The relationship between developing oligodendrocyte units and maturing axons during myelinogenesis in the anterior medullary velum of neonatal rats

Myelinogenesis was investigated in whole-mounted anterior medullary vela from rats aged postnatal day (P) 10–12, using double immunofluorescence labelling with Rip and anti-neurofilament 200 (NF200) antibodies, to identify oligodendrocytes and axons, respectively. A number of discrete phases of maturation of oligodendrocyte units were recognised. (1) Promyelinating oligodendrocytes co-expressed Rip and Myelin basic Protein and formed axonal associations, prior to ensheathment. (2) Transitional oligodendrocytes contained both ensheathing and non-ensheating processes. (3) Myelinating oligodendrocytes were established after a period of remodelling (in which non-ensheathing processes were lost), appearing as oligodendrocyte unit morphological phenotypes with a definitive number of incipient myelin sheaths. (4) Maturation of myelinating oligodendrocytes was defined as the establishment of internodal sheath lengths and the redistrubution of myelin basic protein from the cell somata and radial processes into the myelin sheaths only. Myelination was probably related to the maturational state of the axons, since it was initiated when the latter had attained a critical diameter of between ～0.2 and 0.4 μm, coincident with the expression of NF200. Oligodendrocyte differentiation and myelination of the AMV were asynchronous and multifocal, and at P10: (1) axons which were destined to be of the largest calibre in the adult AMV were already myelinated by early developing oligodendrocytes, whilst those which were destined to be the smallest calibre in the adult were unmyelinated, but ultimately became ensheathed by late developing oligoendrocytes; (2) axons were sequentially ensheathed by early developing myelinating oligodendrocytes and late developing promyelinating oligodendrocytes; (3) all axons were small calibre; (4) oligodendrocyte units exhibited polymorphism. Thus, the development of oligodendrocyte morphological phenotypes was not related solely to either the physical dimension of axon calibre at the time of ensheathment, nor oligodendrocyte birth dates.     

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.     

Extracellular domain of myelin oligodendrocyte glycoprotein (MOG) exhibits solvent-dependent conformational transitions

The conformation of the non-glycosylated recombinant form of the extracellar domain of rat MOG (rMOG(1-125)) dissolved in different solvent conditions was studied by CD spectroscopy. The results show that rMOG(1-125) exhibits a predominantly beta sheet conformation in aqueous buffer solution at pH 7.5 and that this 'beta-form' is stabilized by zwitterionic phospholipids, DPC and LPCP. The alpha helical content of the protein can increase from 9% to up to 20% when TFE or anionic detergent LPAP and SDS are added.     

The regional distribution of myelin oligodendrocyte glycoprotein (MOG) in the developing rat CNS: an in vivo immunohistochemical study

Using an immunohistochemical approach we have characterized the in vivo developmental distribution of myelin oligodendrocyte glycoprotein within the rat CNS. Myelin oligodendrocyte glycoprotein expression emerged in a non-uniform manner during the first 3 postnatal weeks. Although it was absent throughout the CNS of the newborn rat at postnatal day 0(P0), it had appeared in the spinal cord and brainstem by P7. The forebrain and cerebellum remained devoid of immunoreactivity until after P14. Myelin oligodendrocyte glycoprotein emerged at different times within the closely associated fasciculi of the dorsal funiculus. It appeared in the fasciculus cuneatus during the first postnatal week and in the fasciculus gracilis and corticospinal tracts during weeks 2 and 3 respectively. Myelin oligodendrocyte glycoprotein expression developed along a caudo-rostral gradient from spinal cord to forebrain and along an antero-posterior gradient within the CNS in general. The relationship between the onset of myelin oligodendrocyte glycoprotein expression and myelinogenesis was also investigated. In most regions, myelin oligodendrocyte glycoprotein expression lagged behind the initial appearance of myelin basic protein and Luxol Fast Blue-stained myelin by at least 1 week. These observations support the idea that myelin oligodendrocyte glycoprotein is the latest myelin protein to appear in development, only being expressed during the final stages of oligodendrocyte differentiation. Furthermore, the pattern of staggered expression within the dorsal columns indicates that localized, region-specific interactions may comprise a key element in the control of the terminal phases of oligodendrocyte differentiation.     

