The generation and regeneration of oligodendroglia

Journal of the Neurological Sciences, 1986, 72:319-336

319

Elsevier

JNS 2623

The Generation and Regeneration of Oligodendroglia A Short Review P.L. Debbage* Physiologisch-Chemisches Institut der Universitiit. Koellikerstrasse 2, D-8700 Wiirzburg (F.R.G.) (Received 22 July, 1985) (Revised, received 21 October, 1985) (Accepted 21 October, 1985)

SUMMARY

During postnatal development of the higher vertebrate CNS, large populations of oligodendroglia are generated from precursor cells in a very dependable way. In adult lesioned CNS tissues, local populations of oligodendroglia are replenished by proliferation of cells whose identities are not fully elucidated; the completeness of this replenishment varies from one species to another and also from one lesion type to another. Studies on the developmental generation of oligodendroglia are reviewed here, delineating what is known of the early relationships between the CNS glial lineages and of what regulates this development. Contributions from recent cell biological work are considered against the background of morphological and radioautographic results. The quiescent condition of extremely slow turnover in the normal adult CNS is noted, and the dramatic effects of lesions on the neural cell environment are considered. Lesions can trigger proliferation at a much greater rate in the mature oligodendroglial population, as observed both in situ and in tissue culture; in addition to persisting stem cells, the mature cells participate in replenishing the local oligodendroglial population. This regeneration from cells already committed to the oligodendroglial lineage may minimise such disturbing effects of the lesion environment as might distort replenishment of the population from precursor cells.

* Present address: Department of Neurosurgery, Centre of Neurological Medicine, Robert-KochStrasse 40, D-3400 G0ttingen, F.R.G. Abbreviations: CAII= Carbonic anhydrase II (EC 4.2.1.1.); CNS = Central nervous system; CREAE = Chronic relapsing experimental allergic encephalomyelitis; GC = Galactocerebroside; GFAP = Glial fibrillary acidic protein; MBP = Myelin basic protein; MS = Multiple sclerosis; PNS = Peripheral nervous system. 0022-510X/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

320 Key words:

Development

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Regeneration

- Regulation

Glial

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Oligodendroglia

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Precursors

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Proliferation

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INTRODUCTION Myelin-forming cell populations (oligodendroglia in the CNS and Schwann cells in the PNS) are generated and expanded during perinatal and postnatal development, and in adult life in response to damage caused by lesions (Ludwin 1979a). During development, the establishment of functioning oligodendroglial populations rarely fails; an example of such a failure might be certain types of unexplained death in infancy (Foster and Carey 1983). Of the congenital dysmyelinating conditions, only a few may result from failure to generate oligodendroglia; notable examples are the Snell Dwarf mouse (Noguchi et al. 1982) and the Jimpy mouse (Skoff 1982; Privat et al. 1982), although in the Jimpy, it seems more likely that the condition is due to failure to establish, rather than to generate the oligodendroglial population (Skoff 1982; Bologa et al. 1983). In contrast, the generation of a myelinating population as part of recovery of CNS tissue from a lesion (referred to in this review as "regeneration") is much less certain in the diseased adult. In some conditions, regeneration can be extensive (e.g. after viral demyelination: Herndon et al. 1977), but may be partial or fail entirely, as in MS (for review see Hommes 1980). This review considers the cell populations involved in generating or regenerating oligodendroglia, emphasizing differences in the cells involved and also in the conditions under which population expansion occurs. During development, oligodendroglia arises from multipotential stem cells, whereas regeneration in the adult is partly from committed mature oligodendroglia, i.e. from oligodendrocytes; possible implications of this are explored, with particular reference to postlesion recovery in adult CNS tissues. A considerable amount is known about the expansion of Schwann cell populations because their expansion is vigorous, because peripheral nerves are easily accessible and, compared to central nervous tissue, of simple structure. However, we note important differences between the cell biology of Schwann cells and oligodendroglia. They have different embryological origins: Schwann ceils originate from the neural crest, whereas oligodendrogiia originates from neuroectodermal stem cells. In culture, Schwann cells quickly lose their differentiated characteristics in the absence ofneurones (Mirsky et al. 1980), but oligodendroglial cells proceed through several maturation steps, generating myelin-specific proteins and lipids (e.g. Roussel et al. 1983). Furthermore, the myelination mechanisms differ: Schwann cells ensheath single internodes, but an oligodendrocyte can ensheath many axons, not all necessarily in the same tract (e.g. Peters and Vaughn 1970; Sternberger et al. 1978). Thus, whereas simple rotation of the Schwann cell around the axon is all that is required for PNS myelination, such rotation is precluded on geometric grounds in the CNS (Peters et al. 1970); the many processes extended by a single oligodendrocyte evidently recognise and interact with axons individually, so that these important interactions do not occur at the level of the whole

