Myelination by mature ovine oligodendrocytes in vivo and in vitro: evidence that different steps in the myelination process are independently controlled

The ability of isolated mature post-myelination ovine oligodendrocytes to myelinate was investigated in tissue culture and in vivo. In culture, although the cells adhered preferentially to rat dorsal root ganglia (DRG) axons, sent out processes that encircled and wrapped them, proliferated, and synthesised myelin proteins (MBP), no myelination was found. This failure to find myelination occurred despite the fact that the oligodendrocytes both in the present experiments and in previous studies elaborated membranous structures that have been shown chemically and structurally to be similar to normal central nervous system myelin. These findings contrasted with those seen when neonatal rodent glial cells were added to similar DRG neuron cultures, in which myelination readily occurred. When the same adult ovine oligodendrocytes were transplanted into the brains of Shiverer mice, normal compact myelin was formed, proving that the cells were capable of myelination and suggesting that cross-species incompatibility was probably not a major factor in the lack of myelination in vitro. It is possible that the failure of ovine oligodendrocytes to myelinate DRG axons is due either to the relatively low number of supporting glial cells, such as astrocytes or microglia which may be necessary for satisfactory myelination, or that some other factor in the microenvironment is lacking; in any event, these results point to the complexity of oligodendrocyte-axon interactions. It is clear that each of the events, from adherence to proliferation to wrapping and the myelin compaction may be under the control of a different signal and may operate through a distinct mechanism, even though each process is dependent on the other. The results also point to the potential usefulness of this model system for deciphering such signals and mechanisms.     

Myelination in organotypic cultures of sympathetic ganglia

Explants of mouse superior cervical ganglion (SCG), co-cultured with dorsal spinal cord, were grown for up to 4 weeks in vitro. In such cultures, scattered internodes of peripheral nervous system (PNS) myelin were observed, apparently associated with SCG neurites. Although rare, the incidence of PNS myelination in this system might merit further experimentation to provide a model facilitating the evaluation of postganglionic sympathetic myelination, which in vivo may be both extensive and morphologically unusual.     

Overexpression of 2',3'-cyclic nucleotide 3'-phosphodiesterase in transgenic mice alters oligodendrocyte development and produces aberrant myelination

The function of the intracellular protein 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) of oligodendrocytes (ODC) is unknown. We have now generated several homozygous transgenic mouse lines in which the human CNP gene is overexpressed up to sixfold, revealing new insights into early stages of myelinogenesis. Although no behavioral phenotype is immediately apparent, abnormalities of ODC and their myelin sheaths are striking. These are manifested as redundant myelin membrane and intramyelinic vacuoles, as well as lack of myelin compaction concordant with failure of the cytoplasmic leaflets of compact myelin to fuse. Further, ODC that overexpress CNP appear to mature earlier in development, resulting in earlier maximum gene expression for myelin basic proteins and proteolipid protein. These results indicate that CNP is an early expressed regulator of cellular events that culminate in CNS myelination.     

Formation and maintenance of the myelin sheath in the peripheral nerve: roles of cell adhesion molecules and the gap junction protein connexin 32

Based on previous in vitro studies, the cell adhesion molecules L1, N-CAM, MAG, and P0, which all belong to the immunoglobulin (Ig)-superfamily, have been suggested to mediate myelin formation in the peripheral nervous system. Unexpectedly, studies in mice deficient for the corresponding molecules revealed that only P0 plays pivotal roles during the formation of peripheral nerve myelin in vivo, while L1-, N-CAM-, and MAG-deficient mice develop myelin of normal ultrastructure. However, MAG turned out to be important for the maintenance of myelin, as reflected by degeneration of myelin and axons in MAG-deficient mice older than 6 months. The MAG-mediated maintenance of myelin is backed up by N-CAM, since mice deficient in both MAG and N-CAM show an earlier and more prominent myelin degeneration than MAG single mutants. Another peripheral nerve component involved in the maintenance of myelinated fibers is connexin 32 (Cx32), a gap junction channel protein that does not belong to the Ig-superfamily. Mice deficient in Cx32 initially form normal myelin, which then develops blown-up periaxonal collars and abnormally shaped non-compacted regions followed by myelin and axonal degeneration. Our findings strongly support the view that very few myelin components are necessary for myelin formation whereas the maintenance of myelin is much more sensitive to molecular alterations. In addition, it became evident that myelin molecules can fulfill functionally overlapping roles that ensure that myelination takes place even under conditions in which there is a deficiency in the normal molecular components of myelin.     

