[Promoting myelin repair in disorders such as multiple sclerosis and some types of leukodystrophy: current studies]. / Approche expérimentale des stratégies réparatrices des maladies demyélinisantes du système nerveux central.

Several ways of promoting myelin repair in myelin disorders such as multiple sclerosis and certain types of leukodystrophies are currently being investigated. Numerous studies suggest that it is possible to repair the central nervous system (CNS) by cell transplantation or by enhancing endogenous remyelination. Investigations in animal models indicate that cell therapy results in robust anatomical and functional recovery of acute myelin lesions. These models are also used to explore and validate the role of candidate molecules to stimulate endogenous remyelination by activating the myelin competent population or providing neuroprotection. However, in view of the heterogeneity of the lesion environment in MS, it seems more likely that cell therapy alone will not be able to contribute efficiently to the repair of the lesion. Further developments should indicate whether combining multiple approaches will be more powerful to achieve global myelin repair in the CNS than applying these strategies alone.

Molecular cloning and nucleotide sequence of a cDNA clone coding for rat brain myelin proteolipid.

A cDNA library from rat brain was constructed in pBR322 and screened with a 14-mer mixed oligonucleotide probe based on residues 231-235 of bovine proteolipid (PLP). A positive clone was isolated: it contained a 1334-base-pair cDNA insert and was subjected to DNA sequence analysis. The cDNA encoded information for the 276 amino acids of rat PLP. Comparison with bovine PLP sequence showed a complete amino acid sequence homology except for 4 amino acid residues.

Proteolipid DM-20 predominates over PLP in peripheral nervous system.

The major central nervous system (CNS) myelin proteolipid (PLP) is also expressed in the peripheral nervous system (PNS). This paper gives evidence that DM-20, an isoform of PLP, also occurs in rat sciatic nerves, where, in contrast to what is seen in CNS myelin, it predominates over PLP. This conclusion was reached on the basis of results obtained by immunoblot analysis of a crude proteolipid extract from adult peripheral nerve with two site-specific anti-proteolipid (PLP and DM-20) antibodies. This finding was further corroborated by characterization of the products obtained by Polymerase Chain Reaction (PCR) amplification of cDNAs synthesized from total RNA of 14-day-old sciatic nerves. The significance of the occurrence of these proteolipids in PNS remains obscure.

Infantile neuropathy with unstable myelin: study of the P0 protein.

An unusual form of hereditary motor and sensory neuropathy characterized by a prominent disruption of the myelin lamellae is reported. In addition to detailed morphological analysis, we investigated the protein P0, which is the major protein of peripheral myelin involved in adhesion. No major gene rearrangement and no differences in P0 protein expression were observed in the present case.

The Brown Norway opticospinal model of demyelination: does it mimic multiple sclerosis or neuromyelitis optica?

Opticospinal demyelinating diseases in humans are mostly characterized by the opticospinal form of multiple sclerosis (MS) and neuromyelitis optica (NMO). Increasing attention has recently focused on astrocyte markers, aquaporin-4 (AQP4) and glial fibrillary acidic protein (GFAP) in these diseases. We induced opticospinal demyelination in Brown Norway rats with soluble recombinant rat myelin oligodendrocyte glycoprotein (1-116) and incomplete Freund's adjuvant. Clinical, MRI, neuropathological and immunological evaluations were performed, with a focus on AQP4 and GFAP. We confirmed the opticospinal phenotype, including extensive myelitis, but also showed the MRI-characterized involvement of the periventricular area. Expression levels of myelin, AQP4 and GFAP showed the early involvement of astrocytes before demyelination in the optic nerve. The overexpression of AQP4 was particularly pronounced in the spinal cord and was concomitant with demyelination and astrocyte apoptosis. The disability scores were correlated with demyelination and inflammation but not with AQP4/GFAP expression. No antibodies against the linear and conformational epitopes of AQP4 were detected. Whereas a NMO-like phenotype was observed in this model, the AQP4/GFAP expression during the disease process was more closely related to opticospinal MS than NMO. However, this model raises the question of a continuum between opticospinal MS and the seronegative NMO subtype.

Biology of oligodendrocyte and myelin in the mammalian central nervous system.

