I. INTRODUCTION

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Glial cells constitute the large majority of cells in the nervous system. Despite their number and their role during development, their active participation in the physiology of the brain and the consequences of their dysfunction on the pathology of the nervous system have only been emphasized in the recent years.

Virchow (639) first described that there were cells other than neurons. He thought that it was the connective tissue of the brain, which he called "nervenkitt" (nerve glue), i.e., neuroglia. The name survived, although the original concept radically changed.

The characterization of the major glial cell types was the result of microscopic studies, and especially the techniques of metallic impregnation developed by Ramon y Cajal and Rio Hortega. Using gold impregnation, Ramon y Cajal (495) identified the astrocyte among neuronal cells, as well as a third element which was not impregnated by this technique. A few years later, using silver carbonate impregnation, Rio Hortega found two other cell types, the oligodendrocyte (514), first called interfascicular glia, and another cell type that he distinguished from the two macroglial cells (i.e., macroglia), and that he called microglia (513).

The morphological characteristics of macroglia have been reviewed (453). The progress of morphological techniques and the discovery of cellular markers by immunocytochemical techniques indicate the notion of multiple functional macroglial subclasses. Our understanding of the role of glia in central nervous system (CNS) function has made important progress during recent years. Glial cells are necessary for correct neuronal development and for the functions of mature neurons. The ability of glial cells to respond to changes in the cellular and extracellular environment is essential to the function of the nervous system. Furthermore, there is now growing recognition that glia, possibly through a glial network, may have communication skills that complement those of the neurons themselves (Fig. 1). There are currently expanding discoveries of specialization of both neurons and glial cells and their persistent interactions that gives also a new insight to the understanding of pathological outcomes. The association of neuronal and glial expression for some neurotransmitters, their transporters, and receptors contributes to the understanding of their functional cooperation. It now seems likely that oligodendrocytes have functions other than those related to myelin formation and maintenance.

View larger version (42K): [in this window] [in a new window]   Fig. 1. Schematic representation of the different types of glial cells in the central nervous system (CNS) and their interactions, among themselves and with neurons. Astrocytes are stellate cells with numerous processes contacting several cell types in the CNS: soma, dendrites and axons of neurons, soma and processes of oligodendrocytes, and other astrocytes; astrocytic feet also ensheath endothelial cells around blood capillaries forming the blood-brain barrier and terminate to the pial surface of the brain, forming the glia limitans. Oligodendrocytes are the myelinating cells of the CNS; they are able to myelinate up to 50 axonal segments, depending on the region of the CNS. A number of interactions between glial cells, particularly between astrocytes in the mature CNS, are regulated by gap junctions, forming a glial network. [From Giaume and Venance (218). Copyright 1995 Overseas Publishers Association. Permission granted by Gordon and Breach Publishers.]

As an overview, the number of glial cells increases during evolution; glial cells constitute 25% of total cells in the Drosophila, 65% in rodents, and 90% in the human brain (456). The very few morphological studies do not take into account the different glial cell types. Brain white matter tracts are deprived of neuronal cell bodies but contain glial cells; because brain white matter constitutes as big a volume as the cortex gray matter, the overall glia-to-neuron ratio is considerably increased (456).

	II. OLIGODENDROCYTES

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The term oligodendroglia was introduced by Rio Hortega (513) to describe those neuroglial cells that show few processes in material stained by metallic impregnation techniques. The oligodendrocyte is mainly a myelin-forming cell, but there are also satellite oligodendrocytes (449) that may not be directly connected to the myelin sheath. Satellite oligodendrocytes are perineuronal and may serve to regulate the microenvironment around neurons (349). A number of features consistently distinguish oligodendrocytes from astrocytes (reviewed in Ref. 453), in particular their smaller size, the greater density of both the cytoplasm and nucleus (with dense chromatin), the absence of intermediate filaments (fibrils) and of glycogen in the cytoplasm, and the presence of a large number of microtubules (25 nm) in their processes that may be involved in their stability (350). An oligodendrocyte extends many processes, each of which contacts and repeatedly envelopes a stretch of axon with subsequent condensation of this multispiral membrane-forming myelin (82, 84) (Fig. 2). On the same axon, adjacent myelin segments belong to different oligodendrocytes. The number of processes that form myelin sheaths from a single oligodendrocyte varies according to the area of the CNS and possibly the species, from 40 in the optic nerve of the rat (453) to 1 in the spinal cord of the cat (83). Rio Hortega (514) classified oligodendrocytes in four categories, in relation to the number of their processes (also described in Ref. 83). According to their morphology and the size or thickness of the myelin sheath they form, Butt et al. (89) also distinguish four types of myelinating oligodendrocytes, from small cells supporting the short, thin myelin sheaths of 15-30 small diameter axons (type I), through intermediate types (II and III), to the largest cells forming the long, thick myelin sheaths of one 1-3 large diameter axons. Morphological heterogeneity is, in fact, a recurrent theme in the study of interfascicular oligodendrocytes (84). At the electron microscopic level, oligodendrocytes have a spectrum of morphological variations involving their cytoplasmic densities and the clumping of nuclear chromatin. Mori and Leblond (406) distinguish three types of oligodendrocytes: light, medium, and dark. The dark type has the most dense cytoplasm. On the basis of labeling with tritiated thymidine of the corpus callosum of young rats, they suggest that light oligodendrocytes are the most actively dividing cells and that oligodendrocytes become progressively dark as they mature.

View larger version (40K): [in this window] [in a new window]   Fig. 2. The oligodendrocyte and the mature myelin sheath. An oligodendrocyte (G) sends a glial process forming compact myelin spiraling around an axon. Cytoplasm is trapped occasionally in the compact myelin. In transverse sections, this cytoplasm is confined to a loop of plasma membrane, but along the internode length, it forms a ridge that is continuous with the glia cell body. In the longitudinal plan, every myelin unit terminates in a separate loop near a node (N). Within these loops, glial cytoplasm is also retained. [Adapted from Bunge (84).]

Before their final maturation involving myelin formation, oligodendrocytes go through many stages of development. Their characterization is often insufficient by morphological criteria alone both in vivo and in vitro. A number of distinct phenotypic stages have been identified both in vivo and in vitro based on the expression of various specific components (antigenic markers) and the mitotic and migratory status of these cells. They are described in section II, B and C.

