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    • Runx also controls the proliferation and neuronal

    2018-10-26

    Runx1 also controls the proliferation and neuronal differentiation of certain neural progenitor cell populations. In embryonic olfactory bulb progenitor cells in vivo, as well as in cultures of either embryonic olfactory progenitor cells or embryonic cortical NSPCs, Runx1 increases cell proliferation and also increases the expression of the neuronal marker protein NeuroD (Theriault et al., 2005). However, Runx1 inhibits the proliferation of embryonic microglia (Zusso, 2012) and olfactory ensheathing cells (Murthy et al., 2014). Given its ability to regulate stem and progenitor cell proliferation and differentiation, induction of Runx1 in some NSPCs after injury (Logan et al., 2013) suggests that Runx1 could be regulating the injury-induced proliferation or neuronal differentiation of a specific subpopulation of adult NSPCs.
    Materials and methods
    Results
    Discussion The induction of Runx1 in a subpopulation of putative adult NSPCs in the SVZ and hippocampus following traumatic GSK1324726A injury provided the rationale for examining the function of Runx1 in NSPCs in neurosphere culture (Logan et al., 2013). The data we present here indicate that Runx1 could be regulating the post-injury proliferative or neuronal differentiation response in these cells. We demonstrate that endogenous Runx1 expression is highest in proliferating NSPCs in vitro, that it drops on mitogen removal, and that NSPC proliferation is reduced upon Runx1 inhibition. Runx1 expression is further depressed when cells are stimulated to undergo astrocytic differentiation. Runx1 over-expression produced a dramatic morphological change in the neurosphere cultures, causing many cells to acquire a shape reminiscent of immature neurons or neuroblasts. This morphological change was accompanied by a large increase in the expression of the neuronal proteins DCX and Tuj1, with a much smaller increase in the expression of the astrocytic protein S100β. Overall, our results suggest that Runx1 is necessary for NSPC proliferation in cultured adult neurospheres, and promoting a pro-neuronal fate choice on cell differentiation. This model is summarized in Fig. 7. We demonstrate that removing bFGF from the media causes a significant drop in Runx1 protein but not mRNA expression (Fig. 1). There is significant evidence for post-transcriptional regulation of Runx1 protein levels. In the absence of its cofactor CBFβ, Runx1 is continuously degraded by the ubiquitin proteasome pathway, suggesting that any endogenous modification of the interaction with these two proteins could potentially evoke an increase in Runx1 degradation rates (Huang et al., 2001). The Cdk4/cyclin D1 complex can phosphorylate Runx1 and target it for ubiquitination and degradation (Biggs et al., 2006). Additionally, extracellular signal-related kinase (ERK) can also phosphorylate Runx1 at multiple sites, disrupting the interaction between Runx1 and its corepressor protein mSin3A, and thereby facilitating the proteolytic degradation of Runx1 (Imai et al., 2004). Finally Runx1 mRNA is targeted by several miRNAs allowing for significant post-transcriptional regulation of Runx1 protein expression (Fischer et al., 2015; Miao et al., 2015; Rossetti & Sacchi, 2013; Bernardin-Fried, 2004). It is not clear if bFGF signaling potentially maintains Runx1 protein expression through increased translation by regulation of specific miRNAs, or if removal of bFGF signaling leads to increased Runx1 degradation and hence a reduction in Runx1 protein levels. As bFGF is a pro-mitotic factor for cultured NSPCs (Kuhn et al., 1997; Vescovi et al., 1993; Wagner et al., 1999), bFGF signaling links Runx1 levels and neurosphere proliferation. Runx1 expression was further decreased upon addition of 1% FBS, a treatment that induces nearly exclusive astrocytic differentiation (Fig. 1). In these neurosphere growth conditions, there was a non-significant decrease in Runx1 mRNA expression suggesting that the decrease in Runx1 protein could be mediated by transcriptional and post-transcriptional mechanisms. Together, these data indicate that Runx1 is expressed highest in neurosphere cells that are in an undifferentiated, highly proliferative state. As NSPCs are stimulated to undergo astrocytic differentiation, there is a concomitant decrease of Runx1 expression, possibly as part of an overall down-regulation of pro-neuronal differentiation pathways. These findings are in agreement with Bonnert and colleagues (Bonnert et al., 2006), who found the highest Runx1 mRNA expression in neurospheres in growth media. Runx1 mRNA expression dropped upon stimulation of neurospheres to differentiate by either removal of EGF and bFGF, or by treatment with FBS. They also found that Runx1 mRNA expression was enriched in FACS-sorted cells isolated directly from the SVZ that were selected for their high expression of proliferative markers (Bonnert et al., 2006).