In the present study we generated an Eva a

In the present study, we generated an Eva1a knockout (KO) mouse to study the physiological role of EVA1A in vivo. The analysis of Eva1a KO embryos and isolated NSCs revealed the function of EVA1A in self-renewal and neuronal differentiation of NSCs. Our study highlighted a previously unknown role of EVA1A in the CNS.
Results
Discussion In the present study, the role of EVA1A/TMEM166 in the process of neurogenesis was investigated using Eva1a KO mice. We demonstrated that EVA1A regulates NSC self-renewal and neuronal differentiation by modulating autophagy. Genetic disruption of Eva1a caused a deficiency in the autophagic machinery, which consequently impaired the differentiation process, decreasing the number of newborn neurons and impeding their maturation in culture. This effect could be rescued by overexpression of EVA1A or treatment with MP in vitro. Our studies suggest strongly that EVA1A modulates the process of autophagy, which may provide 4 methylumbelliferone for the transition from NSCs to mature neurons. Several studies have suggested that autophagy is activated during nervous system development and cell differentiation (Nassif and Hetz, 2011; Schweichel and Merker, 1973; Zhao et al., 2010). Initially, we observed that the EVA1A levels increased over the course of neurogenesis, along with a concomitant increase in autophagy in wild-type mice. Based on this, we speculated that increased expression of EVA1A in NSCs may play a role in neurogenesis. Here, we present several lines of evidence that demonstrate that the loss of EVA1A leads to substantial decline in NSC self-renewal, as well as in neuronal differentiation in vivo and in vitro. The phenotype changes observed in Eva1a-deleted NSCs could be rescued by a gain-of-function assay in vitro. The mechanism by which EVA1A regulates neurogenesis is unclear. Autophagy plays a critical role in neurogenesis and is mainly regulated by the mTOR pathway; therefore, we examined the effect of Eva1a depletion on the mTOR pathway. The phosphorylation levels of mTOR, RPS6KB1, and EIF4EBP1 were increased in NSCs derived from Eva1a−/− mice. Consistent with these findings, the levels of p-PIK3CA and p-AKT (upstream of mTOR) also increased in Eva1a-depleted cells, which was accompanied by a substantial decrease in p-TSC2 levels. Thus, it can be seen that the PIK3CA-AKT signal negatively regulates TSC1/2 and activates mTOR, and consequently, inhibits autophagy. Importantly, transduction of Ad5-Eva1a in Eva1a−/− NSCs attenuated this PIK3CA/AKT-mTOR signaling pathway, and restored the autophagy level. These results were consistent with those of a previous report, which indicated that overexpression of EVA1A/TMEM166 decreased the activity of mTOR in human tumor cells (Chang et al., 2013). Our studies indicated that EVA1A probably regulates neurogenesis through the PIK3CA/AKT-mTOR pathway. However, further studies are necessary to understand how EVA1A regulates the PIK3CA/AKT axis precisely. Autophagy is a complex catabolic program that functions in the lysosomal degradation of proteins and other subcellular constituents and serves as an effective method of providing metabolic precursors. Previous studies demonstrated that neuroepithelial cells undergo differentiation in an energy-consuming process (Vazquez et al., 2012; Mellen et al., 2008; Qu et al., 2007), and autophagy can provide sufficient energy for the process of neurogenesis. Until recently, only a few studies had demonstrated the effects of autophagy on neurogenesis in vivo. However, how autophagy regulates neurogenesis remains largely unknown. In this study, we observed that NSC self-renewal and differentiation were decreased in Eva1a-deficient mice and autophagy was decreased. Decreased levels of autophagy may lead to a shortage of energy supply and impede the process of neurogenesis; therefore, we treated Eva1a-defective NSCs with MP. Generally, MP treatment restored neuronal differentiation in these cells. Thus, Eva1a deletion-induced defective neuronal differentiation is probably associated with a lack of available energy caused by the inhibition of autophagy.