perspectives With the idea that classical institutes for brain research are still a good solution for small countries, the Ministries of Health and Science in the Republic of Croatia supported the foundation of the Croatian Institute for Brain Research at the School of Medicine, University of Zagreb. The association with the university has two major advantages: (1) the senior scientists working in the institute have permanent teaching appointments at the university, and (2) the association facilitates the recruitment of talented students for research teams in the institute. A novel approach in the organization of the Croatian Institute for Brain Research was the establishment of a system to evaluate scientific programmes through an international Scientific Advisory Board. The members of this board are prominent neuroscientists, among them K. Krnjevid, P. Rakic, P. S. Goldman-Rakic, D. F. Swaab and H. Braak. Furthermore, the institute has a board of trustees that is composed of both foreign and governmental authorities. The scientific programme of the institute is oriented towards the neurobiological basis of the major neurological and mental disorders: developmental disorders, Down's syndrome, Alzheimer's disease and schizophrenia. An essential part of the institute is the Zagreb neuroembryological collection
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and the brain tissue bank. The organizational scheme of the Croatian Institute for Brain Research is based on the positive experience of the Netherlands Institute for Brain Research in Amsterdam. This entire project takes high priority under the sponsorship of the President of the Republic of Croatia, and has a good chance of setting a trend in the organization of neuroscience research all over the Republic of Croatia. Other republics might very soon organize similar neuroscience institutes or revitalize already existing ones. To achieve all these aspirations requires not only hard work but also a fortunate and speedy outcome of the political and economical crisis in Yugoslavia. The international assistance to both the Yugoslav federal state and individual republics is of the greatest value and importance during these transitional stages.
Acknowledgements Wearegratefulto Mr sc.JelkaPetrak, chairmanof the CentraIMedlca/ Library,Schoolof Medicine, University of Zagreb,and to Mr sc.ZoranBuneta, Divisionof Scientometnc Analysis,Schoolof Medicine, University of Zagreb,for their generoushelpin the analysisof published work that is included in thispaper.
Selected references 1 Lackovi(~,Z., Buneta, Z., Relja, M. and Ce~uk, L. (1986) Li/e~. Vjesn. 108, 462--471 2 Lackovi~,Z., Buneta, Z., Relja, M. and C~e~uk,L. (1987) Lije#. Vjesn. 109, 49-56 3 Coles, P. (1990) Nature 344, 616-617 4 Buret, J., Ciganek, L. and Vina[, O. (1991) Trends Neurosci. 14, 11-13 5 Schubert,A., Glanzel,W. and Braun,T. (1989) Scientornetrics 16, 3-478
Thepathogenesisof demyelinatingdisease:insights from cell biology Alastair Compston, Neil Scolding, Damien Wren and Mark Noble Cellularand humoral immune mechanismshavebeen implicated in the pathogenesis of human and experimentaldemyelinating diseases of the CNSI-~. How theseinteractin the complexsequenceof events that culminatesm phagocytosis of myelin by macrophageshas yet to be resolved The relationship between leakage of the blood-brain barrier and demyelination, the reason why recurrent inflammatory demye/ination occurs - seemingly in the absence of an antigenspecific immune response- and the lack of effective remye/inationall require explanation if a coherent account of immuno/ogical/y mediated demyelination Is to be achieved. One approach to these problems is to study in vitro the developmentaland cel/u/arbio/ogy of oligodendrocytes - the gila/cells responsible for the synthesis and maintenanceof CNSmyelin. Thisprovides experimentalopportunities not offered by more direct investigation of the intact nervous system, but carries the clear disadvantage that observations made in vitro cannot necessarilybe extrapolated to humans. Not all inflammatory demyelinating lesions give rise to neurological symptoms 6, and the pathological process is itself sometimes self limiting. In many patients, episodes of demyelination recur- imaging techniques detecting 20 or more new lesions annually - and neurological disability gradually accumulates; this temporal pattern forms the basis for distinguishing multiple sclerosis from the syndromes of isolated demyelination. Classical accounts of the histopathology of human demyelination established that multifocal TINS, Vol. 14, No. 5, 1991
infiltration by perivascular inflammatory cells precedes macrophage-mediated myelin degradation, which in turn is followed by reactive astrocytosis, oligodendrocyte depletion and some axonal loss. Another feature of demyelination is that infiltrating lymphocytes are rarely found in intimate contact with myelin sheaths 7. It is now clear that oligodendrocytes are damaged even in acute lesions, but extensive remyelination can occur 7'8. Immunocytochemical analysis of lesions has shown that (I) the infiltrating cells are predominately of the CD4 phenotype, (2) B lymphocytes, plasma cells, complement activation products and immunoglobulin are also present in acute lesions, and (3) macrophages in contact with myelin sheaths bear surface antibodies and contain aggregates of immunoglobulin and complement 7-11. These findings highlight the need to investigate interactions between oligodendrocytes, macrophages and humoral immune mediators. During development and repair, oligodendrocytes extend elongated processes that contact and then rotate many times around nearby axons, losing their cytoplasm as the segmented, multilamellated sheaths of compact myelin are formed. Myelin is more than an inert insulating material, although its dynamic function is poorly understood. It is rich in a number of specific enzymes, including a basic
© 1991, ElsevierSciencePublishersLtd, (UK) 0166- 2236191/$02.00
A/astairCompston and NellScoldingare at the Universityof CambridgeClinical School,Neurology Dept,Addenbrooke's Hospital, HillsRd, CambridgeCB22QQ, UK, DamienWren is at the lnstituteof Neurology, Queen Square,London WCIN 3BG,UK, and Mark Nobleisat the LudwigInstitutefor CancerResearch, RidingHouseSt, London W1PSBT, UK.
175
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Q 1{ Fig. 1. Acute swelling of oligodendroglia. Various stages of acute swelling of oligodendrocytes in pathological specimens are shown, from normal cell (A) to complete disappearance of the cell body, leaving naked nucleus (H).
protein kinase and cyclic nucleotide phosphodiesterase, and has a variety of membrane ion channels12'13. The parent oligodendrocyte soma supports this anatomically extensive and metabolically active membrane throughout the lifetime of each cell. However, recent work suggests that, among gila, the oligodendrocyte is uniquely vulnerable to immunological injury. The O-2A lineage Much of the current information about the development of oligodendrocytes has emerged from studying the 'simplest' part of the vertebrate CNS, the optic nerve 14. This tissue contains three cell types that are of neuroectodermal origin. Type1 astrocytes (first cell type) and their precursors contribute to the morphogenetic development of the optic nerve by offering a preferred substratum for growing axons. These cells also interact with endothelial cells to induce formation of the bloodbrain barrier, and are a source of mitogen for other cells in the developing nerve. While oligoclendrocytes (second cell type) enwrap axons with myelin sheaths, type-2 astrocytes (third cell type) are thought to extend processes that associate with axons at the nodes of Ranvier- the regions between consecutive myelin segments where ion fluxes occur
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during saltatory conduction. Thus, oligodendrocytes and type-2 astrocytes act together in creating the anatomical specializations that characterize myelinated tracts within the CNS. The three differentiated glial cell types of the optic nerve are generated at specific developmental periods from two distinct cellular lineages. The neuroepithelial cells that form the optic stalk (the embryonic primordium of the optic nerve) seem to give rise only to type-1 astrocytes. The initial differentiation of type-1 astrocytes, at day 16 of rat embryogenesis (E16), is followed developmentally by the appearance within the nerve of oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells, which can be induced to differentiate in vitro into either oligodendrocytes or type-2 astrocytes 15. O-2A progenitors are thought to arise in a germinal zone in or near the optic chiasm, first migrating into the optic nerve at E17. Oligodendrocytes are first seen in the optic nerve at the time of birth [E21, or postnatal day zero (P0)]. Cells with the antigenic phenotype of type-2 astrocytes are present in suspensions of optic nerve at P14 (Ref. 16). Recent studies have demonstrated that although O-2A progenitors can be extracted from both adult and neonatal optic nerve 17, O-2A (adult) progenitors differ in vitro from their perinatal counterparts in antigen expression, morphology, cell-cycle motility, timecourse of differentiation, and immunological behaviour 18. The properties of the O-2A (adult) progenitors might be of particular relevance to studies of human demyelinating diseases. Three procedures yield oligodendrocytes in numbers sufficient for tissue culture studies. The simplest is to dissect optic nerves of rats at P7 and culture the harvested cells. In chemically defined medium, virtually all O-2A progenitors differentiate within 72 hours into oligoclendrocytes, and these cultures routinely are 60-90% pure 15. Alternatively, limited O-2A progenitor division can be induced before oligodendrocyte differentiation by growth on monolayers of type-1 astrocytes or by exposure to astrocyte-conditioned medium or platelet-derived growth factor (PDGF) (the O-2A progenitor mitogen secreted by type-1 astrocytes 14 ). More substantial O-2A progenitor proliferation can be achieved by exposing optic nerve cultures to both PDGF and fibroblast growth factor. The technical details of these and other methods are not important here, but it is worth emphasizing that there is no reason to doubt that the biological and developmental properties of optic nerve glial cells have been conserved across species. However, it is necessary wherever possible to validate hypotheses for the pathogenesis of human demyelinating disease derived from tissue culture studies by the analysis of samples obtained from affected individuals. Toxic effect of serum on oligodendrocytes and O--2A (adult) progenitors The morphological characteristics of oligodendrocytes damaged in vivo were described by Penfield and Cone in 1926, and were common to ischaemic, toxic and inflammatory conditions of animals and TINS, Vol. 14, No. 5, 1991