Oligodendrocytes, the myelin-forming cells of the central nervous system (CNS), and astrocytes constitute macroglia. This review deals with the recent progress related to the origin and differentiation of the oligodendrocytes, their relationships to other neural cells, and functional neuroglial interactions under physiological conditions and in demyelinating diseases. One of the problems in studies of the CNS is to find components, i.e., markers, for the identification of the different cells, in intact tissues or cultures. In recent years, specific biochemical, immunological, and molecular markers have been identified. Many components specific to differentiating oligodendrocytes and to myelin are now available to aid their study. Transgenic mice and spontaneous mutants have led to a better understanding of the targets of specific dys- or demyelinating diseases. The best examples are the studies concerning the effects of the mutations affecting the most abundant protein in the central nervous myelin, the proteolipid protein, which lead to dysmyelinating diseases in animals and human (jimpy mutation and Pelizaeus-Merzbacher disease or spastic paraplegia, respectively). Oligodendrocytes, as astrocytes, are able to respond to changes in the cellular and extracellular environment, possibly in relation to a glial network. There is also a remarkable plasticity of the oligodendrocyte lineage, even in the adult with a certain potentiality for myelin repair after experimental demyelination or human diseases.

Structure and polymorphism of the mouse myelin/oligodendrocyte glycoprotein gene.

We have isolated and characterized genomic clones containing the mouse myelin/oligodendrocyte glycoprotein (MOG) gene. It spans a region of 12.5 kb and consists of eight exons. Its exon-intron structure differs from that of classical MHC-class I genes, with which it is linked in the mouse genome. Nucleotide sequencing of the 5' flanking region reveals that it contains several putative protein-binding sites, some of them in common with other myelin gene promoters. One intragenic polymorphism has been identified: it consists of a GA repeat, defining at least three alleles in mouse inbred strains, and is easily detectable using the polymerase chain reaction method.

Studies on complementation in argininosuccinate synthetase and argininosuccinate lyase deficiencies in human fibroblasts.

Complementation tests after polyethylene glycol fusion have been performed between 10 citrullinemic strains with argininosuccinate synthetase deficiency and between five strains with argininosuccinate lyase deficiency. No complementation was observed between the citrullinemic strains, while two groups of complementation were defined with the argininosuccinate lyase deficients strains. Since the restoration of activity was not total, we assumed that complementation is probably intragenic.

Major Myelin proteolipid: the 4-alpha-helix topology.

Several conflicting models have been proposed for the membrane arrangement of the major myelin proteolipid (PLP). We have compared features of the sequence of PLP with those of other eukaryotic integral membrane proteins, with the view of identifying the most likely transmembrane topology. A new, simple model is suggested, which features four hydrophobic alpha-helices spanning the whole thickness of the lipid bilayer. Its orientation may be such that both the N- and C-termini face the cytosol. None of the biochemical, biophysical or immunological experiments hitherto reported provides incontrovertible evidence against the model. The effect or absence thereof of various PLP mutations is discussed in the frame of the proposed 4-helix topology.

Myelin proteolipid protein (PLP and DM-20) transcripts are deleted in jimpy mutant mice.

The myelin-associated proteolipid protein, PLP, is one of the two major components of the central nervous system (CNS) myelin. We analyze, by using a rat PLP cDNA and S1 nuclease protection experiments, the PLP transcripts in the mouse brain and show that the PLP gene encodes two different but related mRNA transcripts, the PLP and the DM-20 transcripts. On the other hand, we demonstrate that in the jimpy mutant, which is characterized by an abnormal CNS myelination, both these transcripts are partially deleted in the 3' end of their coding region. The deletion is 70 (+/- 5) nucleotides long. Implications of this finding for the synthesis of PLP and DM-20 proteins in the mutant are discussed.

Alexander disease: putative mechanisms of an astrocytic encephalopathy.

Alexander disease (AXD) is the first primary astrocytic disorder. This encephalopathy is caused by dominant mutations in the glial fibrillary acidic protein (GFAP) gene, encoding the main intermediate filament of astrocyte. Pathologically, this neurodegenerative disease is characterised by dystrophic astrocytes containing intermediate filament aggregates associated with myelin abnormalities. More than 20 GFAP mutations have been reported. Many of them cluster in highly conserved regions between several intermediate filaments. Contrary to other intermediate filament-related diseases, AXD seems to be the consequence of a toxic gain of function induced by aggregates. This is supported by the phenotype of mice overexpressing human GFAP. Nevertheless, GFAP null mice display myelin abnormalities and blood-brain barrier dysfunction that are present in AXD. Given the pivotal role of astrocytes in brain physiology, there are many possibilities for astrocytes to dysfunction and to impair the functions of other cells. Physiopathological hypotheses are discussed in the frame of AXD.

