Basic Protein Oligodendrocyte Research Articles

Dual role of the RNA helicase DDX5 in post-transcriptional regulation of myelin basic protein in oligodendrocytes.

In the central nervous system, oligodendroglial expression of myelin basic protein (MBP) is crucial for the assembly and structure of the myelin sheath. MBP synthesis is tightly regulated in space and time, particularly at the post-transcriptional level. We have identified the DEAD-box RNA helicase DDX5 (also known as p68) in a complex with Mbp mRNA in oligodendroglial cells. Expression of DDX5 is highest in progenitor cells and immature oligodendrocytes, where it localizes to heterogeneous populations of cytoplasmic ribonucleoprotein (RNP) complexes associated with Mbp mRNA in the cell body and processes. Manipulation of the amount of DDX5 protein inversely affects the level of MBP. We present evidence that DDX5 is involved in post-transcriptional regulation of MBP protein synthesis, with implications for oligodendroglial development. In addition, knockdown of DDX5 results in an increased abundance of MBP isoforms containing exon 2 in immature oligodendrocytes, most likely by regulating alternative splicing of Mbp Our findings contribute to the understanding of the complex nature of MBP post-transcriptional control in immature oligodendrocytes where DDX5 appears to affect the abundance of MBP proteins via distinct but converging mechanisms.

Inhibition of glial proliferation, promotion of axonal growth and myelin production by synthetic glycolipid: A new approach for spinal cord injury treatment.

After spinal cord injury (SCI) a glial scar is generated in the area affected that forms a barrier for axon growth and myelination, preventing functional recovery. Recently, we have described a synthetic glycolipid (IG20) that inhibited proliferation of human glioma cells. We show now that IG20 inhibited the proliferation of astrocytes and microglial cells, the principal cellular components of the glial scar, and promoting axonal outgrowth and myelin production in vitro. Glial cells were inhibited with IG20 (IC50≈10 μM) and studied by RT-PCR, Western Blotting, immunoprecipitation and fluorescence microscopy. Axonal outgrowth in dorsal root ganglia (DRG) and myelin production by oligodendrocytes were analyzed by immunocytochemistry. Adult rats were assayed in spinal cord contusion model and the recovery of treated animals (n = 6) and controls (n = 6) was followed. The IG20 was localized in the cytosol of glial cells, forming a complex with RhoGDIα, a regulator of RhoGTPases. Treatment of astroglial cultures with IG20 increase the expression of BDNF receptor genes (TrkBT1, TrkB Full). IG20 reduced the astroglial marker GFAP, while increasing production of myelin basic protein in oligodendrocytes and promoted axonal outgrowth from DRG neurons. Local injection of IG20, near a spinal cord contusion, promoted the recovery of lesioned animals analyzed by BBB test (P < 0.05). We propose that inhibition of astrocytes and microglia by IG20 could be diminished the glial scar formation, inducing the re-growth and myelination of axons, these elements constitute a new approach for SCI therapy.

Transcriptional expression of myelin basic protein in oligodendrocytes depends on functional syntaxin 4: a potential correlation with autocrine signaling.

Myelination of axons by oligodendrocytes is essential for saltatory nerve conduction. To form myelin membranes, a coordinated synthesis and subsequent polarized transport of myelin components are necessary. Here, we show that as part of the mechanism to establish membrane polarity, oligodendrocytes exploit a polarized distribution of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) machinery components syntaxins 3 and 4, localizing to the cell body and the myelin membrane, respectively. Our data further reveal that the expression of myelin basic protein (MBP), a myelin-specific protein that is synthesized "on site" after transport of its mRNA, depends on the correct functioning of the SNARE machinery, which is not required for mRNA granule assembly and transport per se. Thus, downregulation and overexpression of syntaxin 4 but not syntaxin 3 in oligodendrocyte progenitor cells but not immature oligodendrocytes impeded MBP mRNA transcription, thereby preventing MBP protein synthesis. The expression and localization of another myelin-specific protein, proteolipid protein (PLP), was unaltered. Strikingly, conditioned medium obtained from developing oligodendrocytes was able to rescue the block of MBP mRNA transcription in syntaxin 4-downregulated cells. These findings indicate that the initiation of the biosynthesis of MBP mRNA relies on a syntaxin 4-dependent mechanism, which likely involves activation of an autocrine signaling pathway.

