Splice variation in the cytoplasmic domains of myelin oligodendrocyte glycoprotein affects its cellular localisation and transport

Although myelin oligodendrocyte glycoprotein is a candidate autoantigen in multiple sclerosis, its function remains unknown. In humans, mRNA expressed by the myelin oligodendrocyte glycoprotein gene is alternatively spliced resulting in at least nine unique protein isoforms. In this study, we investigated the sub-cellular localisation and membrane trafficking of six isoforms by cloning them into mammalian expression vectors. Confocal microscopy revealed that these protein products are expressed in different cellular compartments. While two full-length isoforms (25.6 and 25.1) are expressed at the cell surface, three alternatively spliced forms (22.7, 21.0 and 20.5) have a more intracellular distribution, localising to the endoplasmic reticulum and/or endosomes. Isoform 16.3, which lacks a transmembrane domain, is secreted. A switch in the sub-cellular localisation of myelin oligodendrocyte glycoprotein may have profound effects on receptor:ligand interactions and consequently the function of the protein. The structural features of the alternative isoforms and their differential, sub-cellular expression patterns could dictate the exposure of major immunogenic determinants within the central nervous system. Our findings highlight myelin oligodendrocyte glycoprotein splicing as a factor that could be critical to the phenotypic expression of multiple sclerosis.     

Alternative isoforms of myelin/oligodendrocyte glycoprotein with variable cytoplasmic domains are expressed in human brain

The human myelin/oligodendrocyte glycoprotein (MOG) gene is encoded by 10 exons that exhibit a complex pattern of alternative splicing. This report demonstrates that several MOG-specific alternative splice variants are indeed expressed in human oligodendrocytes (OLs) and myelin during perinatal development and are retained through adulthood. While all forms possess the common extracellular Ig-like domain, these alternative MOG structures differ significantly in their respective cytoplasmic domains. Peptide-specific antibodies were generated to facilitate detection of these different MOG moieties. The fidelity of these antibodies is shown using N20 OLs expressing individual MOG variants. These antibodies also only co-localize with another well-characterized marker of OLs and myelin--PLP/DM20 proteins. Among the human tissue samples tested, very limited expression occurred by 36 weeks gestation for 2-3 MOG variants, and the remaining MOG isoforms were not evident until shortly after birth. This study represents the first evidence of alternative translation products from the MOG gene. To date, it is believed that alternative splicing of MOG is limited to primates. Recent completion of various genome projects has revealed that alternative splicing is much more prevalent than originally estimated, and species-specific alternative splicing is now being shown to be highly relevant to expanding proteomic diversity.     

Genetic study of the myelin oligodendrocyte glycoprotein (MOG) gene in schizophrenia

Schizophrenia (SCZ) is a neuropsychiatric disorder that affects approximately 1% of the general population. The human leukocyte antigen (HLA) system has been implicated in several genetic studies of SCZ. The myelin oligodendrocyte glycoprotein (MOG) gene, which is located close to the HLA region, is considered a candidate for SCZ due to its association with white matter abnormalities and its importance in mediating the complement cascade. Four polymorphisms in the MOG gene (CA)n (TAAA)n, and two intronic polymorphisms, C1334T and C10991T, were investigated for the possibility of association with SCZ using 111 SCZ proband and their families. We examined the transmission of the alleles of each of these polymorphisms with the transmission disequilibrium test. We did not observe significant evidence for biased transmission of alleles at the (CA)n (chi2=2.430, 6 df, P=0.876) (TAAA)n (chi2=3.550, 5 df, P=0.616), C1334T (chi2=0.040, 1 df, P=0.841) and C10991T (chi2=0.154, 1 df, P=0.695) polymorphisms. Overall haplotype analysis using the TRANSMIT program was also not significant (chi2=7.954, 9 df, P=0.539). Furthermore, our results comparing mean age at onset in the genotype groups using the Kruskal-Wallis Test were not significant. Our case-control analyses (182 cases age-, sex- and ethnicity-matched with healthy controls) and combined z-score [(CA)n: z-score=-1.126, P=0.130; (TAAA)n: z-score=-0.233, P=0.408; C1334T: z-score=0.703, P=0.241; C10991T: z-score=0.551, P=0.291] were also not significant. Although our data are negative, the intriguing hypothesis for MOG in SCZ may warrant further investigation of this gene.     

