ST8Sia IV mRNA corresponds with the biosynthesis of alpha2,8sialyl polymers but not oligomers in rat oligodendrocytes.

As oligodendrocytes mature they progress through a series of distinct differentiation steps characterized by the expression of specific markers. One such marker, polysialic acid found on the neural cell adhesion molecule (NCAM), is detected by antibodies and is present on progenitor oligodendrocytes, but is not detected to the same extent on mature oligodendrocytes. Two closely related polysialyltransferases, ST8Sia II (STX) and ST8Sia IV (PST) have been cloned previously and shown to synthesize polysialic acid on NCAM and other glycoproteins. To determine whether or not polyalpha2,8sialyltransferases are downregulated during the differentiation of oligodendrocytes, the enzyme activity and expression of ST8Sia II and ST8Sia IV mRNA at two stages of maturation in JS12/1 and JS3/16 oligodendrocytes were examined. Differentiation in both oligodendroglial cell lines was accompanied by more than a 50% reduction in the biosynthesis of polymers of alpha2,8sialic acid when fetuin was used as substrate. Most interestingly, extracts of JS12/1 mature cells synthesized 60% more short oligomers of alpha2,8sialic acid than the progenitor cells, whereas JS3/16 mature cells synthesized barely detectable amounts of the short oligomers. Transcripts for ST8Sia IV mRNA were present in both JS12/1 and JS3/16 and were reduced when the biosynthesis was markedly reduced. In contrast ST8Sia II mRNA was barely detectable in JS3/16 cells and although detectable in JS12/1 cells, there was no clear modulation with maturation. These results were supported by the examination of the brains of rats from embryonic to Day 21 ages. The enzyme activity and mRNA experiments show that polyalpha2,8sialyltransferase itself is down regulated to cause the reduction in sialyl polymers on mature oligodendrocytes. Moreover, ST8Sia IV is responsible for the polysialylation of NCAM in oligodendrocytes.

Caspase-3 activation in oligodendrocytes from the myelin-deficient rat.

The myelin-deficient (MD) rat has a point mutation in its proteolipid protein (PLP) gene that causes severe dysmyelination and oligodendrocyte cell death. Using an in vitro model, we have shown that MD oligodendrocytes initially differentiate similarly to wild-type cells, expressing galactocerebroside, 2',3'-cyclic nucleotide 3'-phosphodiesterase, and myelin basic protein. However, at the time when PLP expression would normally begin, the MD oligodendrocytes die via an apoptotic pathway involving caspase activation. The active form of caspase-3 was detected, along with the cleavage products of poly-(ADP-ribose) polymerase (PARP) and spectrin, major targets of caspase-mediated proteolysis. A specific inhibitor of casapse-3, Ac-DEVD-CMK, reduced apoptosis in MD oligodendrocytes, but the rescued cells did not mature fully or express myelin-oligodendrocyte glycoprotein. These results suggest that mutant PLP affects not only cell death but also oligodendrocyte differentiation.

Stage-specific effects of bone morphogenetic proteins on the oligodendrocyte lineage.

Oligodendrocyte maturation is regulated by multiple secreted factors present in the brain during critical stages of development. Whereas most of these factors promote oligodendrocyte proliferation and survival, members of the bone morphogenetic protein family (BMPs) recently have been shown to inhibit oligodendrocyte differentiation in vitro. Oligodendrocyte precursors treated with BMPs differentiate to the astrocyte lineage. Given that cells at various stages of the oligodendrocyte lineage have distinct responses to growth factors, we hypothesized that the response to BMP would be stage-specific. Using highly purified, stage-specific cultures, we found that BMP has distinct effects on cultured oligodendrocyte preprogenitors, precursors, and mature oligodendrocytes. Oligodendrocyte preprogenitors (PSA-NCAM+, A2B5-) treated with BMP2 or BMP4 developed a novel astrocyte phenotype characterized by a morphological change and expression of glial fibrillary acidic protein (GFAP) but little glutamine synthetase expression and no labeling with A2B5 antibody. In contrast, treating oligodendrocyte precursors with BMPs resulted in the accumulation of cells with the traditional type 2 astrocyte phenotype (GFAP+, A2B5+). However, many of the cells with an astrocytic morphology did not express GFAP or glutamine synthetase unless thyroid hormone was present in the medium. The addition of fibroblast growth factor along with BMP to either oligodendrocyte preprogenitor or the oligodendrocyte precursor cells inhibited the switch to the astrocyte lineage, whereas platelet-derived growth factor addition had no effect. Treatment of mature oligodendrocytes with BMP elicited no change in morphology or expression of GFAP. These data suggest that as cells progress through the oligodendrocyte lineage, they show developmentally restricted responses to the BMPs.

