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.

[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.

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.

Infantile Alexander disease: spectrum of GFAP mutations and genotype-phenotype correlation.

Heterozygous, de novo mutations in the glial fibrillary acidic protein (GFAP) gene have recently been reported in 12 patients affected by neuropathologically proved Alexander disease. We searched for GFAP mutations in a series of patients who had heterogeneous clinical symptoms but were candidates for Alexander disease on the basis of suggestive neuroimaging abnormalities. Missense, heterozygous, de novo GFAP mutations were found in exons 1 or 4 for 14 of the 15 patients analyzed, including patients without macrocephaly. Nine patients carried arginine mutations (four had R79H; four had R239C; and one had R239H) that have been described elsewhere, whereas the other five had one of four novel mutations, of which two affect arginine (2R88C and 1R88S) and two affect nonarginine residues (1L76F and 1N77Y). All mutations were located in the rod domain of GFAP, and there is a correlation between clinical severity and the affected amino acid. These results confirm that GFAP mutations are a reliable molecular marker for the diagnosis of infantile Alexander disease, and they also form a basis for the recommendation of GFAP analysis for prenatal diagnosis to detect potential cases of germinal mosaicism.

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.

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.

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.

Linkage analysis of candidate myelin genes in familial multiple sclerosis.

Multiple sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system. A complex genetic etiology is thought to underlie susceptibility to this disease. The present study was designed to analyze whether differences in genes that encode myelin proteins influence susceptibility to MS. We performed linkage analysis of MS to markers in chromosomal regions that include the genes encoding myelin basic protein (MBP), proteolipid protein (PLP), myelin-associated glycoprotein (MAG), oligodendrocyte myelin glycoprotein (OMGP), and myelin oligodendrocyte glycoprotein (MOG) in a well-characterized population of 65 multiplex MS families consisting of 399 total individuals, 169 affected with MS and 102 affected sibpairs. Physical mapping data permitted placement of MAG and PLP genes on the Genethon genetic map; all other genes were mapped on the Genethon genetic map by linkage analysis. For each gene, at least one marker within the gene and/or two tightly linked flanking markers were analyzed. Marker data analysis employed a combination of genetic trait model-dependent (parametric) and model-independent linkage methods. Results indicate that MAG, MBP, OMGP, and PLP genes do not have a significant genetic effect on susceptibility to MS in this population. As MOG resides within the MHC, a potential role of the MOG gene could not be excluded.

Increased density of oligodendrocytes in childhood ataxia with diffuse central hypomyelination (CACH) syndrome: neuropathological and biochemical study of two cases.

We report neuropathological, biochemical and molecular studies on two patients with childhood ataxia with diffuse central nervous system hypomyelination (CACH) syndrome, a leukodystrophy recently defined according to clinical and radiological criteria. Both had severe cavitating orthochromatic leukodystrophy without atrophy, predominating in hemispheric white matter, whereas U-fibers, internal capsule, corpus callosum, anterior commissure and cerebellar white matter were relatively spared. The severity of white matter lesions contrasted with the rarity of myelin breakdown products and astroglial and microglial reactions. In the white matter, there was an increase in a homogeneous cell population with the morphological features of oligodendrocytes, in many instances presenting an abundant cytoplasm like myelination glia. These cells were negative for glial fibrillary acidic protein and antibodies PGM1 and MIB1. Some were positive for myelin basic protein, proteolipid protein (PLP), and myelin oligodendrocyte glycoprotein, but the majority were positive for human 2'-3' cyclic nucleotide 3' phosphodiesterase and all were positive for carbonic anhydrase II, confirming that they are oligodendrocytes. Myelin protein and lipid content were reduced. The PLP gene, analyzed in one case, was not mutated or duplicated. The increased number of oligodendrocytes without mitotic activity suggests an intrinsic oligodendroglial defect or an abnormal interaction with axons or other glial cells. This neuropathological study supports the notion that CACH syndrome constitutes a specific entity.

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.

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.

New mutations in the X-linked form of Charcot-Marie-Tooth disease.

Mutations in the gene for connexin 32 are associated with a chromosome X-linked form of Charcot-Marie-Tooth disease. The prevalence of this form is probably underestimated. We screened 12 candidate families and found 7 missense mutations of which 4 are new. These mutations are located in intra- and extramembraneous parts of the protein. Some mutations are probably present with a higher frequency. This study further confirms variation of connexin 32 mutations with scarcity in the second transmembrane domain and, so far, absence in the fourth transmembrane domain and in the carboxy-terminal region.

Identification of a Val 145 Ile substitution in the human myelin oligodendrocyte glycoprotein: lack of association with multiple sclerosis. The Réseau de Recherche Clinique INSERM sur la Susceptibilité Génétique à la Sclérose en Plaques.

Myelin/oligodendrocyte glycoprotein (MOG) is a major target antigen in experimental autoimmune encephalomyelitis and it has been suggested that it may as well play a key role in the demyelination process in multiple sclerosis (MS). As MOG variants could be pathogenic in autoimmune demyelinating diseases of the central nervous system, we analysed the coding sequence of MOG in MS patients and described a G-->A transition occurring in exon 3 of the human MOG gene. The mutation predicts that isoleucine substitutes for a valine at codon 145 (Val 145 Ile) in the transmembrane region of the protein. This is the first aminoacid substitution reported in human MOG. The polymorphism can be detected by restriction enzyme digestion of genomic DNA or reverse-transcribed PCR amplified products, making it a simple tool to detect a potential implication of MOG alleles in susceptibility to MS by association study. The analysis of 83 unrelated MS patients and 82 unrelated healthy controls showed that the polymorphism is found in similar proportions in MS patients (18%) and controls (14.6%). It is therefore unlikely that the MOG Val 145 Ile variant is responsible for genetic susceptibility to MS.