Differential Gene Expression in Oligodendrocyte Progenitor Cells, Oligodendrocytes and Type II Astrocytes

Oligodendrocyte precursor cells (OPCs) are bipotential progenitor cells that can differentiate into myelin-forming oligodendrocytes or functionally undetermined type II astrocytes. Transplantation of OPCs is an attractive therapy for demyelinating diseases. However, due to their bipotential differentiation potential, the majority of OPCs differentiate into astrocytes at transplanted sites. It is therefore important to understand the molecular mechanisms that regulate the transition from OPCs to oligodendrocytes or astrocytes. In this study, we isolated OPCs from the spinal cords of rat embryos (16 days old) and induced them to differentiate into oligodendrocytes or type II astrocytes in the absence or presence of 10% fetal bovine serum, respectively. RNAs were extracted from each cell population and hybridized to GeneChip with 28,700 rat genes. Using the criterion of fold change > 4 in the expression level, we identified 83 genes that were up-regulated and 89 genes that were down-regulated in oligodendrocytes, and 92 genes that were up-regulated and 86 that were down-regulated in type II astrocytes compared with OPCs. The up-regulated genes, such as activating transcription factor 3 and myelin basic protein in oligodendrocytes or claudin 11 in type II astrocytes, might contribute to OPC differentiation and represent constitutive components of oligodendrocytes or type II astrocytes. The down-regulated genes in both oligodendrocytes and type II astrocytes, such as transcription factor 19, might be involved in maintaining self-renewal and/or represent the property of OPCs. These results provide new insights into the elucidation of the molecular mechanisms, by which OPCs differentiate to oligodendrocytes or type II astrocytes.

Histo-clinical variation in multiple sclerosis: Heterogeneous proteolytic immunogenic processing

Multiple sclerosis (MS) presents an incredible histo-clinical variation. It consists of an unpredictable series of relapses, remissions and stationary phases. The initial symptoms vary considerably. Any hypothesis of the pathology of MS must include an explanation of this oddity. Current theory suggests that MS is a collection of diseases which produce generally the same result. However, this is not a satisfactory explanation. MS appears as an enormous continuum of disease paths rather than a finite group of well-defined courses. A hypothesis is presented that histo-clinical variation in MS is due to variable proteolytic processing of several potential immunogens. MS is generally thought to be caused by an autoimmune attack on myelin components. Several myelin proteins, myelin basic protein, lipoprotein, oligodendrocyte related glycoprotein and oligodendrocyte basic protein, are encephalitogenic. Within these proteins are short sequences, which themselves are encephalitogenic. In order for potential immunogens to be "seen" by the immune system they first must be processed. This processing is performed by intracellular and extracellular proteases. A large number of different proteases are located throughout the central nervous system. Their concentrations vary with location and time. Most are under strict control. While myelin has a consistent structure, the action of proteases can present variable concentrations of immunogenic peptides. Because of the differences in location, concentration and control of the central nervous system's (CNS) proteases, the same potential immunogen could be presented to the immune system in different locations within the CNS at different times. At a given time and location, the immune system may be presented with no potential immunogens, one potential immunogen or possibly many immunogens. Therefore, because of the dynamic characteristic of presentation, one would expect to see the initial MS symptoms to be variable. This variability would be continued with subsequent symptoms. This is what is seen in multiple sclerosis. A procedure for testing this hypothesis is presented.

Stage-specific Expression of Myelin Basic Protein in Oligodendrocytes Involves Nkx2.2-mediated Repression That Is Relieved by the Sp1 Transcription Factor

The homeodomain-containing protein Nkx2.2 is critical for the development of oligodendrocyte lineage cells, but the target genes of Nkx2.2 regulation have not been identified. In the present study, we found that the myelin basic protein gene is one of the genes that is regulated by Nkx2.2. Expression of Nkx2.2 represses the expression of myelin basic protein in oligodendrocyte progenitors. Two regulatory elements in the myelin basic protein promoter were identified and found to interact with Nkx2.2 in vitro. Despite their sequence divergence, both sites were involved in the Nkx2.2-mediated repression of the myelin basic protein promoter. Binding of Nkx2.2 also blocked and disrupted the binding of the transcriptional activator Puralpha to the myelin basic protein promoter. Additionally Nkx2.2 recruited a histone deacetylase 1-mSin3A complex to the myelin basic protein promoter. We also found that the transcription factor Sp1 was able to compete off the binding of Nkx2.2 to its consensus binding site in vitro and reversed the repressive effect of Nkx2.2 in vivo. Our data revealed a novel role for Nkx2.2 in preventing the precocious expression of myelin basic protein in immature oligodendrocytes. Based on this study and our previous reports, a model for myelin basic protein gene control is proposed.