perspectives humans 19. These investigators observed swelling of the cell body, nuclear pyknosis, degeneration of the cell processes, the appearance of cytoplasmic granules and eventually lysis (Fig. 1). These features in vivo are closely mimicked when rat oligodendrocytes are exposed to normal syngeneic or xenogeneic serum in vitro (Fig. 2) 2°. Serum cytotoxicity has been widely observed 21,22, but has only recently been shown to result from complement attack. These findings significantly extend the previous observation that purified myelin activates complement 23,24, since fixation and activation are occurring on a living, intact cell membrane and are not processes that have had their normal molecular arrangement disrupted by extensive treatment in vitro. Complement can be activated by the classical (antibody-mediated) or alternative pathways; after the cleavage of (23, the sequential addition of C5b and then of each of the late or terminal components results in formation of the membrane attack complex (Fig. 3), which acts initially as a Ca2+ ionophore, and might cause target cell lysis. Serum obtained from rats depleted of complement by treatment with cobra venom factor lacks cytotoxicity, and this is also true of normal rat serum specifically depleted of either CI or (29 m vitro; reconstitution with the missing component restores cytotoxicity. Blocking activation of the classical pathway with EGTA also abolishes the cytotoxic effect, but specific myelin antibody is not involved since serum that has been preabsorbed with rat myelin retains its cytotoxicity; C9, but not immunoglobulin, can be identified on the surface of oligodendrocytes following exposure to serum. Mature oligodendrocytes and their precursors, the O-2A (adult) progenitor cells, are both able to fix and activate complement by the classical pathway in the absence of antibody 2°'25. If this was proved to be the case in vivo, then myelinating cells (and the pool available to replace them in the event of cell damage) would tend to initiate their own destruction when exposed to normal serum. This is not a property of neonatal progenitors, type-2 astrocytes, type-1 astrocytes, meningeal cells or Schwann cells25. Cells intermediate in differentiation between perinatal progenitors and oligodendrocytes are also susceptible, exhibiting a plateau of maximum sensitivity after developing for approximately six days in vitro 2O . Susceptibility to complement lysis must therefore be acquired during oligodendrocyte maturation, perhaps through loss of protection as perinatal progenitors differentiate (since cells are more sensitive to homologous than heterologous complement), or through failure to express cell surface molecules that normally inhibit lysis by homologous complement, such as decayaccelerating or homologous restriction factors 26. The role of Ca 2+ in recovery
The effect of exposing oligodendrocytes to serum complement in vitro in concentrations that, despite fixation and activation, are not lethal is not only of biological interest, but is also of potential TINS, Vol. 14, No. 5, 1991
on disease
D
A
C
B
Fig. 2. Oligodendrocytes exposed to serum in vitro. Oligodendrocytes were dissociated from rat optic nerve and cultured. Two cells in particular have been studied and are shown prior to exposure (A) and after 5 (B), 15 (C) and 40 (D) minutes' exposure to serum that was diluted one part in five of medium. The figures show similarities between the resulting morphological changes and those of acute swelling of oligodendrocytes (Fig. 1). Scale bar is 10 t~m. (Modified, with permission, from Ref. 20.)
relevance to inflammatory demyelinating disease 27. Scanning electron microscopy shows that 1-2 minutes after exposure to serum diluted by a factor of 30, numerous vesicles, brightly staining for C9, appear on the surface of oligodendrocytes (Fig. 4); these are no longer present after 8-10 minutes. Supernatants from these cultures gradually accumulate vesicular material rich in terminal complement components and galactocerebroside. This suggests that, in common with some other nucleated cells28, oligodendrocytes can resist complement lysis by vesicular removal of membrane attack complexes. These cells remain viable and later resume their normal specialized functions. Intracellular Ca2+ plays an important role in both oligodendrocyte damage and repair. If oligodendrocytes are preloaded with the Ca2+-sensitive photoprotein obelin in vitro, a transient rise in intracellular Ca2+ occurs during sublethal complement attack 29. This rise increases in the presence of oligodendrocyte-specific antibodies, in agreement with observations that such antibodies augment the cytotoxicity of concentrations of complement that are not otherwise lytic. 177
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antigen Cl ..,.......~/C4
C3 factors B, D
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7
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Fig. 3. Complement activation. A simplified representation of the complement system is shown; areas of functional interest and relevance are emphasized. Conventionally, activation of the classical pathway involves antibody-antigen complexes and C1, C4 and C2, while activation of the alternative pathway requires none of these components. The two pathways converge at the stage of C3 convertase generation. The incorporation and activation of C3 produces C5 convertase, with the subsequent sequential recruitment of C5b, C6, C7, C8 and C9 to form membrane attack complexes. Various biologicallyactive byproducts are also released, including soluble C3a and C5a, which have pro-inflammatory activities, and the membranebound opsonin C3b.
Susceptibility and vesicular repair are not unique to oligodendrocyte injury by complement. Identical results are obtained when oligodendrocytes are exposed to perforin derived from T cells3°, which also triggers a transient rise in intracellular Ca2÷, but which has little or no effect on other glia at comparable concentrations. This implies that a single common pathway might explain the identical effects of these pore-forming agents, and the ability of the Ca2+ ionophore A23187 to mimic closely these effects suggests that changes in Ca2+ levels are central in orchestrating the oligodendrocyte response to injury. Moreover, complement cytotoxicity is enhanced, seemingly through inhibition of the oligodendrocyte vesicular repair mechanism, when cultured oligodendrocytes are treated with W7, which blocks the ubiquitous Ca2+-binding protein calmodulin 3~. Macrophage-oligodend rocyte interactions Soluble factors released by activated macrophages, including tumour necrosis factor, leukotrienes, proteases and oxygen radicals, also damage the oligodendrocyte-myelin unit in vitro32,33; however, what induces macrophages to attack myelin in vivo remains unknown. The molecular
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details of this phagocytic cell-cell interaction, which is central to the problem of immune-mediated myelin injury in the adult CNS, can also be investigated in vitro using co-cultures enriched for oligodendroglia and macrophages34. These experiments show that cell-cell interactions are dependent on the presence of humoral immune mediators. Despite expressing C3b receptors, macrophages do not attack oligodendrocytes in the presence of membrane-bound complement activation products (generated by antibody-independent surface activation of complement), perhaps because these receptors are inactive in resting macrophages35. By contrast, immunoglobulin directed against components of the oligodendrocyte surface, in addition to increasing the complement-induced Ca2÷ flux and thereby augmenting cell damage, opsonizes oligodendrocytes in vitro, stimulating macrophage attachment and phagocytosis of oligodendrocytes and their myelin processes via the constitutively active macrophage Fc receptor (Fig. 5). This effect is seen with a variety of antibodies directed against surface components of the oligodendrocyte cell membrane. These findings in vitro therefore suggest that antibodies directed against several surface components TINS, VoL 14, No. 5, 1991
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can enhance both complement-associated and macrophage-mediated mechanisms of oligodendrocyte-myelin injury. Taken together, the cell culture findings emphasize a central role for Ca2+ and calmodulin in oligodendrocyte injury; a surge in intracellular Ca2+ stimulates Ca2+-activated myelin enzymes, including basic protein kinase and a myelin protease ~2,~3 and, at least in the peripheral nervous system, also disturbs conduction in myelinated fibres 36. In vitro, high concentrations of complement, sustained or repeated attack 29, and the availability of specific antibodies overwhelm recovery mechanisms and attract macrophages, thereby sealing the fate of oligodendrocytes.