321 cell in oligodendroglia, in contrast to the whole-ceU response in Schwann cells. Schwann cells produce a basal lamina which plays a role in post-lesion regeneration, but oligodendroglia generates no basal lamina. Again, unlike the Schwann cell population, oligodendroglia are cells without evident reserve (Bunge et al. 1978). Finally, Schwann cells myelinate much more vigorously than oligodendroglia. In a wide range of lesions, Schwann cells myelinate CNS axons in the presence of oligodendroglia (see review by Hommes 1980), and many authors have documented this in MS: a recent demonstration is that by Itoyama et al. (1983). In summary, although Schwann cells and oligodendroglia generate myelin that differs only slightly in morphology (Peters et al. 1970; Bunge 1968) and composition (Hommes 1980), the cell biology of the two cell types is so dissimilar that we must expect that their population kinetics are regulated quite differently. Here we will consider studies of the CNS, and will note only in passing certain relevant results obtained in studies of the independent population kinetics of Schwann cells. THE MATUREOLIGODENDROGLIALPOPULATION Oligodendroglia is numerous in the white matter of the CNS, lying in rows between the axon bundles (interfascicular oligodendroglia), but also occurs in the grey matter as perineuronal or perivascular cells. Their ultrastructure (reviewed by Peters et al. 1970; 1976) exhibits common features independent of sub-type and location: nuclei with chromatin clumped at the nuclear membrane, an organelle-rich cytoplasm with numerous ribosomes, a prominent granular reticulum and Golgi apparatus, and numerous microtubules - especially in the cell processes. Amongst the glia, oligodendroglia is, therefore, easily distinguished from astroglia (pale cytoplasm, sparse organdies, bundles of intermediate filaments) but less readily from resting microglia, which also has a rather dense cytoplasm, the most distinctive difference being the long single strands of granular reticulum in the microglia, together with a less regular morphology. None of the features characterising oligodendroglia are unique, and an overall ultrastructural pattern is required for identification. Histochemical and immunohistochemical markers for oligodendroglia are derived from its generation of myelin, .a huge expanse of membrane with unique protein constituents (for reviews see Norton 1977; Cammer 1984), and from those unusual metabolic properties of oligodendroglia (Bunge 1968) which are related to the generation of lipid-rich myelin (cholesterol, and phospho- and galacto-lipids) (for reviews see Wykle 1977; Robinson and Williamson 1980; Horrocks and Harder 1982; Morell and Toews 1984; Pleasure et al. 1984). Some of these markers are considered below. In the future, markers may be derived from other oligodendroglia functions, in particular from their apparent capacities to take part in modulation of the ionic and neurotransmitter concentrations of the surrounding extracellular medium (e.g. Gilbert et al. 1984; Reynolds and Herschkowitz 1984). Since an oligodendrocyte can ensheath many axons its replacement must involve considerable disturbance, and we may expect the relationship to be stable; any turnover of the adult oligodendroglial population should, therefore, be slow. Autoradiographic