Glial-cell cultures from brains of carbonic anhydrase II-deficient mutant mice: delay in oligodendrocyte maturation.

Carbonic anhydrase II (CAII) is a multifunctional enzyme found in oligodendrocytes and astrocytes in normal mouse brains. We have begun to compare the glial cells in primary cultures from neonatal genetically CAII-deficient (Car) mice to those from normal (con) mice in order to detect developmental defects, if any, in Car glial cells. In con cultures intensely CAII-positive cells costained with antibodies against the oligodendrocytic markers, O4 and myelin basic protein (MBP), respectively. Most (82%) of the CAII-positive cells were O4-positive, but only approximately 60% were MBP-positive. Some clumps of GFAP-positive cells were CAII-positive. At each respective number of days in vitro (DIV) total numbers of O4-positive cells were similar in Car and con cultures, and total numbers of galactocerebroside-positive cells also were similar in Car and con cultures. However, compared to cells in con cultures at 7 DIV, a lower percent of Car cells in the oligodendrocyte lineage expressed MBP, and morphological differentiation also was subnormal in that the Car cells showed fewer processes and membrane sheets. Car and con cultures expressed similar numbers of MBP-positive cells by 10 DIV. The results suggest a temporary delay in the maturation of Car oligodendrocytes.

Chromosomal localization of the myelin-associated oligodendrocytic basic protein and expression in the genetically linked neurological mouse mutants ducky and tippy.

The alternatively spliced cDNAs encoding the myelin-associated/oligodendrocytic basic proteins (MOBPs) have recently been identified in rat. The Mobp gene maps to the distal part of mouse chromosome 9 at a region syntenic with the human chromosome 3p22-p21.3. Two nonallelic mouse mutants, tippy and ducky, with severe neurological phenotypes map to the vicinity of the Mobp locus. We therefore tested whether MOBP malfunction could explain the tippy and ducky defects. In tippy mutant animals, MOBP expression and that of other myelin markers were indistinguishable from wild type. The ultrastructure of tippy myelin was shown to be normal. Ducky animals showed a slight reduction of the brain size, most evident in the spinal cord, but normal progress of myelination. Both MOBP and myelin basic protein expression were lowered only regionally in the CNS, but were mostly normal in the anterior parts of the brain. Ultrastructurally, ducky myelin appeared normal. MOBP transcript sizes and the molecular weights of the encoded proteins were shown to be normal in both mutants. Finally, the nucleotide sequence of the abundant MOBP-81 cDNA was determined and compared with tippy and ducky MOBP-81. Wild-type mouse MOBP-81 protein was 99% identical to the rat homologue, and tippy and ducky MOBP-81 were identical to the wild-type sequence. Our results suggest that alterations in the Mobp gene are not the cause for the severe neurological phenotypes of ducky and tippy mice.

Corticosteroid effects on gene expression of myelin basic protein in oligodendrocytes and of glial fibrillary acidic protein in type 1 astrocytes.

The paper describes the effects of corticosterone and deoxycorticosterone (DOC), used in their native or in their 5 alpha-reduced molecular forms (dihydrocorticosterone, DHC; dihydrodeoxycorticosterone, DHDOC; and tetrahydrodeoxycorticosterone, THDOC) on the gene expression of the myelin basic protein (MBP) and of the glial fibrillary acidic protein (GFAP) in pure cultures, respectively, of oligodendrocytes and type 1 astrocytes obtained from the neonatal rat brain. Among the different steroids tested (corticosterone, DHC, DOC, DHDOC and THDOC), only DHDOC was effective on the gene expression of MBP in the oligodendrocyte cultures; the mRNA levels of this typical oligodendrocyte marker were decreased following exposure to this steroid for 24 h. In the case of the astrocytic marker GFAP, its gene expression was increased by the exposure to corticosterone for 6 and 24 h, while DHC was ineffective; the mineralocorticoid DOC was also ineffective, while its 5 alpha-reduced derivative, DHDOC, strongly inhibited GFAP gene expression, starting at 6 h after beginning of the treatment. In conclusion, the present data show that: (1) adrenal steroids possessing gluco- and mineralocorticoid activities may influence the gene expression of the astrocytic marker GFAP; (2) the 5 alpha-reduced metabolite of DOC, DHDOC is able to influence the gene expression not only of GFAP but also that of MBP, which are, respectively, typical markers of the astrocytes and the oligodendrocytes; (3) the metabolic conversion of hormonal steroids into their 5 alpha-reduced metabolites, which also occurs in the glia, could be implicated in the biochemical control of oligodendrocyte and astrocyte functions.