with improved clinical status, which results from treatment with high doses of intravenous methylprednisolone. Particulate material, identical to that appearing in the supernatant of oligodendrocytes exposed to sublethal complement attack in tissue culture, is also present in spinal fluid from patients with multiple sclerosis but not in controls 27. These vesicles bear C8, C9 and the neoantigen of the membrane attack complex (but not C3), and transmission electron microscopy shows them to have a high concentration of membrane pores. The vesicular nature of this material is consistent with the demonstration of surface staining for galactocerebroside but not myelin basic protein, which is orientated exclusively on the cytoplasmic side of the myelin membrane 39. Myelin damage in vivo in multiple sclerosis It is of course critical to ask whether these Immunocytochemical evidence suggests that experimental findings are relevant to the under- complement is activated in areas of myelin breakstanding of lesion formation in humans and can down. C9 and the neoantigen of the terminal explain otherwise confusing aspects of human complex accumulate in the adventitia surrounding cerebral endothelial cells within acute and chronic demyelinating disease. Intrathecal complement activation occurs in plaques9. Complement deposition on myelin has not patients with isolated or repeated episodes of been demonstrated, but macrophages in acute demyelination. The short-lived activation products lesions show surface staining for antibodies and C3a and C4a can be detected in the cerebrospinal attach to myelin through coated pitsT'l°; myelin fluid of patients with multiple sclerosis37 and, sheets are incorporated within macrophages as conversely, components that are consumed as part elongated vesicles on which complement and antiof the activation process and are not resynthesized bodies, both of which are known to recruit macrowithin the CNS have a reduced concentration in phages, are co-localized 1~. These observations spinal fluid 38. The concentration of C9 increases indicate that the attachment of macrophages
A
C
B
D
Fig. 4. Scanning electron microscopy of cultured rat oligodendrocytes (A) before and (B) one minute after exposure to sublethal concentrations of complement. Numerous vesicles, brightly stained using anti-C9 immunogold labelling, appear on the surface of cells in (B); these are lost after a further six minutes (not shown). A similar vesicular response occurs on exposure of oligodendrocytes in vitro to (C) perforin derived from lymphocytes or to (D) the Ca2+ ionophore A23187. [Rat oligodendrocytes were exposed to previously determined, sublethal concentrations of syngeneic serum as a source of complement (A,B) or perforin (C), or to 5 t~M A23187 (D) for two minutes before washing and fixation for scanning electron microscopy as described27.] Scale bars are 10 l~m in each part of the figure. (Taken, with permission, from Refs 27,30.) TINS, Vol. 14, No. 5, 1991
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The role of lymphocytes in demyelination Magnetic resonance imaging using gadolinium cliethylenetriamine pentaacetic acid (Gd-DTPA) enhancement indicates that the opening of the blood-brain barrier is a consistent and early feature of lesion formation in multiple sclerosis and can recur repeatedly at the same site6. Increased vascular permeability, which occurs in white matter and non-myelinated parts of the nervous system, such as the retina 4°, can be regarded as the primary immunopathological event in demyelinating disease, and might depend critically on T cells4 Activated T cells, regardless of their specificity, penetrate the normal blood-brain barrier 41'42, providing the CNS with a degree of immune surveillance but at the same time potentially exposing it to immune attack. In experimental allergic encephalomyelitis (EAE), encephalitogenic T lymphocytes directly damage the blood-brain barrier , perhaps following interaction with perivascular monocytes that are expressing class II major histocompatibility complex (MHC) products 44. Microglia are the predominant cell type expressing class II products in the normal human CNS45, although endothelial cells might also express them in patients with multiple sclerosis46. EAE can be induced in the Lewis rat by passive transfer of encephalitogenic myelin basic proteinspecific CD4 + T cells47, and activated T cells are present in human and experimental demyelinating lesions7'4°. The associations of multiple sclerosis with MHC products (and more recently with rearrangements of the alpha and beta genes encoding V regions of T-cell receptors) are also consistent with a role for T lymphocytes in the pathogenesis of demyelination. There is, however, little to suggest that infiltrating T cells directly damage the oligodendrocyte-myelin unit. Ultrastructural studies show no intimate contact between lymphocytes and degenerating myelin, and demyelination occurs in areas entirely devoid of lymphocytes 7. The oligodendrocytemyelin unit does not express class I or II MHC products normally or in multiple sclerosis45'46'48, and natural killer T cells are not present in lesions46. Unlike patients with post-infectious encephalomyelitis, direct cloning of T cells from blood, cerebrospinal fluid and brain tissue from patients with multiple sclerosis has failed to show a consistent reaction to myelin basic protein, proteolipid protein or whole myelin 49, although some clones from some patients are responsive to myelin basic protein 5°. •
B
Fig. 5. Scanning electron micrographs of oligodendrocyte-macrophage interactions. Oligodendrocytes are the large, process-bearing cells and macrophages are the small, round cells. (A) No contact occurs in the presence of irrelevant (anti-progesterone) antibody, but incubation with antibodies against surface molecules on oligodendrocytes (MOG) (B) (or against galactocerebroside, not shown) triggers maerophage attachment to oligodendrocytes. Occasional oligodendrocytes show large numbers of adherent macrophages. (Resident rat peritoneal macrophages were added to oligodendrocytes in vitro and the co-cultures were fixed and prepared for scanning electron microscopy as described in Ref. 34.) Scale bars are lO l~m. (Taken, with permission, from Ref. 34.) to intact myelin lamellae is receptor mediated and might depend upon the local availability of antibody and complement. We cannot be certain of the extent to which the tissue culture and immunocytochemical observations accurately reflect mechanisms involved in human demyelinating disease, and we recognize that it is dangerous to extrapolate too much from studies of cultured, neonatal rat oligodendrocytes. Nevertheless, it is striking that properties of O-2A lineage cells offer explanations for several, but otherwise puzzling, clinical and histological observations, and suggest that the development of a demyelinating lesion requires two events - cellmediated damage to the blood-brain barrier, which then permits entry of macrophages and the soluble mediators necessary for their physical attachment to oligodendrocytes and myelin 4. 180
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The blood-brain barrier Activated T lymphocytes crossing the normal blood-brain barrier might interact initially with microglia that are constitutively MHC class II positive, triggering the release of cytokines that act locally to impair blood-brain barrier function further and to induce MHC expression on endothelial cells and possibly astroglia. Thus, the penetrating T lymphocyte might act, Trojan horse-like, to assemble a full repertoire of inflammatory agents within the CNS. TINS, Vol. 14, No. 5, 1991
Several features of demyelination could, however, require repeated blood-brain barrier damage, and depend upon the biological properties of oligodendrocytes and their precursors that are manifested in the presence of complement, antibody and macrophages.
Immune responses in demyelinating disease The finding of antibody-independent complement activation by oligodendrocytes in vitro suggests that the appearance of serum-derived complement within the CNS could be sufficient to injure oligodendrocytes in the absence of anti-myelin antibodies. Experimentally, normal serum not only degrades myelin in organotypic cultures 5° but also causes demyelination of the optic nerve in vivo 51. Membrane vesicles released as part of oligodendrocyte repair contain proteins and lipids that are highly immunogenic and, especially if this sequence of events were to recur, are likely to stimulate secondary cellular and humoral immunization against exposed oligodendrocyte-myelin antigens. The specificity of the resulting humoral responses might be relatively unimportant because as long as antibody is subsequently bound to oligodendrocytes or myelin, the effects of complement activation will be exacerbated and macrophages will be targeted against the cell surface. This interpretation implies that antibody-mediated immune reactions are a secondary phenomenon in multiple sclerosis, and might explain the failure to identify consistently a common antigenic target in the intrathecal antibody response that characterizes multiple sclerosis 1. Perhaps no one antigen is critical since antibodies to several antigens share the ability to enhance the consequences of complement injury and macrophage adherence. This interpretation emphasizes the importance of repeated blood-brain barrier damage and the intrathecal immune responses that ensue in generating demyelinated lesions, since demyelination is not regularly found in association with disruption of the blood-brain barrier following, for example, head injury. The historical observations of Penfield and Cone indicate, however, that oligodendrocyte changes occur more commonly than is generally assumed. These authors suggested that the early morphological changes associated with oligodendrocyte degeneration were reversible; thus, demyelination might not necessarily follow single or even multiple episodes of CNS damage, so long as antibody is not locally available. It hardly needs emphasizing that the presence of intrathecal oligoclonal antibody synthesis has been known for several decades to occur in multiple sclerosis.
Remyelination The evidence for extensive remyelination in acute lesions has already been mentioned 7'8, but it is clear, at least in those areas that subsequently develop into established sclerotic plaques, that remyelinating cells and the lamellae they synthesize are eventually lost. Mature oligodendrocytes do not appear to have proliferative or migratory potential; evidence from phenotypic analysis of glial cells appearing in TINS, Vol. 14, No. 5, 1991
new lesions 8 and from emerging work on glial transplantation ~2 suggests that repair depends on the presence of adult O-2A progenitors. However, these cells have a long cell-cycle time (65 hours) and a slow migration rate (4 [~m per hour) TM in vitro, indicating that even O-2A (adult) progenitors might have a limited capacity for remyelination in vivo; their activity might further be handicapped by the formation of astroglial scars within demyelinated plaques, inhibiting inward progenitor cell migration. Irrespective of these adverse dynamics, the observations we describe from experiments in vitro also suggest that any O-2A (adult) progenitors that were to repopulate oligodendrogliopoenic areas would be vulnerable to complement attack if bloodbrain barrier breakdown recurred.
