Minnetta V. Gardinier

The N-terminal domain of the myelin oligodendrocyte glycoprotein (MOG) induces acute demyelinating experimental autoimmune encephalomyelitis in the Lewis rat

Using a highly purified recombinant protein, mMOG, we demonstrated that autoimmune responses to the N-terminal domain (a.a 1-125) of the myelin oligodendrocyte glycoprotein (MOG) induce an acute demyelinating variant of experimental autoimmune encephalomyelitis (EAE) in the Lewis rat. Immunisation with 100 micrograms of mMOG in adjuvant at the base of the tail induced mild clinical disease in 9 of 11 animals (mean clinical score 1.1). The disease was characterised histopathologically by the presence of inflammation and focal demyelinating lesions in the central nervous system (CNS). Adoptive transfer experiments suggest that this inflammatory demyelinating pathology is mediated by synergy between a weakly encephalitogenic, MOG-specific T cell response and a demyelinating, MOG-specific autoantibody response. Using in vitro selected mMOG-reactive T cell lines, the encephalitogenic T cell response to this domain of MOG was found to recognise two distinct epitopes, MOG1-20 and MOG35-55; whereas ELISA demonstrated that the immunodominant B cell epitope was located within the amino acid sequence MOG1-25. However although active immunisation with synthetic peptides corresponding to the T cell epitopes, MOG1-20 or MOG35-55, induced an inflammatory response in the CNS, this was not associated with demyelination indicating that the demyelinating antibody response recognises other, possibly conformation dependent epitopes. This study unequivocally demonstrates that MOG-specific autoimmune responses are alone sufficient to induce a demyelinating disease of the CNS and supports the proposal that MOG may play an important role in the immunopathogenesis of multiple sclerosis.

Epitope specificity of demyelinating monoclonal autoantibodies directed against the human myelin oligodendrocyte glycoprotein (MOG)

We describe the epitope specificity of a panel of ten demyelinating monoclonal antibodies (mAb) that recognise the extracellular immunoglobulin-like domain of human myelin oligodendrocyte glycoprotein (hMOG(lgd)). All the mAbs bind to the surface of MOG-transfected fibroblasts as assessed in vitro by FACS and immunocytochemistry but failed to recognise overlapping 15-mer MOG peptides when assessed by ELISA. However, increasing peptide length to 25 amino acids revealed that four mAbs recognised epitopes within the amino acid sequence 63-100 of human MOG. In contrast, a non-demyelinating MOG-specific mAb recognised MOG by both ELISA and Western blotting but failed to stain MOG transfected fibroblasts. These observations suggest that assays based on the use of MOG-transfected cell lines will differentiate between pathogenic and non-pathogenic MOG-specific antibody responses in experimental models and human diseases of the nervous system.

An AG→GG transition at a splice site in the myelin proteolipid protein gene in jimpy mice results in the removal of an exon

The myelin proteolipid protein gene was characterized in jimpy mice to identify the specific mutation that produces dysmyelination, oligodendrocyte cell death, and death of the animal by 30 days of age. Exon 5 and flanking intron segments were isolated from jimpy proteolipid protein genomic clones and sequenced. A single nucleotide difference was noted between the normal and jimpy proteolipid protein genes: the conversion of an AG/GT to a GG/GT in the splice acceptor signal preceding exon 5, which apparently destroys the splice signal. Thus, exon 5 of the mouse myelin proteolipid protein gene is skipped during the processing of mRNA, producing a shortened proteolipid protein mRNA.

Purification and Partial Structural and Functional Characterization of Mouse Myelin/Oligodendrocyte Glycoprotein

Abstract: The myelin/oligodendrocyte glycoprotein (MOG) is found exclusively in the CNS, where it is localized on the surface of myelin and oligodendrocyte cytoplasmic membranes. The monoclonal antibody 8‐18C5 identifies MOG. Several studies have shown that anti‐MOG antibodies can induce demyelination, thus inferring an important role in myelin stability. In this study, we demonstrate that MOG consists of two polypeptides, with molecular masses of 26 and 28 kDa. This doublet becomes a single 25‐kDa band after deglycosylation with trifluoromethanesulfonic acid or peptide‐N4‐(N‐acetyl‐β‐glucosaminyl)asparagine amidase, indicating that there are no or few O‐linked sugars and that the doublet band represents differential glycosylation. Partial trypsin cleavage, which also gave a doublet band of lower molecular weight, confirmed this idea. MOG was purified by polyacrylamide gel electrophoresis, followed by electroelution. Three N‐terminal sequences of eight to 26 amino acids were obtained. By western blot analysis, no binding was found between MOG and cerebellar soluble lectin. MOG does not seem to belong to the signal‐transducing GTP‐binding proteins. Reduced MOG concentrations were observed in jimpy and quaking dysmyelinating mutant mice, giving further support to its localization in compact myelin of the CNS.

