Oligodendroglia In Culture Research Articles

MicroRNA miR-204 regulates proliferation and differentiation of oligodendroglia in culture.

Oligodendrocytes wrap and physically shield axons of the central nervous system with myelin sheaths, resulting in rapid signal transduction and accurate neuronal function. The complex oligodendroglial development from immature oligodendrocyte precursor cells (OPCs) to myelinating oligodendrocytes (OLs) is profoundly dependent on the activity of transcription factors of the Sox protein family. Target genes of the crucial regulator Sox10 have recently been expanded to microRNAs. Here, we report miR-204 as a novel transcriptional target of Sox10. Regulatory regions of miR-204 show responsiveness to and binding of Sox10 in reporter gene assays and electromobility shift assays. Once expressed, miR-204 inhibits OPC proliferation and facilitates differentiation into OLs in the presence of Sox10 as evident from overexpression in primary rat and mouse oligodendroglial cultures. Phenotypes are at least in part caused by miR-204-dependent repression of the pro-proliferative Ccnd2 and the differentiation inhibiting Sox4. These findings argue that the transcriptional activator Sox10 forces oligodendroglial cells to exit the cell cycle and start differentiation by gene inhibition via miR-204 induction.

Concentration-dependent effects of CSF1R inhibitors on oligodendrocyte progenitor cells ex vivo and in vivo

Microglia are the principal resident immune cells in the central nervous system (CNS) and play important roles in CNS development, maintenance and repair. The survival and development of microglia depends on colony-stimulating factor 1 receptor (CSF1R), a member of the platelet-derived growth factor receptor (PDGFR) family of tyrosine kinases. Recently pharmacological CSF1R inhibition has been used to investigate the effects of microglial depletion in numerous animal models of CNS disease. However, the effects of CSF1R inhibitors on other cell types in the CNS remains incompletely characterized. In this report, we compared the effect of two commonly used CSF1R inhibitors, PLX5622 and PLX3397, on microglia and oligodendrocyte progenitor cell (OPC) numbers. In ex vivo cerebellar slices and adult mouse brain, both PLX compounds caused robust microglia loss; the kinetics of microglial depletion was more rapid with PLX5622. While high-doses of PLX5622 and PLX3397 reduced OPC number in primary cultures in vitro and ex vivo, low-doses of PLX5622 did not affect the number of OPCs or mature oligodendroglia in culture or in vivo. In adult mice, treatment with PLX5622 had no effect on OPC numbers for 7 days; however, a mild reduction was observed after 21 days in some CNS regions. In contrast, PLX3397 caused significant OPC loss after 7 days of treatment, despite only modest microglia depletion. Neither PLX compound had a remarkable effect on mature oligodendrocytes or myelin protein expression following long-term oral administration. Our results show that CSF1R inhibition with PLX5622 can selectively deplete microglia ex vivo and in vivo without affecting OPC number, demonstrating that microglia are not essential for OPC viability in ex vivo slice cultures or adult CNS tissues.

Identification of G protein-regulated inwardly rectifying K + channels in rat brain oligodendrocytes

Rat brain oligodendroglia in culture express a dominant inwardly rectifying K + current I KIR which can be inhibited through G. proteins by the activation of glial G protein-coupled receptors. Electrophysiologically we have isolated in these cells several conductances of K + inward rectifiers (K IR) between 12 and 175 pS. Experiments on the single channel level with preloading or bath-application of GTPγS revealed the selective suppression of an 18 pS and maybe other K IR conductances, possibly via a direct membrane-delimited mechanism. mRNA amplification from single oligodendrocytes together with polymerase chain reaction resulted in the isolation of IRK-type K + channels which may correspond to the channel species negatively controlled by G proteins.

Glycolipids and transmembrane signaling: antibodies to galactocerebroside cause an influx of calcium in oligodendrocytes.

This is the first study to provide evidence that one function for the surface glycolipid galactocerebroside (GalC) is participation in the opening of Ca2+ channels in oligodendroglia in culture. This glycolipid is a unique differentiation marker for myelin-producing cells; antibodies to GalC have been shown to markedly alter oligodendroglial morphology via disruption of microtubules (Dyer, C. A., and J. A. Benjamins. 1988. J. Neurosci. 8:4307-4318). This study demonstrates that extracellular EGTA blocks anti-GalC-induced disassembly of microtubules in oligodendroglial membrane sheets, demonstrating that an influx of extracellular Ca2+ mediates the cytoskeletal changes. The Ca2+ influx was examined directly by loading oligodendroglia with the fluorescent dye Indo-1 in defined medium, and measuring changes in Ca2+ in individual cells with a laser cytometer. Upon addition of anti-GalC IgG, a marked sustained increase in intracellular Ca2+ occurred in 80% of the oligodendroglia observed. EGTA blocked the increase, indicating the increase is due to an influx of extracellular Ca2+, and not due to release from intracellular stores. The effect is specific, since Ca2+ levels remain normal in oligodendroglia treated with nonimmune IgG; astrocytes do not respond to the anti-GalC. The Ca2+ response in oligodendrocytes is dependent on concentration of antibody and GalC on the oligodendroglial membrane surface. The Ca2+ influx is not mediated by voltage-sensitive Ca2+ channels: it is not blocked by cadmium, and depolarization with K+ does not mimic the response. The kinetics of the response suggest that second messenger-mediated opening of Ca2+ channels is involved.