A cellular biological view of demyelination The hypothesis to emerge 4 from a consideration of the studies in vivo and in vitro is that in the early stages of lesion development activated inflammatory cells, responding to unknown environmental triggers, disrupt the blood-brain barrier, and thus allow complement and other immune mediators to enter the nervous system. Oligodendrocytes are unduly sensitive to contact with complement and other pore-forming molecules, especially when antibody that is directed against surface components of the oligodendrocyte (or its myelin processes) is available as a result of previous oligodendrocyte injury and repair. The interaction of these soluble mediators increases membrane permeability and leads to a rise in intracellular Ca 2÷, the degree and duration of which determine whether or not reversible injury ensues. As complement activation proceeds, and when antibody is also present, macrophages are recruited and contribute to cell injury by releasing cytokines and phagocytosing myelin lamellae. Multiple sclerosis might therefore be regarded as a low grade vasculitis ~3 mediated by activated T lymphocytes that damage the bloodbrain barrier; demyelination then results from the appearance of macrophages and local antibodies, brought into play by the ability of oligodendrocytes to activate complement 4.
Selected references 1 Lisak, R. P. (1986) in Multiple Sclerosis, pp. 74-98, Butterworths 2 Calder,V., Owen, S. and Watson, C. (1989) Immunol. Today 10, 99-103 3 Haffler, D. A. and Weiner, H. L. (1989) Immunol. Today 10, 104-107 4 Scolding,N. J., Linington, C. and Compston, D. A. S. (1989) Autoimmunity 4, 131-142 5 Glyn, P. and Linington, C. (1989) CRC Crit. Rev. NeurobioL 4, 367-385 6 McDonald,W. I. and Barnes,D. (1989) Trends Neurosci. 12, 376-379 7 Prineas, J. W. (1985) Handbook of Clinical Neurology (Vol. 3), pp. 213-257, Elsevier 8 Prineas,J. W., Kwon, E. E. and Goldenberg,P. Z. (1989) Lab. InvesL 61,489-503 9 Compston, D. A. S., Morgan, B. P. and Campbell, A. K. (1989) Neuropathol. App. Neurobiol. 79, 78-85 10 Prineas, J. W. and Graham, J. S. (1981) Ann. Neurol 10, 149-158 11 Gay, D. and Esiri, M. (1991) Brain 114, 557-572 12 Morell, P., ed. (1985) Myelin, Plenum Press 181
13 Campagnoni, A. T. and Macklin, W. B. (1988) Mol. NeurobioL 2, 41-89 14 Miller, R. D., ffrench-Constant, C. and Raft, M. C. (1989) Annu. Rev. Neurosci. 12, 517-534 15 Raft, M. C., Miller, R. H. and Noble, M. (1986) Nature 303, 390--396 16 Fulton, B. and Raft, M. C. Ann. New YorkAcad. Sci. (in press) 17 ffrench-Constant, C. and Raft, M. C. (1986) Nature 319, 499--502 18 Noble, M. et al. (1990) Philos. Trans. R. Soc. London Set. B 327, 127-143 19 Penfield, W. and Cone, W. (1926) Arch. Neurol. Psychiatr. 16, 130-159 20 Scolding, N. J., Morgan, B. P. and Houston, A. (1989) J. Neurol. Sci. 89, 289-300 21 Hirayama, M., Lisak, R. P. and Silberberg, D. H. (1986) Neurology 36, 276-278 22 Wood, P. M. and Bunge, R. P. (1986)J. NeuroL Sci. 74, 153-169 23 Cyong, C-Y., Witkin, C. S. and Reiger, B. (1982) J. Exp. Med. 155, 587-597 24 Vanguri, P., Koski, C. L. and Silverman, B. (1985) Proc. Nat/ Acad. Sci. USA 79, 3290--3294 25 Wren, D. R. and Noble, M. (1989) Proc. NatlAcad. Sci. USA 86, 9025-9029 26 Mollnes, T. E. and Lachmann, P. J. (1988) Scand. J. Immunol. 27, 127-142 27 Scolding, N. J., Morgan, B. P. and Houston, A. (1989) Nature 339, 620-622 28 Morgan, B. P. (1989) Biochem. J. 264, 1-14 29 Scolding, N. J., Houston, W. A. J. and Morgan, B. P. (1989) Immunology 67, 441-446 30 Scolding, N. J., Jones, J., Compston, D. A. S. and Morgan, B. P. (1990) Immunology 70, 6-10 31 Scolding, N. J., Morgan, B. P. and Frith, S. J. (1990) J. NeuroL Neurosurg. Psychiatry 53, 811 32 Selmaj, K W. and Raine, C. S. (1988) Ann. Neurol. 23, 339-346 33 Cammer, W., Bloom, B. R. and Norton, W. T. (1978) Proc.
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to the
editor
Synaptic homeostasis and Parkinson's disease SIR: The article by Zigmond e t al. ~ in the July issue of last year's TINS offers an attractive explanation for the conventional idea that the emergence of neurological disorders of Parkinson's disease requires an almost complete degeneration of the dopaminergic, nigrostriatal bundle: compensatory events in the residual dopaminergic neurones maintain the control over striatal function and, accordingly, prolong the preclinical phase of this disease. According to the authors, frank clinical abnormalities either reflect a disruption of these compensatory processes or signal a degenerative process that exceeds the limits of compensation. The authors provide a reasonable body of data from work on animals in favour of their hypothesis. However, several interesting points are not taken into account. The article contains t w o basic assumptions that are not explicitly formulated. First, it is assumed 182
Natl Acad. Sci. USA 75, 1554-1558 34 Scolding, N. J. and Compston, D. A. S. (1991) Immunology 72, 127-132 35 Wright, S. D. and Silverstein, S. C. (1986) Handbook of Experimental Immunology (Vol. 2, 4th edn), Blackwell 36 Smith, K. J. and Hall, S. M. (1988) J. Neurol. Sci. 83, 37-53 37 Jans, H., Heltberg, A. and Zeeberg, I. (1984) Acta Neurol. Scand. 69, 34-38 38 Sanders, M. E., Koski, C. L. and Robbins, D. (1986) J. ImmunoL 136, 4456-4459 39 Omlin, F. X., Webster, H. F and Palkovitz, C. G. (1982) J. Cell. BioL 95, 242-248 40 Lightman, S., McDonald, W. I~ and Bird, A. C. (1987) Brain 110, 405-414 41 Simmons, R. D., Buzbec, T. M. and Linthicum, D. S. (1987) Acta Neuropathol. 74, 191-193 42 Wekerle, H. eta/. (1986) Trends Neurosci. 9, 271-277 43 Sedgwick,J., Brostoff, S. and Mason, D. (1987) J. Exp. Med. 165, 1058-1075 44 Vass, K., Lassmann, H. and Wekerle, H. (1986) Acta Neuropathol. 70, 149-160 45 Hayes, M., Woodroofe, M. N. and Cuzner, M. L. (1987) J. Neurol. Sci. 80, 25-37 46 Hayashi, T., Morimoto, C. and Burks, J. S. (1988) Ann. NeuroL 24, 523-531 47 Ben Nun, A., Wekerle, H. and Cohen, I. R. (1981) Eur. J. ImmunoL 11, 195-199 48 Traugott, U., Scheinberg, L. C. and Raine, C. S. (1985) J. NeuroimmunoL 8, 1-14 49 Haffler, D. A., Benjamin, D. S. and Burks, J. (1987) J. Immunol. 139, 89-92 50 Silberberg, D. H., Manning, M. C. and Schreiber,A. P. (1984) Ann. NeuroL 15, 575-580 51 Sergott, R. C., Brown, M. J. and Polenta, R. M. D. (1985) Neurology 35, 1438-1442 52 Blakemore,W., Crang, A. J. and Franklin, R. J. M. Cellular and Molecular Biology of Myelination, Springer-Verlag (in press) 53 Adams, C. W. M,, Poston, M. R. and Buk, S. J. (1985) J. Neurol. Sci. 69, 269-283
that a small dopaminergic deficiency within the striatum does not produce any behavioural deficit. Second, the authors assume that neurological deficits appear as soon as the dopaminergic control over striatal function is lost. Although these assumptions are not in conflict with the outcome of more conventional studies on the basal ganglia 2-5, there is now evidence that they need revision. Concerning the first assumption, there is evidence that even a small dopaminergic deficit within the striatum produces subtle changes in behaviour 6-8, such as a reduced ability to show arbitrary switching of ongoing behaviour this small dopamine deficit is not sufficient to produce pure motor disorders 6'7. This change can be manifested at all levels of behaviour. In humans, such a small deficit is evident at the cognitive level, and is labelled as 'a reduced shifting aptitude '9'1°. Today, there is evidence that these cognitive disorders require a smaller dopamine deficiency than
that required for the occurrence of pure neurological disorders 11. Given that dopamine completion therapy might at least partly ameliorate these cognitive deftcits 12, it appears that the postulated synaptic h o m e o s t a s i s has already broken down. These data indicate that the picture painted by Zigmond et a/. ~ represents an over-simplification and indicates that (1) absence of pure motor disorders does not imply that the postulated homeostasis maintains dopaminergic control over striatal function, (2) that the occurrence of pure motor disorders is certainly not the first sign that the postulated synaptic h o m e o s t a s i s starts to break down and (3) that the postulated synaptic homeostasis is unable to compensate a very small dopamine deficiency within the striatum. Given these findings, it is now worthwhile to consider the assessment of neuropsychological tests as a valid tool to diagnose Parkinson's disease at a very early stage. Since this method is noninvasive, it might be better than TINS, Vo/. 14, No. 5, 1991
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and the brain tissue bank. The organizational scheme of the Croatian Institute for Brain Research is based on the positive experience of the Netherlands Institute for Brain Research in Amsterdam. This entire project takes high priority under the sponsorship of the President of the Republic of Croatia, and has a good chance of setting a trend in the organization of neuroscience research all over the Republic of Croatia. Other republics might very soon organize similar neuroscience institutes or revitalize already existing ones. To achieve all these aspirations requires not only hard work but also a fortunate and speedy outcome of the political and economical crisis in Yugoslavia. The international assistance to both the Yugoslav federal state and individual republics is of the greatest value and importance during these transitional stages.
