Transcription factors that are required for neuron lineage direction negatively regulate oligodendrocyte differentiation (25–27). Therefore, suppression of these factors is likely to promote the neuronal to oligodendrocyte fate switch, although the mechanism remains unclear. One of the highly expressed miRNAs in the CNS that regulates neural/glial specification is miR-19. During neural differentiation, miR-19 is undetectable or expressed at low levels. In the current study, bioinformatics results demonstrated that miR-19 has strong evolutionarily conserved target sites on 3′UTR of many key genes in the neurogenesis process including NeuroD1, NeuroD4, Neurogenin, SOX4, SOX5 and SOX6.
Therefore, to address the mechanism of miR-19 action in fate decision, NSCs were transduced with pLEX-miR-19 lentiviruses and evaluated for expression of key markers of neuronal, astrocyte and oligodendrocyte lineages. Our results showed that overexpression of the neurogenic miR-19 in NSCs represses several neuronal genes (NeuroD1, NeuroD4, Neurogenin, SOX4, SOX5 and SOX6) and astrocyte gene (GFAP), while increased OL specific gene markers and function as a promoter to induce oligodendroglial identity.
For the quantitative evaluation of positive cells flow cytometry, instead of the ICC assay, was applied to show the exact number of differentiated cells. The number of differentiated cells toward OPC was about 20% higher in transduced cells than controls.
In this study, luciferase data demonstrates that NeuroD1, NeuroD4, Neurogenin, SOX4, SOX5 and SOX6 that are important for neural differentiation are direct targets of miR-19.
NeuroD1 also called β2, expressed in the nervous system late in development and is, therefore, more likely to be involved in terminal differentiation, neuronal maturation and survival of neurons in the adult SGZ and SVZ. In Xenopus, overexpression of NeuroD1 converts embryonic epidermal cells into fully differentiated neurons and promotes premature cell-cycle exit and differentiation of neural precursor cells (28–30).
Neurogenin along with E-protein binds to E box elements that are thought to be critical for neural differentiation (27). Neurogenin has a dual function that is partly responsible for the timing of neural differentiation that dominates over glial differentiation. Oligogenesis is marked by neurogenin downregulation allowing progenitor cells to respond to glial-inducing factors. In this way, the temporal control of neurogenin expression may orchestrate the sequential onset of cortical neuronal and glial differentiation (31–33). In fact, in the absence of neurogenin, cortical gliogenesis might commence at an earlier time during development. In support of this, in our studies when pLEX-miR-19 is introduced into the NSCs, it results in blocking neurogenesis and premature gliogenesis through neurogenin downregulation.
NeuroD4 (otherwise known as Math3 and NeuroM) is a member of the bHLH family of transcription factors that is being expressed and phosphorylated during primary neurogenesis and acts downstream of Ngn2 in primary neurogenesis. It is alone sufficient to generate functional neurons, although not sufficient to elicit a neuronal subtype identity (34, 35). In the present study, luciferase assay data confirmed NeuroD4 as one of the miR-19 targets. So, blocking neuronal differentiation at an early stage through suppression of NeuroD4 may be beneficial to the development of oligodendrocyte lineage.
SOX4, as one of the SoxC family members, had been implicated in initiating the early stages of neuronal differentiation both in the adult and embryonic neural progenitors. However, SOX4 overexpression inhibits oligodendrocyte differentiation in mice (36, 37). SOX4 activated transcription of genes associated with neural development in NSCs such as βIII-tubulin, MAP2 and doublecortin and reduced the expression of genes promoting oligodendrocyte differentiation. In intermediate neural progenitor cells, SOX4 promotes their maintenance by interacting with Neurgenin2 to activate Tbrain2. On the other hand, SOX4 also functions through HES5 which is a repressor of oligodendrocyte differentiation and myelination and directly activated by SOX4 (36). Therefore, miR-19 may direct NSC differentiation towards oligodendrocytes by repressing the expression of SOX4, shedding light on a novel regulator of oligodendrocyte differentiation.
SOXD group proteins Sox5 and Sox6 are essential during nervous system development and exist in many different cell types, including VZ progenitors, radial glia, oligodendrocyte precursors, and several neuronal subpopulations (37). In OLs, SoxD proteins repressed myelin gene promoters when bound exclusively. Previous studies indicated that in the absence of Sox5 and Sox6, oligodendrocytes start to differentiate prematurely and at higher numbers. In neurons, SOX5 acts as an important brake on WNT–b-catenin mitogenic activity in neural progenitors and its overexpression leads to premature cell cycle exit and prevents terminal differentiation (38).
SOX6, on one hand, could cause the activation of Wnt-1, Mash-1, N-cadherin, and MAP2 genes, leading to neurogenesis. On the other hand, Sox6 induced cell adhesion molecules, such as E-cadherin or N-cadherin, promoting neuronal differentiation by stimulating cellular aggregation or the cell to cell interaction (39, 40). According to our results, miR-19 could target SOX5 and SOX6 genes and so of these targets it can also play a role in inhibiting the neuronal pathway and promoting OL differentiation.
In summary, our results suggesting that miR-19 has several target genes through which neural differentiation is blocked and OL differentiation is facilitated. Since overexpression of miR-19 may restrict differentiation of NPCs to the oligodendrocyte lineage.