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Myelin Basic Protein (myelin + basic_protein)
Selected AbstractsAssociation between parasite infection and immune responses in multiple sclerosisANNALS OF NEUROLOGY, Issue 2 2007Jorge Correale MD Objective To assess whether parasite infection is correlated with a reduced number of exacerbations and altered immune reactivity in multiple sclerosis (MS). Methods A prospective, double-cohort study was performed to assess the clinical course and radiological findings in 12 MS patients presenting associated eosinophilia. All patients presented parasitic infections with positive stool specimens. In all parasite-infected MS patients, the eosinophilia was not present during the 2 previous years. Eosinophil counts were monitored at 3- to 6-month intervals. When counts became elevated, patients were enrolled in the study. Interleukin (IL)-4, IL-10, IL-12, transforming growth factor (TGF)-,, and interferon-, production by myelin basic protein,specific peripheral blood mononuclear cells were studied using enzyme-linked immunospot (ELISPOT). FoxP3 and Smad7 expression were studied by reverse-transcriptase polymerase chain reaction. Results During a 4.6-year follow-up period, parasite-infected MS patients showed a significantly lower number of exacerbations, minimal variation in disability scores, as well as fewer magnetic resonance imaging changes when compared with uninfected MS patients. Furthermore, myelin basic protein,specific responses in peripheral blood showed a significant increase in IL-10 and TGF-, and a decrease in IL-12 and interferon-,,secreting cells in infected MS patients compared with noninfected patients. Myelin basic protein,specific T cells cloned from infected subjects were characterized by the absence of IL-2 and IL-4 production, but high IL-10 and/or TGF-, secretion, showing a cytokine profile similar to the T-cell subsets Tr1 and Th3. Moreover, cloning frequency of CD4+CD25+ FoxP3+ T cells was substantially increased in infected patients compared with uninfected MS subjects. Finally, Smad7 messenger RNA was not detected in T cells from infected MS patients secreting TGF-,. Interpretation Increased production of IL-10 and TGF-,, together with induction of CD25+CD4+ FoxP3+ T cells, suggests that regulatory T cells induced during parasite infections can alter the course of MS. Ann Neurol 2007 [source] Effects of early weaning on anxiety and prefrontal cortical and hippocampal myelination in male and female wistar ratsDEVELOPMENTAL PSYCHOBIOLOGY, Issue 4 2008Yuka Kodama Abstract We investigated developmental changes in myelin formation in the prefrontal cortex and the hippocampus, and behavioral effects of early weaning in Wistar rats. Early-weaned rats showed decreased numbers of open-arm entries in an elevated plus-maze in both sexes at 4 weeks old; this effect persisted in males, but ceased in females after this age. Expression of myelin basic protein (MBP) showed both age-dependent increases and sex differences; 4-week-old males exhibited higher MBP levels in the hippocampus, whereas 7-week-old males showed lower MBP levels in the prefrontal cortex compared to females of the same age. There was a tendency for group differences from weaning for the 21.5-kDa isoform in the prefrontal cortex. Although these results suggest that male rats are more vulnerable than females to early-weaning effects on anxiety-related behaviors, further detailed analysis is needed to clarify the functional relationship between myelination and anxiety-related behaviors. © 2008 Wiley Periodicals, Inc. Dev Psychobiol 50: 332,342, 2008. [source] Memory B cells from a subset of treatment-naïve relapsing-remitting multiple sclerosis patients elicit CD4+ T-cell proliferation and IFN-, production in response to myelin basic protein and myelin oligodendrocyte glycoproteinEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 10 2010Christopher T. Harp Abstract Recent evidence suggests that B- and T-cell interactions may be paramount in relapsing-remitting MS (RRMS) disease pathogenesis. We hypothesized that memory B-cell pools from RRMS patients may specifically harbor a subset of potent neuro-APC that support neuro-Ag reactive T-cell proliferation and cytokine secretion. To test this hypothesis, we compared CD80 and HLA-DR expression, IL-10 and lymphotoxin-, secretion, neuro-Ag binding capacity, and neuro-Ag presentation by memory B cells from RRMS patients to naïve B cells from RRMS patients and to memory and naïve B cells from healthy donors (HD). We identified memory B cells from some RRMS patients that elicited CD4+ T-cell proliferation and IFN-, secretion in response to myelin basic protein and myelin oligodendrocyte glycoprotein. Notwithstanding the fact that the phenotypic parameters that promote efficient Ag presentation were observed to be similar between RRMS and HD memory B cells, a corresponding capability to elicit CD4+ T-cell proliferation in response to myelin basic protein and myelin oligodendrocyte glycoprotein was not observed in HD memory B cells. Our results demonstrate for the first time that the memory B-cell pool in RRMS harbors neuro-Ag specific B cells that can activate T cells. [source] Copolymer effects on microglia and T,cells in the central nervous system of humanized miceEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 12 2005Zsolt Illes The random amino acid copolymers FYAK and VWAK ameliorate EAE in a humanized mouse model expressing both a human transgenic myelin basic protein (MBP)85,99-specific T,cell receptor and HLA-DR2. Here we show that microglia isolated from the central nervous system (CNS) of humanized mice with EAE induced by MBP85,99 and treated with these copolymers had reduced expression of HLA-DR, and thus reduced capacity to present MBP85,99 and activate transgenic T,cells. In vitro microglia up-regulated empty HLA-DR2 upon activation with GM-CSF with or without LPS or IFN-,, but not with IL-4 or IL-10. Correspondingly, gene chip arrays showed that the CNS of untreated and YFAK-treated mice differentially expressed pro- and anti-inflammatory molecules during MBP85,99-induced EAE. Interestingly, microglia expressed the full-length ,,,and ,,,subunits of the tetrameric adaptor protein complexes