The E. coli envY gene encodes a high affinity opioid binding site.

In an attempt to isolate a cDNA encoding an opioid receptor, a cDNA library was constructed in the lambda ZAP vector using NG108-15 mRNA as template. Using an in vitro transcription-translation assay and a sib selection strategy, a single phage was isolated. An RNA transcribed from this cDNA was able to direct in vitro translation of opioid binding sites. The insert was sequenced and comparison with data banks showed a 100% homology with the E. coli envY gene. We assume that the presence of the envY sequence in the NG108-15 cDNA library was due to a contamination of the lambda ZAP vector with E. coli DNA. A search for opioid binding sites on E. coli strains showed that envY+ strains, but not envY- mutants were able to bind opiates. On envY+ cells, the sites are stereospecific, saturable and of high affinity for the opiate ligands. These sites bind opiate agonists and antagonists but neither mu nor delta opioid peptides. In contrast, rabbit reticulocyte lysate primed with RNA transcribed in vitro from the envY sequence elicited the synthesis of an opioid binding site with mixed mu and delta properties. In addition, transfection of the envY sequence into mammalian cells resulted in the expression of opioid binding sites. Depending on the type of cells transfected, these sites were selective for either the mu or delta ligands.

Structure of the human myelin/oligodendrocyte glycoprotein gene and multiple alternative spliced isoforms.

Myelin/oligodendrocyte glycoprotein (MOG), a specific component of the central nervous system localized on the outermost lamellae of mature myelin, is a member of the immunoglobulin superfamily. We report here the organization of the human MOG gene, which spans approximately 17 kb, and the characterization of six MOG mRNA splicing variants. The intron/exon structure of the human MOG gene confirmed the splicing pattern, supporting the hypothesis that mRNA isoforms could arise by alternative splicing of a single gene. In addition to the eight exons coding for the major. MOG isoform, the human MOG gene also contains, in the 3' region, a previously unknown alternatively spliced coding exon, VIA. Alternative utilization of two acceptor splicing sites for exon VIII could produce two different C-termini. The nucleotide sequences presented here may be a useful tool to study further possible involvement of the MOG gene in hereditary neurological disorders.

Membrane topology of the myelin/oligodendrocyte glycoprotein.

Myelin/oligodendrocyte glycoprotein (MOG), a specific component of the mammalian central nervous system, is located on the surface of the oligodendrocyte plasma membrane and the outermost lamellae of mature myelin; it is expressed during the latter steps of myelinogenesis. It has been shown that MOG may play a pathological role in autoimmune demyelinating diseases of the central nervous system, although its physiological function remains unknown. MOG is an integral membrane glycoprotein with an extracellular immunoglobulin-like domain and two hydrophobic segments which were predicted to be membrane-spanning on the basis of hydropathy analysis. As a first step in elucidation of MOG function, we have investigated its membrane topology, combining immunofluorescence studies on cultured oligodendrocytes and MOG-transfected Chinese hamster ovary cells with biochemical analyses, including in vitro translation, membrane insertion and protease-digestion assays. Our results indicate that the C-terminal tail of MOG is located into the cytoplasm, and that only the first hydrophobic region of MOG spans the membrane whereas the second hydrophobic region appears to be semi-embedded in the lipid bilayer, lying partially buried in the membrane with its N-terminal and C-terminal boundaries facing the cytoplasm.

Connexin32 mutations associated with X-linked Charcot-Marie-Tooth disease show two distinct behaviors: loss of function and altered gating properties.

The X-linked form of Charcot-Marie-Tooth disease (CMTX) is associated with mutations in the gene encoding connexin32 (Cx32), which is expressed in Schwann cells. We have compared the functional properties of 11 Cx32 mutations with those of the wild-type protein by testing their ability to form intercellular channels in the paired oocyte expression system. Although seven mutations were functionally incompetent, four others were able to generate intercellular currents of the same order of magnitude as those induced by wild-type Cx32 (Cx32wt). In homotypic oocyte pairs, CMTX mutations retaining functional activity induced the development of junctional currents that exhibited changes in the sensitivity and kinetics of voltage dependence with respect to that of Cx32wt. The four mutations were also capable of interacting in heterotypic configuration with the wild-type protein, and in one case the result was a marked rectification of junctional currents in response to voltage steps of opposite polarity. In addition, the functional CMTX mutations displayed the same selective pattern of compatibility as Cx32wt, interacting with Cx26, Cx46, and Cx50 but failing to do so with Cx40. Although the functional mutations exhibited sensitivity to cytoplasmic acidification, which induced a >/=80% decrease in junctional currents, both the rate and extent of channel closure were enhanced markedly for two of them. Together, these results indicate that the functional consequences of CMTX mutations of Cx32 are of two drastically distinct kinds. The presence of a functional group of mutations suggests that a selective deficit of Cx32 channels may be sufficient to impair the homeostasis of Schwann cells and lead to the development of CMTX.

