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).

B) PROTEOLIPID PROTEINS (PLP AND DM-20). In 1951, Folch and Lees (190) discovered that a substantial amount of protein from brain white matter could be extracted by organic solvent mixtures. Because these proteins were apparently lipid-protein complexes, they were given the generic name of proteolipids to distinguish them from water-soluble lipoproteins. They are mainly myelin constituents. Although other myelin proteins can be solubilized this way, the name has been given to a major component of myelin, named PLP. This protein corresponds to 50% of myelin proteins. The predominant isoform commonly named PLP has an apparent molecular mass on SDS-PAGE of 25 kDa; a second isoform DM-20 (10-20% PLP in the adult) migrates as a 20-kDa band on electrophoresis. The authentic molecular masses appear to be slightly higher as PLP and DM-20 are both acylated by covalent linkage of mainly palmitic, oleic, and stearic acids (3, 606) on numerous cysteine residues localized on the cytoplasmic side of the myelin membrane (659). There is also a nonenzymatic glycosylation of extracytoplasmic domains (660). The complete amino acid sequence of bovine PLP has been elucidated by Stoffel et al. (592) and Lees et al. (326).

C) CNP. CNP represents 4% of total myelin proteins. This protein hydrolyzes artificial substrates, 2',3'-cyclic nucleotides into their 2'-derivatives. However, the biological role of this enzyme activity is obscure since 2',3'-nucleotides have not been detected in the brain (642). The association of CNP with one of the major Wolfgram proteins has been known for a long time (580). CNP appears on SDS-PAGE as a doublet of two peptides of molecular masses varying from 48 to 55 kDa, according to species. In general, there are two closely spaced protein bands at 48 and 46 kDa referred to CNP2 and CNP1, respectively (579).

D) MAG. MAG (487) is quantitatively a minor constituent, representing 1% of the total protein found in myelin isolated from the CNS and 0.1% in the PNS. The MAG gene spans 16 kb in length and includes 13 exons; it is located on chromosome 7 in mouse and 19 in human (35, 142). MAG has an apparent molecular mass of 100 kDa on SDS-PAGE, of which 30% is carbohydrate. Two MAG proteins have been identified (194, 531), large MAG (L-MAG) and small MAG (S-MAG). These proteins correspond to polypeptides of 72 and 67 kDa in the absence of glycosylation. MAG proteins have both a membrane-spanning domain and an extracellular region that contains five segments of internal homology that resemble immunoglobulin domains (531) and are strikingly homologous to similar domains of the NCAM and other members of the immunoglobulin gene superfamily. Both share identical extracellular and transmembrane domains but differ in their cytoplasmic domains, corresponding to the alternative splicing of exon 12 from a single RNA transcript. The deduced amino acid sequence of the human S-MAG COOH terminus shows conservative substitutions of 4 of the 10 residues compared with the rodent peptide (387).

E) MOG. MOG was first identified by a polyclonal antibody directed against an antigen called M2 that induces autoimmune encephalomyelitis in the guinea pig. MOG was first identified as the antigen responsible for the demyelination observed in animals injected with whole CNS homogenate; it was later identified as a minor glycoprotein specific for CNS myelin (324, 325). MOG was further characterized by immunological methods, immunohistochemistry, and Western blot, using a mouse monoclonal antibody against glycoproteins of rat cerebellum (337). Both M2 and MOG migrate at the 52- to 54-kDa and 26- to 28-kDa levels on SDS gels. The purified protein is a minor protein of myelin of 25 kDa with some glycosylation resulting in molecular mass 26-28 kDa that can form dimers of 52-54 kDa (9, 54). MOG is only present in mammalian species (54) and is highly conserved between species. In humans, MOG expresses the L2/HNK1 epitope (86). The NH₂-terminal, extracellular domain of MOG has characteristics of an immunoglobulin variable domain and is 46% identical to the NH₂ terminus of bovine butyrophilin (BT) expressed in the mammary gland, and chick histocompatibility BG antigens (212, 461). These proteins form a subset of the immunoglobulin superfamily. They colocalize to the human major histocompatibility complex (MHC) locus, on chromosome 6 (6p21.3-p22) in the human, and on chromosome 17 in the mouse (461). In both species, the MOG gene is localized at the distal end of the MHC class Ib region (460). The mouse and human MOG genes have been cloned and sequenced (130, 255, 459, 525). The mouse and the human MOG gene are 12.5 and 19 kb long, respectively. There are eight exons and at least six transcripts generated by alternative splicing in the human (459). MOG protein is mostly located on the outermost lamellae of compact myelin sheaths in the CNS (79). The topology of MOG indicates that there may be only one transmembrane domain (138, 316) as other members of the immunoglobulin superfamily. MOG is located on the plasma membrane of the oligodendrocyte, especially on oligodendrocyte processes, and on the outermost lamellae of myelin sheaths (79). It is a surface marker of oligodendrocyte maturation (54, 119, 461, 552). Its presence correlates with late stages of maturation, possibly restricted to the myelinating oligodendrocytes, as shown using the CG4 cell line (570). At present, MOG is the only CNS protein that can induce both a T cell-mediated inflammatory immune reaction and a demyelinating antibody-mediated response in an animal model of demyelinating diseases, the experimental autoimmune encephalomyelitis (EAE) (see sect. IVB2).

F) OTHER PROTEINS OF MYELIN. I) Small basic proteins. Myelin-associated oligodendrocyte basic protein. Myelin-associated/oligodendrocyte basic protein (MOBP) is a small highly basic protein that has been recently isolated (678). Three isoforms are generated by alternative splicing. The three corresponding polypeptides are of 8.2, 9.7, and 11.7 kDa. MOBPs are located in the major dense line of myelin (259, 678) where they could play a similar role to MBP in myelin compaction. MOBP has also been described in the mouse, where the abundance of MOBP transcripts is less than PLP but greater than that of the CNP gene (401). The MOBP mRNA is located initially in the cell bodies of the oligodendrocytes and moves distally into the processes when myelination proceeds, as does the MBP mRNA (401). The gene has been mapped to chromosome 9 in the mouse (372), at a region syntenic with the human chromosome 3 (3p22) (258).

G) OLIGODENDROCYTE/MYELIN ENZYMES. UDP-galactose:ceramide galactosyltransferase (CGT), which catalyzes the transfer of galactose to ceramide to form galactosylceramide, has been found in the myelin fraction. The human gene encoding CGT has five exons distributed over >40 kb; it has been cloned and assigned to human chromosome 4, band q26 (69), and is highly conserved during evolution (70). Expression of CGT transcripts in brain is time regulated and parallels that of MBP (544). In situ hybridization has shown that in cerebrum and cerebellum, CGT is restricted to the oligodendrocyte-containing cell layers that also express MBP (544). However, CGT is also weakly expressed in some subtypes of neurons in the adult CNS (537).

Except for ganglioside GM4, the specific galactolipids of CNS myelin are present in peripheral nerve. Thus leukodystrophies involving the degradation of these sphingolipids such as Krabbe disease, adrenoleukodystrophy, and metachromatic leukodystrophy affect also the peripheral nerve. On the other hand, some glycolipids such as sulfated glucuronyl paragloboside and its derivatives are specific of peripheral nerve myelin (116).

Several major CNS myelin proteins are also present in the peripheral nerve (Fig. 5), generally in smaller quantities, although their function may not be identical.

MBP is present in peripheral nerve myelin, but probably does not play a major role in myelin compaction, as the major dense line of PNS myelin is not affected in the shiverer mutant mouse which is devoid of MBP (302). Moreover, P0 and MBP can play interchangeable roles during the process of myelin formation as shown from the work on P0 knock-out and P0/MBP double knock-outs (363).

The PLP mRNA and protein have been identified in Schwann cells (482). In the sciatic nerve, DM-20 (messengers as well as protein) is present at a much higher level than PLP (458), and both are present in the myelin membrane (2). In the same context, it has recently been shown in humans that PLP/DM-20 protein is necessary in peripheral as well as central myelin, since patients with Pelizaeus-Merzbacher disease caused by absence of PLP gene expression (due to a premature stop codon) also present an associated peripheral neuropathy (209). In contrast, PLP null mice do not present any peripheral defect (235).

MAG is a very minor component of PNS myelin, in which it represents 0.1% total proteins. In rodents, L-MAG is the major isoform during development and declines in the adult CNS myelin, whereas S-MAG is the predominant isoform during development as in the adult PNS myelin (448). In contrast to rodents, the L-MAG splice variant predominates in adult human brain while, like in rodents, S-MAG transcripts are most abundant in peripheral nerve. In contrast to the CNS where MAG is confined to the periaxonal region of the myelin sheath (622), in the adult rat PNS, MAG present a large distribution in myelin internodes and is enriched in paranodal loops, Schmidt-Lanterman incisures, and inner as outer mesaxon membranes (622; see for review Ref. 384).

PMP 22, a peripheral nerve myelin protein, plays an important role in Schwann cell metabolism and PNS myelin compaction (reviewed in Ref. 418); its mRNA are also found in some categories of motoneurons in the CNS (446), even at embryonic stages (445).

Cx32 is not a typical PNS myelin component since it is widely expressed in the organism. Nevertheless, it seems to have a special role in relation to myelin formation and/or maintenance in PNS, as recently shown by the deleterious effect of mutations of Cx32 leading to Charcot-Marie-Tooth neuropathies (46). Recently, Cx32 has also been found in oligodendrocytes of rat brain together with Cx45 (318). Expression in oligodendrocytes of another type of connexin may prevent dysmyelinating effects of Cx32 mutations in the CNS of Charcot-Marie-Tooth X type patients.