Characterization of myelin proteolipid mRNAs in normal and jimpy mice.

A clone specific for the rat myelin proteolipid protein (PLP) was isolated from a cDNA library made in pUC18 from 17-day-old rat brain stem mRNA. This clone corresponded to the carboxyl-terminal third of the PLP-coding region. The clone was used to identify PLP-specific mRNAs in mouse brain and to establish the time course of PLP mRNA expression during mouse brain development. Three PLP-specific mRNAs were seen, approximately 1,500, 2,400, and 3,200 bases in length, of which the largest was the most abundant. During brain development, the maximal period of PLP mRNA expression was from 14 to 25 days of age, and this was a similar time course to that for myelin basic protein mRNA expression. When the jimpy mouse, an X-linked dysmyelination mutant, was studied for PLP mRNA expression, low levels of PLP mRNA were seen which were approximately 5% of wild-type levels at 20 days of age. When jimpy brain RNA was analyzed by Northern blotting, the PLP-specific mRNA was shown to be 100 to 200 bases shorter than the wild-type PLP-specific mRNA. This size difference was seen in the two major PLP mRNAs, and it did not result from a loss of polyadenylation of these mRNAs.

Myelin/oligodendrocyte glycoprotein (MOG) expression is associated with myelin deposition

We investigated the onset of expression of the myelin/oligodendrocyte glycoprotein (MOG) mRNA and protein in the developing mouse central nervous system. In situ hybridization on brain sections at different stages of embryonic and postnatal development showed that MOG transcripts were first detected at birth in the medulla oblongata. During the first week after birth, cells expressing MOG mRNA were located in the ventral longitudinal funiculus. During the second postnatal week, the pattern of MOG mRNA expression extended rostrally to the mid‐forebrain regions and reached completion by the beginning of the third week. MOG transcription was delayed by several days with respect to myelin basic protein (MBP), and it appeared that while the MBP probe labeled both non‐myelinating and myelinating oligodendrocytes, only the latter were MOG‐positive. In vitro, immunocytochemical analysis of MOG protein expression, performed on myelinating cultures derived from mouse brain embryos at 15 days of gestation, confirmed the strict restriction of MOG expression to myelinating oligodendrocytes. In particular, oligodendrocytes lining up their processes along axons, but not yet having started to deposit a myelin sheath, were still MOG negative. However, in the same cultures, pseudo‐myelinating oligodendrocytes (i.e., cells not associated with neurites, but forming whorls of myelin‐like figures) were MOG positive. Similarly, rat CG4 cells, an oligodendrocyte‐like cell line, expressed MOG only after they had extended sheet‐like processes, which suggested that the activation of MOG transcription depends more on an intrinsic oligodendroglial maturation program of myelination than on a neuronal signal.

Expression of a proto-oncogene (proto-myb) in hemopoietic tissues of mice.

This study addressed the possibility that proto-myb (also called c-myb), the cellular homolog of a retroviral transforming gene, plays a role in hemopoiesis, particularly during maturation of T cells. By gel blot hybridization, we confirmed previous reports that proto-myb transcripts are found at much higher levels in thymic lymphocytes and cells of the erythroid lineage than in other tissue sources. Using dot blot hybridizations, we demonstrated further that similar levels of proto-myb expression are found in thymic lymphocytes taken from young mice with active thymuses and from old mice whose thymuses have undergone involution and that the extent of proto-myb expression decreases at least 10-fold as T cells progress from immature cortical thymocytes to the mature, resting T cells taken from lymph nodes. These results suggest that the protein product of proto-myb functions during T-cell differentiation.