Epidermal growth factor receptor agonists increase expression of glutamate transporter GLT-1 in astrocytes through pathways dependent on phosphatidylinositol 3-kinase and transcription factor NF-kappaB.

The glial glutamate transporter GLT-1 may be the predominant Na(+)-dependent glutamate transporter in forebrain. Expression of GLT-1 correlates with astrocyte maturation in vivo and increases during synaptogenesis. In astrocyte cultures, GLT-1 expression parallels differentiation induced by cAMP analogs or by coculturing with neurons. Molecule(s) secreted by neuronal cultures contribute to this induction of GLT-1, but little is known about the signaling pathways mediating this regulation. In the present study, we determined whether growth factors previously implicated in astrocyte differentiation regulate GLT-1 expression. Of the six growth factors tested, two [epidermal growth factor (EGF) and transforming growth factor-alpha] induced expression of GLT-1 protein in cultured astrocytes. Induction of GLT-1 protein was accompanied by an increase in mRNA and in the V(max) for Na(+)-dependent glutamate transport activity. The effects of dibutyryl-cAMP and EGF were additive but were independently blocked by inhibitors of protein kinase A or protein tyrosine kinases, respectively. The induction of GLT-1 in both EGF- and dibutyryl-cAMP-treated astrocytes was blocked by inhibitors targeting phosphatidylinositol 3-kinase (PI3K) or the nuclear transcription factor-kappaB. Furthermore, transient transfection of astrocyte cultures with a constitutively active PI3K construct was sufficient to induce expression of GLT-1. These data suggest that independent but converging pathways mediate expression of GLT-1. Although an EGF receptor-specific antagonist did not block the effects of neuron-conditioned medium, the induction of GLT-1 by neuron-conditioned medium was completely abolished by inhibition of PI3K or nuclear factor-kappaB. EGF also increased expression of GLT-1 in spinal cord organotypic cultures. Together, these data suggest that activation of specific signaling pathways with EGF-like molecules may provide a novel approach for limiting excitotoxic brain injury.

Ectopic bone morphogenetic proteins 5 and 4 in the chicken forebrain lead to cyclopia and holoprosencephaly.

Proper dorsal-ventral patterning in the developing central nervous system requires signals from both the dorsal and ventral portions of the neural tube. Data from multiple studies have demonstrated that bone morphogenetic proteins (BMPs) and Sonic hedgehog protein are secreted factors that regulate dorsal and ventral specification, respectively, within the caudal neural tube. In the developing rostral central nervous system Sonic hedgehog protein also participates in ventral regionalization; however, the roles of BMPs in the developing brain are less clear. We hypothesized that BMPs also play a role in dorsal specification of the vertebrate forebrain. To test our hypothesis we implanted beads soaked in recombinant BMP5 or BMP4 into the neural tube of the chicken forebrain. Experimental embryos showed a loss of the basal telencephalon that resulted in holoprosencephaly (a single cerebral hemisphere), cyclopia (a single midline eye), and loss of ventral midline structures. In situ hybridization using a panel of probes to genes expressed in the dorsal and ventral forebrain revealed the loss of ventral markers with the maintenance of dorsal markers. Furthermore, we found that the loss of the basal telencephalon was the result of excessive cell death and not a change in cell fates. These data provide evidence that BMP signaling participates in dorsal-ventral patterning of the developing brain in vivo, and disturbances in dorsal-ventral signaling result in specific malformations of the forebrain.