Epitope mapping of anti‐myelin oligodendrocyte glycoprotein (MOG) antibodies in a mouse model of multiple sclerosis: microwave‐assisted synthesis of the peptide antigens and ELISA screening

The role of pathologic auto‐antibodies against myelin oligodendrocyte glycoprotein (MOG) in multiple sclerosis is a highly controversial matter. As the use of animal models may enable to unravel the molecular mechanisms of the human disorder, numerous studies on multiple sclerosis are carried out using experimental autoimmune encephalomyelitis (EAE). In particular, the most extensively used EAE model is obtained by immunizing C57BL/6 mice with the immunodominant peptide MOG(35–55). In this scenario, we analyzed the anti‐MOG antibody response in this model using the recombinant refolded extracellular domain of the protein, MOG(1–117). To assess the presence of a B‐cell intramolecular epitope spreading mechanism, we tested also five synthetic peptides mapping the 1–117 sequence of MOG, including MOG(35–55). For this purpose, we cloned, expressed in Escherichia coli and on‐column refolded MOG(1–117), and we applied an optimized microwave‐assisted solid‐phase synthetic strategy to obtain the designed peptide sequences. Subsequently, we set up a solid‐phase immunoenzymatic assay testing both naïve and EAE mice sera and using MOG protein and peptides as antigenic probes. The results obtained disclose an intense IgG antibody response against both the recombinant protein and the immunizing peptide, while no response was observed against the other synthetic fragments, thus excluding the presence of an intramolecular epitope spreading mechanism. Furthermore, as the properly refolded recombinant probe is able to bind antibodies with greater efficiency compared with MOG(35–55), we hypothesize the presence of both linear and conformational epitopes on MOG(35–55) sequence. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Minireview: Autoimmune responses to myelin proteolipid protein

The authors present a brief historical sketch of the development of our understanding of immune responses to myelin proteolipid protein (PLP) and the acceptance of PLP as a potent antigen in the induction of experimental allergic encephalomyelitis (EAE). The distinct characteristics of the PLP molecule that may contribute to complex immune responses to this protein are reviewed and these responses are compared with those to MBP, both in the pathology of EAE and at the level of the T cell. Recent evidence demonstrating differences between T cell responses to PLP and MBP is reviewed. Finally, the potential contribution of immune responses to PLP in human diseases, particularly mutiple sclerosis (MS), that have been identified to date are then summarized.For the authors to write a review on PLP and its role in EAE without Marjorie is like their sailing a ship without a captain, compass or rudder. This review is largely based on work and ideas generated in Marjorie's laboratory, but it was prepared without her input. Consequently, it lacks her meticulous reflection on the structure of each of its sentences and on the use of each word. Papers written with Marjorie are usually honed to near perfection late into the evening at her kitchen table in Newton, where food, ideas, and warmth abound, and where her very patient and accommodating husband Sidney and a demanding but lovable canine are close at hand. Writing this essay gave the authors a chance to recognize our scientific forebears, to consider where we are at this point and to contemplate our future directions in studying immune responses to PLP. We are, indeed, very grateful and indebted to Marjorie for her generous personal and scientific support, wise guidance, inspiration, strength, energy and, most importantly, friendship. Marjorie, we thank you, you are our role model, and we affectionately anticipate many more years of continued collaboration with you.Abbreviations used in this paper CNS central nervous system - EAE experimental allergic encephalomyelitis - MBP myelin basic protein - MHC major histocompatibility complex - MOG myelin oligodendrocyte cyte glycoprotein - MS multiple sclerosis - PLP myelin proteolipid protein - PNS peripheral nervous system - TcR T cell receptor Special issue dedicated to Dr. Marjorie B. Lees.     