P2 protein is a myelin-specific protein of peripheral nerve, although it is found in some areas of the CNS (see sect. IIIC2). It has a molecular mass of 13.5 kDa and contains a high percentage of basic amino acids. P2 is a true fatty acid-binding protein isoform (543).

In humans, Northern blot analysis reveal that Golli-MBP transcripts are also expressed in fetal thymus, spleen, and human B-cell and macrophage cell lines, clearly linking the expressions of genes coding for an encephalitogenic protein to cells and tissues of the immune system (98, 237, 694). Some Golli-MBP proteins have been found expressed in the embryo (365, 421) and have also been found expressed in the thymus (473), in the thymic macrophages. Recently, Kalwy et al. (290) have found that a 25-kDa band, composed at least of four proteins, is present in brain, thymus, and spleen and not in the tissues that do not express the novel MBP transcripts.

PLP transcripts are also expressed in peripheral blood lymphocytes (102), probably by a process of illegitimate transcription, very useful for genetic diagnostic purposes. On the other hand, PLP/DM-20 proteins have also been found in macrophages of the human thymus (473). Thus PLP/DM-20 proteins are expressed in antigen-presenting cells in the human immune system and are not shielded from the immune system behind the blood-brain barrier. These observations have to be interpreted in the context of acquisition of tolerance against encephalitogenic antigens such as PLP and MBP, which may not simply be related to their sequestration from a "naive" immune system (473).

In the rat, an mRNA for CNP has been demonstrated in thymus and in lymphocytes (48). CNP has recently been implicated in the humoral response in MS; indeed, anti-CNP antibodies have been found in large amount in serum and CSF from MS patients, the antibody response being exclusively restricted to CNP1. Both CNP1 and CNP2 isoforms bind the C3 complement fraction, providing a plausible mechanism for opsonization of myelin membrane in the MS brain (647).

There is an amino acid sequence homology between MAG and immunoglobulin-like molecules, especially NCAM (531). The immunoglobulin gene family is a family of proteins that shares a common extracellular subunit termed the immunoglobulin homology unit. Among the members of this superfamily expressed in the nervous system are L1, NCAM, P0, MOG, and CD22. MAG most closely resembles the leukocyte adhesion molecule CD22, which is a sialic acid binding protein (557).

The antibody HNK1 has been used to identify a subpopulation of human lymphocytes with natural killer function. Thirty percent of all MAG molecules isolated from mouse brain carry the L2/HNK1 epitope, which is also expressed in a group of NCAM and L1 molecules (466), on glycolipids (paraglobosides) with sulfated glucuronic acid (116), and on other myelin proteins from CNS, such as MOG, OMgp (see sect. IIIC2), or PNS, such as P0 (643) and PMP-22 (569). Thus several myelin proteins share an epitope with cells of the immune system.

The recently shown association of myelin proteins, MOG and CNP, with different elements of the complement cascade (282, 647), may be relevant to connections between the immune system and the nervous system and may have some physiopathological implications in demyelinating disease (587). Also the MOG gene is located in the MHC locus (460, 461) (see sect. IIIC2E).

In the first step, the preoligodendroglial multiprocessed cells settle along the fiber tracts of the future white matter, maintaining the ability to divide. Indeed, mitoses are present in the interfascicular longitudinal glial rows (507). Second, these preoligodendrocytes become immature oligodendrocytes, characterized by the acquisition of specific markers (see sect. IIIC) and ready for myelination.

Myelination occurs caudorostrally in the brain and rostrocaudally in the spinal cord. The sequence of myelination is a strictly reproducible process for a given species. In the mouse, it starts at birth in the spinal cord. In brain, myelination is achieved in almost all regions around 45-60 days postnatally. In humans, the peak of myelin formation occurs during the first year postnatally, although it starts during the second half of fetal life in the spinal cord. It can continue until 20 years of age in some cortical fibers, especially associative areas (677) (Fig. 6). More precise data on the topographical sequence of myelination in the human have been recently obtained using magnetic resonance imaging (MRI) (630).

View larger version (29K): [in this window] [in a new window]   Fig. 6. Cycles of myelination in the CNS during development. The width and the length of graphs indicate progression in the intensity of staining corresponding to the density of myelinated fibers; the vertical stripes at the end of the graphs indicate approximate age range of termination of myelination estimated from comparison of the fetal and postnatal tissue with tissue from adults in the third and later decades of life. [Adapted from Yakovlev and Lecours (677).]

Myelination requires an extraordinary capacity for oligodendrocytes to synthesize membranes at a given time, specific for a species and a region of the CNS, and concerns only certain nerve fibers and tracts (547). The timing of CNS myelination pathways is so precise and characteristic that the age of human fetuses can be determined accurately simply by assessing which pathways have myelinated. These data suggest that a highly localized signaling mechanism regulates the timing of oligodendrocyte differentiation and myelination.

In vivo, Barres and Raff (30) have shown that proliferation of oligodendrocyte precursor cells depends on electrical activity in ganglion retinal cells. Oligodendrocyte number is also dependent on the number of axons, as has been shown in transgenic mice expressing Bcl2 under the control of a neuron-specific enolase promoter (87). In these mutant mice, the oligodendrocyte cell population is increased due both to the decrease of oligodendrocyte apoptosis and to the increase in the number of axons. Interestingly, it was shown in an early work that sectioning the optic nerve postnatally, a few days after the appearance of mature oligodendrocytes, induces apoptosis in these cells, although astrocytes remain intact (205).

It is well known in vivo that oligodendrocytes myelinate solely axons, although oligodendrocytes are able to form uncompacted myelin or myelin-like figures in the absence of neurons (533, 674). Lubetzki et al. (343) have established long-term dissociated cultures of neurons and oligodendrocytes from mouse embryos that myelinate in culture solely axons after 13-14 days, as shown by the use of specific monoclonal antibodies recognizing differently the somatodendritic domain and axonal neurofilament epitopes.

The signal indicating which axon should be myelinated remains unclear. Studies in the CNS support the idea of a critical diameter for myelination, but individual axons are not myelinated along their entire length simultaneously, indicating that other factors are involved (597) (Fig. 4B). On the other hand, using intracellular dye injections, Butt and Ransom (90) showed that, during the markedly asynchronous development of the whole population, individual oligodendrocytes matured all their processes simultaneously to an equal length. Using MBP immunolabeling, Suzuki and Raisman (596, 597) studied the rat fimbria and found, first, patches of cells of the oligodendrocyte lineage along the axonal tract that finally form unicellular rows. Differentiation of immature oligodendrocytes into myelinating cells occurs in a multifocal mode. Within each cell cluster, a single oligodendrocyte gives rise simultaneously to a complement of varicose myelinating processes. This quantal mode of differentiation of the oligodendrocyte population suggests that over adjacent areas of the fimbria, all the clusters are responding simultaneously to the same overall stimulus but that there exists an internal regulation within each cluster so that only one cell is triggered. Such a highly localized "mosaic-like" pattern of differentiation, and the subsequent asynchronous myelination, could be generated by an interaction between axon maturation and oligodendrocyte differentiation (596, 597) (Fig. 4B). Recently, indirect evidence suggests that, in the rat optic nerve, the timing of oligodendrocytes differentiation and myelination is controlled by the Notch pathway. Whereas the Notch pathway was thought to specifically regulate neuronal development (14), it was also shown that Notch receptors are present on oligodendrocytes and their precursors. Retinal ganglion cells express Jagged 1, a ligand of Notch1 receptor, along their axons (651). Jagged1 is developmentally downregulated in axons of retinal ganglion cells with a time course that parallels myelination, suggesting that Jagged1 signals to Notch1 on surfaces of oligodendrocyte precursors to inhibit their differentiation into oligodendrocytes. The downregulation of Jagged1 expression by retinal ganglion cells correlates well with the onset of myelination in the optic nerve, as well as with the maturation of immature astrocytes. Myelination of axons in a given pathway generally occurs a few days after they reach their target (547), raising the question of whether downregulation of Jagged1 in axons occurs in response to target innervation, subsequently triggering the signal to begin myelination.

The proliferation of oligodendrocyte precursor cells depends in vivo on electrical activity, as shown in ganglion retinal cells by Barres and Raff (30). The role of electrical activity in the myelination process has been suggested by the fact that animals maintained without light since birth have a retardation in myelination (242); conversely, premature opening of the eyelids accelerates myelination in optic nerve (603). Demerens et al. (139) have shown in an in vitro myelination system using dissociated cultures from embryonic brain, that tetrodotoxin (TTX) blocks the initiation of myelination. -Scorpion toxin, which induces repetitive electrical activity by slowing Na⁺ channel inactivation, has an opposite effect. This group has shown also in vivo in the developing optic nerve of mice that the intraocular injection of TTX inhibits myelination. Different results, possibly under slightly different experimental conditions, have been observed by Colello et al. (120) and by Shrager and Novakovic (561).

One of the important steps for triggering myelination is the oligodendrocyte process extension that favors axonal contacts. Recently, it has been shown that astrocytes induce oligodendrocyte process to align with and adhere to axons, providing a novel role for astrocytes in controlling the onset of myelination (385).