Acknowledgements Wearegratefulto Mr sc.JelkaPetrak, chairmanof the CentraIMedlca/ Library,Schoolof Medicine, University of Zagreb,and to Mr sc.ZoranBuneta, Divisionof Scientometnc Analysis,Schoolof Medicine, University of Zagreb,for their generoushelpin the analysisof published work that is included in thispaper.
Selected references 1 Lackovi(~,Z., Buneta, Z., Relja, M. and Ce~uk, L. (1986) Li/e~. Vjesn. 108, 462--471 2 Lackovi~,Z., Buneta, Z., Relja, M. and C~e~uk,L. (1987) Lije#. Vjesn. 109, 49-56 3 Coles, P. (1990) Nature 344, 616-617 4 Buret, J., Ciganek, L. and Vina[, O. (1991) Trends Neurosci. 14, 11-13 5 Schubert,A., Glanzel,W. and Braun,T. (1989) Scientornetrics 16, 3-478
Thepathogenesisof demyelinatingdisease:insights from cell biology Alastair Compston, Neil Scolding, Damien Wren and Mark Noble Cellularand humoral immune mechanismshavebeen implicated in the pathogenesis of human and experimentaldemyelinating diseases of the CNSI-~. How theseinteractin the complexsequenceof events that culminatesm phagocytosis of myelin by macrophageshas yet to be resolved The relationship between leakage of the blood-brain barrier and demyelination, the reason why recurrent inflammatory demye/ination occurs - seemingly in the absence of an antigenspecific immune response- and the lack of effective remye/inationall require explanation if a coherent account of immuno/ogical/y mediated demyelination Is to be achieved. One approach to these problems is to study in vitro the developmentaland cel/u/arbio/ogy of oligodendrocytes - the gila/cells responsible for the synthesis and maintenanceof CNSmyelin. Thisprovides experimentalopportunities not offered by more direct investigation of the intact nervous system, but carries the clear disadvantage that observations made in vitro cannot necessarilybe extrapolated to humans. Not all inflammatory demyelinating lesions give rise to neurological symptoms 6, and the pathological process is itself sometimes self limiting. In many patients, episodes of demyelination recur- imaging techniques detecting 20 or more new lesions annually - and neurological disability gradually accumulates; this temporal pattern forms the basis for distinguishing multiple sclerosis from the syndromes of isolated demyelination. Classical accounts of the histopathology of human demyelination established that multifocal TINS, Vol. 14, No. 5, 1991
infiltration by perivascular inflammatory cells precedes macrophage-mediated myelin degradation, which in turn is followed by reactive astrocytosis, oligodendrocyte depletion and some axonal loss. Another feature of demyelination is that infiltrating lymphocytes are rarely found in intimate contact with myelin sheaths 7. It is now clear that oligodendrocytes are damaged even in acute lesions, but extensive remyelination can occur 7'8. Immunocytochemical analysis of lesions has shown that (I) the infiltrating cells are predominately of the CD4 phenotype, (2) B lymphocytes, plasma cells, complement activation products and immunoglobulin are also present in acute lesions, and (3) macrophages in contact with myelin sheaths bear surface antibodies and contain aggregates of immunoglobulin and complement 7-11. These findings highlight the need to investigate interactions between oligodendrocytes, macrophages and humoral immune mediators. During development and repair, oligodendrocytes extend elongated processes that contact and then rotate many times around nearby axons, losing their cytoplasm as the segmented, multilamellated sheaths of compact myelin are formed. Myelin is more than an inert insulating material, although its dynamic function is poorly understood. It is rich in a number of specific enzymes, including a basic
© 1991, ElsevierSciencePublishersLtd, (UK) 0166- 2236191/$02.00
A/astairCompston and NellScoldingare at the Universityof CambridgeClinical School,Neurology Dept,Addenbrooke's Hospital, HillsRd, CambridgeCB22QQ, UK, DamienWren is at the lnstituteof Neurology, Queen Square,London WCIN 3BG,UK, and Mark Nobleisat the LudwigInstitutefor CancerResearch, RidingHouseSt, London W1PSBT, UK.
175
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protein kinase and cyclic nucleotide phosphodiesterase, and has a variety of membrane ion channels12'13. The parent oligodendrocyte soma supports this anatomically extensive and metabolically active membrane throughout the lifetime of each cell. However, recent work suggests that, among gila, the oligodendrocyte is uniquely vulnerable to immunological injury. The O-2A lineage Much of the current information about the development of oligodendrocytes has emerged from studying the 'simplest' part of the vertebrate CNS, the optic nerve 14. This tissue contains three cell types that are of neuroectodermal origin. Type1 astrocytes (first cell type) and their precursors contribute to the morphogenetic development of the optic nerve by offering a preferred substratum for growing axons. These cells also interact with endothelial cells to induce formation of the bloodbrain barrier, and are a source of mitogen for other cells in the developing nerve. While oligoclendrocytes (second cell type) enwrap axons with myelin sheaths, type-2 astrocytes (third cell type) are thought to extend processes that associate with axons at the nodes of Ranvier- the regions between consecutive myelin segments where ion fluxes occur
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during saltatory conduction. Thus, oligodendrocytes and type-2 astrocytes act together in creating the anatomical specializations that characterize myelinated tracts within the CNS. The three differentiated glial cell types of the optic nerve are generated at specific developmental periods from two distinct cellular lineages. The neuroepithelial cells that form the optic stalk (the embryonic primordium of the optic nerve) seem to give rise only to type-1 astrocytes. The initial differentiation of type-1 astrocytes, at day 16 of rat embryogenesis (E16), is followed developmentally by the appearance within the nerve of oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells, which can be induced to differentiate in vitro into either oligodendrocytes or type-2 astrocytes 15. O-2A progenitors are thought to arise in a germinal zone in or near the optic chiasm, first migrating into the optic nerve at E17. Oligodendrocytes are first seen in the optic nerve at the time of birth [E21, or postnatal day zero (P0)]. Cells with the antigenic phenotype of type-2 astrocytes are present in suspensions of optic nerve at P14 (Ref. 16). Recent studies have demonstrated that although O-2A progenitors can be extracted from both adult and neonatal optic nerve 17, O-2A (adult) progenitors differ in vitro from their perinatal counterparts in antigen expression, morphology, cell-cycle motility, timecourse of differentiation, and immunological behaviour 18. The properties of the O-2A (adult) progenitors might be of particular relevance to studies of human demyelinating diseases. Three procedures yield oligodendrocytes in numbers sufficient for tissue culture studies. The simplest is to dissect optic nerves of rats at P7 and culture the harvested cells. In chemically defined medium, virtually all O-2A progenitors differentiate within 72 hours into oligoclendrocytes, and these cultures routinely are 60-90% pure 15. Alternatively, limited O-2A progenitor division can be induced before oligodendrocyte differentiation by growth on monolayers of type-1 astrocytes or by exposure to astrocyte-conditioned medium or platelet-derived growth factor (PDGF) (the O-2A progenitor mitogen secreted by type-1 astrocytes 14 ). More substantial O-2A progenitor proliferation can be achieved by exposing optic nerve cultures to both PDGF and fibroblast growth factor. The technical details of these and other methods are not important here, but it is worth emphasizing that there is no reason to doubt that the biological and developmental properties of optic nerve glial cells have been conserved across species. However, it is necessary wherever possible to validate hypotheses for the pathogenesis of human demyelinating disease derived from tissue culture studies by the analysis of samples obtained from affected individuals. Toxic effect of serum on oligodendrocytes and O--2A (adult) progenitors The morphological characteristics of oligodendrocytes damaged in vivo were described by Penfield and Cone in 1926, and were common to ischaemic, toxic and inflammatory conditions of animals and TINS, Vol. 14, No. 5, 1991