322 data indicate that in adult mice oligodendroglial turnover is considerably slower than astroglial turnover (Paterson 1983). Oligodendroglia turnover time in adult rats is calculated from autoradiographic data to be between one and two years (Imamoto et al. 1978; Nadler 1978; Kaplan and Hinds 1980). There is apparently a continuous, but very slow, remyelination of the CNS throughout normal adult life. The nature of this turnover is discussed in detail below. OLIGODENDROGLIALGENERATION DURING POSTNATALDEVELOPMENT Oiigodendroglia was visualized reliably by the silver carbonate method developed by Del Rio Hortega (1921; see also Penfield 1924), and early workers applied the method and its variants to postnatal tissues. The general features of oligodendroglial ontogeny emerged from these early studies, and a number of important questions raised at that time still await answer. The precursor cells to oligodendroglia were identified as small round neuroectodermal cells ("spongioblasts"). Later, ultrastructural studies showed these cells to be undifferentiated, their nuclei containing granular chromatin and their cytoplasm few organelles (Vaughn 1969; Privat and Leblond 1972; Sturrock 1975), and the proliferating population in the sub-ventricular layer appears homogenous. For some time it was believed that the stem ceils which generate neuroblasts from this layer later switch to the production of glioblasts (Angevine et al. 1970). However, evidence accumulated that at least some gliogenesis occurs concurrently with neurogenesis, and immunohistochemical studies suggest that distinct glial and neuronal stem cell populations can be discerned already at the start of neurogenesis (Levitt et al. 1981). It is not known whether oligodendroglia already comprises a distinct lineage at this stage, although the evidence n o w to be discussed argues against this. The stem cells proliferate in the sub-ventricular zone and migrate from there to their final locations (Fujita 1965; Privat and Leblond 1972; Choi et al. 1983). A rapid expansion of the oligodendroglial population occurs just prior to myelinogenesis (Vaughn 1969; Skoff et al. 1976b), and this rapidity impedes the analysis of the lineage relationships. Furthermore, glial lineages are hard to distinguish at early stages; ultrastructural features do not provide sensitive criteria, and the more immature the cells the fewer characteristics there are available. At first sight, cytoskeletal features might distinguish the astroglial and oligodendroglial lineages, but immature astroglia can contain varying and sometimes large numbers of microtubules (see Sturrock 1975), and the expression of intermediate filaments is also not a reliable guide. Under certain experimental conditions (e.g. Hirano and Zimmerman 1971) oligodendroglia can express intermediate filaments. Certain immature astroglial cells do not express them (Skoff et al. 1976a), and the extracellular environment can modulate their expression (Herpers et al. 1984). Like oligodendroglia, stem cells and microglia do not usually express them. Distinction between immature glial cell types remains difficult, and the exact relationships between astroglial, oligodendroglial and resting microglial lineages are obscure. That oligodendroglia might derive from the astroglial lineage(s) is often considered (Sturrock 1975; Skoff et al. 1976a; Choi etal. 1983; Choi and Kim 1984). Immature resting microglia is not readily distinguished from immature macroglia on ultrastructural criteria alone (Parnavelas

323 et al. 1983) and although most authors feel that microglia derives from mesenchyme (e.g. Peters et al. 1976), the evidence is not conclusive (for discussion and further references, see Parnavelas et al. 1983). Close relationships between oligodendroglial and microglial precursors have been proposed (Reyners et al. 1982). There is a general consensus that oligodendroglia and astroglia derive from the same stem cell population (e.g. Fujita 1965; Privat and Leblond 1972; Skoff et al. 1976a) and that oligodendroglia does not generate other lineages after its separation from the astroglial lineage. Can the glioblasts generate oligodendroglia directly, or does an intermediate stage, the "oligodendroblast" (Del Rio Hortega 1928; Skoff et al. 1967a, b; Imamoto et al. 1978) intervene? Tissue culture evidence, to be considered below, suggests that in the rat optic nerve a rather unusual kind of glial precursor can differentiate into either astroglia or oligodendroglia without cell division (Temple and Raft 1985), but studies of postnatal tissues have indicated that an oligodendroblast population is precursor to "light" immature oligodendrocytes, and a number of authors describe the ultrastructure of oligodendroblasts (Skoffet al. 1976a; Imamoto et al. 1978; Parnavelas et al. 1983; and others). The oligodendroblast is of considerable importance as a cell fully committed to an oligodendroglial identity yet still in proliferation. It is also the earliest known member of the oligodendroglial lineage. With caution, it is possible to distinguish stages of oligodendroglial maturity. Mori and Leblond (1970) proposed that larger oligodendroglia with relatively electron-lucent cytoplasm are immature, generating smaller cells with darker cytoplasm which are those capable of generating myelin. The mature cells in the sequence might be of either "medium" or "dark" electron density, according to their topographical location and the expanse of myelin they support (Blakemore 1978; Parnavelas et al. 1983). The proliferative rate falls with maturity in the sequence, as shown by autoradiography (Moil and Leblond 1970; Paterson et al. 1973; Imamoto et al. 1978). However, the cytoplasmic density of the cells correlates with variations in the granular reticulum, ribosomes, Golgi apparatus and nuclear cytology, and has often been suggested to reflect the rate or type of biosynthetic activity within the cells (see amongst others: Moil and Leblond 1970; Skoff etal. 1976a; Tennekoon etal. 1977; Parnevelas etal. 1983; Aranella and Herndon 1984) and, thus, the onset of myelinative function. Clearly, this criterion must be used with caution when conditions are conducive to altered glial metabolism, as, for example, in tissue culture. Indeed, "dark" oligodendroglial cells plated into culture have been observed to pass back through the sequence, reverting to pale ceils (e.g. Wollmann et al. 1981). OLIGODENDROGLIALGENERATIONAND REGENERATIONIN ADULTCNS The slow turnover of oligodendroglia in the normal adult brain, already mentioned above, may continue into advanced old age (Korr 1982; Sturrock 1984, 1985). Which cells generate these new oligodendroglia? There is evidence for two sources: the first being stem cells persisting in the adult tissue, and the second being re-entry of mature cells into proliferation. These two sources of oligodendroglial renewal will now be considered. In the forebrain, the subependymal layer, important in gliogenesis during