Interestingly, cells of the oligodendrocyte lineage exclusively express a novel form of microtubule-associated proteins (MAP) called MAP 2c localized in the cell body of preoligodendrocyte that may assist in the initiation of process extension (644). There is a developmental pattern of several other MAPs suggesting that during myelination, there is a reorganization of the cytoskeleton of the oligodendrocyte that may interact with the growing myelin membrane. An isoform of neurofascin, comprising the third fibronectin type 3 (FNIII) repeat but lacking the mucinlike domain, is specifically expressed by oligodendrocytes at the onset of myelinogenesis and decreases rapidly once oligodendrocytes have engaged their targeted axons (121). Given its unique transient pattern of expression in oligodendrocytes at the early stages of myelination, this cell adhesion molecule protein may have a role in mediating axon recognition but also signals axonal contact through its links with the actin cytoskeleton. It is of note that later on a different neurofascin isoform (lacking FNIII but comprising the mucinlike domain) is specifically expressed in the axolemma at the node of Ranvier (135). On the other hand, in MAG-null mutant transgenic mice (400), axons may be myelinated several times, indicating that MAG may be involved in the recognition of myelinated axons from the nonmyelinated ones by the oligodendrocyte processes (330).

The axon probably participates in the regulation of myelin thickness as single oligodendrocytes can myelinate several axons with different diameters; they form thicker myelin along the larger axons (657). This suggests that the axon specifies, in a localized way, the number of myelin lamellae formed by a single oligodendrocyte process. Studies of the female carrier of the jimpy mutation (see sect. IVA1A) exemplify well the plasticity of neuroglial cells and the mechanisms of quantitative production of oligodendrocytes and myelin. Oligodendrocytes have considerable flexibility in the amount of myelin they can make, and a reduction in the number of oligodendrocytes does not necessarily involve a reduction in the amount of myelin (566).

Outside of providing trophic factors to oligodendrocytes, astrocytes may be important in relation to myelination. Suzuki and Raisman (596, 597) have studied the rat fimbria and found, first, patches of cells of the oligodendrocyte lineage along the axonal tract which finally form unicellular rows in which astrocytes are interspersed singly between stretches of 5-10 continuous oligodendrocytes. Mitosis is present in the interfascicular longitudinal glial rows (507). Radial and longitudinal processes of the oligodendrocytes and the astrocytes are interwoven to form a rectilinear meshwork along which the tract axons run (Fig. 4B). The presence of junctional coupling (see sect. IID) between the glial cells suggest that this glial array constitutes a functional as well as an anatomical unit (597).

An increase in glial fibrillary acidic protein (GFAP) level has been found in the mouse CNS during the early stages of myelinogenesis (277); whether it is functionally related to myelinogenesis could not be determined. Recently, mice carrying a null mutation for GFAP were generated (334). These mice display abnormal myelination; nonmyelinated axons are numerous in the optic nerve and spinal cord anterior column; the altered white matter vascularization, possibly linked to abnormal astrocytic processes, may be related to the dysmyelination observed. It is of note that the vimentin-GFAP transition in the rat neuroglia cytoskeleton occurs at the time of myelination (128). Moreover, transgenic mice carrying several copies of the human GFAP gene die by the second postnatal day by a fatal encephalopathy with astrocyte inclusion bodies similar to the Rosenthal fibers found in Alexander's disease (75a). Astrocytes in these mice are hypertrophic and upregulate small heat shock proteins (383). These studies suggest major links between astrocyte function, myelination, and its maintenance. Interestingly, these hypotheses are supported by data obtained in several human demyelinating conditions; e.g., in Alexander's leukodystrophy, the primary target cell is the astrocyte whose dysfunctions are characterized by high levels of expression of the heat shock protein, -B-crystallin (251, 275). In adrenoleukodystrophy (ALD), the mutated protein, adrenoleukodystrophy protein (ALDP), is expressed in white matter oligodendrocytes, but also in astrocytes and microglial cells everywhere in the brain, suggesting that dysfunction of these cells may indirectly affect oligodendrocytes early in the disease (16).

Oligodendrocytes embrace an array of axons while Schwann cells myelinate single axons. In contrast to the CNS node, where there is contact of the axon with the extracellular space (84), the myelinating Schwann cell processes interdigitate over the node. Moreover, the Schwann cell soma is circumferentially covered by a basement membrane. This basal lamina is continuous with that of the adjacent internode; thus the Schwann cell processes and basal lamina cover the PNS node. In fact, whether the Schwann cells myelinate or not, they share a common basal lamina that forms a glial channel along the nerve fiber. PNS myelinated fibers are separated from each other by an extracellular compartment, the endoneurium, whereas the CNS myelinated fibers are in close contact. Interestingly, electron microscopy and immunohistochemistry have shown that rat CNS fibers do not exhibit a strict relation between nodal complexity and fiber size, as in the PNS (56). Furthermore, some of the components of PNS myelin, lipids, and proteins are different (see sect. IIIC3) (Fig. 5).

The internodes are separated along myelinated axons by intervals where the axolemmal membrane is exposed, the node of Ranvier (Figs. 2, 3, 5, and 8). Nodes of Ranvier comprise a membrane specialization (see sect. IIIE) of the axon (Fig. 6) (reviewed in Refs. 253, 655). Premyelinated axons exhibit a low density of sodium channels that appear to be distributed uniformly along the length of the fiber, whereas during maturation of myelinated tracts, sodium channels become segregated into regularly spaced gaps in myelin, i.e., at the nodes of Ranvier, thereby allowing saltatory conduction in myelinated fibers. Moreover, potassium channels are enriched in the paranodal regions where they may contribute to the repolarization and ionic homeostasis. This molecular specialization enables the regeneration of the action potential at the node of Ranvier and is responsible for its unique function. The clustering of sodium channels was first ascribed to the presence of astrocytic processes at the node of Ranvier, since previous studies have demonstrated a close spatial and temporal correlation between astrocyte-axon contact and the appearance of a high density of intramembrane particles (reviewed in Ref. 655). However, it was recently demonstrated, first in the PNS, that the contact with Schwann cells induces clustering of sodium channels along axons of peripheral nerves, in vivo as in vitro (155). In a same way, the remarkable enrichment, i.e., clustering, of voltage-gated sodium channels in the nodal axolemma of myelinated CNS tracts has been shown to be due to signals from the oligodendrocyte, as shown in the optic nerve (293). Ultrastructural studies indicated that the axon begins to differentiate and displays foci of membranes with nodal properties before the onset of myelin compaction (656). Indeed, Kaplan et al. (293) found that clustering of the sodium channel occurs concomitantly as oligodendrocytes wrap axons with insulating myelin sheaths. The induction but not the maintenance of sodium channel clustering along the axons was demonstrated to depend on a protein secreted by oligodendrocytes in coculture with rat retinal ganglion cells. Furthermore, in vivo, mutant md rats (see sect. IVA1A), deficient in oligodendrocytes but not in neurons or astrocytes, develop few axonal sodium channel clusters (293). These data indicate that oligodendrocytes are necessary for clustering of sodium channels in vivo as in vitro. As clusters are regularly spaced even in the absence of direct axon-glial cell contact, with an average intercluster distance similar to that predicted for the Ranvier nodes in vivo, this suggests that there is an intrinsic mechanism, within the axon, that participates in the determination of spacing between nodes (reviewed in Refs. 530, 653). Glia induce ankyrin-G clustering in a pattern identical to that of sodium channel clustering; thus it is likely that cytoskeletal interactions play an important role in the induction and maintenance of sodium channel clusters (312). These data demonstrate that oligodendrocytes are specialized to induce saltatory conduction by inducing clustering of sodium channels and providing myelin insulation, and furthermore by regulation of axonal caliber and other properties (see sect. IIIG) (see discussion in Ref. 293).

E.  Biochemical Aspects of Myelin Assembly and of the Node of Ranvier

1.  Specific processing of myelin proteins during synthesis by oligodendrocytes

Preceding myelination, myelin specific genes are activated. Immature oligodendrocytes express GalC and CNP and are clearly arborized; later, mature nonmyelinating oligodendrocytes express MBP, PLP, and MAG (154, 399). Interestingly, in the postnatal period, the peak of mRNA is probe dependent and not region dependent for CNP, PLP, and MBP, suggesting a common signal occurring simultaneously in all areas, regardless of the caudorostral gradient of myelination in brain (292). Although these proteins appear in oligodendrocytes regardless of the presence of neurons, these data as well as others indicate that the presence of neurons clearly enhances the quantity of myelin specific proteins being synthesized (358). In oligodendrocytes in culture, there is a spatial organization of the protein synthetic machinery in granules that may provide a vehicle for transport and localization of specific mRNAs within the cell (4, 24).

A) MBP. The MBPs are a set of membrane proteins that function to adhere the cytoplasmic leaflet of the myelin bilayer. Because their biophysical properties may render MBP nonspecifically adhesive to any organelle membranes, oligodendrocytes have developed a mechanism for the transport of MBP mRNAs selectively to intracellular regions where MBP proteins will be necessary for myelin compaction to occur (123). Indeed, in situ hybridization has shown that MBP mRNAs are first localized in the cytoplasm of oligodendrocytes, even before myelination (589), and then dispersed in the oligodendrocyte processes at the beginning of myelination; the myelin sheaths stain for MBP mRNAs because the messages are in part transported within the cytoplasmic channels that surround and infiltrate the sheaths. Spatial segregation of MBP messages begins to function only after oligodendrocytes have differentiated into fully myelinating cells, cellular extensions being in place first, before the MBP transport machinery is activated (636). Before that period, MBP is expressed throughout the cytoplasm and surprisingly in the nucleus. All the isoforms do not behave in the same way when individually expressed, as has been shown transfecting individual isoform cDNA in the shiverer oligodendrocyte devoid of functional MBP (6); the 14- and 18.5-kDa forms have a plasma membrane distribution in the oligodendrocyte, whereas the exon 2-containing 17- and 21.5-kDa MBPs (MBP exII) distribute diffusely through the cytoplasm and accumulate in the nuclei. The transport of MBP exII is an active process that may suggest that these karyophilic MBPs are involved in a regulatory function in implementing the myelination program (447). The 14-kDa isoform that lacks exon 2 is sufficient to create myelin compaction in the CNS, as transgenic mice expressing only this form of MBP in the shiverer mutant (see sect. IVA1A) have a compacted myelin (299). Once the cytoplasmic extensions form, the exon 2 lacking MBPs are upregulated, and the mRNA movement mechanism is activated. MBP is exceptionally present in the nuclei of apparently mature oligodendrocytes (247) that may be remodeling or remyelinating some of the myelin sheaths that they support (447). Cytoskeletal elements may be involved in dynamic process of MBP mRNA transport (4).

