Jason C. Dugas

Dicer1 and miR-219 Are required for normal oligodendrocyte differentiation and myelination.

To investigate the role of microRNAs in regulating oligodendrocyte (OL) differentiation and myelination, we utilized transgenic mice in which microRNA processing was disrupted in OL precursor cells (OPCs) and OLs by targeted deletion of Dicer1. We found that inhibition of OPC-OL miRNA processing disrupts normal CNS myelination and that OPCs lacking mature miRNAs fail to differentiate normally in vitro. We identified three miRNAs (miR-219, miR-138, and miR-338) that are induced 10-100x during OL differentiation; the most strongly induced of these, miR-219, is necessary and sufficient to promote OL differentiation, and partially rescues OL differentiation defects caused by total miRNA loss. miR-219 directly represses the expression of PDGFRalpha, Sox6, FoxJ3, and ZFP238 proteins, all of which normally help to promote OPC proliferation. Together, these findings show that miR-219 plays a critical role in coupling differentiation to proliferation arrest in the OL lineage, enabling the rapid transition from proliferating OPCs to myelinating OLs.

Myelin Gene Regulatory Factor Is a Critical Transcriptional Regulator Required for CNS Myelination

The transcriptional control of CNS myelin gene expression is poorly understood. Here we identify gene model 98, which we have named myelin gene regulatory factor (MRF), as a transcriptional regulator required for CNS myelination. Within the CNS, MRF is specifically expressed by postmitotic oligodendrocytes. MRF is a nuclear protein containing an evolutionarily conserved DNA binding domain homologous to a yeast transcription factor. Knockdown of MRF in oligodendrocytes by RNA interference prevents expression of most CNS myelin genes; conversely, overexpression of MRF within cultured oligodendrocyte progenitors or the chick spinal cord promotes expression of myelin genes. In mice lacking MRF within the oligodendrocyte lineage, premyelinating oligodendrocytes are generated but display severe deficits in myelin gene expression and fail to myelinate. These mice display severe neurological abnormalities and die because of seizures during the third postnatal week. These findings establish MRF as a critical transcriptional regulator essential for oligodendrocyte maturation and CNS myelination.

Pro-neural miR-128 is a glioma tumor suppressor that targets mitogenic kinases

MicroRNAs (miRNAs) carry out post-transcriptional control of a multitude of cellular processes. Aberrant expression of miRNA can lead to diseases, including cancer. Gliomas are aggressive brain tumors that are thought to arise from transformed glioma-initiating neural stem cells (giNSCs). With the use of giNSCs and human glioblastoma cells, we investigated the function of miRNAs in gliomas. We identified pro-neuronal miR-128 as a candidate glioma tumor suppressor miRNA. Decreased expression of miR-128 correlates with aggressive human glioma subtypes. With a combination of molecular, cellular and in vivo approaches, we characterize miR-128s tumor suppressive role. miR-128 represses giNSC growth by enhancing neuronal differentiation. miR-128 represses growth and mediates differentiation by targeting oncogenic receptor tyrosine kinases (RTKs) epithelial growth factor receptor and platelet-derived growth factor receptor-α. Using an autochthonous glioma mouse model, we demonstrated that miR-128 repressed gliomagenesis. We identified miR-128 as a glioma tumor suppressor that targets RTK signaling to repress giNSC self-renewal and enhance differentiation.

Identification of a gene regulatory network necessary for the initiation of oligodendrocyte differentiation.

Differentiation of oligodendrocyte progenitor cells (OPCs) into mature oligodendrocytes requires extensive changes in gene expression, which are partly mediated by post-translational modifications of nucleosomal histones. An essential modification for oligodendrocyte differentiation is the removal of acetyl groups from lysine residues which is catalyzed by histone deacetylases (HDACs). The transcriptional targets of HDAC activity within OPCs however, have remained elusive and have been identified in this study by interrogating the oligodendrocyte transcriptome. Using a novel algorithm that allows clustering of gene transcripts according to expression kinetics and expression levels, we defined major waves of co-regulated genes. The initial overall decrease in gene expression was followed by the up-regulation of genes involved in lipid metabolism and myelination. Functional annotation of the down-regulated gene clusters identified transcripts involved in cell cycle regulation, transcription, and RNA processing. To define whether these genes were the targets of HDAC activity, we cultured rat OPCs in the presence of trichostatin A (TSA), an HDAC inhibitor previously shown to inhibit oligodendrocyte differentiation. By overlaying the defined oligodendrocyte transcriptome with the list of ‘TSA sensitive’ genes, we determined that a high percentage of ‘TSA sensitive’ genes are part of a normal program of oligodendrocyte differentiation. TSA treatment increased the expression of genes whose down-regulation occurs very early after induction of OPC differentiation, but did not affect the expression of genes with a slower kinetic. Among the increased ‘TSA sensitive’ genes we detected several transcription factors including Id2, Egr1, and Sox11, whose down-regulation is critical for OPC differentiation. Thus, HDAC target genes include clusters of co-regulated genes involved in transcriptional repression. These results support a de-repression model of oligodendrocyte lineage progression that relies on the concurrent down-regulation of several inhibitors of differentiation.

The T3-induced gene KLF9 regulates oligodendrocyte differentiation and myelin regeneration.