324 development, persists in the adult as a remnant, reduced in volume and extent, around the rostral horns of the lateral ventricles; it continues to generate new glial cells by mitosis from undifferentiated precursors throughout normal adult life (Hubbard and Hopewell 1980; Paterson 1983; Sturrock 1985). The subependymal layer is the most significant site where this type ofglial production occurs, but there are additional local sites such as the corpus callosum, where small populations of immature "glioblasts" or "free subependymal cells" generate glia by mitosis during normal adult life (Paterson 1983). The contribution of these remnant primitive cells is demonstrated dramatically when the cell kinetics of the subependymal plate are irreversibly disrupted, as by X-irradiation (Hubbard and HopeweU 1980). The resulting failure in the supply of fresh glia is manifest in the fatal condition known as delayed white matter necrosis. There is some experimental evidence to suggest that the slow generation of fresh glial cells in the subependymal layer of the normal adult CNS can be accelerated in response to trauma (Willis et al. 1976). Cajal (1909) considered mature oligodendroglia to exhibit a nuclear structure indicating an ability to undergo mitosis. This appears to be true. Of the four types of oligodendroglia described by Del Rio Hortega in 1928, types II-IV exhibit morphologies suggestive of a role in myelin maintenance, but type I cells do not; these are present in both grey and white matter, and although their ultrastructure resembles that of types II-IV and some of them can participate in remyelination (Ludwin 1979b), they proliferate faster than the interfascicular cells and have been suggested to represent a reserve population (Bunge 1968). Recently, autoradiographic evidence has been reported which suggests that in normal adult brain non-proliferating glia, probably including oligodendroglia, can re-enter proliferation (Korr 1980). Mature oligodendroglia can also be triggered into renewed proliferation in response to lesion damage. Although oligodendroglia were long believed to be terminally differentiated, evidence began to accumulate that they can in fact proliferate in response to trauma (Mori and Leblond 1970; Sturrock 1981), and it has now been confirmed (Aranella and Herndon 1984; Ludwin 1984; 1985). Taken in conjunction with evidence that oligodendrocytes can proliferate in response to factors released from T cells (Merrill et al. 1984) this implies that committed, mature oligodendrocytes might be a major source of renewal during regeneration (Manuelidis and Manuelidis 1985). Finally, in traumatised CNS tissue, cells occur which contain a large amount of electron-lucent cytoplasm and which lack intermediate filaments (Peters et al. 1970, 1976), and although these closely resemble immature oligodendroglia they have been classed with the astroglia (Reyners et al. 1982). Reyners et al. consider that these cells may persist from early development as reserve cells, but it has also been suggested (Skoff and Vaughn 1971) that astroglial precursors switch to start producing them. Present in higher vertebrates in very small numbers, they are more common in species noted for their regenerative capacities (see Reyners et al. 1982). CELL B I O L O G I C A L STUDIES

Oligodendroglial cells from perinatal tissues, and also those from adult tissues, can be maintained in culture; gila from adult tissues will be considered first. Bulk