AP-1 and AP-2 respectively, but after treatment with GM-CSF these complexes were cleaved, as had been found in immature dendritic cells derived from bone marrow. Strikingly, in vivo the perivascular lymphocyte infiltration seen in untreated mice immunized with MBP85,99 was composed of equal numbers of hV,2+ MPB85,99-specific transgenic and hV,2, endogenous T,cells, while the much smaller infiltration seen after treatment with YFAK was composed predominantly of hV,2, endogenous T,cells. [source] Exacerbation of experimental autoimmune encephalomyelitis in rodents infected with murine gammaherpesvirus-68EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 7 2003James Abstract Viral infections have long been suspected to play a role in the pathogenesis of multiple sclerosis. In the present study, two different rodent models of experimental autoimmune encephalomyelitis (EAE) were used to demonstrate the ability of murine gammaherpesvirus-68 (,HV-68) to exacerbate development of neurological symptoms. SJL mice received UV-inactivated ,HV-68 or intranasal,HV-68, followed by immunization against proteolipid-protein peptide 139,151. Infected mice became moribund within 10,days post-immunization, whereas mice exposed to UV-inactivated ,HV-68 recovered. In the second model, Lewis rats were exposed to UV-inactivated ,HV-68 or to ,HV-68, followed by passive transfer of encephalitogenic T lymphocytes specific for myelin basic protein. Consistently, infected rats had higher clinical scores, and this result was observed during acute or latent ,HV-68 infection. It is unlikely that this ,HV-68-induced exacerbation was due to significant viral replication within the central nervous system since nested PCR, viral plaque assays, and infectious-centers assays demonstrated no detectable virus in spinal cords or brains of infected rodents undergoing EAE. Taken together, these studies demonstrate increased clinical symptoms of EAE in rodents infected by a gammaherpesvirus that has a limited ability to invade the central nervous system. [source] Minocycline attenuates hypoxia,ischemia-induced neurological dysfunction and brain injury in the juvenile ratEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 2 2006Lir-Wan Fan Abstract To investigate whether minocycline provides long-lasting protection against neonatal hypoxia,ischemia-induced brain injury and neurobehavioral deficits, minocycline was administered intraperitoneally in postnatal day 4 Sprague,Dawley rats subjected to bilateral carotid artery occlusion followed by exposure to hypoxia (8% oxygen for 15 min). Brain injury and myelination were examined on postnatal day 21 (P21) and tests for neurobehavioral toxicity were performed from P3 to P21. Hypoxic,ischemic insults resulted in severe white matter injury, enlarged ventricles, deficits in the hippocampus, reduction in numbers of mature oligodendrocytes and tyrosine hydroxylase-positive neurons, damage to axons and dendrites, and impaired myelination, as indicated by the decrease in myelin basic protein immunostaining in the P21 rat brain. Hypoxic,ischemic insult also significantly affected physical development (body weight gain and eye opening) and neurobehavioral performance, including sensorimotor and locomotor function, anxiety and cognitive ability in the P21 rat. Treatments with minocycline significantly attenuated the hypoxia,ischemia-induced brain injury and improved neurobehavioral performance. The protection of minocycline was associated with its ability to reduce microglial activation. The present results show that minocycline has long-lasting protective effects in the neonatal rat brain in terms of both hypoxia,ischemia-induced brain injury and the associated neurological dysfunction. [source] Delay of myelin formation in arylsulphatase A-deficient miceEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 3 2005Afshin Yaghootfam Abstract Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder caused by the deficiency of arylsulphatase A (ASA). This leads to the accumulation of the sphingolipid 3-O-sulphogalactosylceramide (sulphatide) and progressive demyelination in the nervous system of MLD patients. The mechanisms and development of pathology in the disease are still largely unknown. In this study we investigate how the inability to degrade sulphatide affects the formation of myelin in ASA-deficient (ASA,/,) mice. In mice at 2 weeks of age there was a substantial reduction in myelin basic protein (MBP) mRNA and protein. This was confirmed by an immunohistochemical analysis. MBP mRNA and protein, however, reach normal levels at 3 weeks of age. Proteolipid protein (PLP) and MAL mRNA were also reduced in ASA,/, mice at 2 weeks of age; whereas the level of PLP mRNA was normal at 26 weeks of age, MAL mRNA expression remained reduced up to this age. In situ hybridization revealed no significant changes in the number of myelinating oligodendrocytes or oligodendrocyte precursor cells in ASA,/, mice. These results suggest that oligodendrocyte differentiation was normal in ASA,/, mice. No differences were found in the expression of the sulphatide synthesizing enzymes cerebroside sulphotransferase and UDP-galactose : ceramide galactosyltransferase. Our data demonstrate a delay in myelin formation in ASA,/, mice. This raises the possibility that similar alterations in MLD patients may contribute to the pathology of the disease. [source] Specific Ser-Pro phosphorylation by the RNA-recognition motif containing kinase KISFEBS JOURNAL, Issue 14 2000Alexandre Maucuer We present here a first appraisal of the phosphorylation site specificity of KIS (for ,kinase interacting with stathmin'), a novel mammalian kinase that has the unique feature among kinases to possess an RNP type RNA-recognition motif (RRM). In vitro kinase assays using various standard substrates revealed that KIS has a narrow specificity, with myelin basic protein (MBP) and synapsin I being the best in vitro substrates among those tested. Mass spectrometry and peptide sequencing allowed us to identify serine 164 of MBP as the unique site phosphorylated by KIS. Phosphorylation of synthetic peptides indicated the importance of the proline residue at position +1. We also identified a tryptic peptide of synapsin I phosphorylated