B) PLP. There may be a vesicular transport of myelin PLP and sulfogalactosyl ceramides (78). Studies of DM-20/PLP proteins show that specific sequences of PLP gene and protein are necessary for correct trafficking and that abnormal proteins are readily eliminated in the endoplasmic reticulum (230). Abnormal level of misfolded PLP proteins as seen in Pelizaeus-Merzbacher disease represent a tremendous burden to the oligodendrocyte, as PLP mRNA represents 10% total mRNA of the oligodendrocyte at the time of myelination (see sect. IVA2).

The peak of myelination is extremely precise in rat and mouse, at 18 days postnatal. The enzyme activities, which peak during that particular period, appear to be related to the myelination process. This is the case for enzymes related to lipid metabolism. Mutations in the mouse affecting the myelination process show massive reductions in these enzyme activities, in relation to the degree of reduction in myelin. These aspects were described in the 1980s (38). PAPS cerebroside-sulfotransferase, an enzyme required for the synthesis of sulfated galactosylceramide, is inducible in CNS in relation to myelination; its activity is constitutively stable in the kidney, even in dysmyelinated mutants such as jimpy (534). CGT activity also increases rapidly during myelination and decreases markedly when myelination ceases. Expression of CGT mRNA (544) correlates clearly with myelination and agrees with previously determined enzyme activities. Very-long-chain fatty acid (VLCFA) biosynthesis (C24) follows the same pattern in the endoplasmic reticulum; interestingly, although mitochondria synthesize also VLCFA, there does not seem to be a relation to myelination, in contrast to VLCFA synthesized in the microsomal fraction that comprises the endoplasmic reticulum (72). HMG-CoA reductase seems also to be activated in oligodendrocytes at the time of myelination, as has been shown using a transgenic mouse expressing the promoter of this enzyme colinked to the marker chloramphenicol acetyltransferase (320). Peroxisomal enzymes involved in plasmalogen biosynthesis are necessary for myelination; the key enzymes are acyl CoA:dihydroxyacetone phosphate acetyltransferase (DHAP-AT) and alkyl dihydroxyacetone phosphate (alkyl DHAP) synthase (545).

It has been proposed that proteins destined for the apical surface of polarized epithelial cells cocluster with glycolipid-rich microdomains during sorting and transport from the trans-Golgi network. Glycolipid-rich oligodendrocytes may adopt this mechanism for myelinogenesis. Protein-lipid complexes from oligodendrocytes and myelin were isolated utilizing detergent insolubility and two-dimensional gel electrophoresis, leading to the identification of a developmentally regulated protein, myelin vesicular protein of 17 kDa (MVP17), highly homologous to human T-cell MAL protein. (298). In relation to the processes of myelin deposition and assembly, it is interesting to note that the family of MAL-related proteins (see sect. IIIC2F), highly expressed in myelinating glial cells (362), present a lipidlike behavior and are components of detergent-insoluble myelin membranes (450). Furthermore, it was shown that rMAL, found to interact with glycosphingolipids, may lead to the formation and maintenance of stable protein-lipid microdomains in myelin and apical epithelial membranes, thus contributing to specific properties of these highly specialized plasma membranes (195). In the same context, it was discovered that, in contrast to oligodendrocyte progenitors, in maturing oligodendrocytes and myelin, GPI-anchored proteins form a complex with glycosphingolipids and cholesterol, suggesting sorting of this macromolecular complex to myelin (314). The recently described presence of caveolae, which are membrane microdomains containing high levels of GPI-anchored proteins, in oligodendrocytes as in astrocytes (93) may be relevant to the mechanisms of myelin assembly.

Electron microscopy data visualize myelin as a series of alternating dark and less dark lines that should be protein layers, separated by unstained zones constituted by lipid hydrocarbon chains (404) (Fig. 5). In fact, the currently accepted view is that there are integral membrane proteins embedded in the lipid bilayer, although there are proteins attached to one surface or the other by weaker links. Individual components form macromolecular complexes with themselves and each other (538). Such an orderly structure could require a precise stoichiometric relationship of the individual components so that either increasing or decreasing the amount of one component could perturb the entire structure and therefore perturb myelination (see sect. IV).

The lipid compositions of the two halves of the lipid bilayer of biological membranes, including myelin, are strikingly different. The glycolipids, as in other membranes, are preferentially located at the extracellular surfaces, in the intraperiodic line. Almost all the lipid molecules that have choline in their head group (phosphatidylcholine and sphingomyelin) are in the outer half of the lipid bilayer, i.e., as the glycolipids. Almost all the phospholipid molecules that contain a terminal primary amino group (e.g., phosphatidylethanolamine, mainly plasmalogens, and phosphatidylserine) are in the inner half; they are negatively charged. This lipid asymmetry is functionally important in the compaction mechanisms of the mature myelin.

PLP and MBP are the most abundant proteins of myelin. Thus it is widely believed that the two proteins play complementary roles in myelin and are required for intra- and extracellular myelin compaction; this has been well demonstrated in the double mutant mice for PLP and MBP, viable and fertile (305, 591), which display an absence of fusion at the major dense line (typical of the homozygous shiverer mice) and a more compacted structure at the intraperiodic lines that appears as a single fused line (as in PLP mutants). PLP is an integral membrane protein with four transmembrane domains; in mutant mice that lack expression of the PLP gene, intraperiodic lines have reduced physical stability, indicating that PLP may form a stabilizing membrane junction similar to a zipper (305). It is widely accepted that the positively charged cytoplasmic proteins such as MBP in the CNS, or the cytoplasmic domain of P0 in the PNS, are responsible for compaction at the major dense line of myelin, and most likely as a result of an interaction with the acidic lipids, such as phosphatidylserine, located in the opposing membrane (113, 147).

Details of the molecular organization of the node of Ranvier have recently started to emerge (see Fig. 7). Spacious nodal gaps present in thick spinal axons are labeled by antibodies against HNK-1, chondroitin sulfate, and tenascin, but tiny node gaps along thin callosal axons are not labeled (56). The filamentous network subjacent to the nodal membrane contains the cytoskeletal proteins spectrin and isoforms of ankyrin (135, 322). Among the proteins binding to ankyrin at the node are the sodium channels, the Na⁺-K⁺-ATPase, specific forms of the NCAMs and the neurofascin isoform containing mucin and a third fibronectin III domain (135). Each node is flanked by paranodal regions where the axolemma is in close interaction with the membrane of the terminal loops of myelinating glial cells. Potassium-dependent channels are mainly localized in the axolemma at the level of these paranodal loops (57). Paranodin, a prominent 180-kDa transmembrane neuronal glycoprotein, was purified and cloned from adult rat brain and found in axonal membranes at their junction with myelinating glial cells, in paranodes of central and peripheral nerve fibers (382). Simultaneously, other authors isolated the same protein they called Caspr (171). Paranodin/Caspr may be a critical component of the macromolecular complex involved in the tight interactions between axons and the myelinating glial cells at the level of the paranodal region (163, 468). The glial ligand of Paranodin/Caspr has yet to be determined.

View larger version (73K): [in this window] [in a new window]   Fig. 7. Sequential steps in the formation of the node of Ranvier. Before glial ensheathment, sodium channels are distributed uniformly, and at low density. At the time of glial ensheathment but before the formation of compact myelin, loose clusters of sodium channels develop at sites that will become nodes. With formation of compact myelin and mature paranodal axon-glial cell junctions, well-defined nodal clusters of sodium channels are established, and sodium channels are eliminated from the axon membrane beneath the myelin sheath. [From Waxman (653). Copyright 1997, with permission from Elsevier Science. See also Kaplan et al. (293).]

Hyperthyroidism accelerates the myelination process, and hypothyroidism decreases it (648). Nevertheless, at an advanced age, hyperthyroid animals have a myelin deficit. The differentiation of oligodendrocytes as well as the degree of myelin synthesis are increased by thyroid hormone (7). Hypothyroidism coordinately and transiently affects myelin protein gene expression in most brain regions during postnatal development (269). In culture, oligodendrocyte progenitors stop dividing and differentiate in the presence of 3,3',5-triiodothyronine (T₃); although this process may occur even in the absence of this signal, thyroid hormone may be involved in regulating the timing of differentiation of this cell population (29). T₃ promotes also morphological and functional maturation of postmitotic oligodendrocytes, thus the number of mature oligodendrocytes (19, 269).

The effect of T₃ is mediated by the interaction of this hormone with specific receptor isoforms (TR), three of which, only, are functional, ₁, ₁, and ₂. Progenitor cells express ₁, whereas mature oligodendrocytes express ₁ and ₁, although ₂ may also be expressed on progenitor cells (29). Myelin gene expression correlates with a striking increase in ₁-expression in the developing brain (19). The expression of different receptors in relation to the maturation state of the oligodendrocytes may mean that these effects are independently regulated by thyroid hormone.