325 isolation of viable oligodendrocytes from adult brains of larger animals and Man is now a routine procedure (Kim et al. 1983; and see review by Althaus et al. 1984). Similar procedures can be applied to the brains of smaller animals such as rats (Snyder et al. 1980). The cells are enriched on the basis of their physical properties, and thus are of uniform size and density: nominal enrichments exceed 90Yo. Trypsinization under neutral conditions can then remove selectively certain cell types such as astroglia and neurones (Snyder etal. 1980), although microglia, of,similar size (and, probably, density), might be likely to co-sediment with oligodendroglia and, as discussed above, could be difficult to detect in the preparations. Typical isolates contain small cells resembling "dark" oligodendrocytes: other oligodendroglial populations, including the pale ones considered to be immature, are excluded by the density gradient centrifugations. Oligodendrogha from such isolates can proliferate in tissue culture (Ovadia et al. 1984); these cells are mature oligodendrocytes and evidently not precursors or specialized reserve cells (which comprise very small populations and could not be isolated in bulk). Their capacity to proliferate in cultures confLrms the finding that mature oligodendrocytes can proliferate in adult tissues. Indeed, the traumatic conditions triggering such proliferation in situ are repeated in vitro: the isolation procedures inflict massive trauma on the cells, and reasons why normal culture conditions may resemble a traumatic environment to the cells are given below. The simplest view of this proliferation in culture is, therefore, that it reflects a response to trauma, in which case such cultures are a model system in which the factors modulating oligodendroglial regeneration can be analysed. The evaluation of cultures derived from perinatal tissues is less direct, because the massive trauma inflicted on the cells occurs during the unfolding of their developmental programme: a superimposition of developmental and regenerative modes of behaviour is therefore unavoidable. Nonetheless, the maturation of perinatal oligodendroglia in cultures reflects the onset of myelinogenesis in vivo in many ways. The pattern of incorporation of lipid precursors into myelin-related lipids mimicks the time scale of development (Ayliffe et al. 1984), as does the expression of myelin-related lipid and protein markers: GC (Abney et al. 1981; Bologa et al. 1982; Hirayama et al. 1984); the Wolfgram protein (Roussel et al. 1981, 1983); CAll (Delaunoy et al. 1980; Sarli6ve et al. 1981). The expression of MBP - usually in cells with more elaborate processes is generally considered a further step in maturation (Roussel et al. 1981), although oligodendroblasts have been reported to express MBP (Jacque et al. 1983). In cultures on plane surfaces maturation does not regularly proceed so far as the generation of compact myelin, and this absence of the usual sink for MBP might underlie the protracted expression of MBP in culture for many weeks (Mirsky et al. 1980; Roussel et al. 1981), which does not occur in situ. Unable to generate myelin, the cells also do not express the complex ramifying processes associated with it, and thus cannot be identified easily with the classical neurohistological subtypes. However, Szuchet and Yim (1984) have identified a subpopulation in vitro as Del Rio Hortega's type II. The evaluation of perinatal cell cultures is also complicated by the heterogeneity of the cell populations: not only are different macroghal populations present, but the oligodendrogiia itself is heterogeneous. The simpler forms, not expressing MBP, GC or the Wolf-

326 gram protein, include immature or precursor cells (Bologa et al. 1982; Abney et al. 1983; Espinosa de los Monteros et al. 1985), types which are able to mature and myelinate host tissues (Doering and Federoff 1984). In particular, the G C - population may be heterogeneous, even including cells resembling oligodendroglia but with the ultrastructure of astroglia (Raft et al. 1983). The cells which Greenham et al. (1974) identified as spongioblasts may also be considered amongst such simple G C - cells. Perinatal optic nerve cell cultures contain precursor cells able to differentiate into either astroglia or oligodendroglia without undergoing cell division (Temple and Raft 1985), depending on the presence of serum in the culture medium (Raft et al. 1983); these cells differ from ordinary neuro-epithelial stem cells, and from oligodendroblasts, in that they contain intermediate filaments (Raft et al. 1984). After switching from serum-free medium (in which they differentiate towards oligodendroglia) to serum-containing medium (in which they differentiate towards astroglia), many of them express both GFAP and GC, which is of interest in view of reports (Choi and Kim 1984) that oligodendroglia can express GFAP transiently during development in situ, and that GFAP expression in oligodendroglia occurs in certain neoplasms (Herpers and Budka 1984, who list earlier similar observations). Serum ingress into the CNS is a hallmark of the lesion environment, as discussed below, and in view of the fact that perinatal cell cultures derive from cells in development but also in trauma, the expression of GFAP in response to exposure to serum may reflect a behaviour related to trauma. Should this be so, certain early oligodendroglial precursors may not be phenotypically stable in lesioned tissues. Evidently the perinatal cell culture system is a complex one: do the cell population dynamics of oligodendroglia in such cultures reflect those during development in situ? This might seem unlikely, given the vastly different circumstances. Indeed, Rioux et al. (1980) found that whilst the proliferation of immature oligodendroglia in culture and in situ followed similar patterns, the stabilisation of the mature population that occurs in situ did not occur in culture, perhaps due to an absence of neurones. However, the culture system is able to generate data obtainable only with exceptional difficulty in situ; thus, Barbarese et al. (1983) were able to show that in culture, oligodendroglial precursors undergo approximately 11 divisions, reflecting the population expansion during postnatal development in situ. The pattern observed in situ, that mature oligodendroglia proliferate much more slowly than immature, is repeated in culture: cells expressing both MBP and GC proliferate very slowly in culture (Bologa et al. 1982, 1983; Roussel et al. 1983). Amongst cells expressing only GC, Bologa et al. (1982) and Bologa-Sandru et al. (1984) observe significant proliferation by more than 20~ of the cells, whereas others do not (Abney et al. 1981; Hirayama et al. 1984). The disparity may reflect differences in species or in culture conditions. Tissue culture offers by far the most promising means of analysing the regulation of oligodendroglial proliferation and maturation, therefore it is important to obtain valid comparisons with development in situ, unravelling which events in culture reflect responses to trauma. Three developments are likely to be relevant here. Firstly, an increasing range of serum-free culture media is being described, including some tailored to match oligodendroglial requirements (Eccleston and Silberberg 1984; Kim et al.

