Myelin Oligodendrocyte-specific Protein Research Articles

Calpain inhibition reduces structural and functional impairment of retinal ganglion cells in experimental optic neuritis.

Optic neuritis (ON), inflammation of the optic nerve, is strongly associated with multiple sclerosis. ON pathology is characterized by attack of autoreactive T cells against optic nerve antigens, resulting in demyelination, death of retinal ganglion cells, and cumulative visual impairment. A model of experimental autoimmune encephalomyelitis (EAE) was utilized to study the onset and progression of ON and the neuroprotective efficacy of oral treatment with the calpain inhibitor SNJ 1945. EAE was actively induced in B10.PL mice with myelin basic protein on Days 0 and 2, and mice received twice daily oral dosing of SNJ 1945 from Day 9 until sacrificing (Day 26). Visual function was determined by electroretinogram recordings and daily measurement of optokinetic responses (OKR) to a changing pattern stimulus. Optic nerve and retinal histopathology was investigated by immunohistochemical and luxol fast blue staining. EAE mice manifested losses in OKR thresholds, a measurement of visual acuity, which began early in the disease course. There was a significant bias toward unilateral OKR impairment among EAE-ON eyes. Treatment with SNJ 1945, initiated after the onset of OKR threshold decline, improved visual acuity, pattern electroretinogram amplitudes, and paralysis, with attenuation of retinal ganglion cell death. Furthermore, calpain inhibition spared oligodendrocytes, prevented degradation of axonal neurofilament protein, and attenuated reactive astrocytosis. The trend of early, unilateral visual impairment in EAE-ON parallels the clinical presentation of ON exacerbations associated with multiple sclerosis. Calpain inhibition may represent an ideal candidate therapy for the preservation of vision in clinical ON. As in multiple sclerosis (MS) patients, optic neuritis (ON) and early, primarily monocular loss in spatial acuity is observed in a rodent model (EAE, experimental autoimmune encephalomyelitis). Daily oral treatment with the calpain inhibitor SNJ 1945 preserves visual acuity and preserves retinal ganglion cells (Brn3a, brain-specific homeobox/POU domain protein 3A) and their axons (MOSP, myelin oligodendrocyte-specific protein). Calpain inhibition may represent a candidate therapy for the preservation of vision in ON.

Immunofluorescence: main stages of phenotyping and differential potential of autologous mesenchymal stem cells in vitro

The article contains detailed information about possible use of immunofluorescence for studying the main stages of phenotyping and differentiation of autologous mesenchymal stem cells (AMSCs) in vitro. The authors present facts, which concern origination of cells (3-month-old male Wistar rats), methods of their generation in laboratory conditions (isolation from femoral and tibial diaphyses and epiphyses of animals followed by washing with culture medium, inoculation on Petri dishes, explantation, cleaning from blood corpuscles, incubation in a monolayer, reinoculation). Conditions of phenotyping are described (with use of flow cytofluorometry, the received DNA was polymerised with specific primers and finished with a polymerase chain reaction). The reaction products were analysed by the method of electrophoresis in agarose gel. AMSCs were differentiated in the neuron direction after the second culture reinoculation. In order to induce the differentiation, neurotrophin-3 growth factor was used. AMSCs were grown up to a thick monolayer. The obtained derivates were phenotyped by staining cells with antibodies to protein markers of neurons, neuron-specific nuclear protein (NeuN) (Chemicon), neurofibrillary axonal protein (Tau) (Chemicon), myelin/oligodendrocyte-specific protein (MOSP) (Chemicon), glial fibrillary acidic protein (GFAP) (Chemicon). As a result of the study, it was revealed that the number of CD90+ cells (which actually proved to be the population of AMSCs) was 95-97 %. The phenotype of cells, determined on the standard basis by use of superficial markers, did not change during the first four passages. Incubation of the population of AMSCs during these passages proved that the above cells did not lose their ability to differentiate in three main orthodox directions. Thus, the population of AMSCs remained homogenous.

