The genetic signature of perineuronal oligodendrocytes reveals their unique phenotype.

Oligodendrocytes--best known for assembling central nervous system myelin--can be categorized as precursors, myelin-forming cells and non-myelinating perineuronal cells. Perineuronal oligodendrocytes have been well characterized morphologically and ultrastructurally, but knowledge about their function remains scanty. It has been proposed that perineuronal oligodendrocytes support neurons and, following injury, transform into myelin-synthesizing cells. Recent findings implicating perineuronal oligodendrocytes in cytoarchitectural abnormalities in the prefrontal cortex of schizophrenia and other psychiatric disorders shed new light on these cells. We have obtained the genetic signature of perineuronal oligodendrocytes by identifying gene expression differences between oligodendrocyte subpopulations using cell-specific tags, microarray technology, quantitative time-resolved polymerase chain reaction and bioinformatics tools. We show that perineuronal cells are the progeny of oligodendrocyte progenitors and, hence, are members of the oligodendrocyte lineage. Physiologically they exhibit a novel phenotype. Their expression of PDGFR-αß and its growth factor ligand PDGF-CC sets them apart from members of their lineage as this receptor precludes their response to the same growth factors that act on myelinating cells. Their coordinate expression and context-specific usage of transcription factors Olig2, Ascl1 and Pax6, together with the prominent presence of transcription factors Pea3, Lhx2 and Otx2--not hitherto linked to the oligodendrocyte lineage--suggested a cell with features that blur the boundary between a neuron and a glial cell. But they also maintain a reservoir of untranslated transcripts encoding major myelin proteins presumably for a demyelinating episode. This first molecular characterization of perineuronal oligodendrocytes revealed the striking difference between the myelinating and non-myelinating phenotypes.

Mouse transmembrane BAX inhibitor motif 3 (Tmbim3) encodes a 38 kDa transmembrane protein expressed in the central nervous system.

Cell survival proteins play an important role throughout nervous system development, normal physiological processes, and pathological conditions. Transmembrane BAX inhibitor motif 3 (TMBIM3, also known as GRINA), is a member of a family of proteins that contain a conserved BAX inhibitor-1 motif. This family of proteins includes several members that have been shown to protect cells from apoptosis. In this study, the authors show that TMBIM3 is expressed in the brain including high levels in the hippocampus. Biochemical and sequence analysis of TMBIM3 demonstrates that the rat, murine, and human genes encode an approximately 38 kDa protein with a predicted seven transmembrane domain topology. A Tmbim3 knockout mouse line did not have an obvious phenotype, but may prove useful in future studies of this family of proteins.

A tool for examining the role of the zinc finger myelin transcription factor 1 (Myt1) in neural development: Myt1 knock-in mice.

The Myt1 family of transcription factors is unique among the many classes of zinc finger proteins in how the zinc-stabilized fingers contact the DNA helix. To examine the function of Myt1 in the developing nervous system, we generated mice in which Myt1 expression was replaced by an enhanced Green Fluorescent Protein fused to a Codon-improved Cre recombinase as a protein reporter. Myt1 knock-in mice die at birth, apparently due to improper innervation of their lungs. Elimination of Myt1 did not significantly affect the number or distribution of neural precursor cells that normally express Myt1 in the embryonic spinal cord. Nor was the general pattern of differentiated neurons altered in the embryonic spinal cord. The Myt1 knock-in mice should provide an important tool for identifying the in vivo targets of Myt1 action and unraveling the role of this structurally distinct zinc finger protein in neural development.

Integrating microRNA and mRNA expression profiles of neuronal progenitors to identify regulatory networks underlying the onset of cortical neurogenesis.

BACKGROUND: Cortical development is a complex process that includes sequential generation of neuronal progenitors, which proliferate and migrate to form the stratified layers of the developing cortex. To identify the individual microRNAs (miRNAs) and mRNAs that may regulate the genetic network guiding the earliest phase of cortical development, the expression profiles of rat neuronal progenitors obtained at embryonic day 11 (E11), E12 and E13 were analyzed. RESULTS: Neuronal progenitors were purified from telencephalic dissociates by a positive-selection strategy featuring surface labeling with tetanus-toxin and cholera-toxin followed by fluorescence-activated cell sorting. Microarray analyses revealed the fractions of miRNAs and mRNAs that were up-regulated or down-regulated in these neuronal progenitors at the beginning of cortical development. Nearly half of the dynamically expressed miRNAs were negatively correlated with the expression of their predicted target mRNAs. CONCLUSION: These data support a regulatory role for miRNAs during the transition from neuronal progenitors into the earliest differentiating cortical neurons. In addition, by supplying a robust data set in which miRNA and mRNA profiles originate from the same purified cell type, this empirical study may facilitate the development of new algorithms to integrate various "-omics" data sets.