perspectives humans 19. These investigators observed swelling of the cell body, nuclear pyknosis, degeneration of the cell processes, the appearance of cytoplasmic granules and eventually lysis (Fig. 1). These features in vivo are closely mimicked when rat oligodendrocytes are exposed to normal syngeneic or xenogeneic serum in vitro (Fig. 2) 2°. Serum cytotoxicity has been widely observed 21,22, but has only recently been shown to result from complement attack. These findings significantly extend the previous observation that purified myelin activates complement 23,24, since fixation and activation are occurring on a living, intact cell membrane and are not processes that have had their normal molecular arrangement disrupted by extensive treatment in vitro. Complement can be activated by the classical (antibody-mediated) or alternative pathways; after the cleavage of (23, the sequential addition of C5b and then of each of the late or terminal components results in formation of the membrane attack complex (Fig. 3), which acts initially as a Ca2+ ionophore, and might cause target cell lysis. Serum obtained from rats depleted of complement by treatment with cobra venom factor lacks cytotoxicity, and this is also true of normal rat serum specifically depleted of either CI or (29 m vitro; reconstitution with the missing component restores cytotoxicity. Blocking activation of the classical pathway with EGTA also abolishes the cytotoxic effect, but specific myelin antibody is not involved since serum that has been preabsorbed with rat myelin retains its cytotoxicity; C9, but not immunoglobulin, can be identified on the surface of oligodendrocytes following exposure to serum. Mature oligodendrocytes and their precursors, the O-2A (adult) progenitor cells, are both able to fix and activate complement by the classical pathway in the absence of antibody 2°'25. If this was proved to be the case in vivo, then myelinating cells (and the pool available to replace them in the event of cell damage) would tend to initiate their own destruction when exposed to normal serum. This is not a property of neonatal progenitors, type-2 astrocytes, type-1 astrocytes, meningeal cells or Schwann cells25. Cells intermediate in differentiation between perinatal progenitors and oligodendrocytes are also susceptible, exhibiting a plateau of maximum sensitivity after developing for approximately six days in vitro 2O . Susceptibility to complement lysis must therefore be acquired during oligodendrocyte maturation, perhaps through loss of protection as perinatal progenitors differentiate (since cells are more sensitive to homologous than heterologous complement), or through failure to express cell surface molecules that normally inhibit lysis by homologous complement, such as decayaccelerating or homologous restriction factors 26. The role of Ca 2+ in recovery
The effect of exposing oligodendrocytes to serum complement in vitro in concentrations that, despite fixation and activation, are not lethal is not only of biological interest, but is also of potential TINS, Vol. 14, No. 5, 1991
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D
A
C
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Fig. 2. Oligodendrocytes exposed to serum in vitro. Oligodendrocytes were dissociated from rat optic nerve and cultured. Two cells in particular have been studied and are shown prior to exposure (A) and after 5 (B), 15 (C) and 40 (D) minutes' exposure to serum that was diluted one part in five of medium. The figures show similarities between the resulting morphological changes and those of acute swelling of oligodendrocytes (Fig. 1). Scale bar is 10 t~m. (Modified, with permission, from Ref. 20.)
relevance to inflammatory demyelinating disease 27. Scanning electron microscopy shows that 1-2 minutes after exposure to serum diluted by a factor of 30, numerous vesicles, brightly staining for C9, appear on the surface of oligodendrocytes (Fig. 4); these are no longer present after 8-10 minutes. Supernatants from these cultures gradually accumulate vesicular material rich in terminal complement components and galactocerebroside. This suggests that, in common with some other nucleated cells28, oligodendrocytes can resist complement lysis by vesicular removal of membrane attack complexes. These cells remain viable and later resume their normal specialized functions. Intracellular Ca2+ plays an important role in both oligodendrocyte damage and repair. If oligodendrocytes are preloaded with the Ca2+-sensitive photoprotein obelin in vitro, a transient rise in intracellular Ca2+ occurs during sublethal complement attack 29. This rise increases in the presence of oligodendrocyte-specific antibodies, in agreement with observations that such antibodies augment the cytotoxicity of concentrations of complement that are not otherwise lytic. 177
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Susceptibility and vesicular repair are not unique to oligodendrocyte injury by complement. Identical results are obtained when oligodendrocytes are exposed to perforin derived from T cells3°, which also triggers a transient rise in intracellular Ca2÷, but which has little or no effect on other glia at comparable concentrations. This implies that a single common pathway might explain the identical effects of these pore-forming agents, and the ability of the Ca2+ ionophore A23187 to mimic closely these effects suggests that changes in Ca2+ levels are central in orchestrating the oligodendrocyte response to injury. Moreover, complement cytotoxicity is enhanced, seemingly through inhibition of the oligodendrocyte vesicular repair mechanism, when cultured oligodendrocytes are treated with W7, which blocks the ubiquitous Ca2+-binding protein calmodulin 3~. Macrophage-oligodend rocyte interactions Soluble factors released by activated macrophages, including tumour necrosis factor, leukotrienes, proteases and oxygen radicals, also damage the oligodendrocyte-myelin unit in vitro32,33; however, what induces macrophages to attack myelin in vivo remains unknown. The molecular
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details of this phagocytic cell-cell interaction, which is central to the problem of immune-mediated myelin injury in the adult CNS, can also be investigated in vitro using co-cultures enriched for oligodendroglia and macrophages34. These experiments show that cell-cell interactions are dependent on the presence of humoral immune mediators. Despite expressing C3b receptors, macrophages do not attack oligodendrocytes in the presence of membrane-bound complement activation products (generated by antibody-independent surface activation of complement), perhaps because these receptors are inactive in resting macrophages35. By contrast, immunoglobulin directed against components of the oligodendrocyte surface, in addition to increasing the complement-induced Ca2÷ flux and thereby augmenting cell damage, opsonizes oligodendrocytes in vitro, stimulating macrophage attachment and phagocytosis of oligodendrocytes and their myelin processes via the constitutively active macrophage Fc receptor (Fig. 5). This effect is seen with a variety of antibodies directed against surface components of the oligodendrocyte cell membrane. These findings in vitro therefore suggest that antibodies directed against several surface components TINS, VoL 14, No. 5, 1991
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can enhance both complement-associated and macrophage-mediated mechanisms of oligodendrocyte-myelin injury. Taken together, the cell culture findings emphasize a central role for Ca2+ and calmodulin in oligodendrocyte injury; a surge in intracellular Ca2+ stimulates Ca2+-activated myelin enzymes, including basic protein kinase and a myelin protease ~2,~3 and, at least in the peripheral nervous system, also disturbs conduction in myelinated fibres 36. In vitro, high concentrations of complement, sustained or repeated attack 29, and the availability of specific antibodies overwhelm recovery mechanisms and attract macrophages, thereby sealing the fate of oligodendrocytes.
