The oligodendrocyte-enriched orphan G protein-coupled receptor Gpr62 is dispensable for central nervous system myelination.

BACKGROUND: Myelination is a highly regulated process in the vertebrate central nervous system (CNS) whereby oligodendrocytes wrap axons with multiple layers of insulating myelin in order to allow rapid electrical conduction. Establishing the proper pattern of myelin in neural circuits requires communicative axo-glial interactions, however, the molecular interactions that occur between oligodendrocytes and axons during developmental myelination and myelin maintenance remain to be fully elucidated. Our previous work identified G protein-coupled receptor 62 (Gpr62), an uncharacterized orphan g-protein coupled receptor, as being selectively expressed by mature oligodendrocytes within the CNS, suggesting a potential role in myelination or axoglial interactions. However, no studies to date have assessed the functional requirement for Gpr62 in oligodendrocyte development or CNS myelination. METHODS: To address this, we generated a knockout mouse strain lacking the Gpr62 gene. We assessed CNS myelination during both postnatal development and adulthood using immunohistochemistry, electron microscopy and western blot. In addition, we utilized AAV-mediated expression of a tagged Gpr62 in oligodendrocytes to determine the subcellular localization of the protein in vivo. RESULTS: We find that virally expressed Gpr62 protein is selectively expressed on the adaxonal myelin layer, suggestive of a potential role for Gpr62 in axo-myelinic signaling. Nevertheless, Gpr62 knockout mice display normal oligodendrocyte numbers and apparently normal myelination within the CNS during both postnatal development and adulthood. CONCLUSIONS: We conclude that in spite of being well-placed to mediate neuronal-oligodendrocyte communications, Gpr62 is overall dispensable for CNS myelination.

Neuronal SETD2 activity links microtubule methylation to an anxiety-like phenotype in mice.

Gene discovery efforts in autism spectrum disorder have identified heterozygous defects in chromatin remodeller genes, the 'readers, writers and erasers' of methyl marks on chromatin, as major contributors to this disease. Despite this advance, a convergent aetiology between these defects and aberrant chromatin architecture or gene expression has remained elusive. Recently, data have begun to emerge that chromatin remodellers also function directly on the cytoskeleton. Strongly associated with autism spectrum disorder, the SETD2 histone methyltransferase for example, has now been shown to directly methylate microtubules of the mitotic spindle. However, whether microtubule methylation occurs in post-mitotic cells, for example on the neuronal cytoskeleton, is not known. We found the SETD2 α-tubulin lysine 40 trimethyl mark occurs on microtubules in the brain and in primary neurons in culture, and that the SETD2 C-terminal SRI domain is required for binding and methylation of α-tubulin. A CRISPR knock-in of a pathogenic SRI domain mutation (Setd2SRI) that disables microtubule methylation revealed at least one wild-type allele was required in mice for survival, and while viable, heterozygous Setd2SRI/wtmice exhibited an anxiety-like phenotype. Finally, whereas RNA-sequencing (RNA-seq) and chromatin immunoprecipitation-sequencing (ChIP-seq) showed no concomitant changes in chromatin methylation or gene expression in Setd2SRI/wtmice, primary neurons exhibited structural deficits in axon length and dendritic arborization. These data provide the first demonstration that microtubules of neurons are methylated, and reveals a heterozygous chromatin remodeller defect that specifically disables microtubule methylation is sufficient to drive an autism-associated phenotype.

Differential Sox10 genomic occupancy in myelinating glia.

Myelin is formed by specialized myelinating glia: oligodendrocytes and Schwann cells in the central and peripheral nervous systems, respectively. While there are distinct developmental aspects and regulatory pathways in these two cell types, myelination in both systems requires the transcriptional activator Sox10. Sox10 interacts with cell type-specific transcription factors at some loci to induce myelin gene expression, but it is largely unknown how Sox10 transcriptional networks globally compare between oligodendrocytes and Schwann cells. We used in vivo ChIP-Seq analysis of spinal cord and peripheral nerve (sciatic nerve) to identify unique and shared Sox10 binding sites and assess their correlation with active enhancers and transcriptional profiles in oligodendrocytes and Schwann cells. Sox10 binding sites overlap with active enhancers and critical cell type-specific regulators of myelination, such as Olig2 and Myrf in oligodendrocytes, and Egr2/Krox20 in Schwann cells. Sox10 sites also associate with genes critical for myelination in both oligodendrocytes and Schwann cells and are found within super-enhancers previously defined in brain. In Schwann cells, Sox10 sites contain binding motifs of putative partners in the Sp/Klf, Tead, and nuclear receptor protein families. Specifically, siRNA analysis of nuclear receptors Nr2f1 and Nr2f2 revealed downregulation of myelin genes Mbp and Ndrg1 in primary Schwann cells. Our analysis highlights different mechanisms that establish cell type-specific genomic occupancy of Sox10, which reflects the unique characteristics of oligodendrocyte and Schwann cell differentiation. GLIA 2015;63:1897-1914.