Stem Cell Therapy in Spinal Cord Injury: In Vivo and Postmortem Tracking of Bone Marrow Mononuclear or Mesenchymal Stem Cells

The aim of this study was to address the question of whether bone marrow-originated mononuclear cells (MNC) or mesenchymal stem cells (MSC) induce neural regeneration when implanted intraspinally. The study design included 4 groups of mice: Group 1, non-traumatized control group; Groups 2, 3 and 4 spinal cord traumatized mice with 1 g force Tator clips, which received intralesionally either no cellular implants (Group 2), luciferase (Luc) (+) MNC (Group 3) or MSC (Group 4) obtained from CMV-Luc or beta-actin Luc donor transgenic mice. Following the surgery until decapitation, periodical radioluminescence imaging (RLI) and Basso Mouse Scale (BMS) evaluations was performed to monitor neural activity. Postmortem immunohistochemical techniques were used to analyze the fate of donor type implanted cells. All mice of Groups 3 and 4 showed various degrees of improvement in the BMS scores, whereas there was no change in Groups 1 and 2. The functional improvement was significantly better in Group 4 compared to Group 3 (18 vs 8, p=0.002). The immunohistochemical staining demonstrated GFP(+)Luc(+) neuronal/glial cells that were also positive with one or more of these markers: nestin, myelin associated glycoprotein, microtubule associated protein or myelin oligodendrocyte specific protein, which is considered as indicator of donor type neuronal regeneration. Frequency of donor type neuronal cells; Luc + signals and median BMS scores were observed 48-64% and 68-72%; 44-80%; 8 and 18 within Groups III and IV respectively. MSCs were more effective than MNC in obtaining neuronal recovery. Substantial but incomplete functional improvement was associated with donor type in vivo imaging signals more frequently than the number of neuronal cells expressing donor markers in spinal cord sections in vitro. Our results are in favor of functional recovery arising from both donor MSC and MNCs, contributing to direct neuronal regeneration and additional indirect mechanisms.

Myelin oligodendrocyte‐specific protein is expressed in Müller cells of myelinated vertebrate retinas

The retina of nonmammalian vertebrates has a loose myelin that enwraps the large axons of the ganglion cells in all areas, whereas that of mammals lacks myelin, with some exceptions, such as the rabbit retina, which shows compact myelin restricted to the myelinated streak. Electron microscopy studies in chicken retina showed processes of Müller cells (MCs) and oligodendrocytes enwrapping ganglion cell axons. How each of these cells contributes to chicken retina myelination and whether the MC of other myelinated retinas is involved in myelination remain unknown. By immunohistochemistry, with a monoclonal antibody against myelin oligodendrocyte-specific protein (MOSP), we show that MOSP is intensely expressed in the MC and the optic-fiber layer (OFL) in myelinated but not in unmyelinated retinas. By immunocytochemistry with isolated MCs from the chick and rabbit retinas, we show that MOSP is concentrated in the innermost domain of the vitread processes. By immunoblotting, we show that protein extracts from myelinated retinas, but not those from unmyelinated retinas, presented a single band labelled with anti-MOSP of molecular weight similar to that of brain MOSP. In addition, we show that the MC of the embryonic chicken retina starts to express MOSP just before myelination starts. Our results agree with those of electron microscopy studies showing myelin in chick retina formed by MC processes and with those of immunohistochemistry studies in rabbit and human retinas showing expression of other myelin molecules in the MC. Altogether, our results suggest that the MC in myelinated retinas might contribute MOSP to myelin.