Late-onset neurodegeneration in mice with increased dosage of the proteolipid protein gene

Mutations of the proteolipid protein (Plp) gene cause a generalized central nervous system (CNS) myelin deficit in Pelizaeus-Merzbacher disease of man and various tremor syndromes in animal models. X-linked spastic paraplegia is also due to Plp gene mutations but has a different clinical profile and more restricted pathology involving specific tracts and regions. We have shown previously that PLP overexpression in mice homozygous for a Plp transgene results in premature arrest of CNS myelination and premature death. Here, we demonstrate that a low-level increase in Plp gene expression in transgenic mice causes significant axonal degeneration and demyelination with predilection for specific tracts. Following normal motor development, aged mice develop progressive myelin loss, axonal swellings with resultant Wallerian degeneration, and marked vacuolation of the neuropil associated with ataxia, tremor, and seizures. The age of onset and severity of the phenotype is a function of Plp gene dosage. The corticospinal tracts, optic nerve, fasciculus gracilis cerebellum, and brainstem are particularly involved. Although oligodendrocyte cell bodies show little abnormality, their inner adaxonal tongue is often abnormal, suggesting a perturbation of the axon/glial interface that may underlie the axonal changes. We conclude that abnormal expression of an oligodendrocyte-specific gene can cause axonal damage, a finding that is relevant to the pathogenesis of PLP-associated disorders and probably to other myelin-related diseases.     

CD8+ myelin peptide-specific T cells can chemoattract CD4+ myelin peptide-specific T cells: importance of IFN-inducible protein 10

The demyelination process that occurs in the central nervous system (CNS) of patients with multiple sclerosis (MS) is due, in part, to an inflammatory response in which CD4+ and CD8+ T cells and macrophages infiltrate white matter. While it is thought that the inflammatory and demyelination process in MS is the product of Th1-associated cytokines secreted by CD4+ myelin protein-specific T cells present in the CNS, the mechanisms that are responsible for the recruitment and maintenance of these myelin-reactive CD4+ T cells in the CNS have not been elucidated. We have shown previously that CD8+ CTL that recognize peptides derived from sequences of the myelin proteolipid protein (PLP) presented by HLA class I molecules can be generated in vitro, and that these PLP-specific CD8+ CTL secrete the proinflammatory chemokines macrophage-inflammatory protein-1alpha and -1beta, IL-16, and IP-10. In this study, we demonstrate that soluble products of these PLP-specific CD8+ CTL can chemoattract CD4+ T cells that are specific for a myelin basic protein peptide and a PLP peptide, and that the majority of this chemotactic activity is mediated by IFN-inducible protein 10. These results demonstrate that PLP-specific CD8+ T cells can play a role in the recruitment and retention of myelin-derived peptide-specific CD4+ T cells, and indicate that they may play a proinflammatory role in the pathogenesis of MS.     

Oligodendrocyte-type 2 astrocyte progenitors use a metalloendoprotease to spread and migrate on CNS myelin

Oligodendrocyte-type 2 astrocyte (O-2A) progenitors are highly motile cells which migrate in the developing and adult central nervous system (CNS). Adult CNS myelin, however, contains inhibitory proteins, the neurite growth inhibitors NI 35/250, which block neurite outgrowth and spreading of many different cell types, such as astrocytes and fibroblasts. In the present study we investigated the spreading of dissociated cells and migration out of aggregates ('spheres') of primary O-2A cultures and of a glial precursor cell line (CG-4) on purified CNS myelin and on CNS tissue. Primary O-2A progenitors and CG-4 cells quickly attached to and spread on CNS myelin-coated culture dishes, showing no inhibition by the neurite growth inhibitors. CG-4 cells migrated with a velocity of 30 microns/h on a CNS myelin protein extract and at 5.7 microns/h on adult spinal cord tissue. Both cell spreading and migration on a CNS substrate could be specifically blocked by metalloprotease blockers like o-phenanthroline and the tetrapeptide carbobenzoxy-phe-ala-phe-tyr-amide, whereas blockers of the serine, aspartyl and cysteine proteases had no effect. On differentiation to astrocytes, the O-2A progenitors lost their ability to spread on CNS myelin. These results suggest the crucial involvement of a metalloprotease in the mechanism of migration on a CNS substrate. In vivo, migration of oligodendrocyte progenitors may be an important aspect of myelin repair following local traumatic, inflammatory or toxin-induced myelin loss.     