Clinical course of myelin oligodendrocyte glycoprotein 35-55 induced experimental autoimmune encephalomyelitis is aggravated by glia maturation factor

The role of glia maturation factor (GMF) in myelin oligodendrocyte glycoprotein (MOG) 35-55 peptide-induced experimental autoimmune encephalomyelitis (EAE) was investigated using GMF-deficient (GMF-KO) mice. We demonstrate that GMF-KO mice were resistant to the MOG 35-55 peptide-induced EAE as compared to wild type (Wt) mice (two in eight versus 10 in 10). Next, we examined the effect of administration of recombinant human GMF (rGMF) on MOG 35-55 peptide-induced EAE in mice. Daily administration of rGMF, staring days 1-14, resulted in significant exacerbation of clinical symptoms. Following rGMF injections, both GMF-KO (six in eight) and Wt mice (eight in eight) developed severe EAE (maximal clinical score of 3.5-4.0) with high frequency. The histological examination revealed severe infiltration of inflammatory cells in the spinal cord of MOG-immunized Wt mice while the resistance to EAE in GMF-KO mice was characterized by the absence of inflammatory cells. Administration of rGMF in Wt mice and GMF-KO mice resulted in a significant increase in infiltrating cells in the spinal cord following MOG-immunizations. We also evaluated cytokines and chemokines production as parameters of severity of inflammation in the spinal cord of Wt versus GMF-KO mice with and without GMF-reconstitution following MOG-immunizations. Cytokines (TNF-α, IFN-γ, IL-1β, IL-6) and chemokines (CCL2, CCL3, CXCL10, GM-CSF) production were significantly greater in Wt mice than in GMF-KO mice following MOG-immunization. Furthermore, the reconstitution experiment with rGMF showed that the administration of rGMF in both, Wt mice and GMF-KO mice produced significant increase in the GMF-mediated cytokine/chemokine production.     

Myelin-associated glycoprotein (MAG): past, present and beyond

The myelin-associated glycoprotein (MAG) is a type I transmembrane glycoprotein localized in periaxonal Schwann cell and oligodendroglial membranes of myelin sheaths where it functions in glia-axon interactions. It contains five immunoglobulin (Ig)-like domains and is in the sialic acid-binding subgroup of the Ig superfamily. It appears to function both as a ligand for an axonal receptor that is needed for the maintenance of myelinated axons and as a receptor for an axonal signal that promotes the differentiation, maintenance and survival of oligodendrocytes. Its function in the maintenance of myelinated axons may be related to its role as one of the white matter inhibitors of neurite outgrowth acting through a receptor complex involving the Nogo receptor and/or gangliosides containing 2,3-linked sialic acid. MAG is expressed as two developmentally regulated isoforms with different cytoplasmic domains that may activate different signal transduction pathways in myelin-forming cells. MAG contains a carbohydrate epitope shared with other glycoconjugates that is a target antigen in autoimmune peripheral neuropathy associated with IgM gammopathy and has been implicated in a dying back oligodendrogliopathy in multiple sclerosis.     

MiRNA‐145‐5p prevents differentiation of oligodendrocyte progenitor cells by regulating expression of myelin gene regulatory factor

The roles of specific microRNAs (miRNA) in oligodendrocyte (OL) differentiation have been studied in depth. However, miRNAs in OL precursors and oligodendrocyte progenitor cells (OPCs) have been less extensively investigated. MiR‐145‐5p is highly expressed in OPCs relative to differentiating OLs, suggesting this miRNA may serve a function specifically in OPCs. Knockdown of miR‐145‐5p in primary OPCs led to spontaneous differentiation, as evidenced by an increased proportion of MAG⁺ cells, increased cell ramification, and upregulation of multiple myelin genes including MYRF, TPPP, and MAG, and OL cell cycle exit marker Cdkn1c. Supporting this transition to a differentiating state, proliferation was reduced in miR‐145‐5p knockdown OPCs. Further, knockdown of miR‐145‐5p in differentiating OLs showed enhanced differentiation, with increased branching, myelin membrane production, and myelin gene expression. We identified several OL‐specific genes targeted by miR‐145‐5p that exhibited upregulation with miR‐145‐5p knockdown, including myelin gene regulatory factor (MYRF), that could be regulating the prodifferentiation phenotype in both miR‐145 knockdown OPCs and OLs. Indeed, spontaneous differentiation with knockdown of miR‐145‐5p was fully rescued by concurrent knockdown of MYRF. However, proliferation rate was only partially rescued with MYRF knockdown, and overexpression of miR‐145‐5p in OPCs increased proliferation rate without affecting expression of already lowly expressed differentiation genes. Taken together, these data suggest that in OPCs miR‐145‐5p both prevents differentiation at least in part by preventing expression of MYRF and promotes proliferation via as‐yet‐unidentified mechanisms. These findings clarify the need for differential regulation of miR‐145‐5p between OPCs and OLs and may have further implications in demyelinating diseases such as multiple sclerosis where miR‐145‐5p is dysregulated.     

