channel, then the identification of such channels would be of considerable practical benefit to either inhibit the swelling-induced release or prevent the swelling in the first place. The inhibitory neurotransmitters GABA

20

Astrocytes in Brain Aging and Neurodegeneration

and glycine also activate anion channels associated with some of their receptors, and such receptors appear to be present on astrocytes40-43 (see later).

Ion Carriers Carriers are distinct from channels in that a concerted movement of several ions usually occurs, rather than the independent diffusional movement of ions down their electrochemical gradients as in channels.11 A number of carriers have been identified in astrocytes. Na++K++2Cl– cotransporter One important ion carrier is the Na++K++2Cl– uptake system utilized by cells for active uptake of Cl–. This carrier has been found in astrocytes in primary culture, and intracellular Cl– in astrocyte cultures has been found to be several-fold greater than expected from electrochemical equilibrium.14 This carrier has also been localized by immunocytochemistry to Bergmann glia in situ.44 High [Cl–] in astrocytes may serve as a source to maintain extracellular Cl–, based on the finding of GABAA receptors on astrocytes both in vitro and in situ whose activation leads to efflux of Cl–.45 This was proposed as a mechanism to maintain extracellular concentrations at the same time as GABA causes influx of Cl– into neurons, which contain low Cl–. In addition, the high Cl– in astrocytes may also be required for the efflux of KCl in the process of volume regulation, as proposed by Kimelberg and Frangakis.46 Measurements with ion-specific microelectrodes in guinea-pig brain slices, however, have shown intracellular glial Cl– levels that were in equilibrium with the membrane potential and therefore