by KIS and containing a phosphorylatable Ser-Pro motif. Altogether, our results suggest that KIS preferentially phosphorylates proline directed residues but has a specificity different from that of MAP kinases and cdks. [source] Human fetal radial glia cells generate oligodendrocytes in vitroGLIA, Issue 5 2009Zhicheng Mo Abstract Limited knowledge about human oligodendrogenesis prompted us to explore the lineage relationship between cortical radial glia (RG) cells and oligodendrocytes (OLs) in the human fetal forebrain. RG cells were isolated from cortical ventricular/subventricular zone and their progeny was followed in vitro. One portion of RG cells differentiated into cells of OL lineage identified by cell-type specific antibodies, including platelet-derived growth factor receptor-alpha (PDGFR,), NG2, O4, myelin basic protein, and myelin oligodendrocyte glycoprotein. Moreover, using Cre Lox fate mapping (brain lipid binding protein-Cre/Floxed-yellow fluorescent protein) we established a direct link between RG cells and OL progenitors. In vitro generation of RG-derived O4+ OL progenitors was enhanced by addition of sonic hedgehog (SHH) and reduced by the SHH inhibitor, cyclopamine, suggesting the role of SHH signaling in this process. In summary, our in vitro experiments revealed that a portion of cortical RG cells isolated from human forebrain at the second trimester of gestation generates OL progenitors and this suggests a role of SHH in this process. © 2008 Wiley-Liss, Inc. [source] p38 mitogen-activated protein kinase is required for central nervous system myelinationGLIA, Issue 15 2007Gabriela Fragoso Abstract The p38 MAPKs are a family of kinases that regulate a number of cellular functions including cell migration, proliferation, and differentiation. Here, we report that p38 regulates oligodendrocyte differentiation. Inhibition of p38 with PD169316 and SB203580 prevented accumulation of protein and mRNA of cell-stage specific markers characteristic of differentiated oligodendrocytes, including myelin basic protein, myelin-associated glycoprotein, and the glycosphingolipids, galactosylceramide and sulfatide. In addition, the cell cycle regulator p27kip1 and the transcription factor Sox10 were also significantly reduced. Most significantly, p38 inhibitors completely and irreversibly blocked myelination of dorsal root ganglion neurons by oligodendrocytes and prevented the axolemmal organization of the axo-glial adhesion molecule Caspr. Our results suggest a role(s) for this kinase in key regulatory steps in the maturation of OLGs and initiation of myelination. © 2007 Wiley-Liss, Inc. [source] T-cell seeding: neonatal transfer of anti-myelin basic protein T-cell lines renders Fischer rats susceptible later in life to the active induction of experimental autoimmune encephalitisIMMUNOLOGY, Issue 1 2009Ilan Volovitz Summary Fischer strain rats resist active induction of experimental autoimmune encephalomyelitis (EAE) following immunization with guinea-pig myelin basic protein (MBP) in complete Freund's adjuvant (CFA). Nevertheless, we now report that an encephalitogenic CD4+ anti-MBP T-cell line could be developed from actively immunized Fischer rats. Adoptive transfer of the activated line mediated acute EAE in adult Fischer rats, but not in 1-day-old rats. Moreover, we found that both resting and activated anti-MBP T cells injected 1 day post-natally rendered these rats susceptible later in life to the active induction of EAE by immunization with MBP/CFA. The actively induced EAE manifested the accelerated onset of a secondary, memory-type response. Resting anti-MBP T cells injected even up to 2 weeks post-natally produced no clinical signs but seeded 50,100% of the recipients for an active encephalitogenic immune response to MBP. An earlier T-cell injection (1,2 days) produced a higher incidence and stronger response. The transferred resting T cells entered the neonatal spleen and thymus and proliferated there but did not change the total anti-MBP precursor number in adults. Splenocytes harvested from rats that were injected neonatally but not exposed to MBP in vivo proliferated strongly and produced significant amounts of interferon-, to MBP in vitro. Similar results were observed in rats injected with resting T-cell lines reactive to ovalbumin, suggesting that the neonatal injection of resting T cells specific for a self or for a foreign antigen can seed the immune system with the potential for an enhanced effector response to that antigen later in life. [source] T helper cell type 1 (Th1), Th2 and Th17 responses to myelin basic protein and disease activity in multiple sclerosisIMMUNOLOGY, Issue 2 2008Chris J. Hedegaard Summary Autoreactive T cells are thought to play an essential role in the pathogenesis of multiple sclerosis (MS). We examined the stimulatory effect of human myelin basic protein (MBP) on mononuclear cell (MNC) cultures from 22 patients with MS and 22 sex-matched and age-matched healthy individuals, and related the patient responses to disease activity, as indicated by magnetic resonance imaging. The MBP induced a dose-dependent release of interferon-, (IFN-,), tumour necrosis factor-, (TNF-,) and interleukin-10 (IL-10) by patient-derived MNCs. The patients' cells produced higher amounts of IFN-, and TNF-,, and lower amounts of IL-10, than cells from healthy controls (P < 0·03 to P < 0·04). Five patients with MS and no controls, displayed MBP-induced CD4+ T-cell proliferation. These high-responders exhibited enhanced production of IL-17, IFN-,, IL-5 and IL-4 upon challenge with MBP, as compared with the remaining patients and the healthy controls (P < 0·002 to P < 0·01). A strong correlation was found between the MBP-induced CD4+ T-cell proliferation and production of IL-17, IFN-,, IL-5 and IL-4 (P < 0·0001 to P < 0·01) within the patient group, and the production of IL-17 and IL-5 correlated with the number of active plaques on magnetic resonance images (P = 0·04 and P = 0·007). These data suggest that autoantigen-driven CD4+ T-cell proliferation and release of IL-17 and IL-5 may be associated with disease activity. Larger studies are needed to confirm this. [source] Affinity and catalytic heterogeneity of polyclonal myelin basic protein-hydrolyzing