Polymorphic tri and tetranucleotide repeats in exons 1 and 8 of the myelin oligodendrocyte glycoprotein (MOG) gene

Polymorphic (CTC)n and (TAAA)n sequences were identified in exons 1 and 8 of the myelin oligodendrocyte glycoprotein (MOG) gene. The different alleles were detected by a method combining fluorescence labeling of polymerase chain reaction (PCR) products and use of an automated DNA sequencer. Although only two alleles differing by the number of leucine residues encoded by the (CTC)n array were detected at the first locus, seven alleles were identified at the second. The high degree of polymorphism (75%) of the tetranucleotide repeat makes this marker informative for association or linkage studies with diseases such as hemochromatosis or multiple sclerosis.     

Cell surface targeting of myelin oligodendrocyte glycoprotein (MOG) in the absence of endoplasmic reticulum molecular chaperones

Myelin oligodendrocyte glycoprotein (MOG) is a type I integral membrane glycoprotein that localizes to myelin sheaths in the central nervous system. MOG has important implications in multiple sclerosis, as pathogenic anti-MOG antibodies have been detected in the sera of multiple sclerosis patients. As a membrane protein, MOG achieves its native structure in the endoplasmic reticulum where its folding is expected to be controlled by endoplasmic reticulum chaperones. Calnexin, calreticulin, and ERp57 are essential components of the endoplasmic reticulum quality control where they assist in the proper folding of newly synthesized glycoproteins. In this study, we show that expression of MOG is not affected by the absence of the endoplasmic reticulum quality control proteins calnexin, calreticulin, or ERp57. We also show that calnexin forms complexes with MOG and these interactions might be glycan-independent. Importantly, we show that cell surface targeting of MOG is not disrupted in the absence of the endoplasmic reticulum chaperones. This article is part of a special issue entitled: 11th European Symposium on Calcium.     

Antibody cross-reactivity between myelin oligodendrocyte glycoprotein and the milk protein butyrophilin in multiple sclerosis

The etiology of multiple sclerosis (MS) is believed to involve environmental factors, but their identity and mode of action are unknown. In this study, we demonstrate that Ab specific for the extracellular Ig-like domain of myelin oligodendrocyte glycoprotein (MOG) cross-reacts with a homologous N-terminal domain of the bovine milk protein butyrophilin (BTN). Analysis of paired samples of MS sera and cerebrospinal fluid (CSF) identified a BTN-specific Ab response in the CNS that differed in its epitope specificity from that in the periphery. This effect was statistically significant for the Ab response to BTN(76-100) (p = 0.0026), which cosequestered in the CSF compartment with Ab to the homologous MOG peptide MOG(76-100) in 34% of MS patients (n = 35). These observations suggested that intratheccal synthesis of Ab recognizing BTN peptide epitopes in the CNS was sustained by molecular mimicry with MOG. Formal evidence of molecular mimicry between the two proteins was obtained by analyzing MOG-specific autoantibodies immunopurified from MS sera. The MOG-specific Ab repertoire cross-reacts with multiple BTN peptide epitopes including a MOG/BTN(76-100)-specific component that occurred at a higher frequency in MS patients than in seropositive healthy controls, as well as responses to epitopes within MOG/BTN(1-39) that occur at similar frequencies in both groups. The demonstration of molecular mimicry between MOG and BTN, along with sequestration of BTN-reactive Ab in CSF suggests that exposure to this common dietary Ag may influence the composition and function of the MOG-specific autoimmune repertoire during the course of MS.     

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.     

Differential gene expression of 36-kDa microfibril-associated glycoprotein (MAGP-36/MFAP4) in rat organs

By using quantitative Western blot analysis and the real time polymerase chain reaction technique, we investigated the differential gene expression of microfibril-associated glycoprotein (MAGP-36) in rat organs. The gene was expressed highly in sites rich in elastic fibers, such as aorta, skin, and esophagus. However, MAGP-36 was also expressed highly in some other sites containing no elastic fibers. In lung and trachea, the expression levels of MAGP-36 mRNA were about seven times higher than those in other elastic tissues, although the protein abundances were almost at the same levels as other elastic tissues. MAGP-36 seemed to be secreted outside these organs. In brain, kidney, and spleen, although the expression levels of MAGP-36 mRNA were low, substantial amounts of MAGP-36 protein were detected. An immunohistochemical study revealed that MAGP-36 was present at the brush border of the S3 segment of proximal tubules in kidney. Since MAGP-36 is known to bind to mannan, MAGP-36 might be involved in mannose transport in the S3 segment. Thus, MAGP-36 might be multifunctional and present in a wide variety of sites in various organs.     

