Judith B. Grinspan

Pannexin1 is expressed by neurons and glia but does not form functional gap junctions

Pannexins are a newly described family of proteins that may form gap junctions. We made antisera against mouse pannexin1 (Panx1). HeLa cells expressing Panx1 have cell surface labeling, but not gap junction plaques, and do not transfer small fluorescent dyes or neurobiotin in a scrape‐loading assay. Neuro2a cells expressing Panx1 are not electrophysiologically coupled. Intracellular Panx1‐immunoreactivity, but not gap junction plaques, is seen in cultured oligodendrocytes, astrocytes, and hippocampal neurons. Thus, at least in these mammalian cells lines, Panx1 does not form morphological or functional gap junctions, and it remains to be demonstrated that Panx1 forms gap junction‐forming protein in the CNS.

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

Abstract: 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 µM kainate or 5 mMl‐glutamate, but not by a 24‐h exposure to up to 10 mMα‐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 Ca2+‐dependent excitotoxicity and that AMPA‐induced cytotoxicity is blocked by desensitization of AMPA‐preferring GluRs.

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.

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.

Kv3.1b Is a Novel Component of CNS Nodes

We herein demonstrate that Kv3.1b subunits are present at nodes of Ranvier in the CNS of both rats and mice. Kv3.1b colocalizes with voltage-gated Na+ channels in a subset of nodes in the spinal cord, particularly those of large myelinated axons. Kv3.1b is abundantly expressed in the gray matter of the spinal cord, but does not colocalize with Na+ channels in initial segments. In the PNS, few nodes are Kv3.1b-positive. During the development of the CNS, Kv3.1b clustering at nodes occurs later than that of Na+ channels, but precedes the juxtaparanodal clustering of Kv1.2. Moreover, in myelin-deficient rats, which have severe CNS dysmyelination, node-like clusters of Kv3.1b and Na+ channels are observed even in regions devoid of oligodendrocytes. Ankyrin G coimmunoprecipitates Kv3.1b in vivo, indicating that these two proteins may interact in the CNS at nodes. 4-Aminopyridine, a K+ channel blocker, broadened the compound action potential recorded from adult rat optic nerve and spinal cord, but not from the sciatic nerve. These effects were also observed in Kv3.1-deficient mice. In conclusion, Kv3.1b is the first K+ channel subunit to be identified in CNS nodes; but Kv3.1b does not account for the effects of 4-aminopyridine on central myelinated tracts.

Wnt signaling is sufficient to perturb oligodendrocyte maturation.

The development of oligodendrocytes, the myelinating cells of the central nervous system, is temporally and spatially controlled by local signaling factors acting as inducers or inhibitors. Dorsal spinal cord tissue has been shown to contain inhibitors of oligodendrogliogenesis, although their identity is not completely known. We have studied the actions of one family of dorsal signaling molecules, the Wnts, on oligodendrocyte development. Using tissue culture models, we have shown that canonical Wnt activity through beta-catenin activation inhibits oligodendrocyte maturation, independently of precursor proliferation, cell death, or diversion to an alternate cell fate. Mice in which Wnt/beta-catenin signaling was constitutively activated in cells of the oligodendrocyte lineage had equal numbers of oligodendrocyte precursors relative to control littermates, but delayed appearance of mature oligodendrocytes, myelin protein, and myelinated axons during development, although these differences largely disappeared by adulthood. These results indicate that activating the Wnt/beta-catenin pathway delays the development of myelinating oligodendrocytes.

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

Abstract: 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 µM in cortical glial cells and 66 ± 23 µM 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 ∼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‐α‐aminoadipate, all of the compounds examined were ∼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. The pharmacology of transport measured in these glial cells differs from the reported pharmacology of the cloned Glu transporters, suggesting the existence of additional uncloned Glu transporters or Glu transporter subunits.

Oxidative stress disrupts oligodendrocyte maturation

Periventricular white matter injury (PWMI) is the leading cause of chronic neurologic injury among survivors of preterm birth. The hallmark of PWMI is hypomyelination and a lack of mature, myelinating oligodendrocytes. Oligodendrocytes undergo a well‐characterized lineage progression from neural stem cell to mature oligodendrocyte. Oligodendrocyte precursors have increased susceptibility to oxidative and free radical‐mediated injury compared with mature oligodendrocytes as a result of lower levels of antioxidant enzymes and free radical scavengers. In this study, we show that oxidative stress disrupts oligodendrocyte differentiation by two mechanisms. First, oxidizing agents decrease the expression of key genes that promote oligodendrocyte differentiation from neural stem cells and increase the expression of genes known to inhibit differentiation. Second, global histone acetylation persists under conditions of oxidative stress, further contributing to the prevention of oligodendrocyte differentiation. Both of these mechanisms result in the arrest of oligodendrocyte differentiation without an increase in cell death.