Hypothyroidism is a well-described cause of hypomyelination. In addition, thyroid hormone (T3) has recently been shown to enhance remyelination in various animal models of CNS demyelination. What are the ways in which T3 promotes the development and regeneration of healthy myelin? To begin to understand the mechanisms by which T3 drives myelination, we have identified genes regulated specifically by T3 in purified oligodendrocyte precursor cells (OPCs). Among the genes identified by genomic expression analyses were four transcription factors, Kruppel-like factor 9 (KLF9), basic helix-loop-helix family member e22 (BHLHe22), Hairless (Hr), and Albumin D box-binding protein (DBP), all of which were induced in OPCs by both brief and long term exposure to T3. To begin to investigate the role of these genes in myelination, we focused on the most rapidly and robustly induced of these, KLF9, and found it is both necessary and sufficient to promote oligodendrocyte differentiation in vitro. Surprisingly, we found that loss of KLF9 in vivo negligibly affects the formation of CNS myelin during development, but does significantly delay remyelination in cuprizone-induced demyelinated lesions. These experiments indicate that KLF9 is likely a novel integral component of the T3-driven signaling cascade that promotes the regeneration of lost myelin. Future analyses of the roles of KLF9 and other identified T3-induced genes in myelination may lead to novel insights into how to enhance the regeneration of myelin in demyelinating diseases such as multiple sclerosis.

Odorant receptor gene regulation: implications from genomic organization.

Odorant receptor genes comprise the largest known family of G-protein-coupled receptors in vertebrates. These receptor genes are tightly clustered in the genomes of every vertebrate organism investigated, including zebrafish, mice and humans, and they appear to have expanded and duplicated throughout evolution. In a mechanism that has yet to be elucidated, each olfactory neuron expresses a single receptor gene. This highly restricted expression pattern underlies the ability to distinguish between a wide variety of odorants. Here, we address the evolutionary expansion of odorant receptor genes and the role genomic organization of these genes might have in their tightly regulated expression.

MicroRNAs in Oligodendrocyte and Schwann Cell Differentiation

MicroRNAs (miRNAs) are a class of small (approx. 22 nt) noncoding RNAs that are capable of post-transcriptionally silencing mRNAs that contain sequences complementary to the miRNAs’ 7- to 8-bp ‘seed’ sequence. As single miRNAs are often predicted to target up to hundreds of individual transcripts, miRNAs are able to broadly affect the overall protein expression state of the cell. This can translate into global effects on cellular health and differentiation state. Recently, several reports have identified crucial roles for miRNAs in controlling the production, differentiation, and health of myelinating cells of the mammalian nervous system. In this review, we will discuss how individual miRNAs regulate these various processes, and also how miRNA production in general is required for several stages of myelin generation and maintenance.

Purification of Oligodendrocyte Lineage Cells from Mouse Cortices by Immunopanning

Oligodendrocytes are the myelinating cells of the vertebrate central nervous system, responsible for generating the myelin sheath necessary for saltatory conduction. The use of increasingly sophisticated genetic tools, particularly in mice, has vastly increased our understanding of the molecular mechanisms that regulate development of the oligodendrocyte lineage. This increased reliance on the mouse as a genetic model has led to a need for the development of culture methods to allow the use of mouse cells in vitro as well as in vivo. Here, we present a protocol for the isolation of different stages of the oligodendrocyte lineage, oligodendrocyte precursor cells (OPCs) and/or postmitotic oligodendrocytes, from the postnatal mouse cortex using immunopanning. This protocol allows for the subsequent culture or biochemical analysis of these cells.

Purification of Oligodendrocyte Precursor Cells from Rat Cortices by Immunopanning

This protocol describes how to purify oligodendrocyte precursor cells (OPCs) from postnatal rodent brains. The method utilizes an immunopanning technique to first remove unwanted cells by negative selection and then purify OPCs by positive selection and subsequent enzymatic release from the final panning plate. Included are modifications that allow for purification and culturing of OPCs from mouse instead of rat tissue and for use of optic nerves instead of whole brains. The method for isolating OPCs from whole brain can be used for isolating OPCs from any specific region of the brain, provided that the area can be dissected away from the rest of the tissue. Suggested culture media for maintaining proliferating OPCs or inducing oligodendrocyte (OL) differentiation are also described.

Rbfox proteins regulate alternative splicing of neuronal sodium channel SCN8A

The SCN8A gene encodes the voltage-gated sodium channel Na(v)1.6, a major channel in neurons of the CNS and PNS. SCN8A contains two alternative exons,18N and 18A, that exhibit tissue specific splicing. In brain, the major SCN8A transcript contains exon 18A and encodes the full-length sodium channel. In other tissues, the major transcript contains exon 18N and encodes a truncated protein, due to the presence of an in-frame stop codon. Selection of exon 18A is therefore essential for generation of a functional channel protein, but the proteins involved in this selection have not been identified. Using a 2.6 kb Scn8a minigene containing exons 18N and 18A, we demonstrate that co-transfection with Fox-1 or Fox-2 initiates inclusion of exon 18A. This effect is dependent on the consensus Fox binding site located 28 bp downstream of exon 18A. We examined the alternative splicing of human SCN8A and found that the postnatal switch to exon 18A is completed later than 10 months of age. In purified cell populations, transcripts containing exon 18A predominate in neurons but are not present in oligodendrocytes or astrocytes. Transcripts containing exon 18N appear to be degraded by nonsense-mediated decay in HEK cells. Our data indicate that RBFOX proteins contribute to the cell-specific expression of Na(v)1.6 channels in mature neurons.