Identification of dynamically regulated microRNA and mRNA networks in developing oligodendrocytes.

MicroRNAs (miRNAs) play important roles in modulating gene expression at the posttranscriptional level. In postnatal oligodendrocyte lineage cells, the miRNA expression profile ("microRNAome") contains 43 miRNAs whose expression dynamically changes during the transition from A2B5(+) oligodendrocyte progenitor cells to premyelinating GalC(+) cells. The combination of microRNAome profiling with analyses of the oligodendrocyte transcriptome reveals a target bias for a class of miRNAs which includes miR-9. We show that miR-9 is downregulated during oligodendrocyte differentiation. In addition, miR-9 expression level inversely correlates with the expression of its predicted targets, among which is the peripheral myelin protein PMP22. We found that PMP22 mRNA but not protein is detectable in oligodendrocytes, whereas Schwann cells producing PMP22 protein lack miR-9. We demonstrate that miR-9 interacts with the 3' untranslated region of PMP22 and downregulates its expression. Our results support models in which miRNAs can act as guardians of the transcriptome.

Identification of a novel oligodendrocyte cell adhesion protein using gene expression profiling.

Oligodendrocytes undergo extensive changes as they differentiate from progenitors into myelinating cells. To better understand the molecular mechanisms underlying this transformation, we performed a comparative analysis using gene expression profiling of A2B5+ oligodendrocyte progenitors and O4+ oligodendrocytes. Cells were sort-purified ex vivo from postnatal rat brain using flow cytometry. Using Affymetrix microarrays, 1707 transcripts were identified with a more than twofold increase in expression in O4+ oligodendrocytes. Many genes required for oligodendrocyte differentiation were upregulated in O4+ oligodendrocytes, including numerous genes encoding myelin proteins. Transcriptional changes included genes required for cell adhesion, actin cytoskeleton regulation, and fatty acid and cholesterol biosynthesis. At the O4+ stage, there was an increase in expression of a novel proline-rich transmembrane protein (Prmp). Localized to the plasma membrane, Prmp displays adhesive properties that may be important for linking the extracellular matrix to the actin cytoskeleton. Together, our results highlight the usefulness of this discovery-driven experimental strategy to identify genes relevant to oligodendrocyte differentiation and myelination.

Myt1 family recruits histone deacetylase to regulate neural transcription.

The myelin transcription factor 1 (Myt1) gene family is comprised of three zinc finger genes [Myt1, Myt1L (Myt1-Like) and NZF3] of the structurally unique CCHHC class that are expressed predominantly in the developing CNS. To understand the mechanism by which this family regulates neural differentiation, we searched for interaction partners. In both yeast and a mammalian two-hybrid system, Myt1 and Myt1L interacted with Sin3B, a protein that mediates transcriptional repression by binding to histone deacetylases (HDACs). Myt1-Sin3B complexes were co-immunoprecipitated from transfected mammalian cells and included HDAC1 and HDAC2. Myt1 and Myt1L could partner with all three Sin3B isoforms, the long form (Sin3B(LF)) that includes the HDAC-binding domain, and the two short forms (Sin3B(SF293) and Sin3B(SF302)) that lack this domain and may consequently antagonize Sin3B(LF)/HDAC-mediated co-repression. Myt1 or Myt1L interactions with the HDAC-binding form of Sin3B conferred repression on a heterologous promoter. Oligodendrocytes were shown to express transcripts encoding each of the Sin3B isoforms. We present a model in which the Myt1 family of zinc finger proteins, when bound to a neural promoter, can recruit Sin3B. Depending on the relative availability of Sin3B isoforms, the Myt1 gene family may favor the silencing of genes during neural development.

Myelin transcription factor 1 (Myt1) modulates the proliferation and differentiation of oligodendrocyte lineage cells.