hnRNP A1 and secondary structure coordinate alternative splicing of Mag

Myelin-associated glycoprotein (MAG) is a major component of myelin in the vertebrate central nervous system. MAG is present in the periaxonal region of the myelin structure, where it interacts with neuronal proteins to inhibit axon outgrowth and protect neurons from degeneration. Two alternatively spliced isoforms of Mag mRNA have been identified. The mRNA encoding the shorter isoform, known as S-MAG, contains a termination codon in exon 12, while the mRNA encoding the longer isoform, known as L-MAG, skips exon 12 and produces a protein with a longer C-terminal region. L-MAG is required in the central nervous system. How inclusion of Mag exon 12 is regulated is not clear. In a previous study, we showed that heteronuclear ribonucleoprotein A1 (hnRNP A1) contributes to Mag exon 12 skipping. Here, we show that hnRNP A1 interacts with an element that overlaps the 5′ splice site of Mag exon 12. The element has a reduced ability to interact with the U1 snRNP compared with a mutant that improves the splice site consensus. An evolutionarily conserved secondary structure is present surrounding the element. The structure modulates interaction with both hnRNP A1 and U1. Analysis of splice isoforms produced from a series of reporter constructs demonstrates that the hnRNP A1-binding site and the secondary structure both contribute to exclusion of Mag exon 12.     

Cytokine Control of Inflammation and Repair in the Pathology of Multiple Sclerosis

Cytokines are secreted signaling proteins that play an essential role in propagating and regulating immune responses during experimental autoimmune encephalomyelitis (EAE), the mouse model of the neurodegenerative, autoimmune disease multiple sclerosis (MS). EAE pathology is driven by a myelin-specific T cell response that is activated in the periphery and mediates the destruction of myelin upon T cell infiltration into the central nervous system (CNS). Cytokines provide cell signals both in the immune and CNS compartment, but interestingly, some have detrimental effects in the immune compartment while having beneficial effects in the CNS compartment. The complex nature of these signals will be reviewed.     

Predominant contribution of cis‐regulatory divergence in the evolution of mouse alternative splicing

Divergence of alternative splicing represents one of the major driving forces to shape phenotypic diversity during evolution. However, the extent to which these divergences could be explained by the evolving cis‐regulatory versus trans‐acting factors remains unresolved. To globally investigate the relative contributions of the two factors for the first time in mammals, we measured splicing difference between C57BL/6J and SPRET/EiJ mouse strains and allele‐specific splicing pattern in their F1 hybrid. Out of 11,818 alternative splicing events expressed in the cultured fibroblast cells, we identified 796 with significant difference between the parental strains. After integrating allele‐specific data from F1 hybrid, we demonstrated that these events could be predominately attributed to cis‐regulatory variants, including those residing at and beyond canonical splicing sites. Contrary to previous observations in Drosophila, such predominant contribution was consistently observed across different types of alternative splicing. Further analysis of liver tissues from the same mouse strains and reanalysis of published datasets on other strains showed similar trends, implying in general the predominant contribution of cis‐regulatory changes in the evolution of mouse alternative splicing.     

Effects of peptide fraction and counter ion on the development of clinical signs in experimental autoimmune encephalomyelitis

The most commonly used immunogen to induce experimental autoimmune encephalomyelitis is MOG_35‐55, a 21‐residue peptide derived from myelin oligodendrocyte glycoprotein (MOG). In most studies, mice exhibit a chronic disease; however, in some studies mice show a transient disease. One variable that is not often controlled for is the peptide fraction of the purified MOG material, which can vary from less than 50% to over 90%, with the remainder of mass primarily comprised of the counter ion used for peptide purification. We compared the development of clinical signs in female C57Bl6 mice immunized with two commercially available MOG_35‐55 peptides of similar purity but different peptide fraction (MOG‐A being 45%; MOG‐B being 72%). A single immunization with MOG‐A induced a chronic disease course with some recovery at later stages, whereas immunization with MOG‐B induced a similar course of disease but with significantly lower average clinical scores despite a higher peptide content. The addition of a booster immunization significantly increased clinical severity with both preparations, and significantly reduced the average day of onset using MOG‐A. To determine if the counter ion could influence disease, we compared MOG‐B‐containing trifluoroacetate with MOG‐B‐containing acetate. Although disease incidence and severity were similar, the average day of disease onset occurred approximately 5 days earlier with the use of MOG‐B‐containing trifluoroacetate. These results demonstrate that differences in peptide fraction influence the course of encephalomyelitis disease, which may be due in part to the levels of counter ions present in the purified material. These findings underscore the fact that a knowledge of peptide fraction is as critical as knowledge of peptide purity when using peptides from different sources.