Hydrocortisone induction during oligodendroglial differentiation

Steroid hormones are important for the normal development of brain. The addition of hydrocortisone to oligodendroglia in culture increases the level of mRNA for proteolipid protein, increases the synthesis of cerebrosides, and provides an induction of activities for the ketone body metabolizing enzymes. While proteolipid protein and cerebrosides are major components of myelin membranes, ketone bodies serve as precursors for lipid synthesis during development. Thus, hydrocortisone is important during the differentiation of oligodendroglia and during initial myelin production.

Antibody to galactocerebroside alters organization of oligodendroglial membrane sheets in culture

Antibodies to galactocerebroside (GalC) cause major changes in the organization of the membrane sheets elaborated by murine oligodendroglia in culture. Exposure of oligodendroglia to rabbit anti-GalC IgG for 15 min followed by fluoresceinated second antibodies results in patches of surface GalC staining; when second antibodies are applied after 2 hr of anti-GalC, the pattern of staining on membrane sheets is solid and wrinkled. Anti-GalC exposure for 24 hr results in contracted membrane sheets. No membrane contraction is detected in cultures treated with anti-sulfatide IgM or anti-proteolipid protein IgG. In cultures exposed to anti-GalC continuously for 4-7 d, there is a marked decrease in numbers of extended membrane sheets with an accompanying increase in contracted sheets. This effect is reversible upon removal of anti-GalC from the culture media. By scanning electron microscopy, normally flat membrane sheets appear ruffled after 2 hr of anti-GalC treatment; by 24 hr, contracted membrane sheets consist entirely of bulbous protrusions. Oligodendrocyte membranes exposed to anti-sulfatide for 24 hr are not contracted but are covered with bulbous protrusions. The organization of underlying membrane structures was examined in relation to membrane patching and sheet contraction. In membranes with patching induced by exposure to anti-GalC for 15 min, the anti-GalC: GalC complexes are localized over cytoplasmic MBP domains, with the unstained areas located above cytoplasmic microtubular structures. Membrane sheets that are contracted in response to anti-GalC exposure for 6-24 hr show intense GalC staining over microtubular structures. Anti-GalC exposure does not change metabolism of GalC; in cultures incubated with 3H-galactose and anti-GalC for 24 hr, there are no alterations in GalC labeling compared with control cultures. In summary, these results provide direct evidence that interaction between surface glycolipids and external antibodies can initiate a sequence of events leading to dramatic changes within the oligodendrocyte.

Effects of lead on viability and intracellular metal content of C6 rat glioma cells.

Cultured C6 rat glioma cells were exposed to lead (Pb) acetate (0, 1, 10, or 100 microM) for 3-4 d. Cells were analyzed for changes in viability and intracellular lead, iron, and copper concentrations after Pb treatment was discontinued. The results were compared with previous findings on astroglia and oligodendroglia in culture in order to evaluate C6 cultures as a model for Pb toxicity in glia. Viability was measured by three methods on the day Pb was removed from the cells (designated d 0), and 2 and 9 d after Pb treatment was discontinued (designated d 2 and 9). The methods used were trypan blue dye exclusion, total cell counts, and incorporation of [3H]-L-leucine into proteins. Small, dose-dependent reductions were observed on d 2 in the percentages of cells excluding dye. Decreased cell numbers were seen at all three Pb doses only on d 0. With respect to these two viability measurements, C6 cells responded like astroglia in culture to Pb, but not like oligodendroglia, which are more Pb-sensitive. We expected decreased amino acid incorporation to accompany the decreased viability of the cultures. Instead, increased amino acid incorporation, which indicates increased protein synthesis, was seen on d 0 and 2 at all three Pb doses, though total cellular protein did not increase. A similar response has been reported previously in oligodendroglial cultures. C6 cells treated for 3 with 1 or 100 microM Pb acetate were analyzed for intracellular metal content by atomic absorption aspectroscopy on d 4 and 11 after exposure to Pb was discontinued. The cells were found to take up large amounts of Pb intracellularly and store it for at least 11 d. Cells treated with FeCl2 instead of Pb took up Fe, but required a higher extracellular Fe concentration to achieve an intracellular Fe level comparable to that of Pb in Pb-treated cells. Pb uptake did not affect intracellular Fe or Cu concentrations. With respect to Pb and Fe uptake, C6 cells closely resembled immature astroglia in culture. Unlike C6 cells, however, astroglia showed elevations of intracellular Fe and Cu after Pb treatment. Thus, Pb effects on C6 cells resembled those on cultured oligodendroglia and astroglia in some respects but not in others. C6 cells appear to be an adequate model for selected events in glial toxicosis, such as Pb-stimulated protein synthesis in oligodendroglia and Pb uptake in astroglia, but not Pb-induced alterations of intracellular Cu and Fe in astroglia. Their use as a model for glial progenitor cells in Pb toxicity studies remains to be determined.