Characterization of a number of specific biochemical markers has increased our knowledge on the stages of oligodendrocyte maturation, both in vivo and in vitro. These components, although they may be present in other cell types or other tissues, or in specific cells only at certain stages, must be viewed as modular elements that generate, with other elements, complex and unique surface patterns (502). All the biochemical and molecular characteristics of oligodendrocytes are not described here. We limit ourselves to those that have given insights into our understanding of this cell type. Although some markers are very useful in culture to determine the sequences of maturation, and ultimately their mechanism, they may be more difficult to use in situ, because some of them are present on other cell types in vivo.

Oligodendrocytes originate from migratory and mitotic precursors, then progenitors, and mature progressively into postmitotic myelin-producing cells (see sect. IIC5). The sequential expression of developmental markers, identified by a panel of cell specific antibodies, divide the lineage into distinct phenotypic stages (249, 455; reviewed in Ref. 344) characterized by proliferative capacities, migratory abilities, and dramatic changes in morphology (Fig. 3). Many of these markers have been at first identified in tissue cultures. Some of them are characteristic myelin components (see sect. IIIC). Myelination requires a number of sequential steps in the maturation of the oligodendroglial cell lineage (249, 455) accompanied by a coordinated change in the expression of cell surface antigens often recognized by monoclonal antibodies. Differentiation involves the loss of certain surface or intracellular antigens and the acquisition of new ones. Some of the surface antigens are lineage markers; discrete phases in these lineages are marked by differential expression of additional antigens or phase markers (502).

View larger version (35K): [in this window] [in a new window]   Fig. 3. Schematic representation of the developmental stages of cells of the oligodendrocyte lineage. Schematic drawing of the morphological and antigenic progression from precursor cells to myelinating mature oligodendrocytes, through progenitors, preoligodendrocytes, and immature nonmyelinating oligodendrocytes. Timing of neuronal and astrocytic signaling is indicated. Stage-specific markers are boxed. RNAs are in italics. [From Lubetzki et al. (344). See also Hardy and Reynolds (249) and Pfeiffer et al. (455).]

1.  Markers of maturation of the oligodendrocyte lineage

A) NESTIN. Nestin is a protein recognized by the rat-401 monoclonal antibody (257), whose expression specifically distinguishes neuroepithelial stem cells (from which the name nestin is originated) from other more differentiated cells in the neural tube (327). Nestin defines a distinct sixth class of intermediate filament protein, closely related to neurofilaments. Nestin is also expressed by glial precursors (327), such as radial glia (257), and in the cerebellum by immature Bergmann fibers, and also by adult Bergmann fibers recapitulating developmental stages when placed in the presence of embryonic neurons (573). Using cortical- or CG-4-derived oligodendrocyte lineage cells, culture experiments have shown that high levels of nestin protein are also expressed in proliferating oligodendrocyte progenitors, but the protein is downregulated in differentiated oligodendrocytes (206).

B) PROTEOLIPID PROTEIN. A special note should be made concerning the proteolipid protein (PLP) gene expression, as a marker of developmental maturation of myelinating glial cells, in the CNS as in the peripheral nervous system (PNS). By RT-PCR and in situ hybridization, the mRNA of DM-20, coding for an isoform of PLP, can be detected in the developing CNS at a very early stage of development, before the onset of myelination (272, 454, 610, 611). With the use of PLP-Lac Z transgenic mice, expression of the splicing variant DM-20 of the PLP gene is detected in the mouse embryo within very discrete regions of the CNS and rather extensively throughout the PNS (576, 666). The DM-20 protein is detected precociously in different regions of the nervous system, in oligodendrocyte precursors from the spinal cord (144), or in ensheathing cells from the olfactory bulb (145).

C) PLATELET-DERIVED GROWTH FACTOR -RECEPTOR. The platelet-derived growth factor -receptor (PDGFR-) transcripts are also detected at very early stages of the developmental maturation of myelinating glial cells. Although PDGF R- and PLP/DM-20 cells are usually found in a close vicinity, either in the same or in adjacent territories, they are rarely coexpressed by the same cells, raising the question of single or multiple oligodendrocyte lineage (reviewed in Refs. 510, 577).

D) PSA-NCAM. The embryonic polysialylated form of neural cell adhesion molecule (NCAM), PSA-NCAM, defines in the absence of expression of GD3, the precursor stage (239, 248), from which oligodendrocyte progenitors arise.

E) GANGLIOSIDE GD3. The ganglioside GD3 has been identified by the use of two monoclonal antibodies: R24 (483) and LB1 (127). In fact, R24 recognizes also minor gangliosides. In vitro, there is a high expression of GD3 on oligodendrocyte progenitors in culture (248); thus it is a good marker for oligodendrocyte cell culture; GD3 expression disappears as the cell matures. Nevertheless, in vivo it is also expressed in other glial cell types such as immature neuroectodermal cells, subpopulations of neurons and astrocytes during development, resting ameboid microglia (174, 671), reactive microglia, and astrocytes (223). Thus particular caution should be used when extrapolating from in vitro investigations to the CNS in situ (223).

F) THE MONOCLONAL ANTIBODY A2B5. The monoclonal antibody A2B5 (172) recognizes several gangliosides (198) that remain as yet uncharacterized. It is expressed both on neurons and glial cells in vivo; it is used essentially in oligodendrocyte cultures to follow the maturation of oligodendrocyte progenitors. In culture, ganglioside GT3 and its O-acetylated derivative are the principal A2B5-reactive gangliosides (153, 181). Both antigens are downregulated as the cell differentiates into the mature oligodendrocyte. This corresponds to the disappearance of cell surface immunostaining by A2B5. Like GD3 monoclonal antibodies, A2B5 binding in vivo is not cell specific.

G) THE RAT NG2 PROTEOGLYCAN. The rat NG2 proteoglycan is an integral membrane chondroitin sulfate proteoglycan with a core protein of 260 kDa. In the mature CNS, cells express this antigen together with PDGFR- (426, 427, 504). These cells have extensive arborizations of their cell processes and are found ubiquitously long after oligodendrocytes are generated. They do not express antigens specific to mature oligodendrocytes, astrocytes, microglia, and neurons, suggesting that they are a novel population of glial cells which undergoes proliferation with morphological changes in response to stimuli such as inflammation or demyelination (reviewed in Ref. 136a, 426).