with improved clinical status, which results from treatment with high doses of intravenous methylprednisolone. Particulate material, identical to that appearing in the supernatant of oligodendrocytes exposed to sublethal complement attack in tissue culture, is also present in spinal fluid from patients with multiple sclerosis but not in controls 27. These vesicles bear C8, C9 and the neoantigen of the membrane attack complex (but not C3), and transmission electron microscopy shows them to have a high concentration of membrane pores. The vesicular nature of this material is consistent with the demonstration of surface staining for galactocerebroside but not myelin basic protein, which is orientated exclusively on the cytoplasmic side of the myelin membrane 39. Myelin damage in vivo in multiple sclerosis It is of course critical to ask whether these Immunocytochemical evidence suggests that experimental findings are relevant to the under- complement is activated in areas of myelin breakstanding of lesion formation in humans and can down. C9 and the neoantigen of the terminal explain otherwise confusing aspects of human complex accumulate in the adventitia surrounding cerebral endothelial cells within acute and chronic demyelinating disease. Intrathecal complement activation occurs in plaques9. Complement deposition on myelin has not patients with isolated or repeated episodes of been demonstrated, but macrophages in acute demyelination. The short-lived activation products lesions show surface staining for antibodies and C3a and C4a can be detected in the cerebrospinal attach to myelin through coated pitsT'l°; myelin fluid of patients with multiple sclerosis37 and, sheets are incorporated within macrophages as conversely, components that are consumed as part elongated vesicles on which complement and antiof the activation process and are not resynthesized bodies, both of which are known to recruit macrowithin the CNS have a reduced concentration in phages, are co-localized 1~. These observations spinal fluid 38. The concentration of C9 increases indicate that the attachment of macrophages
A
C
B
D
Fig. 4. Scanning electron microscopy of cultured rat oligodendrocytes (A) before and (B) one minute after exposure to sublethal concentrations of complement. Numerous vesicles, brightly stained using anti-C9 immunogold labelling, appear on the surface of cells in (B); these are lost after a further six minutes (not shown). A similar vesicular response occurs on exposure of oligodendrocytes in vitro to (C) perforin derived from lymphocytes or to (D) the Ca2+ ionophore A23187. [Rat oligodendrocytes were exposed to previously determined, sublethal concentrations of syngeneic serum as a source of complement (A,B) or perforin (C), or to 5 t~M A23187 (D) for two minutes before washing and fixation for scanning electron microscopy as described27.] Scale bars are 10 l~m in each part of the figure. (Taken, with permission, from Refs 27,30.) TINS, Vol. 14, No. 5, 1991
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The role of lymphocytes in demyelination Magnetic resonance imaging using gadolinium cliethylenetriamine pentaacetic acid (Gd-DTPA) enhancement indicates that the opening of the blood-brain barrier is a consistent and early feature of lesion formation in multiple sclerosis and can recur repeatedly at the same site6. Increased vascular permeability, which occurs in white matter and non-myelinated parts of the nervous system, such as the retina 4°, can be regarded as the primary immunopathological event in demyelinating disease, and might depend critically on T cells4 Activated T cells, regardless of their specificity, penetrate the normal blood-brain barrier 41'42, providing the CNS with a degree of immune surveillance but at the same time potentially exposing it to immune attack. In experimental allergic encephalomyelitis (EAE), encephalitogenic T lymphocytes directly damage the blood-brain barrier , perhaps following interaction with perivascular monocytes that are expressing class II major histocompatibility complex (MHC) products 44. Microglia are the predominant cell type expressing class II products in the normal human CNS45, although endothelial cells might also express them in patients with multiple sclerosis46. EAE can be induced in the Lewis rat by passive transfer of encephalitogenic myelin basic proteinspecific CD4 + T cells47, and activated T cells are present in human and experimental demyelinating lesions7'4°. The associations of multiple sclerosis with MHC products (and more recently with rearrangements of the alpha and beta genes encoding V regions of T-cell receptors) are also consistent with a role for T lymphocytes in the pathogenesis of demyelination. There is, however, little to suggest that infiltrating T cells directly damage the oligodendrocyte-myelin unit. Ultrastructural studies show no intimate contact between lymphocytes and degenerating myelin, and demyelination occurs in areas entirely devoid of lymphocytes 7. The oligodendrocytemyelin unit does not express class I or II MHC products normally or in multiple sclerosis45'46'48, and natural killer T cells are not present in lesions46. Unlike patients with post-infectious encephalomyelitis, direct cloning of T cells from blood, cerebrospinal fluid and brain tissue from patients with multiple sclerosis has failed to show a consistent reaction to myelin basic protein, proteolipid protein or whole myelin 49, although some clones from some patients are responsive to myelin basic protein 5°. •
B
Fig. 5. Scanning electron micrographs of oligodendrocyte-macrophage interactions. Oligodendrocytes are the large, process-bearing cells and macrophages are the small, round cells. (A) No contact occurs in the presence of irrelevant (anti-progesterone) antibody, but incubation with antibodies against surface molecules on oligodendrocytes (MOG) (B) (or against galactocerebroside, not shown) triggers maerophage attachment to oligodendrocytes. Occasional oligodendrocytes show large numbers of adherent macrophages. (Resident rat peritoneal macrophages were added to oligodendrocytes in vitro and the co-cultures were fixed and prepared for scanning electron microscopy as described in Ref. 34.) Scale bars are lO l~m. (Taken, with permission, from Ref. 34.) to intact myelin lamellae is receptor mediated and might depend upon the local availability of antibody and complement. We cannot be certain of the extent to which the tissue culture and immunocytochemical observations accurately reflect mechanisms involved in human demyelinating disease, and we recognize that it is dangerous to extrapolate too much from studies of cultured, neonatal rat oligodendrocytes. Nevertheless, it is striking that properties of O-2A lineage cells offer explanations for several, but otherwise puzzling, clinical and histological observations, and suggest that the development of a demyelinating lesion requires two events - cellmediated damage to the blood-brain barrier, which then permits entry of macrophages and the soluble mediators necessary for their physical attachment to oligodendrocytes and myelin 4. 180
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The blood-brain barrier Activated T lymphocytes crossing the normal blood-brain barrier might interact initially with microglia that are constitutively MHC class II positive, triggering the release of cytokines that act locally to impair blood-brain barrier function further and to induce MHC expression on endothelial cells and possibly astroglia. Thus, the penetrating T lymphocyte might act, Trojan horse-like, to assemble a full repertoire of inflammatory agents within the CNS. TINS, Vol. 14, No. 5, 1991
Several features of demyelination could, however, require repeated blood-brain barrier damage, and depend upon the biological properties of oligodendrocytes and their precursors that are manifested in the presence of complement, antibody and macrophages.
Immune responses in demyelinating disease The finding of antibody-independent complement activation by oligodendrocytes in vitro suggests that the appearance of serum-derived complement within the CNS could be sufficient to injure oligodendrocytes in the absence of anti-myelin antibodies. Experimentally, normal serum not only degrades myelin in organotypic cultures 5° but also causes demyelination of the optic nerve in vivo 51. Membrane vesicles released as part of oligodendrocyte repair contain proteins and lipids that are highly immunogenic and, especially if this sequence of events were to recur, are likely to stimulate secondary cellular and humoral immunization against exposed oligodendrocyte-myelin antigens. The specificity of the resulting humoral responses might be relatively unimportant because as long as antibody is subsequently bound to oligodendrocytes or myelin, the effects of complement activation will be exacerbated and macrophages will be targeted against the cell surface. This interpretation implies that antibody-mediated immune reactions are a secondary phenomenon in multiple sclerosis, and might explain the failure to identify consistently a common antigenic target in the intrathecal antibody response that characterizes multiple sclerosis 1. Perhaps no one antigen is critical since antibodies to several antigens share the ability to enhance the consequences of complement injury and macrophage adherence. This interpretation emphasizes the importance of repeated blood-brain barrier damage and the intrathecal immune responses that ensue in generating demyelinated lesions, since demyelination is not regularly found in association with disruption of the blood-brain barrier following, for example, head injury. The historical observations of Penfield and Cone indicate, however, that oligodendrocyte changes occur more commonly than is generally assumed. These authors suggested that the early morphological changes associated with oligodendrocyte degeneration were reversible; thus, demyelination might not necessarily follow single or even multiple episodes of CNS damage, so long as antibody is not locally available. It hardly needs emphasizing that the presence of intrathecal oligoclonal antibody synthesis has been known for several decades to occur in multiple sclerosis.
Remyelination The evidence for extensive remyelination in acute lesions has already been mentioned 7'8, but it is clear, at least in those areas that subsequently develop into established sclerotic plaques, that remyelinating cells and the lamellae they synthesize are eventually lost. Mature oligodendrocytes do not appear to have proliferative or migratory potential; evidence from phenotypic analysis of glial cells appearing in TINS, Vol. 14, No. 5, 1991
new lesions 8 and from emerging work on glial transplantation ~2 suggests that repair depends on the presence of adult O-2A progenitors. However, these cells have a long cell-cycle time (65 hours) and a slow migration rate (4 [~m per hour) TM in vitro, indicating that even O-2A (adult) progenitors might have a limited capacity for remyelination in vivo; their activity might further be handicapped by the formation of astroglial scars within demyelinated plaques, inhibiting inward progenitor cell migration. Irrespective of these adverse dynamics, the observations we describe from experiments in vitro also suggest that any O-2A (adult) progenitors that were to repopulate oligodendrogliopoenic areas would be vulnerable to complement attack if bloodbrain barrier breakdown recurred.
A cellular biological view of demyelination The hypothesis to emerge 4 from a consideration of the studies in vivo and in vitro is that in the early stages of lesion development activated inflammatory cells, responding to unknown environmental triggers, disrupt the blood-brain barrier, and thus allow complement and other immune mediators to enter the nervous system. Oligodendrocytes are unduly sensitive to contact with complement and other pore-forming molecules, especially when antibody that is directed against surface components of the oligodendrocyte (or its myelin processes) is available as a result of previous oligodendrocyte injury and repair. The interaction of these soluble mediators increases membrane permeability and leads to a rise in intracellular Ca 2÷, the degree and duration of which determine whether or not reversible injury ensues. As complement activation proceeds, and when antibody is also present, macrophages are recruited and contribute to cell injury by releasing cytokines and phagocytosing myelin lamellae. Multiple sclerosis might therefore be regarded as a low grade vasculitis ~3 mediated by activated T lymphocytes that damage the bloodbrain barrier; demyelination then results from the appearance of macrophages and local antibodies, brought into play by the ability of oligodendrocytes to activate complement 4.