327 1984; Koper et al. 1984); this eliminates one of the agents causing the culture environment to appear abnormal and traumatic to the cells. Further, any biologically active factors appearing in the media must derive from the cultured cells; consequently, such media are a means of analysing such intercellular interactions as are mediated by soluble factors. Secondly, there is an increasing range of markers for oligodendroglial development. Some of these have been mentioned above, but others relate to the cell surfaces, important for cell biological studies in which the sorting or selective killing of cells is required. Surface differentiation markers are also more likely to offer sensitive indices of developmental changes than intracellular markers. Cell surface microheterogeneity is best dissected by use of the monoclonal hybridoma technique, which has already generated several antibodies capable of recognising developmental stages of oligodendroglia (Schachner 1982). Gangliosides are likely to be important surface determinants and the range of these increases during the proliferation and maturation of glia (Sarlirve et al. 1981; Asou and Brunngraber 1983), whereas another ganglioside antigen, recognised by antibody A2B5, is expressed on immature oligodendroglia but not on the more mature cells expressing GC (Abney et al. 1983). Cerebroside expression also changes during development: immature oligodendroglia expresses surface glucocerebrosides whereas mature glia expresses galactocerebroside (Ayliffe et al. 1984), a switch which involves the insertion of new molecules into the cell membrane. Thirdly, long-term cell lines enriched in phenotypically stable non-transformed glial cells have been generated: astroglia (Alliott and Pessac 1984; Pilkington et al. 1983) and oligodendrogiia (Meller and Waelsch 1984; Debbage 1985a). From such lines clones of def'med glial types can be obtained (Alliott and Pessac 1984; Pilkington et al. 1983; Debbage 1985b), which are free of cells such as macrophages that might modulate glial behaviour. Such cultures should facilitate the analysis of interglial interactions, by simplifying the culture system to include only the interacting cell types. REGULATIONOF OLIGODENDROGLIALGENERATION Oligodendroglia can be generated and destroyed: the establishment of a functioning population depends, amongst other things, on the balance between these processes, and on the committment of newly generated cells to become oligodendroglia. The determinants of these events are likely to be very different in development and during regeneration in adulthood. We consider firstly postnatal development. Korr (1980) has discussed a low rate of cell death which is associated with proliferation in both prenatal and adult life, and which can be observed in glia and also in other neural cell types (e.g. neuroblasts, endothelia). This proliferation-associated cell loss is steady and small, increasing with age: about 3 ~ of mitotic daughter cells die in young brains. It is not known whether a particular glial type, either oligodendroglia or astroglia, is especially prone to this type of cell loss, but it is clear that this kind of cell loss is not involved in regulating the size of the oligodendroglial population during development. Massive oligodendroglial cell death is not observed during development, and clearly the number of oligodendroglia is not regulated by overproduction corrected consequently by cell death. The population size is evidently controlled by variation in the several stimuli to proliferate, and by the committment of precursors to the oligodendroglial