IgGs from sera of patients with multiple sclerosisJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 3 2010Galina A. Legostaeva Abstract Human myelin basic protein (hMBP)-hydrolyzing activity was recently shown to be an intrinsic property of antibodies (Abs) from multiple sclerosis (MS) patients. Here, we present the first evidence demonstrating a significant diversity of different fractions of polyclonal IgGs (pIgGs) from MS patients in their affinity for hMBP and in the ability of pIgGs to hydrolyze hBMP at different optimal pHs (3,10.5). IgGs containing ,- and ,-types of light chains demonstrated comparable relative activities in the hydrolysis of hMBP. IgGs of IgG1,IgG4 sub-classes were analyzed for catalytic activity. IgGs of all four sub-classes were catalytically active, with their contribution to the total activity of Abzs in the hydrolysis of hMBP and its 19-mer oligopeptide increasing in the order: IgG1 (1.5,2.1%) < IgG2 (4.9,12.8%) < IgG3 (14.7,25.0%) < IgG4 (71,78%). Our findings suggest that the immune systems of individual MS patients generate a variety of anti-hMBP abzymes with different catalytic properties, which can attack hMBP of myelin-proteolipid shell of axons, playing an important role in MS pathogenesis. [source] Transgenic mice exhibiting oligodendrocyte-specific expression of a mutant protein tyrosine phosphatase epsilonJOURNAL OF NEUROCHEMISTRY, Issue 2002N. Muja Reversible tyrosine phosphorylation is integral to oligodendrocyte differentiation, and one participant of the phosphorylation cycle, PTP,, is induced in developing oligodendrocytes (Ranjan and Hudson, 1996). To define the role of PTP,, we generated mice expressing a catalytically inactive, hemagglutinin-epitope tagged PTP, (HA-PTP ,) from the 2,,3,-cyclic nucleotide 3,-phosphodiesterase (CNP) promoter. By competing with endogenous, normal PTP, for substrate binding, HA-PTP, would behave in a dominant negative fashion when overexpressed in these mice. Transgene mRNA peaks at postnatal day 21, coincident with the maximal expression of myelin protein mRNAs. Immunohistochemical analyses using antibodies against the HA epitope tag demonstrate that HA-PTP, is expressed in oligodendrocytes, but not in astrocytes and neurons. HA immunoreactivity was present in all myelinated brain structures including the corpus callosum, anterior commissure, and fornix, as well as in the subcortical, cerebellar, and spinal cord white matter. Gross differences in myelination or oligodendrocyte cell density in these brain regions were not detected using antibodies against CNP, myelin basic protein, and an oligodendrocyte marker, CC1. However, by EM axons of the optic nerve appear smaller and less extensively myelinated in transgenic mice than in wild-type littermates. Studies are underway to determine the functional effects of transgene expression on conduction velocity, on the profile of expressed genes, and on potential phosphorylated protein targets of PTP,. [source] Parameters related to lipid metabolism as markers of myelination in mouse brainJOURNAL OF NEUROCHEMISTRY, Issue 1 2001Evan D. Muse Myelination, during both normal development and with respect to disorders of myelination, is commonly studied by morphological and/or biochemical techniques that assay as their end-points the extent of myelination. The rate of myelination is potentially a more useful parameter, but it is difficult and time-consuming to establish, requiring a complete developmental study with labor-intensive methodology. We report herein development of methodology to assay the absolute rate of myelination at any desired time during development. This involves intraperitoneal injection of 3H2O to label body water pools, followed by determination of label in the myelin-specific lipid, cerebroside. The absolute amount of cerebroside synthesized can then be calculated from the specific radioactivity of body water and knowledge of the number of hydrogens from water incorporated into cerebroside. During development, the rate of cerebroside synthesis correlated well with the rate of accumulation of the myelin-specific components, myelin basic protein and cerebroside. For purposes of control, we also tested other putative, albeit less quantitative, indices of the rate of myelination. Levels of mRNA for ceramide galactosyltransferase (rate-limiting enzyme in cerebroside synthesis) and for myelin basic protein did not closely correlate with myelination at all times. Cholesterol synthesis closely matched the rate of cholesterol accumulation but did not track well with myelination. Synthesis of fatty acids did not correlate well with accumulation of either fatty acids (phospholipids) or myelin markers. We conclude that measurement of cerebroside synthesis rates provides a good measure of the rate of myelination. This approach may be useful as an additional parameter for examining the effects of environmental or genetic alterations on the rate of myelination. [source] Reduction of Dicer impairs Schwann cell differentiation and myelinationJOURNAL OF NEUROSCIENCE RESEARCH, Issue 12 2010Jonathan D. Verrier Abstract The process of Schwann cell myelination requires precisely coordinated gene expression. At the onset of myelination, there is an increase in the expression of differentiation-promoting transcription factors that regulate key Schwann cell genes. Further control of myelin gene expression occurs at the posttranscriptional level and, in part, is mediated by RNA binding proteins and micro-RNAs (miRNAs). miRNAs are small, endogenously derived RNA molecules that repress gene expression by specifically binding to their mRNA targets. In the experiments described here, we tested whether miRNAs were essential in controlling myelination by reducing the levels of Dicer, an essential endoribonuclease in miRNA biogenesis. We decreased the expression of Dicer by about 60% within Schwann cells using a lentiviral vector expressing an shRNA against Dicer. The reduced levels of Dicer led to a decrease in the steady-state expression of selected miRNAs and of the transcription factors Oct6 and Egr2/Krox20, both of which are critical for Schwann cells differentiation and myelination. In contrast, the levels of c-jun and Sox2 were up-regulated by the reduction in