X-linked dominant Charcot-Marie-Tooth neuropathy (CMTX): new mutations in the connexin32 gene.

X-linked dominant Charcot-Marie-Tooth (CMTX) neuropathy has been mapped to the Xq13 region. Subsequently, several mutations that could account for CMTX have been detected in the coding part of the connexin32 (Cx32) gene, which is located within this region. In order to develop more specific diagnostic tools, we have begun a systematic screening of families with dominant CMTX for mutations in the coding region of the Cx32 gene. This report describes a study of ten families and different mutations segregating with the disease were detected in five of them. In addition to the previously reported Arg22stop and Arg215Trp substitutions, three novel mutations are described, including two different missense mutations at codon Arg22 (Arg22Pro and Arg22Gly), and a nonsense mutation at codon Trp133. The identification of new CMTX-causing mutations is a critical step for carrier detection and presymptomatic diagnosis, and should provide essential information on the structure-function relationship of Cx32 in vitro as well as in vivo.

Charcot-Marie-Tooth type 1B neuropathy: third mutation of serine 63 codon in the major peripheral myelin glycoprotein PO gene.

We report studies on two patients (a mother and her daughter) presenting with a Charcot-Marie-Tooth type 1 (CMT1) phenotype: low nerve conduction velocities of 13-15 m/s and an early onset at the age of walking. DNA analysis of the gene coding for the major peripheral myelin protein PO showed a new point mutation in exon 2, which resulted in substitution of a phenylalanine for serine at amino acid position 63 of PO. This is the third mutation reported at this codon, the two previously described leading to CMT1B (serine 63 deletion), or to Dejerine-Sottas disease (cysteine for serine 63 substitution), suggesting that different phenotypes can result from alteration of a single amino acid, depending on the type of the change involved.

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.

Mutations in the myelin protein zero gene associated with Charcot-Marie-Tooth disease type 1B.

Charcot-Marie-Tooth type 1 (CMT1) disease is an autosomal dominant neuropathy of the peripheral nerve. The majority of CMT 1 cases are due to a duplication of an 1.5-Mb DNA fragment on chromosome 17p11.2 (CMT 1a). Micromutations were found in the gene for peripheral myelin protein 22 (PMP22) located in the duplicated region of CMT 1a, and in the peripheral myelin protein zero (PO) located on chromosome 1q21-q23 (CMT 1b). We have characterized two new mutations in the PO gene in two french families presenting CMT disease. Both mutations occur in the extracellular domain of the PO protein. One mutation is a de novo mutation and is from paternal origin.

Physical mapping of the human and mouse MOG gene at the distal end of the MHC class Ib region.

Myelin/oligodendrocyte glycoprotein (MOG) is expressed specifically in the central nervous system (CNS) by myelinating glial cells, the oligodendrocytes. The external location of MOG on myelin sheaths and its late expression during myelinogenesis argue for a role of MOG in the completion of myelin and maintenance of its integrity. MOG is a target autoantigen in demyelinating diseases, such as experimental autoimmune encephalomyelitis (EAE) in animals and multiple sclerosis (MS) in humans. We previously located the gene encoding MOG to the major histocompatibility complex (MHC), both in human, by cytogenetics, and in mouse, by analysis of recombinants. To refine the position, we have now selected yeast artificial chromosome clones (YAC) which contain the MOG gene. Physical mapping of the human MOG and the mouse Mog genes by characterization of these YAC clones indicated that the gene is located at the distal end of the major histocompatibility complex (MHC) class Ib region in both species. The human MOG gene lies 60 kilobases (kb) telomeric to HLA-F in a head-to-head orientation; the mouse Mog gene lies 25 (kb) telomeric to H2-M5 in a tail-to-head orientation. These orthologous genes provide markers for comparative analysis of the evolution of the MHC in the two species. The physical mapping of MOG should facilitate analysis of its role in hereditary neurological diseases, and the YAC clones identified here will permit the identification of new genes in the region.

Rapid screening of myelin genes in CMT1 patients by SSCP analysis: identification of new mutations and polymorphisms in the P0 gene.

Charcot-Marie-Tooth type 1 (CMT1) disease or hereditary motor and sensory neuropathy type I (HMSNI) is an autosomal dominant peripheral neuropathy. In most CMT1 families, the disease cosegregates with a 1.5-Mb duplication on chromosome 17p11.2 (CMT1A). A few patients have been found with mutations in the peripheral myelin protein 22 (PMP-22) gene located in the CMT1A region. In other families mutations have been identified in the major peripheral myelin protein P0 gene localized on chromosome 1q21-q23 (CMT1B). We performed a rapid mutation screening of the PMP-22 and P0 genes in non-duplicated CMT1 patients by single-strand conformation polymorphism analysis followed by direct polymerase chain reaction sequencing of genomic DNA. Six new single base changes in the P0 gene were observed: two missense mutations in, respectively, exons 2 and 3, two nonsense mutations in exon 4, and two silent mutations or polymorphisms in, respectively, exons 3 and 6.