TR are ligand-activated transcription factors that modulate the expression of certain target genes in a developmental and tissue-specific manner. These specificities are determined by the tissue distribution of the TR isoforms, the structure of the thyroid hormone responsive element (TRE) bound by the receptor and heterodimerization partners such as retinoic acid receptor (64). Addition of glucocorticoids, thyroid hormone, or retinoic acid all increase galactocerebroside synthesis in oligodendroglia (465) and sulfolipid biosynthesis (51). Among the known target genes are MBP (182), PLP, MAG, and CNP (269).

Progress has been made in identifying some of the molecules involved in the transcriptional regulation of myelination (265). Huo et al. (267) have identified a nuclear protein that binds to TR isoform ₁, which forms a specific complex on the MBP-TRE. The expression of this brain nuclear factor is restricted to the brain perinatal period when myelination is sensitive to T₃.

2.  Other hormones

A) GROWTH HORMONE. Growth hormone has an effect on proliferation and maturation of both glial and neuronal cells (430).

B) NEUROSTEROIDS. Baulieu and co-workers (287) have shown that steroids can be synthesized in the CNS and PNS. In the CNS, pregnenolone, progesterone, and their sulfated metabolites are synthesized by oligodendrocytes (287) and activate the synthesis of specific proteins.

Factors that may act within the nucleus at early stages of the myelination program in the upregulation of transcription for myelinogenesis have been described. The DNA binding protein MyT1 (297), which binds to the promoter of the PLP gene (265), may play a role in specification toward the glial lineage; as do SCIP (402), cerebellum-enriched nuclear factor 1 (CTF-NF) (274), and TR transcription factor (182). Both CTF-NF and TR activate the MBP promoter. The karyophilic MBPs, containing exon 2, once in the nucleus, may also act as regulatory factors in myelination (447).

G.  Roles of Myelin

1.  Saltatory conduction of nerve impulses

As described in section IIIB, a myelinated fiber appears as myelinated membrane segments, termed internodes, separated by regions of unsheathed (naked) nerve membrane (Figs. 1, 2, 3, 5, and 8). It is this disposition by segments that induces the major characteristic of the so-called "saltatory conduction" of nerve impulses in myelinated tracts. As myelin is found almost exclusively in vertebrates, it can be considered as an essential element in the evolution of higher nervous functions (559).

The extremely dense clustering of sodium channels in the node of Ranvier accounts for the specialization of this region of the axolemma (see sect. IIID6). In myelinated fibers, at the nodes of Ranvier, the sites where impulses are generated, the sodium channels are clustered at a density of some 120,000/µm², the highest density in the nervous system. The myelin sheath has a high resistance and low capacitance that makes the current tend to flow down the fiber to the next node rather than leak back across the membrane. Therefore, the impulse may jump from node to node, and this form of propagation is therefore called saltatory conduction (Fig. 8) (559).

View larger version (28K): [in this window] [in a new window]   Fig. 8. Mechanisms for spread of the nerve impulse. A: continuous conduction in an unmyelinated axon. Amplitude scale is in millivolts. B: discontinuous (saltatory) conduction from node to node in a myelinated axon. In the diagrams, the impulses are shown in their spatial extent along the fiber at an instant of time. The extent of the current is governed by the cable properties of the fiber. [From Shepherd (559).]

2.  Role of myelin on axonal development and maintenance

A) DEVELOPMENT AND REGULATION OF AXONAL CALIBER. As early as 1951, Rushton (527) noted that the conduction velocity in a myelinated fiber is linearly correlated with diameter and proposed that, at a given diameter, there is a particular myelin thickness that maximizes conduction velocity. However, today it is still not yet fully understood how the mechanisms of morphogenesis of the myelinated nerve fibers are regulated. Although it was first proposed that myelination occurs only on fibers with a minimum diameter, it has been actually shown that signals from the myelinating glial cell, independent of myelin formation, are sufficient to induce full axon growth, primarily by triggering local accumulation and organization of the neurofilament network, as this can still occur in dysmyelinated mutant mice (532). Therefore, myelinating cells and myelin itself are important actors in the internal organization of the axonal structure and the subsequent growth of axon caliber (669; reviewed in Ref. 653). The final axonal caliber is influenced by other factors, such as the neurofilaments themselves.

B) MAINTENANCE AND SURVIVAL OF AXONS. In absence of the PLP/DM-20 proteins, in the PLP-deficient mice which make a near-normal myelin sheath (305) (see sect. IIIG2B), morphological abnormalities are surprisingly observed in axons, i.e., axonal swellings and degeneration, and particularly by axonal "spheroids" (235). The axonal spheroids contain numerous membranous bodies and mitochondria and are often located at the distal paranode, suggesting an impairment of axonal transport; these spheroids were detected predominantly in regions of myelinated small-caliber nerve fibers and were never observed in unmyelinated fibers. To demonstrate that the absence of PLP and DM-20 was fully responsible of these axonal abnormalities, PLP null mice were interbred with transgenic mice expressing an autosomal PLP transgene (500): full complementation was observed in offspring that were totally rescued from the defect. To distinguish the ability of each isoform, PLP and DM-20, to rescue a normal phenotype, PLP null mice were crossed with the PLP or DM-20 cDNA-based transgenes (416); the presence of one or the other of these isoforms allowed a near-complete restoration of a normal phenotype for the PLP, and at a lesser level for the DM-20 transgene, suggesting a possible similar role for the two isoforms in maintenance of axonal integrity. It is of note that PLP abnormal overexpression in transgenic lines (288, 500) causes significant axonal degeneration with predilection for specific tracts (13) and is toxic for myelinating oligodendrocytes and leads to different degrees of dysmyelination or demyelination. Altogether, these data demonstrate a pivotal role of glial-axonal communication for PLP in maintenance of axonal integrity and function, although the molecular mechanisms involved have yet to be determined.

A major problem following mammalian CNS injury, and particularly spinal cord traumatic insult in humans, is that lesioned axons do not regenerate. It has been suggested that myelin and oligodendrocytes (reviewed in Refs. 20, 61, 188, 222, 485, 546), as well as reactive astrocytes and the molecules they secrete (reviewed in Ref. 512), participate in inhibition of regeneration within the adult mammalian CNS.

A) INHIBITORY PROPERTIES OF MYELIN FOR LATE-DEVELOPING AXONAL TRACTS. Oligodendrocytes and myelin may play an inhibitory role on neurite growth, e.g., for late-developing tracts and inhibition of plasticity in the adult brain. Because myelin formation starts at different times in different regions and tracts of the CNS, in a so-called "mosaic-like pattern," the inhibitory property of myelin could serve boundary and guidance functions for late-growing fiber tracts. Oligodendrocyte-associated neurite growth inhibitors could channel late-growing CNS tracts by forming boundaries and by exerting a "guard-rail" function (548). The different candidate proteins for a major role in inhibition of neurite growth are described below.

B) MYELIN INHIBITORY ROLES IN CNS REGENERATION. The limited potential for axonal regrowth and regeneration of nerve fibers in the adult CNS from higher vertebrates contrasts with recovery that happens in the PNS of these species. It was long recognized that the permissiveness of the environment was a determinant for CNS axonal repair (496, 604). Later, Aguayo and collaborators demonstrated that most CNS neurons are able to regenerate a lesioned axon in the environment of a peripheral nerve transplant (508), whereas peripheral nerves fail to regenerate in a CNS glia environment (132). Recently, the use of multiple grafts of peripheral nerve tissue, bridging the lesion site and the gray matter, was shown to permit extensive CNS axon growth and to restore partial function to a completely transected spinal cord (reviewed in Refs. 183, 438). Investigations of the biological mechanisms that could be responsible for this difference in neuronal regeneration between CNS and PNS tissue led to a new concept, that of a neurite growth inhibitor (549). Central myelin and differentiated oligodendrocytes in the adult CNS were therefore recognized as responsible for inhibition of neurite outgrowth and shown to cause growth-cone collapse in culture (21).

C) MYELIN-ASSOCIATED INHIBITORY PROTEINS, NI-35 AND NI-250. Two related protein fractions of 35 and 250 kDa (NI-35 and NI-250) were found to contain the myelin-associated inhibitory activity. NI-35 and NI-250 are membrane-bound proteins highly enriched in mammalian CNS myelin and oligodendrocytes, but minor in PNS myelin (105). A monoclonal antibody (MAb IN-1) has been generated and shown in vitro to have neutralizing properties against the inhibitory activity of oligodendrocytes and myelin (106). In vivo, application of MAb IN-1, after the complete bilateral transection of the corticospinal tracts in spinal cord, allowed some regeneration on a short length of a small percentage of cortical tract axons (542). On the other hand, in a recent work (607), IN-1 treatment after unilateral transection of the corticospinal tract in the brain stem, above the pyramidal decussassion, was shown to induce a significant collateral sprouting of both intact and lesioned corticospinal and corticobulbar nerve fibers, above and below the lesion site. This remarkable structural plasticity was associated with a functional recovery of forelimb function contralateral to the lesion (607). A similar form of recovery, through sprouting in gray matter rather than through severed axon regrowth in white matter, has been described in another lesion model (reviewed in Ref. 438).