Dicer and were associated with an increase in Schwann cell proliferation. In dorsal root ganglion cocultures, Schwann cells transduced with Dicer shRNA synthesized less myelin, which was accompanied by significant reductions in the levels of myelin basic protein and protein zero. These findings support a critical role for Dicer and miRNAs in Schwann cell differentiation and myelination. © 2010 Wiley-Liss, Inc. [source] Characterization of CD8-positive macrophages infiltrating the central nervous system of rats with chronic autoimmune encephalomyelitisJOURNAL OF NEUROSCIENCE RESEARCH, Issue 5 2009Keiko Hiraki Abstract CD8+ macrophages appear in the central nervous system (CNS) under various pathological conditions such as trauma and ischemia. Furthermore, macrophages expressing CD8 were found in CNS lesions of chronic, but not acute, experimental autoimmune encephalomyelitis (EAE). To further characterize cells with this phenotype, we examined CD8+ macrophages/monocytes in the CNS and peripheral organs during the course of acute and chronic EAE that had been induced by immunization of rats with myelin basic protein and myelin oligodendrocyte glycoprotein, respectively. Counting CD8+ macrophages in CNS lesions revealed that their numbers increased reaching about 60% of total infiltrating macrophages in chronic EAE, while CD8+ macrophages remained less than 5% throughout the course of acute EAE. Unexpectedly, however, higher abundance of CD8+ monocytes/macrophages in the peripheral blood was found in both acute and chronic EAE. Real-time polymerase chain reaction analysis revealed no significant difference in the levels of chemokines and chemokine receptors of blood CD8+ monocytes between acute and chronic EAE. mRNA expression of perforin, a cytotoxic substance, was up-regulated in CD8+ monocytes compared with that of CD8, monocytes in both acute and chronic EAE. These findings suggest that activated CD8+ macrophages may play a cytotoxic role in chronic EAE lesions and that cells other than CD8+ monocytes/macrophages determined the difference in CNS pathology between acute and chronic EAE. Analysis of CD8+ monocytes/macrophages may provide useful information to permit further dissect the pathomechanisms of multiple sclerosis and to develop effective immunotherapies against autoimmune diseases in the CNS. © 2008 Wiley-Liss, Inc. [source] Signal transduction pathways involved in interaction of galactosylceramide/sulfatide-containing liposomes with cultured oligodendrocytes and requirement for myelin basic protein and glycosphingolipidsJOURNAL OF NEUROSCIENCE RESEARCH, Issue 7 2008Joan M. Boggs Abstract We showed previously that the addition to cultured oligodendrocytes (OLs) of multivalent carbohydrate in the form of liposomes containing the two major glycosphingolipids (GSLs) of myelin, galactosylceramide (GalC) and cerebroside sulfate (Sulf), or galactose conjugated to bovine serum albumin caused clustering of GalC on the extracellular surface and myelin basic protein (MBP) on the cytosolic surface. Multivalent carbohydrate also caused depolymerization of actin microfilaments and microtubules, indicating that interaction of the carbohydrate with the OL surface transmits a transmembrane signal to the cytoskeleton. In the present study we show that inhibition of GSL synthesis with fumonisin B1 prevents clustering of MBP in GalC/Sulf-negative oligodendrocytes, suggesting that GSLs are required for the effect. Because the effects of multivalent carbohydrate resemble those caused by the addition of anti-GalC/Sulf antibodies to OLs and because GalC and Sulf can interact with each other by trans carbohydrate,carbohydrate interactions across apposed membranes, these results support the conclusion that the OL receptor for GalC/Sulf in liposomes is GalC/Sulf in the OL membrane. Inhibition of MBP expression using MBP siRNA inhibited GalC clustering, suggesting that MBP is required for the effect. We also investigate the signal transduction pathways involved using a number of enzyme inhibitors. These indicated that the Akt and p42/p44 MAPK pathways, Rho GTPases, and GSK-3, are involved, consistent with their known involvement in regulation of the cytoskeleton. These interactions between GalC/Sulf-containing liposomes and the OL membrane may mimic interactions between GalC/Sulf-enriched signaling domains when OL cell membranes or the extracellular surfaces of compact myelin come into contact. © 2008 Wiley-Liss, Inc. [source] Insulin-like growth factor-I ameliorates demyelination induced by tumor necrosis factor-, in transgenic miceJOURNAL OF NEUROSCIENCE RESEARCH, Issue 4 2007Ping Ye Abstract Our groups have reported that tumor necrosis factor-, (TNF-,) causes myelin damage and apoptosis of oligodendrocytes and their precursors in vitro and in vivo. We also have reported that insulin-like growth factor-I (IGF-I) can protect cultured oligodendrocytes and their precursors from TNF-,-induced damage. In this study, we investigated whether IGF-I can protect oligodendrocytes and myelination from TNF-,-induced damage in vivo by cross-breeding TNF-, transgenic (Tg) mice with IGF-I Tg mice that overexpress IGF-I exclusively in brain. At 8 weeks of age, compared with those of wild-type (WT) mice, the brain weights of TNF-, Tg mice were decreased by ,20%, and those of IGF-I Tg mice were increased by ,20%. The brain weights of mice that carry both TNF-, and IGF-I transgenes (TNF-,/IGF-I Tg mice) did not differ from those of WT mice. As judged by histochemical staining and immunostaining, myelin content in the cerebellum of TNF-,/IGF-I Tg mice was similar to that in WT mice and much more than that in TNF-, Tg mice. Consistently, Western immunoblot analysis showed that myelin basic protein (MBP) abundance in the cerebellum of TNF-,/IGF-I Tg mice was double that in TNF-, Tg mice. In comparison with WT mice, the number of oligodendrocytes was decreased by ,36% in TNF-, Tg mice, whereas it was increased in IGF-I Tg mice by ,40%. Oligodendrocyte number in TNF-,/IGF-I Tg mice was almost twice that in TNF-, Tg mice. Furthermore, IGF-I overexpression significantly reduced TNF-,-induced increases in apoptotic cell number, active caspase-3 abundance, and degradaion of MBP. Our results indicate that IGF-I is capable