H) THE MONOCLONAL ANTIBODY O4. The monoclonal antibody O4 (571) marks a specific preoligodendrocyte stage of oligodendrocyte maturation. It reacts also with sulfatides and still unidentified glycolipids (23).

2.  Oligodendrocyte/myelin markers

A) GLYCOLIPIDS. There are specific glycolipids in oligodendrocytes and myelin, such as galactosylceramides (GalC) (galactocerebrosides) and sulfogalactosylceramides (sulfatides). Galactosylceramides and sulfogalactosylceramides are early markers that remain present on the surface of mature oligodendrocytes in culture (455, 488) and in vivo (693). It appears often difficult to characterize them on the cell surface of the oligodendrocyte during development with precision, because many of the reagents, i.e., the monoclonal antibodies used, are less specific than thought at first. The main antibody used to identify some of these developmental stages is O1 (571), which recognizes galactocerebrosides, but also monogalactosyldiglycerides and an unidentified antigen present during oligodendrocyte development. This is also the case for the R-MAb (497), which has been mainly used for galactocerebroside identification, but recognizes also sulfatides, monogalactosyldiglycerides, seminolipids, and another unidentified antigen (23). Some polyclonal IgG antibodies to galactocerebrosides alter the organization of oligodendroglial membrane sheets (164).

B) RIP ANTIGEN. RIP antigen has been identified through the use of a monoclonal antibody that was generated against oligodendroglia from rat olfactory bulb (199). It recognizes an unknown cytosolic epitope on oligodendrocytes and labels both oligodendrocyte processes and the myelin sheath (44, 199). Two bands of 23 and 160 kDa have been found by Western Blot, the nature of which have not been elucidated. Interestingly, this marker may serve to determine biochemical subtypes of oligodendrocytes together with carbonic anhydrase II (89).

C) CARBONIC ANHYDRASE II. Among the seven isozymes that are products of different genes, carbonic anhydrase II (CAII) is the only one that is located in the nervous system, in oligodendrocytes. CAII covers all stages of the lineage and is also a marker of adult oligodendrocytes (217); it is a more diffuse marker of glial cells in the developing animal (95). In the anterior medullary velum of the rat, RIP⁺ CAII⁺ oligodendrocytes support numerous myelin sheaths for small diameter axons, whereas RIP⁺ CAII oligodendrocytes support fewer myelin sheaths for large-diameter axons (89). This immunocytochemical classification overlaps the morphological classification of Bunge (84). However, CAII is also reported to be expressed by microglia (671).

D) NI-35/250 PROTEINS. NI-35/250 proteins are transmembrane proteins enriched in mammalian myelin CNS and oligodendrocytes (105). NI-35/250 have been found to be potent inhibitors of axonal regrowth in pathological conditions (see sect. IIIG3C). The bovine NI-220 cDNA has been recently characterized (578). These proteins are recognized by the monoclonal antibody IN-1. The gene has recently been cloned (114). There are three isoforms of the proteins; one of them, NOGO A, is the one with the inhibitory properties and which is mainly expressed on CNS oligodendrocytes and myelin (see sect. IIIG3) (reviewed in Refs. 20, 222, 232).

3.  Other oligodendrocyte protein markers

A) TRANSFERRIN. Transferrin, the iron mobilization protein, is expressed in oligodendrocytes (62) and choroid plexus epithelial cells. Before the establishment of the blood-brain barrier, neural cells are dependent on serum transferrin biosynthesized in the liver. As the blood-brain barrier gets established during development, neural cells become dependent on transferrin produced by oligodendrocytes and choroid plexus epithelial cells. In addition, transferrin acts as a trophic and survival factor for neurons and astrocytes, suggesting an important function for oligodendrocytes besides myelination (176).

B) S100 PROTEINS. S100 proteins are Ca²⁺ and Zn²⁺ binding proteins; they are at high concentration in the mammalian brain. It is now known that the S100 family of calcium-binding proteins contains ~16 members, each of which exhibits a unique pattern of tissue/cell type specific expression. Whereas the most abundant isoform, S100 -protein, is expressed predominantly by astrocytes, it is also present in adult rat brain in a minor subpopulation of oligodendrocytes (511).

C) GLUTAMINE SYNTHETASE. Glutamine synthetase catalyzes the conversion of glutamate to glutamine in the presence of ATP and ammonia. By immunocytochemical methods, its presence has been demonstrated in the cytoplasm of astrocytes. It is also found in a discrete population of oligodendrocytes (129).

For many years, one has mainly inferred lineage relationships by examining patterns of antigen expression and morphological changes in developing glia. Lineage can now more securely be traced with gene transfer techniques using retroviral vectors (109, 352, 476) and transgenic constructs (191, 224, 666). In the former kind of analysis, a replication-deficient retrovirus bearing a reporter gene such as LacZ coding for the bacterial -galactosidase, is introduced into a dividing cell population. After the virus enters a cell, DNA copies of viral RNA are synthesized, and a proportion of cells undergoing DNA replication will integrate viral DNA into genomic DNA. From that point on, viral genes will be passed to all the progeny (633). Moreover, the promotor sequences of genes specifically activated in glial precursors induce the expression of the LacZ reporter gene in the glial lineages. In the transgenic mice bearing this type of transgene, LacZ expression characterizes the subpopulation that expresses the transgene during its differentiation.

Mammalian species acquire their full complement of cortical neurons during the first half of gestation. Radial glia are established early in development at the time of neurogenesis, during the early gestational period, and support neurons during their migration to the cortex (reviewed in Ref. 493). The subventricular zone (SVZ), which is present in late gestational and early postnatal mammalian brain, is a major source of astrocytes and oligodendrocytes, although astrocytes may also develop from radial glia. Maturation of oligodendrocytes and astrocytes takes place in the mammalian CNS largely in early postnatal life.

In the Drosophila CNS, during neurogenesis, there is a binary genetic switch between neuronal and glial determination (261, 285). The mutation of a gene encoding a nuclear protein, glial cells missing (gcm), causes presumptive glial cells to differentiate into neurons, whereas its ectopic expression using transgenic constructs forces virtually all CNS cells to become glial cells. Thus, in the presence of gcm protein, presumptive neurons become glia, whereas in its absence, presumptive glia become neurons. However, a gene with an identical function has not as yet been identified in the vertebrate nervous system.

