Alexander disease (AxD) is a rare neurodegenerative disorder characterized by large cytoplasmic aggregates in astrocytes and myelin abnormalities and caused by dominant mutations in the gene encoding glial fibrillary acidic protein (GFAP), the main intermediate filament protein in astrocytes. We tested the effects of three mutations (R236H, R76H and L232P) associated with AxD in cells transiently expressing mutated GFAP fused to green fluorescent protein (GFP). Mutated GFAP-GFP expressed in astrocytes formed networks or aggregates similar to those found in the brains of patients with the disease. Time-lapse recordings of living astrocytes showed that aggregates of mutated GFAP-GFP may either disappear, associated with cell survival, or coalesce in a huge juxtanuclear structure associated with cell death. Immunolabeling of fixed cells suggested that this gathering of aggregates forms an aggresome-like structure. Proteasome inhibition and immunoprecipitation assays revealed mutated GFAP-GFP ubiquitination, suggesting a role of the ubiquitin–proteasome system in the disaggregation process. In astrocytes from wild-type-, GFAP-, and vimentin-deficient mice, mutated GFAP-GFP aggregated or formed a network, depending on qualitative and quantitative interactions with normal intermediate filament partners. Particularly, vimentin displayed an anti-aggregation effect on mutated GFAP. Our data indicate a dynamic and reversible aggregation of mutated GFAP, suggesting that therapeutic approaches may be possible.

Myelin/oligodendrocyte glycoprotein (MOG) is a minor integral membrane protein specific to CNS myelin, encoded by a gene located in the major histocompatibility complex. MOG is an highly encephalitogenic autoantigen and a target for autoaggressive immune responses in CNS inflammatory demyelinating diseases. We performed transcriptomic analyses for a gene expressed only in mammalian CNS, myelin oligodendrocyte glycoprotein (MOG). Complex splicing patterns were exclusively found in primates and not in mice, unlike patterns found for other myelin protein genes. In addition to those shared with rodents, these multiple MOG isoforms likely support functions unique to the primate order, in particular maintenance of myelin structure, intracellular signaling, and modulation of CNS autoimmunity via exposure of specific MOG determinants. Developmentally, in human brain the splice variants of MOG appear at a late stage compared to the major isoform, coincidental with myelination and myelin maturation, unlike other myelin proteins. These findings are discussed within the framework of a biological basis for phenotype diversity in recent mammalian evolution and for the notoriously variable clinical expression of diseases such as multiple sclerosis.

Myelin oligodendrocyte glycoprotein (MOG) is a protein of great neuroimmunological interest, whose function is not known. This chapter examines the structural functional relationships of MOG in myelin. Two different strategies are used to clone MOG mRNAs. One uses a mixture of MOG mAbs to screen a rat ƛgt11 expression library, whereas other used degenerate primers developed from the N-terminal amino acid sequence of bovine MOG to screen bovine, rat, and mouse brain ƛgt10 phage libraries. Subsequently, the mouse gene is isolated and its organization characterized. Similar approaches are used to characterize the human MOG cDNA and the human gene. Sequence analyses indicate that the mature protein is highly conserved between species and consists of 218 amino acids comprising a putative signal peptide, an immunoglobulin (Ig)-like N-terminal domain, two highly hydrophobic segments separated by a short hydrophilic loop, and a short hydrophilic C-terminal domain. The presence of two putative membrane-spanning domains, if verified, would make MOG a unique member of the Ig super-gene family (IgSF).

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.

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.

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.

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.

The complete 129-amino-acid sequences of two rainbow trout lysozymes (I and II) isolated from kidney were established using protein chemistry microtechniques. The two sequences differ only at position 86, I having aspartic acid and II having alanine. A cDNA clone coding for rainbow trout lysozyme was isolated from a cDNA library made from liver mRNA. Sequencing of the cloned cDNA insert, which was 1 kb in length, revealed a 432-bp open reading frame encoding an amino-terminal peptide of 15 amino acids and a mature enzyme of 129 amino acids identical in sequence to II. Forms I and II from kidney and liver were also analyzed using enzymatic amplification via PCR and direct sequencing; both organs contain mRNA encoding the two lysozymes. Evolutionary trees relating DNA sequences coding for lysozymes c and alpha-lactalbumins provide evidence that the gene duplication giving rise to conventional vertebrate lysozymes c and to lactalbumin preceded the divergence of fishes and tetrapods about 400 Myr ago. Evolutionary analysis also suggests that amino acid replacements may have accumulated more slowly on the lineage leading to fish lysozyme than on those leading to mammal and bird lysozymes.

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.