of protecting myelin and oligodendrocytes from TNF-,-induced damage in vivo. © 2007 Wiley-Liss, Inc. [source] Characterization of thromboxane A2 receptor signaling in developing rat oligodendrocytes: Nuclear receptor localization and stimulation of myelin basic protein expressionJOURNAL OF NEUROSCIENCE RESEARCH, Issue 7 2006Santosh Ramamurthy Abstract The present work investigates the role of thromboxane A2 (TXA2) receptors in the development of oligodendrocytes (OLGs). The results demonstrate that the proteins of the TXA2 signaling pathway, i.e., cyclooxygenase (COX-1), TXA2 synthase (TS), and TXA2 receptor (TPR) are expressed in the developing rat brain during myelination. Furthermore, culture of OLG progenitor cells (OPCs) revealed that the expression levels of these proteins as well as TXA2 synthesis increase during OLG maturation. Separate studies established that activation of TPRs by the agonist U46619 increases intracellular calcium in both OPCs and OLGs as visualized by digital fluorescence imaging. Immunocytochemical staining demonstrated that TPRs are localized in the plasma membrane and perinuclear compartments in OPCs. However, during OLG differentiation, TPRs shift their localization pattern and also become associated with the nuclear compartment. This shift to nuclear localization was confirmed by biochemical analysis in cultured cells and by immunocytochemical analysis in developing rat brain. Finally, it was found that U46619 activation of TPRs in maturing OLGs resulted in enhanced myelin basic protein (MBP) expression. Alternatively, inhibition of endogenous TPR signaling led to reduced MBP expression. Furthermore, TPR-mediated MBP expression was found to be associated with increased transcription from the MBP promoter using a MBP-luciferase reporter. Collectively, these findings suggest a novel TPR signaling pathway in OLGs and a potential role for this signaling during OLG maturation and myelin production. © 2006 Wiley-Liss, Inc. [source] Quantitation of myelin oligodendrocyte glycoprotein and myelin basic protein in the thymus and central nervous system and its relationship to the clinicopathologic features of autoimmune encephalomyelitisJOURNAL OF NEUROSCIENCE RESEARCH, Issue 3 2006Hiroshi Sakuma Abstract There is controversy whether the amount of autoantigens expressed in the thymus regulates negative selection of autoreactive T cells and determine susceptibility or resistance to experimental autoimmune encephalomyelitis (EAE). In the present study, we have addressed this issue by quantifying neuroantigens in the thymus of two EAE-susceptible (LEW and LEW.1AV1) and one EAE-resistant (BN) rat strains. We further examined whether amounts of neuroantigens in various parts of the central nervous system (CNS) affect the clinical course and lesion distribution of acute and chronic EAE. Real-time PCR and histologic analyses showed that there was no significant difference in the amount and distribution of myelin oligodendrocyte glycoprotein and myelin basic protein in the thymus and CNS among the three strains and that both acute and chronic EAE lesions in the CNS were preferentially distributed in the area where neuroantigens were abundantly present. These findings suggest that susceptibility or resistance to EAE is not regulated by the amount of the neuroantigens expressed in the thymus. Furthermore, the lesion distribution, but not the clinical course, of EAE is related to the neuroantigen expression in the CNS. © 2006 Wiley-Liss, Inc. [source] Myelin proteolipid protein, basic protein, the small isoform of myelin-associated glycoprotein, and p42MAPK are associated in the Triton X-100 extract of central nervous system myelinJOURNAL OF NEUROSCIENCE RESEARCH, Issue 1 2002Dina N. Arvanitis Abstract To further our understanding of the functions of the major myelin proteins, myelin basic protein (MBP) and proteolipid protein (PLP), and other myelin proteins, such as 2,3,-cyclic nucleotide 3,-phosphodiesterase (CNP) and myelin-associated glycoprotein (MAG), bovine brain myelin was extracted with Triton X-100, and protein complexes in the detergent-soluble fraction were isolated by coimmunoprecipitation and sucrose density gradient sedimentation. MBP, PLP, and the small isoform of MAG (S-MAG) were coimmunoprecipitated from the detergent-soluble fraction by anti-PLP, anti-MBP or anti-MAG monoclonal antibodies. Additionally, a 30 kDa phosphoserine-containing protein and two phosphotyrosine-containing proteins (Mr 30 and 42 kDa) were found in the coimmunoprecipitates. The 42 kDa protein is probably p42MAPK, in that MAPK was shown also to be present in the immunoprecipitated complex. CNP, the small PLP isoform DM20, the large MAG isoform L-MAG, MOG, CD44, MEK, p44MAPK, and actin were not present in the immunoprecipitates, although they were present in the detergent-soluble fraction. Lipid analysis revealed that the PLP,MBP,S-MAG coimmunoprecipitated with some phospholipids and sulfatide but not cholesterol or galactosylceramide. However, the complex had a high density, indicating that the lipid/protein ratio is low, and it was retained on a Sepharose CL6B column, indicating that it is not a large membrane fragment. Given that MAG is localized mainly in the periaxonal region of myelin, where it interacts with axonal ligands, the PLP,MBP,S-MAG complex may come from these regions, where it could participate in dynamic functions in the myelin sheath and myelin,axonal interactions. © 2002 Wiley-Liss, Inc. [source] Efficient gene transfer in mouse neural precursors with a bicistronic retroviral vectorJOURNAL OF NEUROSCIENCE RESEARCH, Issue 3 2001Isabelle A. Franceschini Abstract Gene transfer into neural precursors is a powerful approach to study the function of specific gene products during nervous system development. Here we describe a retrovirus-based methodology to transduce foreign genes into mouse neural precursors. We used a high-titer bicistronic retroviral vector that encodes a marker gene, placental alkaline phosphatase (plap), and a selection gene, neomycin phosphotransferase II (neoR), under the translational control of two retroviral internal ribosome entry segments. Transduction efficiency even without selection was up to 95% for multipotential neurospheres