Demonstration of steroid hormone receptors and steroid action in primary cultures of rat glial cells

Primary cultures of rat glial cells were established from newborn rat forebrains. A mixed population of oligodendrocytes and astrocytes was obtained, as confirmed by indirect immunofluorescence staining with specific markers for each cell type. Receptors were measured 3 weeks after primary culture in glial cells cultured in the presence or not of 50 nM estradiol and we have identified progesterone, glucocorticoid, estrogen, and androgen receptors (PR, GR, ER and AR), but only PR was inducible by the estrogen treatment. This estrogen-induction of PR was more dramatic in glial cells derived from female offsprings than from males, as measured by binding studies and by immunohistochemical techniques with the KC 146 anti-PR monoclonal antibody. The antiestrogen tamoxifen inhibited the estrogen induction, but had no effect by itself on PR concentration. Specific binding sites for PR, GR, ER and AR were measured by whole cell assays after labeling cells with, respectively, [ 3H]R5020, [ 3H]dexamethasone, [ 3H]OH-tamoxifen or [ 3H]R1881. PR and GR were also analyzed by ultracentrifugation and after exposure of cells to agonists, both receptors were recovered from cytosol as a 9S form, and from the nuclear high-salt, tungstate ions-containing fraction as a 4–6S form. In contrast, when the antiprogestin- and antiglucocorticosteroid RU486 was used as a ligand, a non-activated 8.5S receptor complex was found for both receptors in this nuclear fraction. The 8.5S complex of the GR was further analyzed in the presense of specific antibodies and, in addition to GR, the presence of the heat shock protein hsp90 and of a 59 kDa protein was found. During primary culture, the effects of progesterone (P) and estradiol (E 2) were tested on glial cell multiplication, morphology and differentiation. Cell growth was inhibited by P and stimulated by E 2. Both hormones induced dramatic morphologic changes in oligodendrocytes and astrocytes and increased synthesis of the myelin basic protein in oligodendrocytes and of the glial fibrillary acidic protein in astrocytes.

Epidermal growth factor inhibits the expression of myelin basic protein in oligodendrocytes

The major function of the oligodendrocyte is to myelinate axons in the central nervous system (CNS). Two of the components of myelin, galactocerebroside (galc) and myelin basic protein (MBP), have been used as markers of oligodendrocyte maturation in the developing CNS, and it has been found that galc+ cells arise initially, which then mature into MBP+ oligodendrocytes several days later. We have been interested in the control of expression of MBP and have followed its appearance in cultures of brain cells isolated from 4 day-old mice. In low serum (0.5% foetal bovine serum), approximately 330 MBP+ cells arise per 2 x 10(5) brain cells after 3 days incubation. We have examined the ability of several growth factors to influence the expression of MBP in these cultures, including epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and the fibroblast growth factors (acidic and basic FGF). EGF was found to suppress strongly the developmental expression of MBP in these cultures, but the suppression was reversible, since the number of MBP+ cells approached control numbers 3 days after removal of EGF from the cultures. It was also found that MBP could be down-regulated in mature MBP+ oligodendrocytes. The action of EGF in these cultures could be mimicked by transforming growth factor-alpha (TGF alpha). The effects of EGF appear to be associated primarily with MBP production in oligodendrocytes since expression of galc is unaffected by EGF.

Expression and Localization of Myelin Basic Protein in Oligodendrocytes and Transfected Fibroblasts

Myelin basic protein (MBP) is a major structural component of myelin. It is expressed exclusively in myelinating glia (oligodendrocytes in the CNS and Schwann cells in the PNS) and is localized to the cytoplasmic surface of the plasma membrane and myelin membrane produced by these cells. The work described here concerns the mechanism of plasma membrane localization of MBP in myelinating glial cells and whether it involves differentiated functions specific to these cells or general functions of plasma membrane assembly common to all cells. To this end, the subcellular localization of endogenous MBP in mouse oligodendrocytes was compared with that of transiently expressed MBP in monkey fibroblasts (Cos-1 cells) transfected with an MBP expression vector containing cDNA for rat 14K MBP. The steady-state levels of MBP-specific RNA and of MBP polypeptide expressed in the transfected fibroblasts were comparable to the levels expressed in oligodendrocytes in primary culture. MBP localization was analyzed in whole cells by immunofluorescence and in specific intracellular compartments by subcellular fractionation. The results show that MBP expressed in wild-type oligodendrocytes is localized to the plasma membrane. In contrast, MBP expressed in transfected fibroblasts appears dispersed in the cytoplasm and is distributed uniformly among the various subcellular fractions.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of myelin basic protein in oligodendrocytes by a soluble neuronal factor