In the mammalian cortex, both neurons and glia arise from the proliferating neuroepithelial cells of the telencephalic ventricular and SVZ (149). The SVZ is a mosaic of multipotential and lineage restricted precursors (329), where environmental cues influence both fate, choice, and all surviving cells (279). A number of trophic factors (see sects. IIC8 and IIIF) influence the actual developmental fate of a progenitor or a multipotent stem cell from the SVZ, which can differ from its developmental potential (91, 241, 281, 484, 682). As a consequence of this growth factor dependency, neurons are not formed postnatally because of the lack of trophic factors necessary for their survival, resulting in their death; this has been shown by a recent clonal analysis of the progeny of single rat SVZ cells using replication deficient retroviral vectors (329).

Because environmental conditions can be provided in vitro when adding specific trophic factors to the culture medium (18, 281, 415, 484, 503), glial cells differentiating in cultures may exhibit a considerable plasticity in the extent of lineage switching, not observed in vivo (565) (see sect. IIC6).

On the other hand, it has been hypothesized that even after development is completed, the Notch pathway may help maintain some mammalian stem cells and precursor cells in adult tissues by preventing them from differentiating, thus allowing for the possibility of new cell generation in renewing or damaged tissues (14).

The identity of neural stem cells in the adult CNS is presently debated and could be either ependymal cells (280) or SVZ astrocytes (149, 323a). For a critical review of these data, see Reference 27.

Oligodendrocyte precursors originate from neuroepithelial cells of the ventricular zones, at very early stages during embryonic life. This was first suggested by following the expression of specific markers of oligodendrocytes (127, 248, 249, 328, 455), some of which are transcripts of future protein components of myelin (CNP, MBP, PLP) (272, 454, 478, 610, 611, 690).

In spinal cord, oligodendrocytes initially arise in ventral regions of the neural tube. Miller and co-workers (652) have shown, using a special dye (DiI) associated with specific markers of the oligodendrocyte lineage, that oligodendrocyte precursors arise in the ventral spinal cord and then migrate dorsally during development. During subsequent development, the dorsal regions acquire the capacity for oligodendrogenesis, probably both intrinsically (94) and through the ventral to dorsal migration of oligodendrocyte precursors. This has been confirmed using cultures of the thoraco-lumbar area of the rat spinal cord, which showed that the ability to give rise to oligodendrocytes is restricted to the ventral region of this area of the spinal cord until E14 (652). Signals from the notochord/floor plate, involving the morphogenetic protein sonic hedgehog, are necessary to induce the development of ventrally derived oligodendroglia (392, 441, 442, 467, 479).

In situ hybridization for mRNAs of proteins involved in oligodendrocyte maturation has been used to characterize early oligodendrocyte precursors. Ventral ventricular cells express mRNA for PDGFR- (478). Similarly, a discrete population of cells expressing mRNA for the myelin protein CNP (690) and DM20 (PLP gene) (610) has been localized to the ventral ventricular zone of the developing mammalian spinal cord. A group of cells in the ventricular and mantle zone of the ventral diencephalon of the E13 rat express mRNA for the PDGFR- (478). A similar distribution of cells expressing DM-20 mRNA has also been described in the mouse (610). Nevertheless, this issue still remains unclear as both markers are not expressed by the same population of oligodendrocyte precursors, indicating a diversity in this population already at this stage. DM-20 protein is also expressed on embryonic cells that may become oligodendrocyte progenitors as shown by Dickinson et al. (144) in the spinal cord.

The initial restricted localization of oligodendrocyte precursors in the ventral plate of the neural tube appears not to be limited to the spinal cord (690) but is also observed in mid- and forebrain (576, 610). In addition, a similar restricted localization of the sites of oligodendrogliogenesis has been described in other vertebrate species, such as human (244), chick (440), and Xenopus (688). The population of oligodendrocytes derived from the restricted loci of cells appears to be regionally distributed in the brain. For instance, in the chick embryo, some of the oligodendrocytes of the optic nerve generate suprachiasmatic foci of precursors located in the medio-ventral epithelium of the third ventricle (440). The developing cerebral cortex of murine embryos could be populated by oligodendrocytes originating from precursor cells located in the laterobasal plate of the diencephalon and probably also in the telencephalon (475).

For comprehensive reviews of the different hypotheses concerning origin and development of cells of the oligodendrocyte lineage, see References 245, 510, 577.

The SVZ is a germinal matrix of the forebrain that first appears during the later third of murine embryonic development (149). It enlarges during the peak of gliogenesis, between P5 and P20, and then shrinks but persists into adulthood. Lineage tracing studies of perinatal SVZ cells using stereotactically injected retrovirus support the view that the majority of progenitors within this germinal matrix are glial precursors that generate astrocytes on the one hand and oligodendrocytes on the other hand (352, 476). Although the majority of cells give rise to homogeneous progeny, some SVZ cells give rise to both oligodendrocytes and astrocytes, and a rare cell will develop into both neurons and glia (329). The assumption that each cluster is a clone, i.e., the complete progeny of a single precursor, was based on the thought that the dispersion of cells in structures like embryonic cortex was purely radial. However, other retroviral studies, together with other techniques, have shown that cells disperse considerably and also tangentially during postnatal corticogenesis and embryonic development (475). Thus two clones could occupy an overlapping space. Therefore, this is not yet a completely settled issue. Nevertheless, it does seem from tritiated thymidine, immunocytochemistry, and retroviral studies that the majority and possibly all oligodendrocytes originate from different cells in the SVZ than those that give rise to astrocytes. In the neonatal rat cerebrum, oligodendrocytes arise postnatally from the SVZ of the lateral ventricles (329, 695). Similarly, immunocytochemical studies have indicated that oligodendrocytes of the cerebellum arise postnatally from the SVZ of the fourth ventricle (505). Oligodendrocyte progenitors then migrate long distances away from these zones and populate the developing brain to form white matter throughout the brain, as shown by developmental (200, 329) and transplantation studies (177, 321). Because mature oligodendrocytes cannot migrate, preventing premature differentiation of progenitors is crucial for ensuring that they successfully make it to their final destination. Premature oligodendrocyte differentiation is effectively prevented by an inhibition mechanism recently shown to occur in gliogenesis, the Notch pathway (651) (see sect. IIID2).