The protective role of squalene in alcohol damage in the chick embryo retina

The developing CNS, and in particular the visual system, is very sensitive to the effects of alcohol. Alcohol causes lipid peroxidation. Squalene, the major olive oil hydrocarbon, is a quencher of singlet oxygen and prevents the corresponding lipid peroxidation. We presumed that squalene can protect against the alcohol-induced damage already observed during the development of the chick retina. Alcohol+squalene was administered directly into the yolk sac of the egg of White Leghorn chicks at day 6 of incubation. The lipid composition of the retina was analyzed in embryos at E7, E11, E15 and E18. The proportions of phospholipids, free and esterified cholesterol, diacylglycerides and free fatty acids were estimated using the Iatroscan TLC/FID procedure. Gas chromatography and mass spectrometry were used to determine the fatty acid composition. The morphological study was carried out at E11 using semithin sections, and by means of immunohistochemical techniques at E19. Comparing the results obtained in control embryos, the administration of alcohol+squalene reduces the effects of alcohol on the total lipid composition of the retina during development. The effects were, in fact, of less magnitude than in embryos treated only with alcohol. The major phospholipid species of alcohol+squalene-treated embryos exhibited total recuperation at E15. As far as fatty acids are concerned, no significant changes were observed with regard to control embryos during development. From a morphological point of view, the retinas of alcohol+squalene-treated embryos show at E11 fewer cellular alterations than the retinas of alcohol-treated embryos. In this respect, the retinas of alcohol+squalene-treated embryos exhibited: a columnar cell arrangement similar to that observed in control retinas; few pycnotic cells and very few alterations in ganglion cell layers and in the optic nerve fibers layer. At E19 the recuperation of the expression of myelin oligodendrocyte specific protein (MOSP) in alcohol+squalene-treated embryos was recorded. Since squalene reduces the deleterious effects caused by alcohol on the lipid composition and the structure of the retina, squalene could act as a naturally occurring agent for the prevention of damage caused by abusive alcohol ingestion during pregnancy.

Effects of interferon-gamma and tumor necrosis factor-alpha on survival and differentiation of oligodendrocyte progenitors.

There is strong evidence from recent clinical studies that ascending intrauterine infection is associated with an increased incidence of periventricular leukomalacia in very premature fetuses. Periventricular leukomalacia is characterized by disrupted myelination from a loss of oligodendrocyte progenitors. We investigated the effects of proinflammatory cytokines on the survival and differentiation of this cell type. Cultures of more than 90% A2B5-positive progenitors were prepared from neonatal rats and kept for 3 days in medium supplemented with factors that stimulate cell proliferation. After 1 day in proliferation medium, cells were treated with interferon-gamma (100 U/mL) and tumor necrosis factor-alpha (100 ng/mL) for 48 hours triggering an increase in apoptotic A2B5 progenitor cells from 3.2 +/- 2.3% to 11.0 +/- 2.6%. After cytokine treatment cultures were transferred to medium containing factors to promote differentiation of progenitors into the myelinating phenotype. In cytokine pretreated cultures, only 2.6 +/- 1.1% of total cells survived after a total of 9 days in vitro, whereas in untreated cultures most cells differentiated as shown by expression of myelin basic protein, myelin-associated glycoprotein, 2,3-cyclic nucleotide 3-phosphodiesterase, and myelin oligodendrocyte-specific protein. Using ten-fold reduced concentrations of combined interferon-gamma (10 U/mL) and tumor necrosis factor-alpha (10 ng/mL) pretreatment resulted in a survival to 11.2 +/- 4.9% of total cells with 36.3 +/- 11.6% A2B5-positive cells at day 9. This indicates a major enrichment of undifferentiated cells compared with untreated controls which harbored only 1.0 +/- 0.3% A2B5-positive cells. Inflammatory cytokines not only induced apoptotic cell death but also prevented the differentiation of immature A2B5 oligodendrocyte progenitors into the myelinating phenotype.

Age-dependent myelin degeneration and proteolysis of oligodendrocyte proteins is associated with the activation of calpain-1 in the rhesus monkey.

Myelin provides important insulating properties to axons allowing for propagation of action potentials over large distances at high velocity. Disruption of the myelin sheath could therefore contribute to cognitive impairment, such as that observed during the normal aging process. In the present study, age-related changes in myelin, myelin proteins and oligodendrocyte proteins were assessed in relationship to calpain-1 expression and cognition in the rhesus monkey. Isolation of myelin fractions from brain white matter revealed that as the content of the intact myelin fraction decreased with age, there was a corresponding increase in the floating or degraded myelin fraction, suggesting an increased breakdown of intact myelin with age. Of the myelin proteins examined, only the myelin-associated glycoprotein decreased with age. Levels of the oligodendrocyte-specific proteins 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) and myelin/oligodendrocyte-specific protein (MOSP) increased dramatically in white matter homogenates and myelin with age. Age-related increases in degraded CNPase also were demonstrable in white matter in association with increases in activated calpain-1. Degraded CNPase was also detectable in myelin fractions, with only the floating fraction containing activated calpain-1. The increases in the activated enzyme in white matter were much greater than those found in myelin fractions suggesting a source other than the myelin membrane for the marked overexpression of activated calpain-1 with age. In addition, CNPase was demonstrated to be a substrate for calpain in vitro. In summary, changes in myelin and oligodendrocyte proteins occur with age, and they appear to have a significant relationship to cognitive impairment. The overexpression of CNPase and MOSP suggests new formation of myelin by oligodendrocytes, which may occur in response to myelin degradation and injury caused by proteolytic enzymes such as calpain.