Cell cycle arrest induced by ectopic expression of p27 is not sufficient to promote oligodendrocyte differentiation.

Oligodendrocyte differentiation is accompanied by dramatic changes in gene expression as well as cell cycle arrest. To determine whether cell cycle arrest is sufficient to induce the changes in cell phenotype associated with differentiation, we inhibited oligodendrocyte precursor proliferation in vitro by overexpressing p27, a cyclin kinase inhibitor, using a recombinant adenovirus. Ectopic expression of p27 efficiently inhibited oligodendrocyte precursor cell division, even in the presence of exogenous mitogens, by blocking the activity of the cyclin-dependent kinase, cdk2. Although the cells had stopped dividing, they did not express galactocerebroside (GalC) or myelin basic protein (MBP), changes associated with oligodendrocyte differentiation, suggesting that they had not differentiated. After removal of exogenous mitogens, however, adenovirus-expressing oligodendrocyte precursors differentiated with a temporal profile similar to that of control, uninfected oligodendrocytes, as indicated by expression of GalC and MBP. We conclude that cell cycle arrest is not sufficient to induce differentiation of dividing oligodendrocyte precursors, and that modulation of additional, as yet unknown, signaling pathways is required for this to occur.

Maturation-dependent apoptotic cell death of oligodendrocytes in myelin-deficient rats.

Mutations in the proteolipid protein gene (PLP/plp), which encodes the major intrinsic membrane protein in central nervous system (CNS) myelin, cause inherited dysmyelination in mammals. One of these mutants, the myelin-deficient (md) rat, has severe dysmyelination that is associated with oligodendrocyte cell death. Using the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labeling (TUNEL) assay, which labels apoptotic cells, we find that cell death is increased in multiple white matter tracts of md rats. The tracts that myelinate the earliest show the earliest increase in cell death, and cell death persists for at least 22 days, the lifespan of these mutant animals. In all tracts, and at all developmental ages examined, apoptotic cells expressed the markers of mature oligodendrocytes, such as myelin basic protein, myelin-associated glycoprotein, and the Rip antigen, but not chondroitin sulfate proteoglycan, a marker of oligodendrocyte precursors. Mature oligodendrocytes fail to accumulate in md brain because they die before they fully mature.

MEBA derepresses the proximal myelin basic protein promoter in oligodendrocytes.

The central nervous system expression of myelin basic protein (MBP) is restricted to oligodendrocytes and is developmentally regulated; these regulatory features are transcriptionally mediated. We have previously shown that the proximal 149 nucleotides of the MBP promoter were both necessary and sufficient to activate the transcription of MBP in cultured oligodendrocytes, but not in other cell types. Sequences within the distal portion of this promoter, which contains a nuclear factor 1 (NF1) binding site, repressed activation of the MBP promoter in Cos-7 cells, but not in oligodendrocytes. We now describe a sequence upstream of and partially overlapping the NF1 site that activates the MBP promoter in oligodendrocytes, but not in Cos-7 cells. A protein complex binds to this site, designated MEBA (myelinating glia-enriched DNA binding activity), and is enriched in nuclear extracts prepared from the brain, oligodendrocytes, and Schwann cells. The amount of MEBA parallels MBP expression and myelinogenesis in the developing brain and parallels new MBP expression as purified oligodendrocytes differentiate. Mutational analyses of binding and function distinguish MEBA, an activator, from NF1, a repressor of MBP transcription, and suggest that MEBA consists of at least two proteins. Because the binding sites of MEBA and NF1 overlap, we suggest that MEBA may either compete with or modify NF1 binding, thereby activating the MBP promoter in oligodendrocytes.

Re-entry into the cell cycle is required for bFGF-induced oligodendroglial dedifferentiation and survival.