Oligodendrocytes, first found in the optic nerve around birth, continue to increase in number for six postnatal weeks in rodents (31, 567). Indirect evidence suggests that optic nerve oligodendrocytes are derived from precursor cells that migrate into the nerve from the brain rather than from neuroepithelial cells of the original optic stalk. For example, during late embryonic development in the rat (at about E15), cultures from the chiasma but not from the retinal end of the nerve contain significant numbers of oligodendrocyte progenitor cells (568).

Oligodendrocyte progenitors migrate extensively throughout the CNS before their final differentiation into myelin-forming oligodendrocytes (568). Moreover, as in other developmental systems, oligodendrocytes have to extend processes in a similar fashion to the extension of neurites from neuronal cell bodies. As for neurons, a number of extracellular matrix (ECM) molecules play an instructive role in the control of migrating oligodendrocytes. For example, tenascin-C is expressed at high levels at the rat retina-optic nerve junction, an area through which oligodendrocytes do not migrate, so preventing access of myelin-forming cells to the retina (187). Therefore, it has been proposed that tenascin-C might provide a barrier to oligodendrocyte migration at the retinal end of the optic nerve (37). The inhibitory effect of tenascin-C is dependent on the substrate: tenascin-C inhibits migration in response to fibronectin but not in response to merosin (200). Moreover, to find their way and to regulate the extension of their processes along the astrocyte-produced ECM, oligodendrocytes use metalloproteinases (MMP). In vivo, elevation of MMP protein expression in tissues rich in oligodendrocytes and myelin coincides with the temporal increase in myelination that occurs postnatally. Process formation is retarded significantly in oligodendrocytes cultured from MMP-9 null mice, as compared with controls, providing genetic evidence that MMP-9 is necessary for process outgrowth (434). On the contrary, MMP inhibitors decrease process extension of oligodendrocytes, supporting the key role of MMPs (623).

With the use of explant cultures of newborn rat neurohypophysis, it was shown that migrating oligodendrocyte progenitors express a polysialylated (PSA) form of NCAM (649). Removal of PSA-NCAM from the surface of progenitors by an endoneuraminidase treatment results in a complete blockade of the dispersion of the oligodendrocyte progenitor population from the explant. As shown for neurons and astrocytes, the expression of PSA-NCAM does not itself provide a specific signal for migration, but its expression appears to open a permissive gate (528) that allows cells to respond to external cues at the appropriate time and space. In contrast, in the chick, the migration of oligodendrocyte precursors along the optic nerve axons appears unaffected by removal of PSA-NCAM (440). Thus migrating oligodendrocyte progenitors may use distinct and discrete mechanisms to navigate specific migrational pathways.

Oligodendrocyte progenitors express a distinctive array of integrin receptors (394) that may mediate specific interactions with ECM components, such as thrombospondin-1, which could also be involved in the regulation of migration (553).

As described in section II, A and B, oligodendrocyte progenitors have been characterized in rodent species by their bipolar morphology and by the presence of specific markers: 1) glycolipids:GD3 (248) recognized by the monoclonal antibodies (MAbs) R24 and LB1; other glycolipids, not as yet fully characterized (198) recognized by the MAb A2B5; and 2) a chondroitin sulfate proteoglycan called NG2 (427). In vitro studies (127, 455) as well as in vivo experiments through transplantation techniques (26, 177, 226, 321) show that these cells are actively proliferating and possess migratory properties. They proliferate in vitro, in response to growth factors such as fibroblast growth factor (FGF) and PDGF (211, 249, 393, 489, 509) (see sect. IIC8).

After their migration in the mammalian CNS, progenitors settle along fiber tracts of the future white matter and then transform into preoligodendrocytes (Fig. 4), multiprocessed cells which keep the property of cell division and acquire the marker O4 (571). At this stage, they are less motile (442), or even postmigratory (455), and lose their mitogenic response to PDGF (208, 250, 478).

View larger version (57K): [in this window] [in a new window]   Fig. 4. A: migration and differentiation of cells of astrocyte and oligodendrocyte lineages, and multifocal pattern of development of glial rows in the fimbria between embryonic day 15 (E15) and postnatal day 60 (P60). (Lower surface, ependyma; upper surface, pia.) There is a relatively small increase in the thickness of the fimbria. The multicellular ventricular layer (small open circles at lower surface at E15 and P0) becomes reduced to a unicellular adult ependyma (triangles on ependymal surface). Cells migrating into the body of the fimbria, and supplemented by division of precursors there, become arranged in progressively longer interfascicular glial rows. The astrocytes (large, pale circles) change from predominantly radial processes to predominantly longitudinal. Oligodendrocytes are represented by small, black, filled contiguous circles. Myelination (not represented) largely occurs between P10 and P60. Axons (not represented) are present from the earliest developmental stage. [From Suzuki and Raisman (596). Copyright 1992, reprinted by permission of Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc.] B: organization of the glial framework of central white matter tracts. Arrangement of radial and longitudinal processes of oligodendrocytes (Og) and astrocytes (As) forming a continuous meshwork of processes intermingled within the axonal tracts in the fimbria. Astrocytes are linked with each other by gap junctions, and also form gap junctions with oligodendrocytes, thus providing an indication for a functional as well as an anatomical functional syncytium. Myelination occurs on an asynchronous mode, with individual oligodendrocytes maturing independently within a cluster of adjacent oligodendrocytes, suggesting an interaction between axon maturation and oligodendrocyte differentiation. [From Raisman (492).]

The preoligodendrocyte becomes an immature oligodendrocyte, characterized in the rat by the appearance of the marker GalC, and the loss of expression of GD3 and A2B5 antigens on the cell surface. CNP is the earliest known myelin-specific protein to be synthesized by developing oligodendrocytes (505, 506, 579, 642). Other markers appear at this stage, such as RIP (199), CAII, and MBP (89) (see sect. IIB). In vivo there seems to be a GD3/GalC intermediate stage (127). In rat cerebellum, CNP is expressed at the same time as GalC (505) and MBP is expressed 2-3 days later along with PLP, immediately before myelin formation. The same sequence occurs also in vitro; CNP is expressed at the same time as GalC in cultured oligodendrocytes (455). MBP, MAG, and PLP appear sequentially both in vivo and in vitro (154, 249, 399, 455) and signify a mature oligodendrocyte.