Myelin basic protein (MBP) is one of the most important myelin components. Based on our previous studies, we hypothesized that neurons might have regulatory effects on the production of MBP by oligodendrocytes, and we conducted studies designed to verify this hypothesis. Oligodendroglia-rich cultures from total brain of neonatal rats or mice and pure cultures of embryonic rats or chicks were prepared. Cultures of mouse fibroblasts and astrocytes were prepared as well. We show here that MBP production by oligodendrocytes was greatly enhanced by treatment with either pure neurons, rat neuronal conditioned medium, or chick neuronal conditioned medium, while chemically defined, hormonally supplemented medium or medium conditioned by astrocytes and fibroblasts had no effect on MBP expression. We conclude that the production of MBP by oligodendrocytes is regulated by a nonspecies specific soluble neuronal factor. The conservation of this phenomenon from avian to rodent species implies its critical role in myelination and suggests its potential application as a treatment in demyelination.

Development and maturation of central nervous system myelin: comparison of immunohistochemical localization of proteolipid protein and basic protein in myelin and oligodendrocytes.

The immunohistochemical localization of two myelin specific proteins-basic protein (BP) and proteolipid protein (PLP)-was compared during the process of myelination. Although both proteins were present in oligodendrocytes, (i) neither protein was observed in oligodendrocytes not already closely associated with nerve fibers exhibiting a fluorescent coating; (ii) in any discrete anatomical area oligodendrocytes were positive for BP before PLP was visible; and (iii) as myelination progressed, immunoreactivity for BP in oligodendrocytes appeared to decrease and simultaneously PLP immunofluorescence became visible in this cell type. During the period of active myelination, fibers exhibited a distinct varicose appearance. As myelination progressed, the myelin sheath increased in thickness and these varicosities became less prominent, eventually completely disappearing. Therefore, the nature and the appearance of varicosities can be used as an index of the relative stage of maturation of myelin in an individual fiber. In general, PLP appeared in fibers at a later stage of maturation than did BP based on the above criteria. However, in a relatively small number of fine fibers PLP was observed at a very early stage. In fully mature myelin, very large fibers were frequently more intensely fluorescent for BP than PLP, whereas fine myelinated fibers were more intensely stained for PLP. These observations are consistent with the following interpretations. (i) Substantial differentiation of oligodendrocytes occurs prior to appearance of either of these proteins by immunofluorescence. (ii) BP is added to the myelin sheath prior to PLP and there appears to be a shift in priority of synthesis from BP to PLP in individual oligodendrocytes during the process of myelination. (iii) Very small fibers often contain low concentrations of BP relative to PLP, and conversely, very large fibers may contain a high concentration of BP relative to PLP. Thus, the relative concentration of these proteins in myelin appears not to be constant but may vary as a function of the size of the myelinated fiber.

Immunocytochemical method to identify basic protein in myelin-forming oligodendrocytes of newborn rat C.N.S.

An immunocytochemical method for detecting myelin basic protein in oligodendrocytes and myelin of newborn rat C.N.S. is described. C.N.S. tissue is perfused and fixes in HgCl2--formaldehyde and 20 micron Vibratome sections are treated with antibodies to myelin basic protein using the peroxidase--antiperoxidase method. Oligodendrocytes in the newborn rat are intensely stained by antiserum to basic protein and multiple stained processes extend from the perikaryon to myelin sheaths. With this procedure it is possible to demonstrate the geometric relationships between a single oligodendrocyte and multiple myelin sheaths. Stained oligodendrocytes and myelin are present in newborn cervical spinal cord, medulla oblongata, pons and midbrain. By 25 days of age, staining in oligodendrocytes is less intense than in newborn rats and differences in amount of staining can be detected in areas that are myelinating at different rates. With anticerebroside serum, cerebroside, of newborn and developing rat C.N.S. tissue is localized only in myelin. In the developing P.N.S., myelin basic protein is localized in Schwann cell cytoplasm and myelin sheaths of the trigeminal ganglion. Cerebroside is found only in myelin.