A metalloprotease activity from C6 glioma cells inactivates the myelin-associated neurite growth inhibitors and can be neutralized by antibodies

Glioblastoma cells infiltrate brain tissue and migrate preferentially along white matter fibre tracts, an environment that is highly inhibitory to the migration of astrocytes and the growth of neurites because of the presence of specific inhibitory proteins. In vitro, spreading and migration of rat C6 glioma cells on a CNS (central nervous system) myelin substrate is correlated with and dependent on the presence of a metalloprotease. This membrane-bound metalloendoprotease exhibits a blocker profile different from known proteases. Pretreatment of CNS myelin or of a highly purified CNS myelin component, the inhibitory protein bNI-220, with C6 metalloproteolytic activity converts these non-permissive substrates into permissive environments for astrocytes and fibroblasts, indicating that this C6 cell-derived metalloprotease may inactivate myelin-associated inhibitory proteins. Antibodies were raised in chicken against fractions enriched in metalloproteolytic activity; these antibodies were able to inhibit specifically spreading and migration of C6 glioma cells on a CNS myelin substrate, as well as the invasion of C6 cells into adult rat optic nerve explants in vitro. These results suggest a crucial involvement of a membrane-bound metalloprotease in the mechanisms of C6 glioma migration and infiltration of brain tissue by proteolytic inactivation of the neurite growth inhibitory proteins present in CNS myelin.     

Amino acids within residues 181-200 of the nicotinic acetylcholine receptor alpha1 subunit involved in nicotine binding

Hematogenous macrophages and resident brain microglia are agents of demyelination in multiple sclerosis (MS) and paradoxically may also participate in remyelination. In vitro studies have shown that macrophage enrichment of aggregate brain cultures promotes myelination per se and enhances the capacity to remyelinate following a demyelinating episode. It has been hypothesized that remyelination in MS is implemented by surviving dedifferentiated oligodendrocytes or by newly recruited progenitors that migrate, proliferate and synthesize myelin in response to signalling molecules in the local environment. We postulate that macrophage-derived cytokines or growth factors may directly or indirectly promote oligodendroglial proliferation and differentiation, contributing to myelin repair in inflammatory demyelinating disease.     

Neurofibromatosis 1 and multiple sclerosis

Neurofibromatosis 1 is a common autosomal dominant disease that principally involves the skin and peripheral nervous system. The gene for the disorder has been located on chromosome 17q11.2 and there are three embedded genes within the neurofibrosis gene. One of these genes codes for oligodendrocyte-myelin glycoprotein, is found in the CNS during myelination, and may have a role in myelin formation. The case histories of five patients, including two siblings, who have both neurofibromatosis 1 and multiple sclerosis are reported. All five had the primary progressive form of multiple sclerosis, which forms only 15% of multiple sclerosis in population surveys. The coincidence of neurofibromatosis 1 and multiple sclerosis might be due to a mutation in the embedded oligodendrocyte-myelin glycoprotein gene.     

Developmental changes in neuronal responsiveness to the CNS myelin-associated neurite growth inhibitor NI-35/250

The extent of fibre regeneration in the adult injured vertebrate nervous system appears to be primarily determined by the local environment. Thus, the failure of axon regrowth in the central nervous system (CNS) is crucially influenced by the presence of the myelin-associated neurite growth inhibitor NI-35/250 and possibly also by molecules such as the myelin-associated glycoprotein and the proteoglycans. Developmental time course studies have shown that the capacity for regeneration declines sharply with the appearance of mature oligodendrocytes and myelin, which indicates a role of NI-35/250 in restricting CNS regeneration and plasticity. However, recent in vitro and in vivo studies showed that embryonic neurons are capable of extending fibres on and in adult CNS tissue apparently unaffected by myelinated areas. A possible explanation is that very immature neurons have yet to express the appropriate receptors and response mechanisms for factors that normally induce growth inhibition at a later stage of development. Here we report that embryonic rat dorsal root ganglion and chick retinal ganglion cells display different sensitivity to bovine NI-35/250 compared with mature neurons. In older neurons NI-35/250 could evoke long-lasting collapse responses accompanied by a large increase in the intracellular calcium level, persisting for several minutes. In contrast, their embryonic counterparts collapsed only transiently when exposed to NI-35/250, and increases in intracellular calcium concentration were small and transient. Calcium influx induced experimentally by the calcium ionophore A23187 revealed that it was not the maximal size of the calcium increase but rather the duration of elevated calcium concentration that was the most important determinant for subsequent morphological alterations of the growth cone. Our data further suggest that developing neurons acquire their complete sensitivity for NI-35/250 around the time of myelination.     