D) MAG, ANOTHER INHIBITORY MYELIN PROTEIN. A well-characterized myelin protein produced by oligodendrocytes but also by Schwann cells (albeit at 10-fold less level), MAG (see sect. IIIC2), has been recently identified as a major CNS inhibitory protein (376, 414), for a number of different types of neurons. MAG is an inhibitory rather than a nonpermissive protein; MAG is able to cause growth cone collapse in vitro (333), and soluble extracellular domain of MAG (dMAG), released in abundance from myelin and found in vivo (398, 583), has been shown to inhibit axonal regeneration in vitro (602; reviewed in Ref. 485).

H.  Plasticity of the Oligodendrocyte Lineage: Demyelination and Remyelination

1.  Fate of glial precursors and progenitors in the adult CNS

In the adult, the subventricular germinative zone persists around the brain lateral ventricles, along the striatum. These cells, although mostly quiescent, can be amplified in vitro in the presence of epidermal growth factor (EGF) and give rise to neurons, and to astrocytes and oligodendrocytes, when grown in the presence of FGF2, LIF or BDNF (339, 503), or T₃ (281). The intrathecal infusion of EGF and FGF-2 activates the mitotic and migratory potential of these cells (126). CNS stem cells are also present in the adult spinal cord and ventricular neuroaxis (661). Recently, a major glial cell population, suggested to be adult oligodendrocyte progenitors, has been described in the CNS; these cells can be identified by the expression of two cell surface molecules, the NG2 proteoglycan and the PDGF- receptor (reviewed in Ref. 426). They could represent a third type of glial cells in the CNS, with still unknown functions (reviewed in Ref. 339a).

Recently, mouse oligodendroglial cells derived from totipotent embryonic stem cells were transplanted in a myelin-deficient rat model; these oligodendrocytes were able to interact with host neurons and efficiently myelinate axons in brain and spinal cord of the mutant animal, suggesting that similar transplantations might help patients with Pelizaeus-Merzbacher disease or other demyelinating conditions (80).

Very little is known about spontaneous remyelination, as only a few early studies deal with the subject of myelin turnover. It appears that under normal conditions, the turnover of myelin is very slow and that each component has a different turnover rate, possibly different according to its location in myelin and in the myelinating cell (404).

Spontaneous remyelination was observed for the first time by Bunge et al. (83), after an experimental lesion in the cat spinal cord. It has also been reported in other experimental models (347, 349). The ways to stimulate the endogenous oligodendrocyte population to expand and regenerate myelin may be an important strategy. For the moment, the growth factor responses have not as yet been fully determined in humans, and no myelinating cell line has as yet been obtained analogous to the CG4 cell line isolated from rodents (340). So far, no studies have identified growth factor stimulation of division in the human oligodendrocyte lineage. Studies determining the effect of growth factors on remyelinating capacities in vivo are few (379, 675). In immune-mediated demyelination, related to Theiler's virus infection, monoclonal antibodies of natural origin (266) may stimulate remyelination through a mechanism as yet unknown. Interestingly, inflammation seems to enhance migration of oligodendrocyte and thus remyelination in EAE (615).

Interestingly, in chronic MS (see sect. IVB1) lesions, oligodendrocyte precursor cells are present (671, 672); however, they appear to be quiescent, not expressing a nuclear proliferation antigen. In MS, remyelination also occurs, but it is incomplete and poorly sustained (477, 491). After a demyelinating lesion, remyelinated myelin never regains its normal thickness, and the normal linear relationship between axon and sheath thickness is also never regained (349). It is not clear whether the mechanism of remyelination is identical to myelination. At least in part, it could be similar to mechanisms at work in myelinogenesis; for example, it appears that MBP transcripts containing exon 2 are widely expressed in remyelinating oligodendrocytes in vivo and in vitro (101, 221). The molecular basis of the remyelination process has not been studied extensively.

The origin and identity of endogenous cells that affect remyelination is still an unsolved issue. There are immature cycling cells endogenous to white matter, which respond to experimental demyelination, by differentiating into myelinating oligodendrocytes (216). Proliferation of mature oligodendrocytes can also occur after a trauma of the nervous system (348) and in an experimental model of stab wound (8). However, no one has demonstrated that mature oligodendrocytes become involved in remyelination.

Glial transplantation techniques have proven to be a useful tool for the study of glial cell biology, as it is possible to label the transplanted cells and follow them throughout the whole myelination (321) and remyelination process. It is also possible to determine the amount of myelin being formed by transplanting myelin-producing cells into the CNS of myelin-deficient mutants. With the use of such techniques, it is also possible to determine the influence of the in vivo environment on these capacities. The experimental conditions, myelin-deficient models, markers for transplanted cells, and use of myelinating cell lines have been extensively reviewed recently (156). Even adult brain retains the potential to generate oligodendrocyte progenitors with extensive myelination capacity (696). The potentiality for remyelination can be exerted by many types of cells of the oligodendrocyte lineage. Cells present in the SVZ could be able to migrate toward a demyelinating lesion and to differentiate into myelinating oligodendrocytes (420). It appears that migration is reduced or very scarce under normal conditions (196, 197), except in genetic dysmyelinating mutants (321). There seem to be signals in a demyelinated lesion that attract myelinating cells (638). PSA-NCAM, which is a migration-supporting signal, has been observed in areas of demyelination (420). Inflammation may favor migration (615). Analogous results have not been obtained by other groups (278) in the rat without X-irradiation (197). It is possible to enhance the proliferating capacities of progenitor cells to be transplanted by producing oligospheres (18, 415), or using the oligodendrocyte progenitor cell line, i.e., the CG4 cells (612). Nevertheless, up to now, no extensive remyelination has been obtained experimentally, although, in some cases, a functional electrophysiological recovery has been obtained (627).

Remyelination by transplantation of potentially myelinating cells may be a therapeutic approach. Although oligodendrocytes derived from fetal human white matter implanted into the dysmyelinating rodent CNS can synthesize myelin even after prolonged cryopreservation (554), harvesting aborted fetuses on a therapeutic scale is unlikely to be ethically or socially acceptable (550). Furthermore, there is a risk of immunological rejection. Thus it could be envisaged to use tissues from the patient itself, to obtain donor cells, such as Schwann cells (17, 260) or olfactory glia which both can be expanded (273, 494). The ability of a transplanted cell to migrate is obviously also of great importance in its remyelinating capacity after a lesion (196, 197).

Recent experimental data show that axonal loss or perturbations may be more responsible than myelin deficiency for permanent disability (133). This has recently been observed in an experimental model: mice sensitive to Theiler's virus develop secondary demyelination. In a transgenic mouse deficient in MHC gene class I, Theiler's virus demyelination still occurs, but the animal does not present any functional deficit (515). This may be as a result of the persistence of sodium channels at the site of the Ranvier node, implicating possibly MHC class I molecules in the development of neurological deficits after demyelination.

Loss of myelin, even from a single internode, is sufficient to block conduction. If sufficient axons are affected in this way, severe symptoms can arise. The loss of insulation along the axon has as consequence that the action current is no longer focused at the very excitable nodal membrane. The axon may adapt to its demyelinated state over several days, by a redistribution of sodium channels. Possibly, cell contact involving astrocytes may be required to initiate channel rearrangement and permit restoration of nerve function (155), although, under these conditions, conduction is very slow and highly susceptible to external disturbances. Fast potassium channels are located in the internodal axon membrane; being unmasked, they may also interfere with conduction in demyelinated axons (reviewed in Ref. 653).

	IV. EXPERIMENTAL AND HUMAN DISEASES OF MYELIN

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A.  Genetic Diseases of Myelin

1.  Myelin mutants

The recent characterization of a number of mutants and determination of physical maps for mammalian genomes have greatly improved our ability to characterize a number of animal models for human diseases. The term myelin mutants designates animal mutants in which myelin formation or maintenance is affected. Myelin mutants are often named according to their phenotypes, e.g., shiverer, rumpshaker, twitcher, etc. They allow not only the identification and cloning of genes possibly related to human diseases, but also analysis of myelin development and biology of myelin-forming cells. They are furthermore essential for the development of therapeutic strategies useful for human diseases.

A) MUTATIONS OF MYELIN STRUCTURAL PROTEINS. I) PLP. Because they are recessive X-linked diseases, the PLP mutation disorders are expressed only in males that are hemizygous (they possess only one allele). Because they are "mosaic" at the cellular level as a result of the random inactivation of one of the two X-chromosomes in each cell (355), heterozygous females present generally with a wild-type phenotype. This group of mutants contains the higher number of alleles within and across species. The PLP mutations occur in different regions of the PLP protein, yet they produce qualitatively similar phenotypes.

B) MUTATIONS IN A REGULATORY PROTEIN: THE QUAKING MOUSE. The quaking mutation comprises two classes of recessive alleles, with strikingly different phenotypes. Indeed, quaking mutations present pleiotropic effects on myelination and embryogenesis. The original "quaking viable" mutation is a spontaneous, recessive mutation, presenting a hypomyelination of both CNS and PNS, associated with a large deletion of one megabase on chromosome 17 (167). Homozygous quaking viable mice survive to adulthood and exhibit a characteristic tremor or "quaking" of the hindlimbs (562). The identification of oligodendrocytes as the affected cells, the abnormal composition and compaction of myelin, with pockets of oligodendrocyte cytoplasm, suggested that an arrest of myelin assembly is causative of the quaking viable mutation. Interestingly, myelin is not reduced in all fascicles (43). Other developmental abnormalities have also been observed, such as an increased number of noradrenergic neurons in the locus ceruleus that is likely to be involved in the convulsions of this mutant mouse (370).