328 lineage. Sturrock (1983) suggests that division by mature oligodendrocytes contributes little to this postnatal expansion. Precursor proliferation might proceed for a period governed by an internal clock, although this would not fit in well with the results obtained in Jimpy mice, where oligodendroblast proliferation is protracted (Skoff 1982; Bologa et al. 1983). The more likely circumstance is that proliferation continues until curtailed. Proliferation apparently depends upon the presence of axons (Rioux et al. 1980; Privat et al. 1981). It is rapid prior to myelination, but then the population stabilises (Skoffet al. 1976b; Privat et al. 1982), and we have noted that there are only low levels of proliferation in the adult. This pattern is explicable in terms of oligodendroglial response to axons or to factors they might release (Skoffet al. 1976b; Privat et al. 1981), a possibility consistent with tissue culture results (Hanson and Partlow 1977; Pettmann et al. 1980; Sensenbrenner et al. 1982). An analogous stimulation of Schwann cells has been known and studied for several years (Wood and Bunge 1975; Bray et al. 1981; Bunge 1982). Ensheathment of axons by the newly generated oligodendroglia would reduce such stimulation and curtail proliferation. A breakdown in axonooligodendroglial interaction might underlie the failure to establish a mature myelinating population in the Jimpy mouse, so that proliferation is not curtailed. A different parameter that might govern oligodendroglial proliferative rate is the density of the generated population, again consistent with tissue culture results (Bologa et al. 1982), although aging of the precursor cells in these cultures may have played a role (BologaSandru et al. 1984). Regulation via population density would imply some substrate (axons or axonal factors?) for which oligodendroglial precursors compete, or the secretion by the oligodendroglia themselves of a factor inhibiting generation or commitment. Astroglia has been implicated in the proliferation of the optic nerve bipotential precursor mentioned above (Noble and Murray 1984), an interesting result implying that astroglia responds in some way to the density of the oligodendroglial population. An important class of soluble factors involved in regulating glial proliferation and maturation are the hormones; some acting at the level of proliferation whereas others influence maturation. Thus, growth hormone stimulates oligodendroglial proliferation (Noguchi et al. 1982), whereas glucocorticoids (Preston and McMorris 1984) and thyroid hormones (Shanker et al. 1984) promote aspects of maturation related to myelinogenesis. Insulin, by stimulating glucose uptake into the brain and its metabolism there (Baskin et al. 1983), modulates a quantity which studies in vitro have shown to be important for oligodendroghal survival and development (Zuppinger et al. 1981). In addition to soluble factors, the nature of the extracellular substrate is likely to be important, as has been shown for Schwann cell proliferation (Bunge et al. 1978; Bunge 1982; Wood and Bunge 1984); these authors have suggested that fibrous astrocytes may play an important role of a similar nature in the CNS. A lesion creates entirely new conditions within the CNS, and the importance of these for oligodendroglial proliferation is as yet incalculable; an early consequence of most lesions is that the blood-brain barrier opens. The lesion may be sited within the CNS a recent study in MS has been reported by Takeoka et al. (1983) - but disturbances to the blood-brain barrier can also result from disease processes outside the CNS (Du Moulin et al. 1985). Breach of the blood-brain barrier dramatically alters -

329 the composition of the fluid bathing the neural cells; the CNS is normally a serum-free compartment to which serum hormones and any other regulatory factors, inflammatory agents, and enzymes have access only within closely controlled limits, and often not at all. The influx of serum, therefore, represents a hitherto unknown modification to the ceils' environment (a consideration relevant to tissue culture studies, see above); the cells' responses must now occur against a background of factors which may act upon them as signals, yet most likely as uncoordinated and contradictory ones. This is particularly likely to occur when a factor subserves regulatory roles independently in the CNS and outside it; insulin is an example of this. The brain contains high concentrations of insulin (or polypeptides closely similar to it), which binds to receptors probably located on glial cells (Clarke et al. 1984). These receptors show structural differences to non-CNS insulin receptors (Hendricks et al. 1984), are regulated differently (Zahniser et al. 1984), and appear able to mediate the regulation of cellular events, since they span the cell membrane and are coupled to tyrosine kinase activity (Rees-Jones et al. 1984), similarly to the non-CNS receptors. Insulin binding stimulates glucose uptake by glial cells (Clarke et al. 1984), though not by neurones. Regulation of brain insulin and its receptors seems entirely independent of insulin and receptor concentrations outside the CNS (Havrankova et al. 1979), consistent with insulin playing independent roles in the two compartments. In brain, insulin may act as a growth hormone (Lenoir and Honegger 1983) in addition to regulating glucose metabolism. Clearly, breakdown of the blood-brain barrier, which separates the two compartments, could result in confusion of signals to the brain glia, due to pancreatic insulin entering the brain. Such disturbances are obviously strongest locally, where the blood-brain barrier fails, and do not form part of a globally coordinated development. Further examples of confused regulation might occur in connection with the immune system; both oligodendroglia (Merrill et al. 1984) and astroglia (Fontana 1982) respond to T-ceil factors, so that interactions between these glial populations seem vulnerable to derangement by immune processes occurring within the CNS. Lesions result not only in the influx of serum, but also in the ingress of immunocompetent ceils into the CNS. After the resealing of the blood-brain barrier, these guests may remain in the nervous tissue (Prineas 1979), modifying the extracellular fluids or interacting with the glial cells: the nervous tissue does not necessarily revert to a normal condition. Intrathecal antibodies are produced in a range of neurological diseases (Gorny et al. 1983), and although their pathogenic significance is debated, there is no doubt about their capacity to disturb oligodendroglial function in tissue culture (review: Raine 1984; and for serum-borne antibodies: Bornstein and Grundke-Iqbal 1982). Modulation of the immune response in CREAE (Raine and Traugott 1983; Raine 1984) permits or even stimulates oligodendroglial proliferation, indicating the disturbance present in the untreated CNS subjected to immune aggression. While such a disturbance may be a purely secondary response in demyelinating diseases, it may still be very important if it hinders the re-establishment of a functioning oligodendroglial population. Thus although oligodendroglia have been reported to survive and even proliferate in demyelinating lesions (Raine et al. 1981; Moore et al. 1984) they do not express myelin-related antigens in the zone of active demyelination (Itoyama et al.