Selected references 1 Lisak, R. P. (1986) in Multiple Sclerosis, pp. 74-98, Butterworths 2 Calder,V., Owen, S. and Watson, C. (1989) Immunol. Today 10, 99-103 3 Haffler, D. A. and Weiner, H. L. (1989) Immunol. Today 10, 104-107 4 Scolding,N. J., Linington, C. and Compston, D. A. S. (1989) Autoimmunity 4, 131-142 5 Glyn, P. and Linington, C. (1989) CRC Crit. Rev. NeurobioL 4, 367-385 6 McDonald,W. I. and Barnes,D. (1989) Trends Neurosci. 12, 376-379 7 Prineas, J. W. (1985) Handbook of Clinical Neurology (Vol. 3), pp. 213-257, Elsevier 8 Prineas,J. W., Kwon, E. E. and Goldenberg,P. Z. (1989) Lab. InvesL 61,489-503 9 Compston, D. A. S., Morgan, B. P. and Campbell, A. K. (1989) Neuropathol. App. Neurobiol. 79, 78-85 10 Prineas, J. W. and Graham, J. S. (1981) Ann. Neurol 10, 149-158 11 Gay, D. and Esiri, M. (1991) Brain 114, 557-572 12 Morell, P., ed. (1985) Myelin, Plenum Press 181
13 Campagnoni, A. T. and Macklin, W. B. (1988) Mol. NeurobioL 2, 41-89 14 Miller, R. D., ffrench-Constant, C. and Raft, M. C. (1989) Annu. Rev. Neurosci. 12, 517-534 15 Raft, M. C., Miller, R. H. and Noble, M. (1986) Nature 303, 390--396 16 Fulton, B. and Raft, M. C. Ann. New YorkAcad. Sci. (in press) 17 ffrench-Constant, C. and Raft, M. C. (1986) Nature 319, 499--502 18 Noble, M. et al. (1990) Philos. Trans. R. Soc. London Set. B 327, 127-143 19 Penfield, W. and Cone, W. (1926) Arch. Neurol. Psychiatr. 16, 130-159 20 Scolding, N. J., Morgan, B. P. and Houston, A. (1989) J. Neurol. Sci. 89, 289-300 21 Hirayama, M., Lisak, R. P. and Silberberg, D. H. (1986) Neurology 36, 276-278 22 Wood, P. M. and Bunge, R. P. (1986)J. NeuroL Sci. 74, 153-169 23 Cyong, C-Y., Witkin, C. S. and Reiger, B. (1982) J. Exp. Med. 155, 587-597 24 Vanguri, P., Koski, C. L. and Silverman, B. (1985) Proc. Nat/ Acad. Sci. USA 79, 3290--3294 25 Wren, D. R. and Noble, M. (1989) Proc. NatlAcad. Sci. USA 86, 9025-9029 26 Mollnes, T. E. and Lachmann, P. J. (1988) Scand. J. Immunol. 27, 127-142 27 Scolding, N. J., Morgan, B. P. and Houston, A. (1989) Nature 339, 620-622 28 Morgan, B. P. (1989) Biochem. J. 264, 1-14 29 Scolding, N. J., Houston, W. A. J. and Morgan, B. P. (1989) Immunology 67, 441-446 30 Scolding, N. J., Jones, J., Compston, D. A. S. and Morgan, B. P. (1990) Immunology 70, 6-10 31 Scolding, N. J., Morgan, B. P. and Frith, S. J. (1990) J. NeuroL Neurosurg. Psychiatry 53, 811 32 Selmaj, K W. and Raine, C. S. (1988) Ann. Neurol. 23, 339-346 33 Cammer, W., Bloom, B. R. and Norton, W. T. (1978) Proc.
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Synaptic homeostasis and Parkinson's disease SIR: The article by Zigmond e t al. ~ in the July issue of last year's TINS offers an attractive explanation for the conventional idea that the emergence of neurological disorders of Parkinson's disease requires an almost complete degeneration of the dopaminergic, nigrostriatal bundle: compensatory events in the residual dopaminergic neurones maintain the control over striatal function and, accordingly, prolong the preclinical phase of this disease. According to the authors, frank clinical abnormalities either reflect a disruption of these compensatory processes or signal a degenerative process that exceeds the limits of compensation. The authors provide a reasonable body of data from work on animals in favour of their hypothesis. However, several interesting points are not taken into account. The article contains t w o basic assumptions that are not explicitly formulated. First, it is assumed 182
Natl Acad. Sci. USA 75, 1554-1558 34 Scolding, N. J. and Compston, D. A. S. (1991) Immunology 72, 127-132 35 Wright, S. D. and Silverstein, S. C. (1986) Handbook of Experimental Immunology (Vol. 2, 4th edn), Blackwell 36 Smith, K. J. and Hall, S. M. (1988) J. Neurol. Sci. 83, 37-53 37 Jans, H., Heltberg, A. and Zeeberg, I. (1984) Acta Neurol. Scand. 69, 34-38 38 Sanders, M. E., Koski, C. L. and Robbins, D. (1986) J. ImmunoL 136, 4456-4459 39 Omlin, F. X., Webster, H. F and Palkovitz, C. G. (1982) J. Cell. BioL 95, 242-248 40 Lightman, S., McDonald, W. I~ and Bird, A. C. (1987) Brain 110, 405-414 41 Simmons, R. D., Buzbec, T. M. and Linthicum, D. S. (1987) Acta Neuropathol. 74, 191-193 42 Wekerle, H. eta/. (1986) Trends Neurosci. 9, 271-277 43 Sedgwick,J., Brostoff, S. and Mason, D. (1987) J. Exp. Med. 165, 1058-1075 44 Vass, K., Lassmann, H. and Wekerle, H. (1986) Acta Neuropathol. 70, 149-160 45 Hayes, M., Woodroofe, M. N. and Cuzner, M. L. (1987) J. Neurol. Sci. 80, 25-37 46 Hayashi, T., Morimoto, C. and Burks, J. S. (1988) Ann. NeuroL 24, 523-531 47 Ben Nun, A., Wekerle, H. and Cohen, I. R. (1981) Eur. J. ImmunoL 11, 195-199 48 Traugott, U., Scheinberg, L. C. and Raine, C. S. (1985) J. NeuroimmunoL 8, 1-14 49 Haffler, D. A., Benjamin, D. S. and Burks, J. (1987) J. Immunol. 139, 89-92 50 Silberberg, D. H., Manning, M. C. and Schreiber,A. P. (1984) Ann. NeuroL 15, 575-580 51 Sergott, R. C., Brown, M. J. and Polenta, R. M. D. (1985) Neurology 35, 1438-1442 52 Blakemore,W., Crang, A. J. and Franklin, R. J. M. Cellular and Molecular Biology of Myelination, Springer-Verlag (in press) 53 Adams, C. W. M,, Poston, M. R. and Buk, S. J. (1985) J. Neurol. Sci. 69, 269-283
that a small dopaminergic deficiency within the striatum does not produce any behavioural deficit. Second, the authors assume that neurological deficits appear as soon as the dopaminergic control over striatal function is lost. Although these assumptions are not in conflict with the outcome of more conventional studies on the basal ganglia 2-5, there is now evidence that they need revision. Concerning the first assumption, there is evidence that even a small dopaminergic deficit within the striatum produces subtle changes in behaviour 6-8, such as a reduced ability to show arbitrary switching of ongoing behaviour this small dopamine deficit is not sufficient to produce pure motor disorders 6'7. This change can be manifested at all levels of behaviour. In humans, such a small deficit is evident at the cognitive level, and is labelled as 'a reduced shifting aptitude '9'1°. Today, there is evidence that these cognitive disorders require a smaller dopamine deficiency than
that required for the occurrence of pure neurological disorders 11. Given that dopamine completion therapy might at least partly ameliorate these cognitive deftcits 12, it appears that the postulated synaptic h o m e o s t a s i s has already broken down. These data indicate that the picture painted by Zigmond et a/. ~ represents an over-simplification and indicates that (1) absence of pure motor disorders does not imply that the postulated homeostasis maintains dopaminergic control over striatal function, (2) that the occurrence of pure motor disorders is certainly not the first sign that the postulated synaptic h o m e o s t a s i s starts to break down and (3) that the postulated synaptic homeostasis is unable to compensate a very small dopamine deficiency within the striatum. Given these findings, it is now worthwhile to consider the assessment of neuropsychological tests as a valid tool to diagnose Parkinson's disease at a very early stage. Since this method is noninvasive, it might be better than TINS, Vo/. 14, No. 5, 1991