GFAP-positive and myelin marker-positive glia in normal and pathologic environments.

The data herein demonstrate that in addition to the well-characterized myelin marker-positive, glial fibrillary acidic protein (GFAP)-negative, membrane sheet-bearing oligodendrocytes, another type of myelin marker-positive, process-bearing glia exists in normal and pathologic conditions. This second type of myelin marker-positive glia expresses GFAP, and therefore these cells have been referred to as mixed phenotype glia. Although mixed phenotype glia have been documented previously, their identity and function have remained a mystery. The goal of this immunocytochemical study was to further characterize these cells. Using the MBPlacZ transgenic mouse in which beta-galactosidase is under the control of the myelin basic protein (MBP) gene promoter, GFAP-positive/beta-galactosidase-positive and myelin/oligodendrocyte-specific protein (MOSP)-positive/beta-galactosidase-positive cells were detected in subcortical white matter and in perivascular locations within cerebral white and gray matter. In cultures prepared from highly enriched myelin marker-positive immature glia, mixed phenotype glia were detected that were GFAP-positive and either MOSP-, MBP-, O1-, and O4-positive. The expression of multiple myelin markers by mixed phenotype glia may suggest that these cells are of oligodendrocyte origin. Increased numbers of MOSP-positive/GFAP-positive mixed phenotype glia were detected in sections from adult hypomyelinated brain from shiverer, quaking, and PKU mice compared to myelinated control adult mouse brain. Similarly, cultures from control brain exposed to elevated pH for 2-3 weeks showed dramatically increased numbers of mixed phenotype glia (80%) compared to control (<10%). Increased numbers of mixed phenotype glia also were detected in shiverer cultures (40%). Since increases in the number of mixed phenotype glia occur in shiverer, quaking, and PKU mouse brain, these data suggest that mixed phenotype glia contribute to gliosis in pathologic white matter.

Evidence for central nervous system glial cell plasticity in phenylketonuria.

Phenylketonuria (PKU) is caused by mutation(s) in the phenylalanine hydroxylase (PAH) gene which lead to deficient PAH activity and an accumulation of phenylalanine in the blood. The primary pathologic finding is hypomyelination and gliosis of central nervous system white matter. Similar white matter pathology is observed in the Pahenu2 mouse, a genetic model for PKU. We studied this mouse to examine the basis for these neuropathologic changes in PKU and to determine if hypomyelination and gliosis occur independently or are interrelated. Although white matter tracts within PKU brains are hypomyelinated, immunostaining and Western blot analyses revealed that these tracts contain abundant amounts of myelin markers, i.e. myelin basic protein (MBP), 2',3'-cyclic nucleotide 3'phosphohydrolase, and myelin/oligodendrocyte-specific protein (MOSP). However, Western blot analyses also showed that MBP isoform expression was aberrant. Investigation of individual cells was performed by extraction of tissue sections with Triton X-100. Most of the MOSP was extracted, with the remaining MOSP clearly visible in dual labeled cells, i.e. MOSP was colocalized along glial fibrillary acidic protein (GFAP) filaments. Cells expressing both MBP and GFAP were also identified in optic tract. Double labeling with a riboprobe for MBP and antibodies specific for GFAP revealed that the majority of GFAP-positive cells expressed MBP mRNA. Our in vitro studies examined the response of cultured wild type oligodendrocytes to elevated phenylalanine for 4 weeks (wk). Under these conditions, about 50 these conditions, about 50% of the oligodendrocytes expressed GFAP filaments and failed to elaborate membrane sheets. Proliferation of astrocytes appears not to be the source of gliosis, since the nuclei of GFAP-positive cells in the PKU brains did not immunostain for proliferating cell nuclear antigen. Dual-labeled cells were detected in normal mouse brain sections; however, PKU mouse white matter tracts were found to contain about twice the number of dual-labeled cells compared to normal tissue. Taken together, these data suggest that both myelinating and nonmyelinating oligodendrocytes are present in the normal adult brain, and that in response to a toxic factor such as elevated phenylalanine, myelinating oligodendrocytes adopt a nonmyelinating phenotype that expresses GFAP. Since myelinating Schwann cells and GFAP-positive nonmyelinating Schwann cells are normally present in adult peripheral nerve, and the myelinating Schwann cells react to pathologic situations by switching to GFAP-positive nonmyelinating cells, it may be that oligodendrocytes and Schwann cells are more similar than previously thought.