Investigation of Myelin/Oligodendrocyte Glycoprotein Membrane Topology

Abstract: Myelin/oligodendrocyte glycoprotein (MOG) is a CNS‐specific integral membrane protein that is an atypical member of the immunoglobulin (Ig) superfamily with two potential transmembrane domains based upon hydropathy analysis. With only one other exception, all Ig family members possess a single or no membrane spanning region. In order to analyze MOG membrane topology, we prepared stably transfected cells that express mouse MOG and used three domain‐specific antisera to ascertain the localization of these hydrophilic domains. As expected, MOGs glycosylated N‐terminal Ig‐like domain was identified as extracellular, because membrane permeabilization was not required for immunoreactivity with the MOG1–125 antiserum. In contrast, both MOG154–169 and MOG198–218 antisera stained cells only upon permeabilization. These data indicate that only MOGs N‐terminal hydrophobic domain spans the lipid bilayer, and we propose that MOGs C‐terminal hydrophobic domain associates with the cytoplasmic face of the plasma membrane. As for MOGs second hydrophobic domain, it is clear that either orientation (transmembrane versus membrane‐associated) would be unique among Ig‐like proteins, and the implications of our proposed topology for MOG in oligodendroglial plasma membrane are discussed.

Mutations in the Myelin Proteolipid Protein Gene Alter Oligodendrocyte Gene Expression in Jimpy and Jimpymsd Mice

Abstract: The mouse myelin proteolipid protein (PLP) gene has been studied in normal and jimpymsd mice. Potential upstream regulatory regions of the normal gene have been cloned and mapped, but when these regions were studied in jimpymsd mice by Southern blots, no alterations were observed, relative to the normal gene. To assess whether the low ratio of PLP to DM20 proteins in this mutant reflected an altered PLP/DM20 ratio mRNAs, S1 nuclease analyses were undertaken, which demonstrated that at all ages studied in both jimpy and jimpymsd mice, PLP mRNA was elevated above DM20 mRNA. When exon 3 (the site of the alternative splice signal for DM20 mRNA) of the jimpymsd PLP gene was sequenced, no mutation was identified. The transcription of the PLP gene in normal and mutant animals was studied. The transcription rate increases in normal animals with development, and in very young jimpymsd or jimpy mice, the transcription rate of the PLP gene was close to that of agematched normal animals. However, by 10 days of age, the transcription rate of this gene in both mutants was significantly below that of age‐matched controls. The transcription rate of the myelin basic protein (MBP) gene was also reduced, indicating that expression of both genes is affected by this mutation. In contrast, the transcription rate of the glycerol phosphate dehydrogenase (GPDH) gene, an early marker of oligodendrocytes, is equal to or greater than normal in both mutants. We have confirmed an earlier report of a point mutation in exon 6 of the jimpymsd PLP gene, which converts an alanine to a valine. This mutation apparently alters oligodendrocyte metabolism such that the cell can differentiate to express early oligodendrocyte genes such as GPDH, but it cannot differentiate to a stage where it expresses the PLP and MBP genes at normal high levels.

Myelin/oligodendrocyte glycoprotein is alternatively spliced in humans but not mice

Myelin/oligodendrocyte glycoprotein (MOG) is an integral membrane protein expressed on the oligodendrocyte cell surface and the outermost surface of myelin sheaths. Due to this localization, MOG is a primary target antigen involved in immune‐mediated demyelination. We previously reported that MOG is a unique member of the immunoglobulin (Ig) superfamily in that it possesses two large hydrophobic domains. MOG is highly conserved between deduced peptide sequences of rodent and human MOG (≈89% identity). We have completed an investigation of alternative splicing within the human and mouse MOG genes. Reverse transcriptase‐polymerase chain reaction (RT‐PCR) analysis of total cellular RNA isolated from both fetal and adult human central nervous system (CNS) tissues reveals a complex array of alternatively spliced MOG‐specific variants and the presence of two novel exons. Exon 3 encodes a short hydrophilic domain containing multiple in‐frame termination codons that would result in truncation of MOG prior to translation of its transmembrane domain. Exon 7 encodes an additional hydrophilic domain that replaces MOGs second hydrophobic domain in one splice variant. We also observed that five of our eight MOG variants exhibited an alternative internal 3′ splice acceptor within MOGs terminal exon. Surprisingly, no splicing was observed in a developmental study using mouse brainstem RNA.