Absence of the myelin-associated glycoprotein (MAG) and the neural cell adhesion molecule (N-CAM) interferes with the maintenance,but not with the formation of peripheral myelin

We have previously shown that mice deficient in the gene for the myelin-associated glycoprotein (MAG) develop normal myelin in the peripheral nerves, but show axon and myelin degeneration at eight months of age, suggesting that MAG is involved in the maintenance of axon-Schwann cell integrity. The search for molecules that might replace MAG during myelination revealed an overexpression of the neural cell adhesion molecule (N-CAM) at those aspects where MAG is detectable in wild type mice. To test whether N-CAM might compensate for MAG during myelination in MAG-deficient mice, double mutants deficient in both MAG and N-CAM (MAG⁻/N-CAM⁻mice) were generated by cross-breeding the single mutants. Whereas alterations of myelin development were not detectable in either of the single or double mutants, degeneration of myelin and axons occurred approximately 4 weeks earlier in MAG⁻/N-CAM⁻than in MAG⁻mutants. Furthermore, at 8 weeks of age, single fiber preparation and electron microscopy revealed that the number of profiles indicative of degeneration was substantially increased in MAG⁻/N-CAM⁻mutants when compared to MAG⁻mice. These data suggest that in MAG-deficient mice N-CAM does not compensate for MAG in myelin formation but partially substitutes for it in the maintenance of axon-myelin integrity. Received: 20 May 1996 / Accepted: 19 July 1996     

Generation and characterization of mouse monoclonal antibodies to the myelin-associated glycoprotein (MAG)

A panel of mouse monoclonal antibodies to rat and human myelin-associated glycoprotein (MAG) was developed. Normal mice were unresponsive to rat MAG, and successful immunization with rat MAG was only achieved in autoimmune NZB mice. By contrast, all strains of mice were responsive to human MAG. The monoclonal antibodies developed differ with respect to immunoglobulin type, their specificity for human and/or rat MAG, and their recognition of protein or carbohydrate epitopes in MAG. In general, the antibodies that react with the protein backbone recognize both rat and human MAG, whereas a large number of the monoclonal antibodies recognize a carbohydrate determinant in human MAG that is not in rat MAG. Immunocytochemical staining of adult human spinal cord with the monoclonal antibodies resulted in periaxonal staining of myelin sheaths, similar to that produced by well-defined, rabbit, polyclonal anti-MAG serum. In addition, the antibodies recognizing, carbohydrate determinants in human MAG strongly stained oligodendrocyte cytoplasm. These monoclonal antibodies will be of value for the further chemical and biological characterization of MAG.     

Selective unresponsiveness to conformational B cell epitopes of the myelin oligodendrocyte glycoprotein in H-2b mice

Autoantibodies directed against conformation-dependent epitopes of the extracellular domain of the myelin oligodendrocyte glycoprotein (MOG(Igd)) play a major role in the immunopathogenesis of demyelination in experimental autoimmune encephalomyelitis. We now demonstrate that one or more genes encoded within the MHC selectively censor the ability of H-2(b) mice to mount this conformation-dependent autoantibody response, while leaving T and B cell responses to linear MOG(Igd) epitopes intact. This novel form of selective B cell unresponsiveness discriminates between pathogenic and nonpathogenic Ab responses to MOG and determines whether or not Ab-dependent effector mechanisms play an important role in the pathogenesis of MOG-induced experimental autoimmune encephalomyelitis in the mouse.     

Single-step purification and refolding of recombinant mouse and human myelin oligodendrocyte glycoprotein and induction of EAE in mice

The extracellular domain of human and rat MOG (ED-MOG) induces experimental autoimmune encephalomyelitis (EAE) when injected into susceptible animals. EAE is a T cell-mediated disease of the central nervous system commonly used as an animal model for human multiple sclerosis. Here, we describe a straightforward procedure for the purification and refolding of mouse and human ED-MOG overexpressed in Escherichia coli as inclusion bodies. Following solubilization and purification using Ni-NTA resin chromatography under denaturing conditions, a column-based refolding proceeded in renaturation buffer supplemented with a glutathione redox buffer system. Using this approach up to 33 mg of highly pure soluble proteins was obtained per liter of expression culture. The ability of purified proteins to induce EAE was evaluated in three strains of mice. We believe that the strategy described here would facilitate researchers to carry out encephalitogenic as well as structure-function studies of this autoantigen. Additionally, we show for the first time that mouse ED-MOG induces severe disease in mice.     