In vitro analyses suggest that maturation of oligodendrocytes from the precursor stage to the mature cell is identical in culture, even without neurons, as in intact tissue. Thus the capacity of oligodendrocyte progenitors to differentiate into oligodendrocytes is intrinsic to the lineage (605). In the absence of neurons, oligodendrocytes can clearly make a myelin-like membrane (533); nevertheless, coculture with neurons increases myelin gene expression, such as PLP, MBP, and MAG (358, 366). The presence of MOG correlates with late stages of maturation of the oligodendrocyte (570) (see sect. IIIC2).

A similar myelin-gene induction by neuronal contact with oligodendrocytes is also observed in vivo (295); the switch to PLP isoform expression in DM20-expressing premyelinating oligodendrocytes indicates the beginning of the process of myelination in individual myelinating processes (620). This myelin-gene induction parallels morphological modifications, i.e., in vivo, when axonal contact occurs, there is a dramatic modification of oligodendrocyte morphology with loss of oligodendrocyte processes that have not contacted axons (246).

From the early 1980s glial research was centered around an oligodendrocyte-type 2 astrocyte progenitor (O-2A) first discovered in culture of the optic nerve (reviewed in Ref. 475). O-2A progenitors in culture generate oligodendrocytes constitutively but can give rise to process-bearing astrocytes (so-called type 2 astrocytes) when treated with 10% fetal calf serum. Attempts to find cells in vivo with the type 2 astrocyte phenotype during normal development have failed (248, 505), even after grafting O-2A cells into brain during development (177). Combination of tritiated thymidine and specific cell type immunocytochemistry indicate that most astrocytes are generated prenatally and during the first postnatal week, before oligodendrocyte formation (565), suggesting that there is not a second wave of type 2 astrocytes in vivo as suggested by the previous in vitro studies. "The apparent discrepancies between the data obtained in vitro and in vivo highlights an important aspect of the approach in vitro; when a progenitor cell is grown in tissue culture, the environments to which it can be exposed are restricted only by the imagination of the experimenter. By exposing the cell to a variety of signals, the full differentiation potentiality of the cell can be explored. In contrast, during development, a progenitor cell is exposed to a restricted sequence of signals that are spatially and temporally programmed" (196).

The final number of mature myelinating oligodendrocytes is determined by the proliferative rate of their progenitors and by the process of programmed cell death that occurs during development (reviewed in Ref. 29). As oligodendrocytes differentiate late in the CNS, their development may be influenced by signals derived from other neural cell types, such as astrocytes and neurons (249, 510). Axon-to-oligodendrocyte signaling results in the generation of the precise numbers necessary to myelinate entirely a given population of axons (reviewed in Ref. 32).

Confocal microscopy analysis of rat brain tissue has clearly demonstrated that premyelinating oligodendrocytes that express DM-20 have two fates, programmed cell death (PCD) or myelination. PCD occurs during a process of axon matching to eliminate surplus cells. The cells that make contact with axons survive and begin myelination (620), as hypothesized earlier (28). The appearance of MOG (see sect. IIIC2E) in oligodendrocytes (119, 368, 455, 552), as in the CG4 oligodendrocyte cell line (570), is a reliable marker for fully differentiated myelin-forming oligodendrocytes.

8.  Factors necessary for oligodendrocyte maturation and survival

A) GROWTH FACTORS. Many growth factors have been found to be involved in the proliferation, differentiation, and maturation of the oligodendrocyte lineage (29, 99, 100, 248, 249, 377, 378, 455, 489, 509, 684). Most of these studies have been performed in vitro. It is extremely difficult to extrapolate to in vivo conditions, as multiple factors may act in concert to achieve the exquisitely fine regulation of the complex process of oligodendrocyte development and myelination. Combinations of factors often produce effects that are significantly different from those seen with any one factor alone (379). Furthermore, these factors have multiple effects during development. In contrast to experiments on rodent cells, growth factors that act on human cells have not as yet been fully determined.

B) NEUROTRANSMITTERS AND OLIGODENDROCYTE DEVELOPMENT. Oligodendrocyte progenitors are equipped with a variety of ligand- and voltage-gated ionic channels that are expressed in vitro as in vivo (572, 586). They are not detailed here.

In a review of cell junctions among the supporting cells of the CNS, E. Mugnaini wrote in 1986 (413): "Cell junctions require careful analysis because they reflect not only the biology of individual cells, but also their sociology; that is, the cooperativity with other cells, and the relation to the environment." In situ, morphological studies have shown that astrocyte gap junctions are localized between cell bodies, between processes and cell bodies, and between astrocytic end-feet that surround brain blood vessels (679). In vitro, junctional coupling between astrocytes has also been observed (189, 635). Although less frequently observed than junctions between astrocytes, gap junctions also occur between oligodendrocytes, as observed in situ (90, 519, 574) and in vitro (634). Moreover, astrocyte-to-oligodendrocyte gap junctions have been identified between cell bodies, cell bodies and processes, and between astrocyte processes and the outer myelin sheath (654; reviewed in Ref. 218). Thus the astrocytic syncytium extends to oligodendrocytes, allowing glial cells to form a generalized glial syncytium (413), also called "panglial syncytium," a large glial network that extends radially from the spinal canal and brain ventricles, across gray and white matter regions, to the glia limitans and to the capillary epithelium (498) (Fig. 1).

Gap junctions are channels that link the cytoplasm of adjacent cells and permit the intercellular exchange of small molecules with a molecular mass <1-1.4 kDa, including ions, metabolites, and second messengers (reviewed in Refs. 81, 218). In addition to homologous coupling between cells of the same general class, heterologous coupling has been observed not only between astrocytes and oligodendrocytes, but also between ependymal cells and retinal Muller glia. Homologous coupling could serve to synchronize the activities of neighboring cells that serve the same functions. Such coupling would extend the size of a functional compartment from a single cell to a multicellular syncytium, acting as a functional network (reviewed in Ref. 218).

Gap junctions are now recognized as a diverse group of channels that vary in their permeability, voltage sensitivities, and potential for modulation by intracellular factors; thus heterotypic coupling may also serve to coordinate the activities of the coupled cells by providing a pathway for the selective exchange of molecules below a certain size. In addition, some gap junctions are chemically rectifying, favoring the transfer of certain molecules in one direction versus the opposite direction. The main gap junction protein of astrocytes is connexin (Cx) 43 (review in Ref. 218), whereas Cx32 is expressed in oligodendrocytes in the CNS (539) as well as another type of connexin, Cx45 (140, 141, 318, 332, 419). Heterologous astro-oligodendrocyte gap junctions may be composed of Cx43/Cx32, if these connexins form functional junctions. Heterocoupling between astrocytes and oligodendrocytes has been proposed to serve K⁺ buffering around myelinated axons (332; reviewed in Ref. 692).