Remyelination in the CNS following demyelinating disease may be accomplished by surviving mature oligodendrocytes that dedifferentiate, proliferate, migrate, and finally regenerate myelin. We previously reported that basic fibroblast growth factor (bFGF) induces oligodendrocytes in primary mixed glial cultures to dedifferentiate and synthesize DNA (Grinspan et al.: J Neurosci Res 36:672-680, 1993). We now show that this effect is direct and not mediated through the effects of bFGF on other cell types, because we were able to demonstrate similar changes in oligodendrocyte phenotype in enriched oligodendrocyte cultures prepared by immunopanning. The bFGF-induced reversion to the precursor stage of the oligodendroglial lineage can be blocked by agents that inhibit entry to the cell cycle; thus oligodendroglial dedifferentiation is dependent on proliferation. We also report that 2 days of bFGF treatment inhibits oligodendroglial apoptosis. However, when oligodendroglia are prevented from entering the cell cycle in the presence of bFGF, apoptotic cell death is increased. Thus, bFGF induces oligodendroglial dedifferentiation if oligodendroglial DNA synthesis can occur but causes oligodendroglial apoptosis when oligodendroglial DNA synthesis is prevented.

Axonal interactions regulate Schwann cell apoptosis in developing peripheral nerve: neuregulin receptors and the role of neuregulins.

Programmed cell death during development resulting from the lack of appropriate survival factors has been demonstrated in both neurons and oligodendrocytes and occurs mostly in the form of apoptosis. We now demonstrate that Schwann cells in the rat sciatic nerve undergo apoptosis during early postnatal development and that the amount of apoptosis is markedly increased by axotomy. The apoptotic Schwann cells express the low-affinity nerve growth factor receptor but not myelin-related proteins, indicating that they are in the premyelinating state. Apoptosis resulting from normal development or from axotomy can be inhibited markedly by exogenous neuregulin. Consistent with this, the neuregulin receptor components erbB2 and erbB3 are expressed and phosphorylated in developing sciatic nerve. These data suggest that Schwann cell number in developing peripheral nerve is regulated by apoptosis through competition for axonally derived neuregulin.

Connexin32 is a myelin-related protein in the PNS and CNS.

We have examined the expression of a gap junction protein, connexin32 (Cx32), in Schwann cells and oligodendrocytes. In peripheral nerve, Cx32 is found in the paranodal myelin loops and Schmidt-Lanterman incisures of myelinating Schwann cells, and the levels of Cx32 protein and mRNA change in parallel with those of other myelin-related genes during development, Wallerian degeneration, and axonal regeneration. In the central nervous system, Cx32 is found in oligodendrocytes and their processes, but not in compact myelin, and the levels of Cx32 protein and mRNA increase during development in parallel with those of the other myelin genes. Thus, Cx32 is expressed as part of the myelinating phenotype of both Schwann cells and oligodendrocytes, indicating that this gap junction protein plays in important role in the biology of myelin-forming cells.

Myelin gene expression in glia treated with oligodendroglial trophic factor.

Oligodendroglia synthesize myelin in the CNS. In vitro, oligodendroglia may be identified by the binding of monoclonal antibodies against galactocerebroside, a myelin-specific galactolipid. Oligodendroglial trophic factor is a protein mitogen for cells of the oligodendroglial lineage. When oligodendroglia in cerebral white matter cultures are treated with oligodendroglial trophic factor, galactocerebroside-positive cells undergo mitosis but fail to express the myelin structural proteins, myelin basic protein and proteolipid protein. Oligodendroglia treated with oligodendroglial trophic factor, however, do express 2',3'-cyclic nucleotide 3'-phosphodiesterase and myelin-associated glycoprotein in a manner similar to oligodendroglia treated with platelet-derived growth factor. Oligodendroglial trophic factor, therefore, generates a population of somewhat 'immature' oligodendroglia, which are galactocerebroside, myelin-associated glycoprotein and 2', 3'-cyclic nucleotide 3' phosphodiesterase positive but myelin basic protein and proteolipid protein negative.

Platelet-derived growth factor is a survival factor for PSA-NCAM+ oligodendrocyte pre-progenitor cells.