     

Determining Immune System Suppression versus CNS Protection for Pharmacological Interventions in Autoimmune Demyelination

A major hallmark of the autoimmune demyelinating disease multiple sclerosis (MS) is immune cell infiltration into the brain and spinal cord resulting in myelin destruction, which not only slows conduction of nerve impulses, but causes axonal injury resulting in motor and cognitive decline. Current treatments for MS focus on attenuating immune cell infiltration into the central nervous system (CNS). These treatments decrease the number of relapses, improving quality of life, but do not completely eliminate relapses so long-term disability is not improved. Therefore, therapeutic agents that protect the CNS are warranted. In both animal models as well as human patients with MS, T cell entry into the CNS is generally considered the initiating inflammatory event. In order to assess if a drug protects the CNS, any potential effects on immune cell infiltration or proliferation in the periphery must be ruled out. This protocol describes how to determine whether CNS protection observed after drug intervention is a consequence of attenuating CNS-infiltrating immune cells or blocking death of CNS cells during inflammatory insults. The ability to examine MS treatments that are protective to the CNS during inflammatory insults is highly critical for the advancement of therapeutic strategies since current treatments reduce, but do not completely eliminate, relapses (i.e., immune cell infiltration), leaving the CNS vulnerable to degeneration.     

Pax基因功能及其选择性剪接的研究进展

Pax基因家族编码的蛋白是一组极为重要的转录调控因子,在胚胎发育的器官形成中扮演重要角色,其主要功能包括：调控细胞增殖、促进细胞自我更新、诱导前体细胞定向转移以及改变特异细胞系的分化方向。目前已知,Pax基因的非正常表达是多种先天性疾病的主要诱因。Pax基因的选择性剪接体通常具有一定的空间特异性,每种剪接体都有其主要作用的靶位和信号通路。文章简述了Pax基因的相关背景知识,详细介绍Paxl—Pax9调控在胚胎组织发育中的各项功能,并列举了现已确定的Pax基因在不同物种中的选择性剪接产物。     

Cleavage and polyadenylation specificity factor 1 (CPSF1) regulates alternative splicing of interleukin 7 receptor (IL7R) exon 6

Interleukin 7 receptor, IL7R, is expressed exclusively on cells of the lymphoid lineage, and its expression is crucial for the development and maintenance of T cells. Alternative splicing of IL7R exon 6 results in membrane-bound (exon 6 included) and soluble (exon 6 skipped) IL7R isoforms. Interestingly, the inclusion of exon 6 is affected by a single-nucleotide polymorphism associated with the risk of developing multiple sclerosis. Given the potential association of exon 6 inclusion with multiple sclerosis, we investigated the cis-acting elements and trans-acting factors that regulate exon 6 splicing. We identified multiple exonic and intronic cis-acting elements that impact inclusion of exon 6. Moreover, we utilized RNA affinity chromatography followed by mass spectrometry to identify trans-acting protein factors that bind exon 6 and regulate its splicing. These experiments identified cleavage and polyadenylation specificity factor 1 (CPSF1) among protein-binding candidates. A consensus polyadenylation signal AAUAAA is present in intron 6 of IL7R directly downstream from the 5′ splice site. Mutations to this site and CPSF1 knockdown both resulted in an increase in exon 6 inclusion. We found no evidence that this site is used to produce cleaved and polyadenylated mRNAs, suggesting that CPSF1 interaction with intronic IL7R pre-mRNA interferes with spliceosome binding to the exon 6 5′ splice site. Our results suggest that competing mRNA splicing and polyadenylation regulate exon 6 inclusion and consequently determine the ratios of soluble to membrane-bound IL7R. This may be relevant for both T cell ontogeny and function and development of multiple sclerosis.