Myelin transcription factor 1 (Myt1) is a zinc finger DNA-binding protein that is expressed in neural progenitors and oligodendrocyte lineage cells. This study examines the role of Myt1 in oligodendrocyte lineage cells by overexpressing putative functional domains, a four-zinc finger DNA-binding region (4FMyt1) or a central protein-protein interaction domain (CDMyt1), without the predicted transcriptional activation domain. In the presence of mitogens, overexpression of 4FMyt1 inhibited proliferation of oligodendrocyte progenitors, but not cell types (astrocytes and NIH3T3 cells) lacking endogenous Myt1. Expression of 4FMyt1 inhibited the differentiation of oligodendrocyte progenitors into oligodendrocytes as assessed by morphology, immunostaining, and myelin gene expression. Progenitor differentiation was similarly inhibited by expression of CDMyt1 but only partially suppressed by overexpression of the intact Myt1. These data indicate that Myt1 may regulate a critical transition point in oligodendrocyte lineage development by modulating oligodendrocyte progenitor proliferation relative to terminal differentiation and up-regulation of myelin gene transcription.

PDGF and FGF2 regulate oligodendrocyte progenitor responses to demyelination.

Acute demyelination of adult CNS, resulting from trauma or disease, is initially followed by remyelination. However, chronic lesions with subsequent functional impairment result from eventual failure of the remyelination process, as seen in multiple sclerosis. Studies using animal models of successful remyelination delineate a progression of events facilitating remyelination. A universal feature of this repair process is extensive proliferation of oligodendrocyte progenitor cells (OPs) in response to demyelination. To investigate signals that regulate OP proliferation in response to demyelination we used murine hepatitis virus-A59 (MHV-A59) infection of adult mice to induce focal demyelination throughout the spinal cord followed by spontaneous remyelination. We cultured glial cells directly from demyelinating and remyelinating spinal cords using conditions that maintain the dramatically enhanced OP proliferative response prior to CNS remyelination. We identify PDGF and FGF2 as significant mitogens regulating this proliferative response. Furthermore, we demonstrate endogenous PDGF and FGF2 activity in these glial cultures isolated from demyelinated CNS tissue. These findings correlate well with our previous demonstration of increased in vivo expression of PDGF and FGF2 ligand and corresponding receptors in MHV-A59 lesions. Together these studies support the potential of these pathways to function in vivo as critical factors in regulating remyelination.

Nuclear organization in differentiating oligodendrocytes.

Many studies have suggested that the 3D organization of chromatin and proteins within the nucleus contributes to the regulation of gene expression. We tested multiple aspects of this nuclear organization model within a primary cell culture system. Oligodendrocyte lineage cells were examined to facilitate analysis of nuclear organization relative to a highly expressed tissue-specific gene, proteolipid protein (PLP), which exhibits transcriptional upregulation during differentiation from the immature progenitor stage to the mature oligodendrocyte stage. Oligodendrocyte lineage cells were isolated from brains of neonatal male rodents, and differentiation from oligodendrocyte progenitors to mature oligodendrocytes was controlled with culture conditions. Genomic in situ hybridization was used to detect the single copy of the X-linked PLP gene within each interphase nucleus. The PLP gene was not randomly distributed within the nucleus, but was consistently associated with the nuclear periphery in both progenitors and differentiated oligodendrocytes. PLP and a second simultaneously upregulated gene, the myelin basic protein (MBP) gene, were spatially separated in both progenitors and differentiated oligodendrocytes. Increased transcriptional activity of the PLP gene in differentiated oligodendrocytes corresponded with local accumulation of SC35 splicing factors. Differentiation did not alter the frequency of association of the PLP gene with domains of myelin transcription factor 1 (Myt1), which binds the PLP promoter. In addition to our specific findings related to the PLP gene, these data obtained from primary oligodendrocyte lineage cells support a nuclear organization model in which (1). nuclear proteins and genes can exhibit specific patterns of distribution within nuclei, and (2). activation of tissue-specific genes is associated with changes in local protein distribution rather than spatial clustering of coordinately regulated genes. This nuclear organization may be critical for complex nucleic-acid-protein interactions controlling normal cell development, and may be an important factor in aberrant regulation of cell differentiation and gene expression in transformed cells.