Mature oligodendroglia, which synthesize and express lipids and proteins characteristic of myelin, are generated from precursor cells which are formed in germinal matrix, then migrate widely through the neuraxis. We now demonstrate that these precursor cells can be recognized at a very early stage by their surface expression of polysialylated neural cell adhesion molecules (PSA-NCAM), and only later bind anti-ganglioside antibodies that had previously been used to recognize "O-2A" oligodendroglial precursor cells. PSA-NCAM expression by these cells is likely to be of functional significance, since a recent study demonstrated that O-2A cells become immobile when stripped of PSA-NCAM. Platelet-derived growth factor (PDGF) proved to be a survival factor for these PSA-NCAM+cells, and in a defined medium, PDGF was sufficient to ensure maturation of immunopurified PSA-NCAM+cells to oligodendroglia.

Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors mediate excitotoxicity in the oligodendroglial lineage.

We demonstrate by reverse transcriptase-polymerase chain reaction and Southern blotting that an immortalized rat oligodendroglial cell line (CG-4) expresses the non-N-methyl-D-aspartate (non-NMDA) glutamate receptor (GluR) genes GluR2-7, KA-1, and KA-2 and that nonimmortalized cells of the rat oligodendroglial lineage express the GluR1-3, GluR5-7, KA-1, and KA-2 genes. Lactic dehydrogenase release assays show that both immortalized and nonimmortalized cells of the oligodendroglial lineage are damaged by a 24-h exposure to 500 microM kainate or 5 mM L-glutamate, but not by a 24-h exposure to up to 10 mM alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA). Damage is prevented by the non-NMDA GluR channel inhibitor 6-cyano-7-nitroquinoxaline-2,3-dione and is also averted if Ca2+ is removed from the culture medium. Cyclothiazide, which blocks desensitization of AMPA-preferring GluRs, increases cytotoxicity of kainate as well as inducing toxicity of AMPA. We conclude that cells of the oligodendroglial lineage express a population of AMPA-preferring and possibly also kainate-preferring GluR channels that are capable of mediating Ca(2+)-dependent excitotoxicity and that AMPA-induced cytotoxicity is blocked by desensitization of AMPA-preferring GluRs.

Pharmacology of sodium-dependent high-affinity L-[3H]glutamate transport in glial cultures.

Pharmacological and molecular biological studies provide evidence for subtypes of sodium-dependent high-affinity glutamate (Glu) transport in the mammalian CNS. At least some of these transporters appear to be selectively expressed in different brain regions or by different cell types. In the present study, the properties of L-[3H]Glu transport were characterized using astrocyte-enriched cultures prepared from cerebellum and cortex. In both brain regions, the kinetic data for sodium-dependent transport were consistent with a single site with Km values of 91 +/- 17 microM in cortical glial cells and 66 +/- 23 microM in cerebellar glial cells. The capacities were 6.1 +/- 1.6 nmol/mg of protein/min in cortical glial cells and 8.4 +/- 0.9 nmol/mg of protein/min in cerebellar glial cells. The potencies of approximately 40 excitatory amino acid analogues for inhibition of sodium-dependent transport into glial cells prepared from cortex and cerebellum were examined, including compounds that are selective inhibitors of transport in synaptosomes prepared from either cerebellum or cortex. Of the analogues tested, 14 inhibited transport activity by > 50% at 1 mM concentrations. Unlike L-[3H]Glu transport in synaptosomes prepared from cerebellum or cortex, there were no large differences between the potencies of compounds for inhibition of transport measured in glial cells prepared from these two brain regions. With the exception of (2S,1'R,2'R)-2-(carboxycyclopropyl)glycine and L-alpha-aminoadipate, all of the compounds examined were approximately 10-200-fold less potent as inhibitors of L-[3H]Glu transport measured in glial cells than as inhibitors of transport measured in synaptosomes prepared from their respective brain regions.(ABSTRACT TRUNCATED AT 250 WORDS)

Partial purification of a novel mitogen for oligodendroglia.