derived from embryonic striata and grown with basic fibroblast growth factor 2. Expression of plap and neoR was sustained with time in culture and upon differentiation into neurons, astrocytes, and oligodendrocytes, as shown by double immunofluorescence labeling with cell type-specific markers, Western blotting, and neomycin resistance. However, levels of plap were decreased in differentiated oligodendrocytes. Transduction with the same vector of neonatal oligodendrocyte precursors grown in oligospheres consistently resulted in a lower proportion of plap-immunoreactive cells and enhanced cell death in the absence of neomycin. However, plap expression was maintained in some differentiated oligodendrocytes expressing galactocerebroside or myelin basic protein. In that neurospheres can be easily expanded in vitro and factors enabling their differentiation into the three main central nervous system cell types are being elucidated, this methodology could be used in the future to produce large number of transduced, differentiated neural cells. J. Neurosci. Res. 65:208,219, 2001. © 2001 Wiley-Liss, Inc. [source] A CALCIUM-DEPENDENT PROTEIN KINASE FUNCTIONS IN WOUND HEALING IN VENTRICARIA VENTRICOSA (CHLOROPHYTA)JOURNAL OF PHYCOLOGY, Issue 6 2000Koh-ichi Sugiyama The cytoplasm around a wound made in the multinucleate unicellular green alga Ventricaria ventricosa ( J. Agardh) Olsen et West formed an aggregation-ring surrounding the wound immediately after injury. A contraction of the ring then brought about wound healing in culture medium containing Ca2+. Involvement of a calcium-dependent protein kinase (CDPK) as a regulator of wound healing was examined using an anti- Dunaliella tertiolecta CDPK antibody. A 52-kDa protein cross-reacting with the antibody was detected by Western blotting. Protein kinases of 60 kDa and 52 kDa, which were markedly activated by Ca2+, and a 40-kDa Ca2+ -independent protein kinase were detected by an in-gel protein kinase assay using myelin basic protein as the substrate. A 52-kDa band with Ca2+ -dependent protein kinase activity was immunoprecipitated from the cytoplasmic extract, indicating that these 52-kDa proteins are identical and possess CDPK activity. Microscopic observation showed that the contraction of the aggregation ring was suppressed by application of the anti-CDPK to the culture medium. A protein kinase inhibitor, K-252a, and the calmodulin inhibitors, calmidazolium and compound 48 / 80, which inhibit CDPK activity, also suppressed the contraction of the aggregation-ring. Immunofluorescence microscopy showed a similar distribution of 52-kDa CDPK to the distribution of f-actin, which was randomly distributed in an intact cell and formed a bundle during wound healing. Further, f-actin was not recruited after injury in the presence of the antibody to CDPK. These results suggest that the 52-kDa CDPK functions as a Ca2+ receptor in wound healing and simultaneously participates in the organization and contraction of f-actin to heal the wound. [source] Ultrastructural identification of peripheral myelin proteins by a pre-embedding immunogold labeling methodJOURNAL OF THE PERIPHERAL NERVOUS SYSTEM, Issue 1 2003Marie-Hélène Canron Abstract Ultrastructural immunolabeling of peripheral nervous system components is an important tool to study the relation between structure and function. Owing to the scarcity of certain antigens and the dense structure of the peripheral nerve, a pre-embedding technique is likely appropriate. After several investigations on procedures for pre-embedding immunolabeling, we propose a method that offers a good compromise between detection of antigenic sites and preservation of morphology at the ultrastructural level, and that is easy to use and suitable for investigations on peripheral nerve biopsies from humans. Pre-fixation by immersion in paraformaldehyde/glutaraldehyde is necessary to stabilize the ultrastructure. Then, ultrasmall gold particles with silver enhancement are advised. Antibodies against myelin protein zero and myelin basic protein were chosen for demonstration. The same technique was applied to localize a 35 kDa myelin protein. [source] Two Ser/Thr protein kinases essential for efficient aggregation and spore morphogenesis in Myxococcus xanthusMOLECULAR MICROBIOLOGY, Issue 6 2006Emily A. Stein Summary Myxococcus xanthus has a complex life cycle that involves vegetative growth and development. Previously, we described the espAB locus that is involved in timing events during the initial stages of fruiting body formation. Deletion of espA caused early aggregation and sporulation, whereas deletion of espB caused delayed aggregation and sporulation resulting in reduced spore yields. In this study, we describe two genes, pktA5 and pktB8, that flank the espAB locus and encode Ser/Thr protein kinase (STPK) homologues. Cells deficient in pktA5 or pktB8 formed translucent mounds and produced low spore yields, similar in many respects to espB mutants. Double mutant analysis revealed that espA was epistatic to pktA5 and pktB8 with respect to aggregation and fruiting body morphology, but that pktA5 and pktB8 were epistatic to espA with respect to sporulation efficiency. Expression profiles of pktA5,lacZ and pktB8,lacZ fusions and Western blot analysis showed that the STPKs are expressed under vegetative and developmental conditions. In vitro kinase assays demonstrated that the RD kinase, PktA5, autophosphorylated on threonine residue(s) and phosphorylated the artificial substrate, myelin basic protein. In contrast, autophosphorylation of the non-RD kinase, PktB8, was not observed in vitro; however, the phenotype of a pktB8 kinase-dead point mutant resembled the pktB8 deletion mutant, indicating that this residue was important for function and that it likely functions as a kinase in vivo. Immunoprecipitation of Tap-tagged PktA5 and PktB8 revealed an interaction with EspA during development in M. xanthus. These results, taken together, suggest that PktA5 and PktB8 are STPKs that function during development by interacting with EspA and EspB to regulate M. xanthus development. [source] Germinal vesicle materials are not required for the activation of MAP kinase in porcine oocyte maturationMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 