An immortalized jimpy oligodendrocyte cell line: defects in cell cycle and cAMP pathway.

Normal and jimpy oligodendrocytes in secondary cultures were transfected with plasmids containing the SV40 T-antigen gene expressed under the control of the mouse metallothionein-I promoter. Two immortalized stable cell lines, a normal (158N) and jimpy (158JP) cell line, expressed transcripts and proteins of oligodendrocyte markers, including proteolipid protein (PLP), myelin basic protein (MBP), and carbonic anhydrase II (CAII). Galactocerebroside and sulfatide were also detected with immunocytochemistry. Immunoelectron microscopy using gold particles showed that the truncated endogenous jimpy PLP was distributed throughout the cytoplasm and in association with the plasma membrane of cell bodies and processes. The length of the cell cycle in the jimpy oligodendrocytes in the absence of zinc was 31 h, about a 4-h longer cell cycle than the normal line. In the presence of 100 microM zinc, the cell cycle became 3 h shorter for both cell lines, with the jimpy cell cycle duration remaining 4 h longer than the normal line. Interestingly, the jimpy cell line showed a significant deficiency in stimulation via the cAMP pathway. While the level of oligodendrocyte markers (PLP, MBP, and CAII) were significantly increased by dibutyryl cAMP (dbcAMP) treatment in the normal cell line, no changes were observed in the jimpy cell lines. This observation, together with previous results showing jimpy oligodendrocyte's failure to respond to basic fibroblast growth factor (bFGF), suggests a role for PLP in a signal transduction pathway. Jimpy and normal oligodendrocytes transfected with the SV40T antigen gene, driven by the wild-type promoter of mouse metallothionein-I, continue to express properties of oligodendrocytes and therefore provide a powerful model to explore the function of myelin proteins and to dissect the complexity of the jimpy phenotype.

Development of myelin oligodendrocyte glycoprotein autoreactive transgenic B lymphocytes: receptor editing in vivo after encounter of a self-antigen distinct from myelin oligodendrocyte glycoprotein.

We explored mechanisms involved in B cell self-tolerance against brain autoantigens in a double-transgenic mouse model carrying the Ig H-chain (introduced by gene replacement) and/or the L-chain kappa (conventional transgenic) of the mAb 8.18C5, specific for the myelin oligodendrocyte glycoprotein (MOG). Previously, we demonstrated that B cells expressing solely the MOG-specific Ig H-chain differentiate without tolerogenic censure. We show now that double-transgenic (THkappa(mog)) B cells expressing transgenic Ig H- and L-chains are subjected to receptor editing. We show that in adult mice carrying both MOG-specific Ig H- and L-chains, the frequency of MOG-binding B cells is not higher than in mice expressing solely the transgenic Ig H-chain. In fact, in THkappa(mog) double-transgenic mice, the transgenic kappa(mog) L-chain was commonly replaced by endogenous L-chains, i.e., by receptor editing. In rearrangement-deficient RAG-2(-) mice, differentiation of THkappa(mog) B cells is blocked at an immature stage (defined by the B220(low)IgM(low)IgD(-) phenotype), reflecting interaction of the autoreactive B cells with a local self-determinant. The tolerogenic structure in the bone marrow is not classical MOG, because back-crossing THkappa(mog) mice into a MOG-deficient genetic background does not lead to an increase in the proportion of MOG-binding B cells. We propose that an as yet undefined self-Ag distinct from MOG cross-reacts with the THkappa(mog) B cell receptor and induces editing of the transgenic kappa(mog) L-chain in early immature B cells without affecting the pathogenic potential of the remaining MOG-specific B cells. This phenomenon represents a particular form of chain-specific split tolerance.