MiRNA-145-5p prevents differentiation of oligodendrocyte progenitor cells by regulating expression of myelin gene regulatory factor.

The roles of specific microRNAs (miRNA) in oligodendrocyte (OL) differentiation have been studied in depth. However, miRNAs in OL precursors and oligodendrocyte progenitor cells (OPCs) have been less extensively investigated. MiR-145-5p is highly expressed in OPCs relative to differentiating OLs, suggesting this miRNA may serve a function specifically in OPCs. Knockdown of miR-145-5p in primary OPCs led to spontaneous differentiation, as evidenced by an increased proportion of MAG+ cells, increased cell ramification, and upregulation of multiple myelin genes including MYRF, TPPP, and MAG, and OL cell cycle exit marker Cdkn1c. Supporting this transition to a differentiating state, proliferation was reduced in miR-145-5p knockdown OPCs. Further, knockdown of miR-145-5p in differentiating OLs showed enhanced differentiation, with increased branching, myelin membrane production, and myelin gene expression. We identified several OL-specific genes targeted by miR-145-5p that exhibited upregulation with miR-145-5p knockdown, including myelin gene regulatory factor (MYRF), that could be regulating the prodifferentiation phenotype in both miR-145 knockdown OPCs and OLs. Indeed, spontaneous differentiation with knockdown of miR-145-5p was fully rescued by concurrent knockdown of MYRF. However, proliferation rate was only partially rescued with MYRF knockdown, and overexpression of miR-145-5p in OPCs increased proliferation rate without affecting expression of already lowly expressed differentiation genes. Taken together, these data suggest that in OPCs miR-145-5p both prevents differentiation at least in part by preventing expression of MYRF and promotes proliferation via as-yet-unidentified mechanisms. These findings clarify the need for differential regulation of miR-145-5p between OPCs and OLs and may have further implications in demyelinating diseases such as multiple sclerosis where miR-145-5p is dysregulated.

Cytoskeletal Linker Protein Dystonin Is Not Critical to Terminal Oligodendrocyte Differentiation or CNS Myelination.

Oligodendrocyte differentiation and central nervous system myelination require massive reorganization of the oligodendrocyte cytoskeleton. Loss of specific actin- and tubulin-organizing factors can lead to impaired morphological and/or molecular differentiation of oligodendrocytes, resulting in a subsequent loss of myelination. Dystonin is a cytoskeletal linker protein with both actin- and tubulin-binding domains. Loss of function of this protein results in a sensory neuropathy called Hereditary Sensory Autonomic Neuropathy VI in humans and dystonia musculorum in mice. This disease presents with severe ataxia, dystonic muscle and is ultimately fatal early in life. While loss of the neuronal isoforms of dystonin primarily leads to sensory neuron degeneration, it has also been shown that peripheral myelination is compromised due to intrinsic Schwann cell differentiation abnormalities. The role of this cytoskeletal linker in oligodendrocytes, however, remains unclear. We sought to determine the effects of the loss of neuronal dystonin on oligodendrocyte differentiation and central myelination. To address this, primary oligodendrocytes were isolated from a severe model of dystonia musculorum, Dstdt-27J, and assessed for morphological and molecular differentiation capacity. No defects could be discerned in the differentiation of Dstdt-27J oligodendrocytes relative to oligodendrocytes from wild-type littermates. Survival was also compared between Dstdt-27J and wild-type oligodendrocytes, revealing no significant difference. Using a recently developed migration assay, we further analysed the ability of primary oligodendrocyte progenitor cell motility, and found that Dstdt-27J oligodendrocyte progenitor cells were able to migrate normally. Finally, in vivo analysis of oligodendrocyte myelination was done in phenotype-stage optic nerve, cerebral cortex and spinal cord. The density of myelinated axons and g-ratios of Dstdt-27J optic nerves was normal, as was myelin basic protein expression in both cerebral cortex and spinal cord. Together these data suggest that, unlike Schwann cells, oligodendrocytes do not have an intrinsic requirement for neuronal dystonin for differentiation and myelination.