C) MUTATIONS INVOLVING ENZYMES OF LIPID METABOLISM. The twitcher mouse was early recognized as an authentic model of Krabbe's disease (599). The twitcher mouse exhibits a leukodystrophy characterized by ataxia ("twitching") and abnormal periodic acid-Schiff (PAS)-positive cells, often multinucleated ("globoid cells"), in both CNS and PNS. On C57BL/6j background, death occurs at 40-45 days. Loss of myelin and accumulation of globoid cells, often clustered around blood vessels, are prominent in the white matter spinal cord and brain stem of twitcher mice. However, compacted myelin appears structurally normal, with an orderly periodicity of the dense major line and intraperiodic line. Crystalloid or slender tubular inclusions (GLD inclusions) are present in macrophages, oligodendrocytes, and astrocytes. Extensive astrocytosis is detected both in white and gray matter. Axonal loss is not prominent. The PNS is also severely affected. Infiltration of macrophages, similar to those seen in the CNS, is apparent between nerve fibers. Many nerves are demyelinated, and the presence of thinly myelinated fibers indicates remyelination. Schwann cells of myelinated fibers contain the characteristic GLD inclusions, whereas nonmyelinating Schwann cells present unusually elongated and branching processes. Both Schwann cell types possess numerous GFAP-immunoreactive processes.

D) MYELIN MUTANTS WITH UNKNOWN GENETIC DEFECTS. The taiep rat presents a developmental defect in CNS myelination, leading to a progressive neurological disorder (160). "Taiep" is an acronym for trembling (t), ataxia (a), immobility episodes (i), epilepsy (e), and paralysis (p). The myelin defect correlates with an abnormal and progressive accumulation of microtubules associated with endoplasmic reticulum membranes in taiep oligodendrocytes. These morphological abnormalities suggest a blockage of protein trafficking (125). Glial cell number is normal (351). Myelin yield decreases continuously from 2 wk until it reaches a stable level of ~10-15% in the affected brain and 20-25% in the spinal cord, compared with control. Thus the myelination defect in taiep has features of both hypomyelination and demyelination.

Inherited myelin diseases in humans, leukodystrophies, may be the result of dysmyelination, hypomyelination, or demyelination. Dysmyelination and hypomyelination are failure to myelinate occurring during fetal life or early infancy, as observed in different forms of Pelizaeus-Merzbacher disease. Demyelination, breakdown of myelin, is characteristic of metabolic leukodystrophies, such as Krabbe's disease, metachromatic leukodystrophy, ALD, Canavan disease, Alexander disease, orthochromatic leukodystrophy, or mitochondrial disorders (reviewed in Ref. 309). Dysmyelination and demyelination can be combined in some forms of leukodystrophies.

Myelin formation and maintenance require also a balance between synthesis and degradation of lipids. Although alterations of cerebroside, sulfatide, and long-chain fatty acid degradation possibly involve other cell types than oligodendrocytes, they give rise to leukodystrophies (reviewed in Ref. 309). These complex lipids and their VLCFAs are also present in the PNS. Therefore, symptoms associate both central demyelination visible by MRI and PNS demyelination with slowing of nerve conduction velocity. These leukodystrophies associate defective lysosomal enzymes or lipid transporters and accumulation of the undegraded lipid which is easily tested in fibroblast cell cultures.

In Krabbe's disease (1916), an autosomal recessive disorder, there is a defect in the lysosomal galactocerebrosidase (598), the enzyme which degrades GalC into ceramide and galactose. This disease occurs mainly during infancy, but some adult cases manifesting only as spastic paraplegia have been diagnosed (reviewed in Ref. 310). The degeneration of myelin and oligodendrocytes is due to the accumulation of psychosine, a toxic metabolite also normally degraded by this lysosomal enzyme (reviewed in Ref. 595). Exogenous macrophages, the globoid cells, invade the CNS and have given the name of globoid cell leukodystrophy to this disease. The gene is located on chromosome 14 and contains 17 exons (353, 354). There is a spontaneous mouse mutant, the twitcher mouse (see sect. IVA1), which constitutes an experimental model of Krabbe's disease (reviewed in Ref. 599).

Metachromatic leukodystrophy is also an autosomal recessive inherited lysosomal disorder. It is caused by a defect in the arylsulfatase A (ASA) gene (reviewed in Ref. 219). ASA, together with an activator (saposin B), degrades sulfogalactosylceramide into GalC and sulfate. In most of the cases, it is the deficiency of ASA that leads to the accumulation of sulfogalactosylceramide and provokes a lethal progressive demyelination. The ASA gene has been cloned; the coding sequence is divided into eight exons (315); the entire coding sequence is of 1.5 kb. The leukodystrophy generally develops during infancy, and there is a destruction of the newly formed myelin, but it can also occur later, even at adulthood (40). The phenotype of ASA-deficient mice shows morphological, biochemical, but not functional relationships to human metachromatic leukodystrophy (252), possibly in relation to other compensating metabolic pathways in the mouse.

Sphingolipid activator proteins (saposins) are necessary to the enzymatic hydrolysis of myelin galactolipids. They originate from a prosaposin gene. Targeted disruption of the mouse sphingolipid activator protein gene gives rise to a complex phenotype including a severe leukodystrophy (204).

In X-linked adrenoleukodystrophy (ALD), there is an impairment in VLCFA -oxidation that is normally degraded in peroxisomes. There is an accumulation of VLCFA cholesterol esters and other lipids mainly in white matter of the brain and in the adrenal gland. There are two major forms (410): 1) cerebral childhood ALD (which is associated with inflammation in the brain) and multifocal demyelination and 2) adrenomyeloneuropathy which affects mainly the spinal cord and peripheral nerves. The ALD locus has been mapped to Xq28. The gene involved is called the ALD protein (ALDP) gene and consists of 21 kb and 10 exons. ALDP is a 75-kDa protein (411) that belongs to the ATP-binding cassette (ABC) protein family. ALDP has been localized to the peroxisomal membrane (411) and might be involved in the import of cofactors necessary to VLCFA-CoA synthase activity or import of VLCFA-CoA into the peroxisomes. In the brain, ALDP is expressed in oligodendrocytes but also in astrocytes and microglia (16). Mice defective in the ALDP gene have been generated (192, 308, 342). Unexpectedly, there are no neurological symptoms even in aged mice, although there is impaired -oxidation of VLCFA that accumulate in CNS white matter, adrenal cortex, and testis. Therefore, the accumulation of VLCFA alone is probably not sufficient to cause demyelination in the nervous system (reviewed in Refs. 16, 410).

Zellweger disease is a complex peroxisomal syndrome (reviewed in Ref. 409) that also affects white matter in brain and gives rise to an accumulation of VLCFA; there is also a defect in plasmalogen biosynthesis (545), compounds known to be enriched in myelin and other abnormalities of this cerebro-hepato-renal syndrome. It is a group of genetically heterogeneous disorders that affects peroxisome peroxidation.

Another enzyme, aspartoacylase, which degrades N-acetylaspartic acid, is possibly also a myelin enzyme (284). The enzyme deficiency is the cause of Canavan's disease, which is also characterized biochemically by N-acetylaspartic aciduria. Canavan's disease gives rise to dysmyelination and spongy degeneration of the brain (reviewed in Ref. 364). Significant levels of N-acetylaspartate (NAA) are detected in cultures of immature oligodendrocytes; these metabolic abnormalities in the development of oligodendroglia may be related to the physiopathology of this disease (626). In the adult, NAA is neuronal and a useful neuronal marker in NMR spectroscopy. Recently, it has been shown that mature oligodendrocytes can express NAA in vitro (50) and possibly in vivo contribute to the NAA signals observed in spectroscopy.

Mutations in the PLP gene cause a rare leukodystrophy, the Pelizaeus-Merzbacher disease (PMD). PMD presents in several forms: connatal, infantile, and more recently a different phenotype with spastic paraplegia type 2 (SPG-2), either isolated or associated to clinical features of the classical PMD (reviewed in Refs. 422, 555). More than 30 point mutations have been found in the PLP gene from PMD and SPG-2 patients.

Another rare leukodystrophy, the 18q-syndrome, presents a deletion which includes the MBP gene (213, 309, 520).

An astrocyte pathology may also give rise to leukodystrophies such as Alexander disease. Recently, sequence analysis of DNA samples from patients representing different Alexander disease phenotypes revealed that most cases are associated with nonconservative mutations in the coding region of GFAP. Alexander disease represents the first example of a primary genetic disorder of astrocytes, one of the major cell types in the CNS (75a).

A number of leukodystrophies, most of which are genetically inherited, have also no known etiology. A new leukoencephalopathy termed "childhood ataxia with diffuse central nervous system hypomyelination" (CACH syndrome) or "leukoencephalopathy with vanishing white matter" was recently identified on clinical and neuroradiological grounds (540, 629). In addition to the cavitating white matter lesions found in this orthochromatic leukodystrophy, neuropathological data recently obtained showed that this new characterized entity is associated with an unusually increased oligodendrocyte density (521, 522).

White matter is vulnerable to ischemic damage (143, 186). White matter diseases can also be posttoxic, postinfectious, and postradiological.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an autosomal dominant cerebral arteriopathy. MRI evidences multiple subcortical infarcts, with a demyelination of white matter that can be more or less extensive (616; reviewed in Ref. 136).

MELAS means mitochondrial myopathy, encephalopathy, lactic acidosis, strokelike episodes. Most of the patients present a lactic acidosis with an increase of the lactate-to-pyruvate ratio in serum and CSF. MRI shows white matter modifications are present together with cortical atrophy (148).