Oligodendrocyte maturation and myelin gene expression in PDGF-treated cultures from rat cerebral white matter

SummaryMyelination in the CNS is accompanied by the differentiation of oligodendrocytes as well as the coordinate expression of a group of myelin-specific genes, including those encoding proteolipid protein and myelin basic protein. In order to compare the timing of the onset of myelin gene expression with the known sequence of oligodendrocyte maturation, we analyzed cerebral white matter cultures grown in the presence of platelet-derived growth factor for expression of the mRNAs encoding these myelin proteins, as well as for the numbers of oligodendrocytes and their precursors. Platelet-derived growth factor treatment increased the rate of oligodendrocyte precursor cell proliferation and the number of mature oligodendrocytes. Platelet-derived growth factor also produced a significant increase in oligodendrocyte precursors prior to an increase in their proliferation rate, suggesting that platelet-derived growth factor may also have an effect on oligodendrocyte precursor survival. Furthermore, steady-state levels of proteolipid protein and myelin basic protein mRNAs increased within 24 h of the addition of platelet-derived growth factor, before any significant change in the numbers of oligodendrocytes or their precursors, demonstrating that platelet-derived growth factor also regulates myelin gene expression. At later times after platelet-derived growth factor addition, however, when the number of oligodendrocytes and their precursors was rapidly increasing, the increase in proteolipid protein and myelin basic protein mRNA levels was proportionally much greater than the increase in oligodendroglial lineage cells, suggesting that platelet-derived growth factor also increased the number of proteolipid protein and myelin basic protein transcripts per cell; this interpretation was confirmed byin situ hybridization analysis. Finally, by examining the co-expression of galactocerebroside using the epitopes recognized by the Ranscht monoclonal antibody and proteolipid protein mRNA in individual cells by a combination ofin situ hybridization and immunohistochemistry, we demonstrated that oligodendrocytes express proteolipid protein and myelin basic protein mRNA. Oligodendrocyte maturation, as measured by surface galactocerebroside expression, is thus contemporaneous with the activation of myelin-specific gene expression.

Bone Morphogenetic Proteins 4, 6, and 7 Are Up-Regulated in Mouse Spinal Cord during Experimental Autoimmune Encephalomyelitis

Although spontaneous remyelination occurs in multiple sclerosis (MS), the extent of myelin repair is often inadequate to restore normal function. Oligodendrocyte precursors remaining in nonremyelinating MS plaques may be restricted by an inhibitory signal. Bone morphogenetic proteins (BMPs) have been implicated as repressors of oligodendrocyte development and inducers of astrogliogenesis. We hypothesized that BMPs are up‐regulated in MS lesions and play a role in demyelination and astrogliosis. We examined expression of BMPs in an animal model of MS, chronic experimental autoimmune encephalomyelitis (EAE) induced by the myelin oligodendrocyte glycoprotein (MOG) peptide in C57BL/6 mice. By 14 days postimmunization, compared to those of control mice, the lumbar spinal cords of MOG‐peptide EAE mice demonstrated prominent astrogliosis, infiltration of inflammatory cells, and disrupted expression of myelin proteins. Quantitative RT‐PCR showed that expression of BMP4, BMP6, and BMP7 mRNA increased 2‐ to 4‐fold in the lumbar spinal cords of animals with symptomatic EAE versus in vehicle‐treated and untreated controls on days 14, 21, and 42 postimmunization. BMP2 mRNA expression was not altered. BMP4 mRNA was much more abundant in the spinal cords of all animals than was mRNA encoding BMP2, BMP6, and BMP7. Immunoblot analysis confirmed the increased expression of BMP4 in the EAE animals. Immunohistochemistry revealed increased BMP4 immunoreactivity in areas of inflammation in MOG‐peptide EAE animals. BMP4 labeling was mostly limited to macrophages but was sometimes associated with astrocytes and oligodendrocytes. These results indicate that members of the BMP family are differentially expressed in adult spinal cord and are up‐regulated during EAE.