Myelination of S1 dorsal root axons in the cat

Samples of S1 dorsal root nerve fibers from cats of different pre- and postnatal ages were examined electron microscopically with regard to axon caliber and number of myelin lamellae. Each root was examined at four different cross-sectional levels. Two levels were situation close to the spinal cord entrance on each side of the peripheral (PNS) and central nervous system (CNS) border. The third and fourth levels were located more distally. The first compact myelin lamella was observed in the CNS part of the root in a 47-day-old fetus. In the 53-day-old fetus the degree of myelination was the same in the CNS as distal in the PNS part of the root. Surprisingly, all axons appeared unmyelinated close to the PNS-CNS border and remained so for a further 10-day period. After this time lag, this part of the root became myelinated and showed a rapid increase in myelin sheath thickness. Calculations of axonal growth, mesaxonal length, and myelin volume indicated a maturation process that progressed discontinuously. Myelination did not proceed in a strict somatofugal direction, but was a regionally differentiated process. The maximal myelin production, expressed as the increase in myelin volume per Schwann cell, was found during the second to fourth postnatal months, i.e., very late in development.     

Membrane-type 1 matrix metalloprotease (MT1-MMP) enables invasive migration of glioma cells in central nervous system white matter

Invasive glioma cells migrate preferentially along central nervous system (CNS) white matter fiber tracts irrespective of the fact that CNS myelin contains proteins that inhibit cell migration and neurite outgrowth. Previous work has demonstrated that to migrate on a myelin substrate and to overcome its inhibitory effect, rat C6 and human glioblastoma cells require a membrane-bound metalloproteolytic activity (C6-MP) which shares several biochemical and pharmacological characteristics with MT1-MMP. We show now that MT1-MMP is expressed on the surface of rat C6 glioblastoma cells and is coenriched with C6-MP activity. Immunodepletion of C6-MP activity is achieved with an anti-MT1-MMP antibody. These data suggest that MT1-MMP and the C6-MP are closely related or identical. When mouse 3T3 fibroblasts were transfected with MT1-MMP they acquired the ability to spread and migrate on the nonpermissive myelin substrate and to infiltrate into adult rat optic nerve explants. MT1-MMP-transfected fibroblasts and C6 glioma cells were able to digest bNI-220, one of the most potent CNS myelin inhibitory proteins. Plasma membranes of both MT1-MMP-transfected fibroblasts and C6 glioma cells inactivated inhibitory myelin extracts, and this activity was sensitive to the same protease inhibitors. Interestingly, pretreatment of CNS myelin with gelatinase A/MMP-2 could not inactivate its inhibitory property. These data imply an important role of MT1-MMP in spreading and migration of glioma cells on white matter constituents in vitro and point to a function of MT1-MMP in the invasive behavior of malignant gliomas in the CNS in vivo.     

Phosphodiesterase I, a novel adhesion molecule and/or cytokine involved in oligodendrocyte function