Conversion of myelin-associated glycoprotein (MAG) to a smaller derivative by calcium activated neutral protease (CANP)-like enzyme in myelin and inhibition by E-64 analogue

Using the immunoblot technique, we found that an incubation of purified human myelin in 10 mM Tris-HCl buffer at pH 7.5 resulted in the conversion of the myelinassociated glycoprotein (MAG) to a smaller derivative (dMAG). Exogenously added 5 mM CaCl₂ accelerated the conversion of MAG. In buffer containing more than 100 M of EGTA, the conversion was inhibited. In addition, the existence of endogenous calcium in purified myelin was confirmed using atomic absorption spectroscopy. The conversion was also inhibited partially by one of the thiol protease inhibitors, E-64 analogue (E-64-a). These observations suggest that the conversion of MAG is mediated by calcium-activated neutral protease (CANP)-like enzyme.     

Differential expression of receptor tyrosine kinases and Shc in fetal and adult rat fibroblasts: toward defining scarless versus scarring fibroblast phenotypes

The remarkable ability of the fetus to heal early gestation skin wounds without scarring remains poorly understood. Taking advantage of recent advances in signal transduction, the tyrosine phosphorylation patterns of fetal rat fibroblasts, representing the scarless cutaneous repair phenotype, and adult rat fibroblasts, representing scarforming phenotype, were examined whether there were inherent differences in cellular signaling. Specifically, correlation of the phosphorylation patterns with the expression levels of the signaling molecules that transmit information from the plasma membrane receptor to the nucleus was sought. By using three different cell lines of explanted fibroblasts from gestational day 13 fetal rat skin (n = 24) and 1-month-old postnatal adult rat skin (n = 3), immunoblotting was performed to compare tyrosine phosphorylation patterns. The results revealed five major protein bands of interest in fetal rat fibroblasts, but not in the adult rat fibroblasts. These phosphorylated protein bands are of interest because of their possible role in wound repair and may have the potential to regulate cellular responses to the extracellular matrix and their secondary signaling molecules. It was hypothesized that these bands represented receptor tyrosine kinases, epidermal growth factor receptor, and discoidin domain receptor 1, and their downstream adaptor protein Shc that binds receptor tyrosine kinases to transduce signals intracellularly. Furthermore, elevated expression of platelet-derived growth factor receptor-beta in adult compared with fetal fibroblasts was demonstrated, suggesting that decreased expression of certain growth factors may also be important for the scarless phenomenon to occur.     

Differential expression of ionic channels in rat anterior pituitary cells.

Secretory anterior pituitary cells are of the same origin, but exhibit cell type-specific patterns of spontaneous intracellular Ca2+ signaling and basal hormone secretion. To understand the underlying ionic mechanisms mediating these differences, we compared the ionic channels expressed in somatotrophs, lactotrophs, and gonadotrophs from randomly cycling female rats under identical cell culture and recording conditions. Our results indicate that a similar group of ionic channels are expressed in each cell type, including transient and sustained voltage-gated Ca2+ channels, tetrodotoxin-sensitive Na+ channels, transient and delayed rectifying K+ channels, and multiple Ca2+ -sensitive K+ channel subtypes. However, there were marked differences in the expression levels of some of the ionic channels. Specifically, lactotrophs and somatotrophs exhibited low expression levels of tetrodotoxin-sensitive Na+ channels and high expression levels of the large-conductance, Ca2+ -activated K+ channel compared with those observed in gonadotrophs. In addition, functional expression of the transient K+ channel was much higher in lactotrophs and gonadotrophs than in somatotrophs. Finally, the expression of the transient voltage-gated Ca2+ channels was higher in somatotrophs than in lactotrophs and gonadotrophs. These results indicate that there are cell type-specific patterns of ionic channel expression, which may be of physiological significance for the control of Ca2+ homeostasis and secretion in unstimulated and receptor-stimulated anterior pituitary cells.