	III. MYELIN

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The myelin sheath around most axons constitutes the most abundant membrane structure in the vertebrate nervous system. Its unique composition (richness in lipids and low water content allowing the electrical insulation of axons) and its unique segmental structure responsible for the saltatory conduction of nerve impulses allow the myelin sheath to support the fast nerve conduction in the thin fibers in the vertebrate system. High-speed conduction, fidelity of transfer signaling on long distances, and space economy are the three major advantages conferred to the vertebrate nervous system by the myelin sheath, in contrast to the invertebrate nervous system where rapid conduction is accompanied by increased axonal calibers.

The importance of myelin in human development is highlighted by its involvement in an array of different neurological diseases such as leukodystrophies and multiple sclerosis (MS) in the CNS and peripheral neuropathies in the PNS.

In terms of evolution of the nervous system, the myelin sheath is the most recent of nature's structural inventions. Myelin is found in all vertebrate classes except the evolutionarily oldest agnathan cyclostomata, i.e., the jawless fish (hagfish and lampreys) in which axons, however, are surrounded by glial cells. All other vertebrates, including cartilaginous fish, bony fish, and tetrapods as well as lungfish and coelacanth manifest extensive myelination in both the CNS and PNS. So, the first myelin-like ensheathed axons may have appeared about 400 million years ago. Although compact myelin is a uniquely vertebrate feature, a form of glial ensheathment of axons exists in invertebrates (such as annelids, crustaceans, mollusks, and arthropods), forming a superficially resembling vertebrate myelin sheath (reviewed in Refs. 122, 645). Ontogenetically, PNS myelin appears before CNS myelin; however, it has not been demonstrated to occur phylogenetically.

B.  Morphological Structure of the Myelin Sheath and of the Node of Ranvier

1.  Myelin

Myelin, named so by Virchow (640), is a spiral structure constituted of extensions of the plasma membrane of the myelinating glial cells, the oligodendrocytes in the CNS (82, 452). These cells send out sail-like extensions of their cytoplasmic membrane (Figs. 2 and 3), each of which forms a segment of sheathing around an axon, the myelin sheath (reviewed in Refs. 41, 404, 453, 457). Several structural features characterize myelin. Its periodic structure, with alternating concentric electron-dense and light layers, was already shown in 1949, in the optic nerve myelin of guinea pig (563). The major dense line (dark layer) forms as the cytoplasmic surfaces of the expanding myelinating processes of the oligodendrocyte are brought into close apposition. The fused two outer leaflets (extracellular apposition) form the intraperiodic lines (or minor dense lines) (Figs. 2 and 5). The periodicity of the lamellae is 12 nm. Each myelin sheath segment or internode appears to be 150-200 µm in length (90); internodes are separated by spaces where myelin is lacking, the nodes of Ranvier (84). It does seem that oligodendrocytes form thicker myelin around larger axons (657).

View larger version (43K): [in this window] [in a new window]   Fig. 5. Myelinating glial cells, myelin structure, and composition in the peripheral nervous system (PNS) and in the CNS. In the PNS, the myelinating Schwann cell myelinates only one segment of axon (top left corner), whereas in the CNS (top right corner), the oligodendrocyte is able to myelinate several axons. The compact myelin is formed by the apposition of the external faces of the membrane of the myelinating cell, forming the "double intraperiodic line"; the apposition of the internal faces followed by the extrusion of the cytoplasm, form the "major dense line." The myelin proteins are schematically described; they differ between PNS and CNS. [Adapted from Pham-Dinh (457).]

A) THE SCHMIDT-LANTERMAN CLEFTS. The Schmidt-Lanterman clefts (also called Schmidt-Lanterman incisures) correspond to cytoplasmic faces of the myelin sheath that have not compacted to form the major dense line; thus they constitute pockets of uncompacted glial cell cytoplasm within the compact internode myelin. They are oblique, funnel-like clefts that extend across the entire thickness of the sheath, providing a pathway through which cytoplasm on the outside of the sheath is confluent with that of the inside part. Schmidt-Lanterman clefts are common in the PNS, but rare in the CNS (Fig. 5). The myelin sheath is separated from the axonal membrane by a narrow extracellular cleft, the periaxonal space.

B) THE PARANODAL LOOPS. Myelin is less compacted at the inner and outer end of the spiral, forming inner and outer loops that retain small amounts of oligodendrocyte cytoplasm. The myelin lamellae end near the node of Ranvier in little expanded loops containing cytoplasm. The loops are arranged on a roughly regular and symmetric pattern on each side of the node. This sequence of loops is named the paranodal region or paranode. Each paranodal loop makes contact with the axon. Freeze-fracture studies have shown distinct membrane morphologies in the axolemma (reviewed in Ref. 655), corresponding to the different domains of the myelinated fibers: the internode, the paranodal region, and the node of Ranvier. These anatomically different regions of the axolemma are formed by specific interactions between the axon and the myelinating glial cell. The interface between the lateral loops and the axon membrane exhibits a row of 15 nm regularly spaced densities, the transverse band (256). The transverse bands, which comprise rows of regularly spaced particles in glial and axonal membranes, are associated with cytoskeletal filaments and appear to tighten the axonal-paranodal apposition (270). Molecular structure and organization of the axolemma at the paranode and node of Ranvier have recently come to light (135, 171, 293, 382; reviewed in Refs. 530, 653) (see below and sect. IIIE).

Along myelinated fibers, adjacent internodes are separated by the nodes of Ranvier where the axolemma is exposed to the extracellular milieu (84) (Fig. 2). The nodes of Ranvier play a major role in nerve impulse conduction. They allow the fast saltatory conduction, the impulse jumping from node to node, rather than progressing slowly along the axon as in unmyelinated or demyelinated fibers. Thin axons appear to possess tiny node gaps, and thick axons exhibit spacious node gaps, as observed at nodes of Ranvier in rat white matter (56). Astrocytic processes may be seen in close proximity to the axon membrane at the node of Ranvier (58) (see sect. IIIE for the molecular organization of the node). Another cell type characterized by the NG2 antigen has recently been shown to contact the node of Ranvier in adult CNS white matter (88), and this cell is thought to be a glial precursor.