Refsum disease is characterized biochemically by an accumulation of phytanic acid in tissues. This acid is only present in food (green vegetables, and also grass-eating animals) and normally degraded by phytanate oxidase (585). MRI evidences demyelination in the brain stem and the cerebellum.

Some neuronal genetic diseases can affect myelin (GM2 gangliosidoses, Wilson's disease, and degenerative diseases of CNS). Oligodendrocyte express AMPA/kainate-type receptors, and they share with neurons a high vulnerability to the AMPA/kainate receptor-mediated death, a mechanism that may contribute to white matter CNS disease (369, 373, 435).

Breakdown of the blood-brain barrier is a primary event in pathological manifestations of demyelinating disease of the CNS, such as MS, demyelinating forms of EAE (reviewed in Ref. 663), and virus-induced demyelination (reviewed by Ref. 184). Neural and even nonneural activated T cells play a pivotal role in this process (336, 665). Moreover, the opening of the blood-brain barrier allows the entry of circulating antibodies into the CNS, in particular demyelinating antibodies, such as anti-MOG antibodies (214, 215, 336; see also review in Ref. 77). Access of activated T cells to the CNS is responsible for release by inflammatory cells, macrophages, and microglia and of proinflammatory cytokines, such as TNF- and interferon- (reviewed in Ref. 77). However, the toxic action of TNF- on oligodendrocytes (556) remains debatable. Activated macrophages engulf myelin debris or internalize myelin by Fc receptor and complement receptor-mediated phagocytosis after binding to myelin-specific antibodies (403, 628).

Other different mechanisms may be involved in the process of demyelination. Oligodendrocytes have a high iron content, making them especially prone to oxidative stress by reactive oxygen species (240). Neurotransmitters, especially glutamate, may also exert a direct deleterious effect on oligodendrocytes and lead to demyelination (369, 464; reviewed in Ref. 588). Heat shock proteins may also be involved (77), among which -B crystallin, which may be a candidate encephalitogenic antigen in demyelination (631). Matrix metalloproteinases (MMP) are a group of zinc-dependent enzymes that can degrade extracellular matrix components (reviewed in Ref. 687). Among other activities, they can degrade MBP (111). MMPs are increased in active and chronic MS (361), especially MMP-7 and MMP-9 (124). More and more, there is thought to be a viral triggering of autoimmune diseases (664). More than one mechanism may be present in the same disease or even in the same lesion (349).

There are many causes of demyelination in human diseases. The main causes of primary demyelination are genetic (such as in ALD, see sect. IVA2), immune mediated, viral such as HIV, and toxic (reviewed in Ref. 349); they may also be secondary to neuronal dysfunction. In some cases, white matter thinning is visualized on MRI; it is called leuko-araiosis, and it can be seen in normal, even young individuals, and more frequently in aging; its significance remains obscure. There may be a relationship between leuko-araiosis at MRI, myelin pallor, and white matter atrophy, which are as yet poorly understood (637). Diffuse myelin pallor is seen in some dementia patients (471).

MS is a chronic and inflammatory demyelinating disease of the CNS (112). Storch and Lassmann (593) have shown that there is a profound heterogeneity of pathology and immunopathogenesis of the lesions. There is a high interindividual but a low intraindividual variety of MS lesions. The spectrum of pathology appears to be the following (344, 345): 1) primary demyelination with little oligodendrocyte damage; 2) extensive oligodendrocyte loss in the course of demyelination; 3) primary oligodendrocyte damage with secondary demyelination; and 4) extreme macrophage activation together with rather nonselective tissue damage, involving not only myelin and oligodendrocytes, but also axons and astrocytes. Axonal loss is also a prominent feature of MS and may be the pathological correlate of the irreversible neurological impairment in this disease (185, 341, 375, 621). Current evidence shows that the etiology of MS and other demyelinating diseases may involve a combination of viral and autoimmune factors.

Oligodendrocyte progenitors are present in the normal adult human CNS and in the lesions of MS but fail to repair demyelinated regions (551, 671, 672).

There are several experimental models of demyelination: toxic, viral, and immune mediated. Among the toxic models, ethidium bromide (676) and cuprizone (59) alter mitochondrial DNA leading to abnormal respiration and necrosis (347); lysophosphatidylcholine (60) is also widely used.

B) EAE. EAE, also called experimental allergic encephalomyelitis, is provoked by injection of CNS tissue, whole spinal cord, purified myelin, or myelin proteins or peptides in susceptible animals (reviewed in Ref. 73). The incubation period between sensitization and onset of the disease as well as the severity and course of the clinical disease are variable and depend on the genetic background of the animal (species and strain differences); on environmental factors, like the mode of sensitization; the dose and nature of the sensitizing antigen; or exogenous influences on immune functions, such as intercurrent infections or treatment with immunosuppressive or immunomodulatory drugs. In many cases, it is also possible to transfer EAE by sensitized T lymphocytes to a naive recipient. For a long time, MBP has been considered as the main encephalitogenic antigen (296). Later, it was recognized that PLP may lead to a very similar disease (92). Immunodominant encephalitogenic epitopes of MBP and PLP have been characterized and found to be different in various animal species and strains, in relation to MHC phenotypes (12, 304, 646; review in Ref. 268).

The most common CNS gliomas traditionally are thought to arise from mature astrocytes and oligodendrocytes. Oligodendrogliomas, which appear to arise from oligodendroglial cells, have been thought for long times to be uncommon gliomas (<10% of all intracranial tumors) (254). However, reappraisal of recent data indicates that they may be more common than generally thought (193).

Recently, the possibility that gliomas may arise from a population of glia that has properties of oligodendrocyte progenitors has been examined. These glial cells express the NG2 chondroitin sulfate proteoglycan and the -receptor of PDGFR- in vivo. NG2 and the PDGFR- high levels of expression have been identified in tissue from seven of seven oligodendrogliomas, three of three pilocytic astrocytomas, and one of five glioblastoma multiform, raising the possibility that the NG2+/PDGF R+ cells in the mature CNS contribute to glial neoplasm. These data provide evidence that glial tumors arise from glial progenitor cells. Molecules expressed by these progenitor cells should be considered as targets for novel therapeutics (426, 560).

	V. CONCLUSIONS

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The importance of glia has become more and more clear with the development of techniques of molecular biology and cell cultures. Culturing neurons and glia separately or together has contributed to the understanding of the role of oligodendrocytes in the development of neuronal pathways as well as in synaptic functions. The study of spontaneous mutations, as well as transgenic mice with overexpression or no expression of specific markers, has shown the role of specific glial components in brain constitution and function. More and more, the molecules involved, in developmental processes and in the adult, are being identified. Molecular studies of developmental mutants and human pathology have led to the identification of the involvement of glia in numerous developmental diseases.

Axonal guidance seems in many cases to involve preformed glial pathways that may remain and create glial boundaries. Myelin plays an inhibitory role to growing axons and participates in the stability of the mature, intact nervous system, by preventing random regrowth of axon sprouts once pathways have been established and myelinated. Although it may present the unwanted side effect of impairing axonal reparation after CNS injury, it gives new areas of research for brain repair (134, 607). Without glia, and especially the oligodendrocyte, nerve conduction velocity would not develop normally because these glial cells are necessary for the clustering of sodium channels at the Ranvier node essential to fast nerve conduction velocity in myelinated fibers (293). The role of the oligodendrocyte and neuronal pairing is not only essential in relation to myelin formation but also in demyelinating diseases. If neurons are needed for myelination, impaired myelin function may induce profound cytoskeletal alterations leading to axonal degeneration, as observed in myelin protein-deficient animal models (235, 686). Altogether, these data enlighten the role of myelinating glial cells-axon interactions, the "functional axon-glial unit," in the pathophysiology of myelin-related disorders. Furthermore, these studies and other works related to remyelination show the unexpected intrinsic plasticity of glial cells in the CNS, commonly recognized in neonates, but surprisingly still present in adults (reviewed in Ref. 61). Furthermore, experimental models such as EAE and studies of cellular markers in human brain seem to indicate that there are different ways of inducing demyelination, thus possibly different types of MS.

More and more these neuroglial interactions are being identified in relation to neuronal functions. By their mobility and plasticity, glial cells appear to be intrically involved in the functions of the neuronal network. As mentioned by Ullian and Barres (624), "glia are not listening to synaptic transmission but participating in the conversation as well." Synapses throughout the brain are ensheathed by glial cells. Astrocytes help to maintain synaptic functions by buffering ion concentrations, clearing released neurotransmitters, and providing metabolic substrates to synapses. Recent findings suggest that oligodendrocytes may have such a role as well.

The dysfunction of glial cells is possibly at the origin of many of the degenerative diseases of the nervous system and of the major brain tumors (glioma). Nevertheless, there are still many mysteries that are not unraveled. Although growth factors and cytokines are secreted by astrocytes and oligodendrocytes in vitro, their relevance in vivo remains to be elucidated, as neurons are also able to synthesize them. Some cytokines may be useful or harmful. Receptors for these substances have not as yet been well identified in vivo, especially in pathological states.

Although myelin repair and synaptic remodeling and regeneration can occur, many enigmas are still to search for, especially in the human in which growth factors may be different from the rodent species. Thus studies in primates, and in vivo systems, cannot be omitted at this stage, in view of therapeutic implications.

No doubt we will understand more and more the language between glial cells and neurons in normal and pathological states. Already abnormal astrocytes and oligodendrocytes appear to be involved in cognitive functions as seen in the leukodystrophies.

As mentioned by Peschanski (451), time has come for "neurogliobiology" in which neurons and glia (including microglia) in the nervous system are inseparable partners.