Glycoproteins in the whorls of membrane produced by oligodendroglia in culture

Two glycoproteins of 99 kDa and 77 kDa which exhibit intense binding to wheat germ agglutinin have been purified from the whorls of membrane produced by oligodendroglia in culture. The whorls of membrane were isolated by gradient centrifugation from purified bovine oligodendroglia maintained in culture. The two glycoproteins were solubilized from the membranes using a non-ionic detergent and purified by Sephadex LH-60 chromatography, wheat germ agglutinin affinity chromatography, and SDS-polyacrylamide pore gradient gel electrophoresis. HPLC peptide mapping of the 99-kDa and 77-kDa glycoproteins revealed structural differences between the two proteins. Peptide mapping suggested that the 99-kDa glycoprotein from the whorls of membrane may be homologous to that from the plasma membranes. The 77-kDa glycoproteins from both sets of membrane may also be structurally related. Lectin binding studies showed that both glycoproteins from the whorls of membrane bound to wheat germ agglutinin, succinylated wheat germ agglutinin, concanavalin A, and lentil lectin, indicating the presence of high mannose and hybrid type oligosaccharide side-chains.

Production and characterization of high titer antibodies to galactocerebroside

High titer antibodies primarily of the IgG class were produced against galactocerebroside (GalC) by including keyhole limpet hemocyanin (KLH) and supplemental M. tuberculosis in the adjuvant mixture used for immunization of rabbits. Antibody titers were determined by an ELISA in which microtiter wells were coated with liposomes containing lecithin, cholesterol and GalC. The antibodies showed reactivity with GalC and psychosine, but not glucocerebroside, sulfatide, mixed gangliosides or asialo G M1. Specificity was further demonstrated by absorption of antibodies with GalC. Binding was inhibited by galactose, but only at high concentrations. Further, the antibodies did not bind to any brain proteins on immunoblots, indicating lack of reactivity with glycoproteins which might contain a terminal galactose. Antibodies to GalC are directed against different determinants than those reacting with peanut agglutinin since the lectin will not react with GalC, and the antibodies will not react with asialo G M1. The antibodies raised to GalC by this method show specific staining for oligodendroglia in culture. Peanut agglutinin binds intensely to process-bearing GalC + oligodendroglia, but very poorly to the membrane sheets elaborated by oligodendroglia after longer times in culture. Other process-bearing GalC −, GFAP − cells were also stained with peanut agglutinin; these cells may represent glial precursors.

Proteins and glycoproteins in plasma membranes and in the membrane lamellae produced by purified oligodendroglia in culture

Oligodendroglial plasma membranes are complex structures composed of a heterogeneous mixture of proteins and glycoproteins. The Coomassie stained gel patterns showed a maximum of 40 proteins with molecular weights ranging from > 200 000 to 12 500 . Autoradiography was used to detect binding of radioiodinated lectins to glycoproteins. With concanavalin A, 5 major glycoproteins were seen; with wheat germ agglutinin, 2 major glycoproteins with approximate molecular weights of 95 000 and 78 000 were found; with Ulex europaeus, 7 major glycoproteins were observed. Additional minor bands were also seen. The impermeant probe diazodi[ 125I]iodosulfanilic acid was used to radiolabel intact cells. It was found that 5 major proteins were radiolabeled in the plasma membranes. In all cases, the whorls of membrane lamellae produced in culture by oligodendroglia tend to have a somewhat less complicated pattern with fewer proteins and glycoproteins than the plasma membranes. However, the whorls of membrane lamellae have far more complicated protein patterns than myelin.