MYRF is a membrane-associated transcription factor that autoproteolytically cleaves to directly activate myelin genes.

The myelination of axons is a crucial step during vertebrate central nervous system (CNS) development, allowing for rapid and energy efficient saltatory conduction of nerve impulses. Accordingly, the differentiation of oligodendrocytes, the myelinating cells of the CNS, and their expression of myelin genes are under tight transcriptional control. We previously identified a putative transcription factor, Myelin Regulatory Factor (Myrf), as being vital for CNS myelination. Myrf is required for the generation of CNS myelination during development and also for its maintenance in the adult. It has been controversial, however, whether Myrf directly regulates transcription, with reports of a transmembrane domain and lack of nuclear localization. Here we show that Myrf is a membrane-associated transcription factor that undergoes an activating proteolytic cleavage to separate its transmembrane domain-containing C-terminal region from a nuclear-targeted N-terminal region. Unexpectedly, this cleavage event occurs via a protein domain related to the autoproteolytic intramolecular chaperone domain of the bacteriophage tail spike proteins, the first time this domain has been found to play a role in eukaryotic proteins. Using ChIP-Seq we show that the N-terminal cleavage product directly binds the enhancer regions of oligodendrocyte-specific and myelin genes. This binding occurs via a defined DNA-binding consensus sequence and strongly promotes the expression of target genes. These findings identify Myrf as a novel example of a membrane-associated transcription factor and provide a direct molecular mechanism for its regulation of oligodendrocyte differentiation and CNS myelination.

Myelin gene regulatory factor is required for maintenance of myelin and mature oligodendrocyte identity in the adult CNS.

Although the transcription factors required for the generation of oligodendrocytes and CNS myelination during development have been relatively well established, it is not known whether continued expression of the same factors is required for the maintenance of myelin in the adult. Here, we use an inducible conditional knock-out strategy to investigate whether continued oligodendrocyte expression of the recently identified transcription factor myelin gene regulatory factor (MRF) is required to maintain the integrity of myelin in the adult CNS. Genetic ablation of MRF in mature oligodendrocytes within the adult CNS resulted in a delayed but severe CNS demyelination, with clinical symptoms beginning at 5 weeks and peaking at 8 weeks after ablation of MRF. This demyelination was accompanied by microglial/macrophage infiltration and axonal damage. Transcripts for myelin genes, such as proteolipid protein, MAG, MBP, and myelin oligodendrocyte glycoprotein, were rapidly downregulated after ablation of MRF, indicating an ongoing requirement for MRF in the expression of these genes. Subsequently, a proportion of the recombined oligodendrocytes undergo apoptosis over a period of weeks. Surviving oligodendrocytes gradually lose the expression of mature markers such as CC1 antigen and their association with myelin, without reexpressing oligodendrocyte progenitor markers or reentering the cell cycle. These results demonstrate that ongoing expression of MRF within the adult CNS is critical to maintain mature oligodendrocyte identity and the integrity of CNS myelin.

Cholesterol synthesis in the lactating cow: Induced expression of candidate genes.

Despite the extensive knowledge for other species, cholesterol metabolism in ruminants is nowadays still not clear. Huge differences in milk cholesterol concentration are observed between breeds, managing strategies, individuals and moment of the lactating cycle, but the genetic actors working in the process of cholesterol secretion into milk have not been identified. As ruminant diet contains no cholesterol, understanding the mechanisms and regulation of synthesis, transport and secretion into milk is crucial when trying to reduce the amount of this metabolite in dairy products. The present work aims to study the expression of candidate genes for these processes in the liver of Bos taurus during the lactating cycle. Liver biopsies were obtained from 16 adult brown Swiss cows at different time points (2 weeks pre-partum and 0, 2, 4 and 8 weeks post-partum). After RNA extraction and reverse transcription, gene expression of candidate genes was studied using quantitative RT-PCR. Key enzymes of the cholesterol synthesis (3-hydroxy-methyglutaryl-coenzyme-A (HMG-CoA) synthase, HMG-CoA reductase and farnesyldiphosphat-farnesyltransferase (FDFT)) and gene expression feed-back regulators involved in lipid metabolism (sterol regulatory element binding proteins (SREBP1and 2) SREBP-cleavage activating protein (Scap) were selected as candidate genes. HMG-CoA-reductase and FDFT showed a huge expression increase until week 2 post-partum (p<0.01), most probably in response to the new requirements in the mammary gland. As well, and as a possible explanation for such modifications, an increase in the expression of the regulators SREBP1 and Scap was observed (p<0.01 and p<0.05 respectively). Most important, the whole synthesis machinery showed a coordinated regulation, as highly significant positive correlations were found between the expression levels of the above mentioned enzymes (p<0.01). The increase of milk and blood cholesterol levels in B. taurus after parturition might be the result of a coordinated induction in the expression of key liver enzymes and their regulating factors.