Transcriptlional and post-transcriptional regulation of adult and fetal mouse neural stem cell properties: role of repressor element 1-silencing transcription factor and microRNAs in neuronal and glia differentiation

Neural stem cells (NSCs) are a self-renewing population of cells that generate the neurons and glia of both the developing and adult central nervous system (CNS). The maintenance versus differentiation of NSCs relies on an intricate network of signalling regulators that finely control gene expression program in a spatial and temporal defined sequence. One factor that has been implicated in neuronal differentiation is the repressor element-1 silencing transcription factor (REST). Recent evidences suggest that REST plays a much broader role that varies according to cell type and developmental stage. In this study, we address the role of REST in regulating the properties of NSCs derived from the mouse adult Subventricular Zone (SVZ). Rest silencing in a transiently or stable transfected adult NSC line (aNSC1) promoted cell differentiation into neurons and glia, despite the presence of growth factors in the culture medium. Interestingly REST silencing did not modify the neurogenic and gliogenic potential of aNSC1 induced to differentiate by growth factors withdrawal. These data suggest that REST is crucial to the maintenance of aNSC1 in an undifferentiated proliferative state but it does not affect their lineage-specific differentiation program. Consistently, knockdown of REST in primary neurospheres impaired their growth and clonogenic potential, as evidenced in proliferation- and colony-forming assays. Furthermore, genome- and transcriptome-wide analyses have shown that REST targets different genes implicated in fundamental biological regulatory networks. REST binds to several neuronal gene and microRNA regulatory regions and its inhibition affects the expression of various genes implicated in the control of cell growth and differentiation including Fgf1, Wnt5a and Notch3. The analysis of the regulatory network governed by REST may elucidate the molecular mechanisms involved in the regulation of the aNSC1 properties. Posttranscriptional control by microRNAs (miRNAs), in addition to transcriptional regulation, has emerged as critical regulator of NSC biology. Although several miRNAs have been identified as specific regulators of neuronal and oligodendrocyte differentiation, the miRNAs involved in astrogliogenesis remain still unknown. Here we aimed to identify specific miRNAs involved in astrocyte fate specification of neural progenitor cells derived from mouse embryonic cortex (eNPCs). ENPCs in culture can fully differentiate into GFAP-positive astrocyte in the presence of FBS. We performed a large-scale analysis of more then 350 miRNAs on eNPCs under proliferating and differentiating condition. This global analysis has revealed that many miRNAs are differently expressed during differentiation in vitro. Among these, northern blot analysis showed that miR-23a/b, miR-27a/b, miR-24, miR-26a and miR-125b increased almost linearly during differentiation, instead the family members of miR-29 (miR-29a, miR-29b and miR-29c) are characterized by a late induction. These data suggest a potential role of different microRNAs at different times during the process of astrocyte differentiation. Furthermore, we demonstrated that miR-23a and miR-125b suppresses the expression of Musashi-1 (Msi1), a RNA-binding protein critical regulator of NSC proliferation and self-renewal. The miRNAs mediated suppression of Msi1 could play an important role in maintaining the NSCs in an undifferentiated state preventing astrocyte differentiation.