A protein with a MWapp of 50-70 kDa isolated from the salt extract of crude membranes from neonatal rat brain increases the numbers of oligodendroglia in mixed glial cultures prepared from neonatal rat cerebral white matter. After partial purification by ion exchange and gel exclusion chromatography, and elution from an SDS-polyacrylamide gel, this protein ("oligodendroglial trophic factor," OTF) elicited half-maximal oligodendroglial recruitment at a concentration of 5 ng/mL. OTF is a mitogen for oligodendroglia, and to a lesser extent, for oligodendroglial progenitor (O2A) cells, but does not stimulate proliferation of astroglia, Schwann cells, or endoneurial fibroblasts. OTF, unlike platelet-derived growth factor (PDGF), is not an oligodendroglial survival factor. Antibodies against PDGF and basic fibroblast growth factor (bFGF) do not interfere with the accumulation of oligodendroglia induced by OTF. When OTF is given simultaneously with either PDGF or bFGF, there is an additive increase in the numbers of cells of the oligodendroglial lineage.

Protein growth factors as potential therapies for central nervous system demyelinative disorders.

Demyelinative diseases are frequently accompanied by loss of oligodendroglia; in such instances, oligodendroglial regeneration must precede remyelination. Recent studies indicate that extracellular proteins such as platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) profoundly influence the oligodendroglial lineage. PDGF stimulates the formation of oligodendroglia from partially differentiated progenitor cells, whereas bFGF induces mature oligodendroglia to proliferate and dedifferentiate. Manipulations of the central nervous system concentrations of these and other protein growth factors may prove of therapeutic value in multiple sclerosis.

Trophic effects of basic fibroblast growth factor (bFGF) on differentiated oligodendroglia: a mechanism for regeneration of the oligodendroglial lineage.

We have investigated the effect of basic fibroblast growth factor (bFGF) on the proliferation and phenotype of differentiated oligodendroglia. Using primary cell cultures enriched in oligodendrocytes but containing few O2A-oligodendrocyte progenitor cells, we demonstrate that bFGF treatment greatly increases the proportion of O2A cells while decreasing the proportion of galactocerebroside +(GalC+), myelin basic protein +(MBP+) oligodendrocytes, and the steady state levels of MPB mRNA. Complement mediated cell lysis experiments using the A2B5 antibody to deplete existing O2A cells or the R-Mab antibody to deplete existing oligodendroglia show that bFGF elicits a rapid increase in the number of O2A cells in cultures previously depleted of O2A cells, but does not cause an early increase in O2A cells in cultures from which oligodendroglia had been removed, indicating that the oligodendrocytes are the source of the newly recruited O2A cells. This bFGF-mediated transition from oligodendrocyte to O2A cells occurs with a time course similar to the bFGF-induced increase of the proliferation rate of the GalC+ oligodendrocytes. Studies with purified, passaged cells of the oligodendroglial lineage show that bFGF augments oligodendroglial dedifferentiation and proliferation in chronologically adult oligodendrocytes and in the virtual absence of other cell types. We have thus demonstrated that mature oligodendrocytes are induced by bFGF to dedifferentiate and proliferate, suggesting a mechanism for regeneration of the oligodendroglial lineage following demyelinating disease.

Cerebral white matter contains PDGF-responsive precursors to O2A cells.

Cells dissociated from the cerebral white matter of immature rats were maintained in monolayer culture. Treatment with platelet-derived growth factor (PDGF) caused a large increase in the numbers of "O2A" oligodendroglial precursor cells (which bind the monoclonal antibody A2B5) and subsequently in the numbers of galactocerebroside (galC)-positive oligodendroglia. A2B5-negative "pre-O2A cells" in cerebral white matter cultures in which O2A cells and oligodendroglia had been killed by antibody-dependent complement-mediated cytolysis were induced by PDGF to proliferate and to differentiate into O2A cells and subsequently into oligodendroglia and type 2 astroglia. The most mature pre-O2A phenotype in these cultures was a small, round, process-bearing cell which expressed vimentin but not glial fibrillary acidic protein or galC. Cells of this phenotype were not observed upon PDGF treatment of immature rat optic nerve monolayer cultures from which O2A cells and oligodendrocytes had been depleted, and PDGF also failed to elicit the accumulation of O2A cells and oligodendroglia in such cultures.