2 2001K. Sugiura Abstract The requirement of the germinal vesicle (GV) for the normal kinetics of mitogen-activated protein (MAP) kinase activity during porcine oocyte maturation was investigated. Porcine follicular oocytes were enucleated, and the locations of their extracellular signal-regulated kinases 1 and 2 (ERK1/2), major MAP kinases in maturating porcine oocytes, were detected by indirect immunofluorescent microscopy. The MAP kinase activity was assayed as myelin basic protein (MBP) kinase activity, and the phosphorylation states of ERK1/2 were detected by immunoblotting analyses. Translocation of MAP kinase into the GV and association with the spindle were observed in intact oocytes, while MAP kinase in enucleated oocytes was distributed almost uniformly in cytoplasm throughout the culturing period. The phosphorylation and the activation of MAP kinase were induced, and the activity was comparable with that of control denuded oocytes. The high level of activity was maintained through maturation, even in the absence of spindle formation. These results indicate that the presence of nuclear material and translocation into the GV are dispensable for the activation of MAP kinase and that associating with the spindle is not required for maintenance of its activity though porcine oocyte maturation. Mol. Reprod. Dev. 59:215,220, 2001. © 2001 Wiley-Liss, Inc. [source] A growth hormone-secreting adenoma with incomplete nerve bundle formationNEUROPATHOLOGY, Issue 3 2008Hidetoshi Ikeda We present a unique case of an adenoma secreting growth hormone (GH), showing incomplete nerve bundle formation without ganglion cells. A 47-year-old man presenting with acromegaly was revealed to have high serum GH and IGF-1 levels. The concentrations of the other adenohypophysial hormones were within the normal range. Histology revealed an unusual pituitary adenoma containing many nerve bundle-like structures. Adenoma cells with ovoid or round hyperchromatic nuclei and eosinophilic cytoplasms lacked the typical features of ganglion cells. The nerve bundles consisted of slender elongated cells. These fibers were arranged into groups in a roughly parallel fashion. By immunohistochemistry, many adenoma cells were positive for GH, prolactin, thyrotropin beta, synaptophysin and chromogranin. Fibrous bodies revealed by keratin immunostaining were found only in adenoma cells. Scattered star-shaped adenoma cells showed the same immunoreactivity as folliculo-satellite cells. Adenoma cells, but not the bundle-like structures, were also positive for Pit-1. Immunostaining for neurofilament protein, GFAP, vimentin, and S-100 protein revealed variable amounts of fibrils within the bundle-like structures. Scattered immunoreactivity for myelin basic protein and synaptophysin was also found in the bundle area. Our case is the first GH-secreting pituitary adenoma showing incomplete nerve bundle differentiation and lacking mature ganglion cells. [source] Sequential myelin protein expression during remyelination reveals fast and efficient repair after central nervous system demyelinationNEUROPATHOLOGY & APPLIED NEUROBIOLOGY, Issue 1 2008M. Lindner To understand the mechanisms of remyelination and the reasons for regeneration failure is one of the major challenges in multiple sclerosis research. This requires a good knowledge and reliable analysis of experimental models. This work was undertaken to characterize the pattern of myelin protein expression during experimental remyelination. Acute demyelination of the corpus callosum was induced by feeding of 0.3% cuprizone for 6 weeks, followed by a 10-week remyelination period. We used a combination of Luxol fast blue (LFB) myelin staining, electron microscopy (EM) and immunohistochemistry for the myelin proteins 2,,3,-cyclic nucleotide 3, phosphodiesterase (CNPase), myelin basic protein (MBP), proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG). Early remyelination was detected by the re-expression of CNPase, MBP and PLP as early as 4 days. MOG, as a marker for late differentiation of oligodendrocytes, was not detectable until 2 weeks of remyelination. EM data correlated well with the LFB myelin staining and myelin protein expression, with 50% of the axons being rapidly remyelinated within 2 weeks. While particularly MBP but also PLP and CNPase are re-expressed very early before significant remyelination is observed by EM, the late marker MOG shows a lag behind the remyelination detected by EM. The presented data indicate that immunohistochemistry for various myelin proteins expressed early and late during myelin formation is a suitable and reliable method to follow remyelination in the cuprizone model. Furthermore, investigation of early remyelination confirms that the intrinsic repair programme is very fast and switched on within days. [source] Amelioration of experimental autoimmune encephalomyelitis in Lewis rats treated with fucoidanPHYTOTHERAPY RESEARCH, Issue 3 2010Heechul Kim Abstract We examined whether fucoidan affected the clinical symptoms of experimental autoimmune encephalomyelitis (EAE) in rats. EAE was induced in Lewis rats that were immunized with guinea-pig myelin basic protein (MBP) and complete Freund's adjuvant. Fucoidan (50,mg/kg, daily) was administered to rats with EAE intraperitoneally, either in the EAE induction phase from either 1 day before immunization to day 7 post-immunization (PI), or the effector phase from day 8 to 14 PI, to test which phase of rat EAE is affected by fucoidan treatment. The onset, severity and duration of EAE paralysis in the fucoidan-treated group in the days 8,14 PI-treated rats, but not in days ,1,7 PI-treated rats, were significantly delayed, suppressed and reduced, respectively, compared with the vehicle-treated controls. Treatment with fucoidan reduced the encephalitogenic response and TNF-, production during EAE. Moreover, the clinical amelioration coincided with decreased infiltration of inflammatory cells in the EAE-affected spinal cord. The ameliorative effect of fucoidan on clinical paralysis in EAE-affected rats may be mediated, in part, by the suppression of the autoreactive T cell response and inflammatory cytokine production. 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