330 1983), suggesting a failure of function. One possible cause for such failure would be a disturbance of axono-oligodendroglial interaction, already discussed above in relation to perinatal development. Alternatively, derangement of astroglial-oligodendroglial interactions might underlie such failure. A further possibility is that the proliferating oligodendroglia might express cell surface determinants reflecting immaturity, triggering an immune aggression against later oligodendroglial proliferation; thus it could be immature oligodendroglia that becomes a target in demyelinating diseases. It is also interesting to note in this connection that MS has been suggested to reflect a heightened congenital cellular susceptibility to radiation damage (Gipps and Kidson 1984), and that immature oligodendroglia (Hirayama et al. 1984), as also the "stem cells" of the subependymal plate (Hubbard and Hopewell 1980), are peculiarly susceptible to radiation damage. In lesioned CNS tissue it is not only the cells' environment that differs from that of postnatal development: the proliferating population of cells is also different. The repair capacity of the stem cell populations persisting in adult CNS has been discussed above; in addition, lesions can trigger mature oligodendrocytes into a rate of proliferation much higher than the usual turnover rate (Raine and Traugott 1983; Aranella and Herndon 1984; Manuelidis and Manuelidis 1985): these cells must be expected to display different kinetics to the precursors proliferating during development. In particular, the cell losses due to patho-physiological processes occurring concomitant with proliferation must be made good; this is not required during development. In addition to its influence on the kinetics, the disturbed environment of the damaged tissue could also distort the course of precursor cell development by favouring differentiation towards a certain cell lineage, as mentioned above with respect to the effect of serum on optic nerve glial precursors. In a disturbed environment the regeneration of oligodendroglia might be more assured if it originated in cells already committed to the oligodendroglial lineage. The l~malmatter to be discussed here is, therefore, the stability associated with such committment to the oligodendroglial lineage. Committment to becoming an oligodendrocyte presupposes that reversion to a more primitive (presumably multipotential) state is blocked. In oligodendroglia the relationship between differentiation and division is not exclusive, since mitosis occurs in cells that express myelin-related products and are capable of initiating myelination (Skoff et al. 1976a; Jacque et al. 1983), a point made by Levitt et al. (1981). The term "oligodendroblast" applies, therefore, to cells already committed to the oligodendroglial lineage but capable both of generating further similar cells and of differentiating into oligodendrocytes. As mentioned above, most authors imply that oligodendroblasts do not revert to a more primitive state, in contrast to Schwann cells, which rapidly do so in culture if neurones are absent (Mirsky et al. 1980). Oligodendrocytes generate differentiated cells when plated into culture without neurones (review: Althaus et al. 1984). The characteristics of the blood-nerve barrier have not been richly documented, and we do not know whether the endoneurial tissues are as well screened off from the blood compartment as are most CNS tissues; it is interesting to speculate that some of the cell biological differences between Schwann cells and oligodendrocytes might relate to possibly different exposures of these two cell types to the blood serum.

331 CONCLUSION In normal adult C N S , the oligodendroglial population is renewed in a process of slow turnover, n o w partially documented in rodents, and but poorly documented elsewhere. The two processes contributing to this turnover are the proliferative activities of remnant precursor cells, and the re-entry o f mature oligodendrocytes into proliferation. H o w the balance o f these two processes is maintained is yet to be determined. The exact sequence o f each process also still has to be elucidated, in particular: how immature are the daughter cells produced by oligodendroglial mitosis? In the damaged C N S either o f these processes appears capable o f adjusting to greater activity to replenish depleted oligodendroglial populations; for both processes a more extensive documentation will be required for assessment o f the factors involved in regulation of this greater activity. At present it seems likely that some hormones, and some immunerelated factors, are involved. Whether the balance between precursor proliferation and oligodendrocyte proliferation is the same in post-lesion recovery as in normal circumstances is an important question, in view of the possibility that immature precursors may be diverted from an oligodendroglial destiny by conditions within a lesion, whereas committed cells of the oligodendroglial lineage m a y well resist this.

ACKNOWLEDGEMENTS I thank Dr. M. Brammer, Dr. A . R . Lieberman, Dr. M. Sensenbrenner, Dr. P. Spoerri and Dr. R. Sturrock for their critical reading o f the manuscript, and Professor E. Helmreich, Professor B. H a m p r e c h t and E. Karlitzky for encouragement and advice.

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