One of the more complex developmental processes occurring postnatally in the CNS is the formation of the myelin sheath by oligodendrocytes. To examine the molecular events that take place during myelination, we isolated oligodendrocyte-derived cDNA clones, one of which (p421.HB) represents a putative alternatively spliced isoform of rat brain-specific phosphodiesterase I (PD-Ialpha) and a species homolog of the human cytokine autotaxin. Analysis of the structural composition of the p421.HB/PD-Ialpha protein suggests a transmembrane-bound ectoenzyme, which, in addition to the phosphodiesterase-active site contains presumed cell recognition and Ca2+-binding domains. Consequently, it may be involved in extracellular signaling events. Expression of p421.HB/PD-Ialpha is enriched in brain and spinal cord, where its mRNA can be detected in oligodendrocytes and in cells of the choroid plexus. Expression in the brain increases during development with an intermediate peak of expression around the time of active myelination and maximal expression in the adult. We have identified four presumably alternatively spliced isoforms, two of which appear to be CNS-specific. Decreased levels of p421.HB/PD-Ialpha mRNA in the dysmyelinating mouse mutant jimpy, but not shiverer, suggest a role for p421.HB/PD-Ialpha during active myelination and/or late stages of oligodendrocyte differentiation. Furthermore, p421.HB/PD-Ialpha mRNA levels were reduced in the CNS at onset of clinical symptoms in experimental autoimmune encephalomyelitis. These data together implicate the importance of p421.HB/PD-Ialpha in oligodendrocyte function, possibly through cell-cell and/or cell-extracellular matrix recognition.     

The proteolipid protein gene

Proteolipid protein (PLP) is the major myelin protein of the CNS and is believed to have a structural role in maintaining the intraperiod line of compact myelin. An isoform, DM-20, produced by alternative splicing of exon 3B is expressed earlier than PLP in the CNS and may be involved in glial cell development. DM-20 is also present in myelin-forming and non-myelin-forming Schwann cells, olfactory nerve ensheathing cells, some glial cell lines and cardiac myocytes. Molecular studies suggest the existence of a PLP gene family with sequence similarities between molecules of different species. Such studies also lend credence to the suggestion that PLP and/or DM-20 may function as a membrane pore. Mutations in the PLP gene occur in several animal species and cause severe pleiotropic effects on myelination. In man this presents as Pelizaeus-Merzbacher disease (PMD). The phenotype of such mutants is characterized by dysmyelination with myelin of abnormal periodicity, paucity of mature oligodendrocytes and astrocytosis. Duplication of the PLP gene in transgenic animals or in one form of PMD also results in dysmyelination. X-linked spastic paraplegia (SPG2) is allelic to PMD and is associated with PLP mutations in which the levels of the DM-20 isoform are probably relatively normal. The effects of PLP gene dosage on CNS myelination can be compared in many ways to the variety of phenotypes in the PNS in hereditary neuropathies of the Charcot-Marie-Tooth type in which the peripheral myelin-22 gene is mutated.     

Ex vivo and in vitro testis and ovary explants: utility for identifying steroid biosynthesis inhibitors and comparison to a Tier I screening battery

Retinal ganglion cell (RGC) axons in lizards (reptiles) were found to regenerate after optic nerve injury. To determine whether regeneration occurs because the visual pathway has growth-supporting glia cells or whether RGC axons regrow despite the presence of neurite growth-inhibitory components, the substrate properties of lizard optic nerve myelin and of oligodendrocytes were analyzed in vitro, using rat dorsal root ganglion (DRG) neurons. In addition, the response of lizard RGC axons upon contact with rat and reptilian oligodendrocytes or with myelin proteins from the mammalian central nervous system (CNS) was monitored. Lizard optic nerve myelin inhibited extension of rat DRG neurites, and lizard oligodendrocytes elicited DRG growth cone collapse. Both effects were partially reversed by antibody IN-1 against mammalian 35/250 kD neurite growth inhibitors, and IN-1 stained myelinated fiber tracts in the lizard CNS. However, lizard RGC growth cones grew freely across oligodendrocytes from the rat and the reptilian CNS. Mammalian CNS myelin proteins reconstituted into liposomes and added to elongating lizard RGC axons caused at most a transient collapse reaction. Growth cones always recovered within an hour and regrew. Thus, lizard CNS myelin and oligodendrocytes possess nonpermissive substrate properties for DRG neurons--like corresponding structures and cells in the mammalian CNS, including mammalian-like neurite growth inhibitors. Lizard RGC axons, however, appear to be far less sensitive to these inhibitory substrate components and therefore may be able to regenerate through the visual pathway despite the presence of myelin and oligodendrocytes that block growth of DRG neurites.     

The jimpy mouse

Ultrastructural study of the Jimpy mice shows lack of myelin in the central nervous system: low density of glial cells, defect of maturation of oligodendrocytes and deficiency in axonal growth. The lack of myelin is more proeminent in late myelinating structures: corpus callosum and corticospinal fasciculus. Failure in myelin is rather the consequence of an early interruption of myelination as that of demyelination (a destruction of the sheats).