Myelin is the essential constituent of white matter in the CNS which contains ~40-50% myelin on a dry weight basis. The composition of myelin was widely studied more than 20 years ago, when methods of myelin isolation became available (431, 432); its low density allows its easy preparation as a fraction after sucrose gradient centrifugation. Although developed for fresh rat brain, it has been widely used for human specimens and gives a well-defined fraction, even from frozen myelin (39). Contaminants may increase when myelin is in low quantities, such as during development, and in pathological conditions such as hypomyelination and demyelination (689). Most biochemical analyses refer to the work of the group of Norton (reviewed in Ref. 404). Myelin is a poorly hydrated structure containing 40% water in contrast to gray matter (80%). Myelin dry weight consists of 70% lipids and 30% proteins. This lipid-to-protein ratio is very peculiar to the myelin membrane. It is generally the reverse in other cellular membranes. The insulating properties of the myelin sheath, which favor rapid nerve conduction velocity, are largely due to its structure, its thickness, its low water content, and its richness in lipids.

The specific constituents of myelin, glycolipids, and proteins are formed in the oligodendrocyte.

Lipids found in oligodendrocytes and myelin are also present in other cellular membranes, but in a different proportion. In every mammalian species, myelin contains cholesterol, phospholipids, and glycolipids in molar ratios ranging from 4:3:2 to 4:4:2 (reviewed in Ref. 404). Therefore, the molar ratio of cholesterol is greater than that of any other lipid. Cholesterol esters are not present in normal myelin. There are no major peculiarities in myelin phospholipids that represent 40% of total lipids, except for the high proportion of ethanolamine phosphoglycerides in the plasmalogen form which account for about one-third of the phospholipids. Diphosphoinositides and triphosphoinositides are nonnegligible components, respectively, 1-1.5 and 4-6% of myelin phosphorus.

One of the major characteristics of oligodendrocyte and myelin lipids is their richness in glycosphingolipids, in particular galactocerebrosides, i.e., galactosylceramides (GalC) and their sulfated derivatives, sulfatides, i.e., sulfogalactosylceramides. These lipids have been immunolocalized in oligodendrocytes and tissue sections of myelin (490, 693). Even though there are no "myelin lipids," GalC are the most typical lipids of myelin in which they are specifically enriched (reviewed in Ref. 404) and represent 20% lipid dry weight in mature myelin. These glycosphingolipids represent a family of components, as there is a great variability in the ceramide moiety, both in its sphingosine content (18-20 carbon atoms) and in its long-chain fatty acids content. The ceramide core of these glycosphingolipids is particularly rich in very-long-chain fatty acids, 22-26 carbon atoms, saturated, mono-unsaturated, and -hydroxylated. During development, the concentration of galactocerebrosides in brain is proportional to the amount of myelin present (432). This is also the case for the very-long-chain fatty acids (276). The biosynthesis for myelin very-long-chain fatty acids occurs in the microsomal compartment and not in mitochondria (72). Abnormalities of lysosomal enzymes related to degradation of sulfatides or galactocerebrosides, or proteins involved in the transport of these fatty acids to the peroxisomes, give rise to leukodystrophies (see sect. IVA2).

There are also several minor galactolipids (3% of total galactose) in myelin such as fatty esters of cerebroside, i.e., acylgalactosylceramides (608) and galactosyldiglycerides (463). Monogalactosyl diglyceride appears also to be a marker for myelination (541). This may be also the case for acylgalactosylceramides (608).

A sialylated derivative of galactocerebroside (sialosylgalactosylceramide), the ganglioside GM4, is present mainly in murine and primate myelin, including human, but not, for instance in bovine brain (117). Ganglioside GM1, although a minor component of myelin, is greatly enriched in myelin, compared with polysialogangliosides. It has been localized in myelin and some glial cells using specific monoclonal antibodies (313).

Furthermore, a number of major CNS myelin proteins, such as PLP, MAG, CNP (see below) are also covalently acylated (3, 448) (see sect. IIIC2), which gives them hydrophobic properties.

Myelin proteins, which comprise 30% dry weight of myelin, are for most of the known ones, specific components of myelin and oligodendrocytes (97). The major CNS myelin proteins MBP and PLP (and isoform DM-20) are low-molecular-weight proteins and constitute 80% of the total proteins. Another group of myelin proteins, insoluble after solubilization of purified myelin in chloroform-methanol 2:1, have been designated as the Wolfgram proteins, since their existence was suspected already in 1966 by Wolfgram (670). These proteins comprise the CNP and other proteins.

Several glycoproteins are present in myelin (reviewed in Ref. 486), among which are MAG and MOG. Other proteins have also been identified, some of which have enzyme activities.

A) MBP. MBP (296) is one of the major proteins of CNS myelin and constitutes as much as 30% of protein. In fact, it is a family of proteins, as there are many isoforms of different molecular masses. The amino acid composition of the major MBPs was determined by Eylar et al. (178) from bovine brain, and by Carnegie (104) in humans. The major protein isoforms can be separated by SDS-PAGE. The molecular masses of the major forms are 21.5, 18.5, 17, and 14 kDa in the mouse and 21.5, 20.2, 18.5, and 17.2 kDa in humans (for review, see Ref. 97). In the adult, the major isoforms are 18.5- and 17.2-kDa isoforms in humans and 18.5 and 14 kDa in the mouse, constituting ~95% of the MBPs (582). The MBP isoforms are coded by alternative transcripts from the MBP gene which consists of seven exons (516). The MBP gene is distributed over a 32-kb stretch in the mouse on chromosome 18. In the human, the MBP gene has been assigned to 18q22-qter (535, 575) and is distributed over a length of 45 kb (291). In the mouse, at least seven transcripts are expressed from this gene through alternative splicing of exons 2, 5A, 5B, and 6 (15, 137, 424, 600). A number of other mRNA splicing variants of the mouse MBP gene predominantly expressed in embryonic stage have also been characterized (365, 421). One of these variants is expressed at the protein level in embryonic nervous system at a time when other MBP isoforms are not detected. By in situ hybridization, the expression of the MBP gene has been observed as early as E14.5 in the spinal cord in the mouse (454).