Developmental expression of MOSP in cultured oligodendrocytes

Myelin/oligodendrocyte specific protein (MOSP) is a recently characterized 48 kDa surface membrane protein that is expressed exclusively by oligodendrocytes in the CNS. In this report, evidence is presented for the identification of the stage in the oligodendrocyte lineage when MOSP is first expressed. MOSP initially appears on immature oligodendrocytes about four to five days postnatal, which is about one to two days after the appearance of galactocerebroside and sulfatide. The initial expression of MOSP occurs at the stage in development when oligodendrocytes are elaborating processes and just beginning to form membrane sheets. Since 1) MOSP is capable of signaling increases in microtubular structures in oligodendrocytes and 2) microtubular structures may be essential for extension of growing processes and the formation of membrane sheaths, MOSP may play an important role in differentiation of oligodendrocytes and the formation of myelin.

Antibodies to myelin/oligodendrocyte-specific protein and myelin/oligodendrocyte glycoprotein signal distinct changes in the organization of cultured oligodendroglial membrane sheets.

Cultured murine oligodendrocytes elaborate extensive membrane sheets that, unlike multilamellar myelin in vivo, allow the study of interactions between myelin proteins and cytoskeletal elements. This article describes the events that occur due to the interaction of specific antibodies with their respective antigens, myelin/oligodendrocyte-specific protein (MOSP) and myelin/oligodendrocyte glycoprotein (MOG), which are expressed uniquely by oligodendrocytes. After antibody binding, surface anti-MOSP:MOSP complexes redistribute over those cytoplasmic microtubular veins that have 2',3'-cyclic nucleotide 3'-phosphohydrolase colocalized along them. In contrast, surface anti-MOG-MOG complexes redistribute over internal myelin basic protein domains. Long-term anti-MOSP IgM exposure results in an apparent increase in number as well as thickness of microtubular structures in oligodendrocyte membrane sheets, whereas long-term anti-MOG exposure causes depolymerization of microtubular veins in membrane sheets. These data suggest that antibody binding to these two surface proteins elicits signals that have opposite effects on the cytoskeleton in oligodendroglial membrane sheets. Thus, it is possible that signals transduced via antibody binding may contribute to the pathogenesis of diseases affecting CNS myelin.

Novel oligodendrocyte transmembrane signaling systems. Investigations utilizing antibodies as ligands.

Antibodies are increasingly being used as tools to study the function of cell surface markers. Several types of responses may occur upon the selective binding of an antibody to an epitope on a receptor. Antibody binding may trigger signals that are normally transduced by endogenous ligands. Moreover, antibody binding may activate normal signals in a manner that disrupts a sequence of events that coordinates either differentiation, mitogenesis, or morphogenesis. Alternately, it is possible that binding elicits either a modified signal or no signal. This article focuses on the cascade of events that occur following specific antibody binding to myelin markers expressed by cultured murine oligodendrocytes. Binding of specific antibodies to the oligodendrocyte membrane surface markers myelin/oligodendrocyte glycoprotein (MOG), myelin/oligodendrocyte specific protein (MOSP), galactocerebroside (GalC), and sulfatide on cultured murine oligodendrocytes results in different effects with regard to phospholipid turnover, Ca2+ influxes, and antibody:marker distribution. The consequence of each antibody-elicited cascade of events appears to be the regulation of the cytoskeleton within the oligodendroglial membrane sheets. The antibody binding studies described in this article demonstrate that these myelin surface markers are capable of transducing signals. Since endogenous ligands for these myelin markers have yet to be identified, it is not known if these signals are normally transduced or are a modification of normally transduced signals.