Runx (Runt-related) family members are transcription factors, evolutionarily conserved, which regulate expression of genes involved in differentiation and cell cycle progression both in vertebrates and in invertebrates. RUNX proteins act in the activation and repression of gene transcription depending on the cellular context and interaction with a variety of nuclear effectors of great importance for signaling pathways. Deregulation of RUNX activity is related to the appearance of various types of cancers and other diseases. In a previous work of the lab it was shown that the three Runx family members (Runx1, Runx2 and Runx3) are expressed by cerebellar neuronal precursors and that this expression is modulated during postnatal development. Thus, aim of this study is to determine if the expression of Runx family members by neural stem cells from the subventricular zone (SVZ) of adult mice is associated with maintaining the proliferative capacity and with differentiation of these cells into neurons. Therefore, the neural stem cells will be extracted from the SVZ of adult mice and grown as neurospheres. RNA will be extracted and RT-PCR will be used to evaluate gene transcripts for members of the Runx family (Runx1, Runx2 and Runx3). The identification of the expression of these genes by the SVZ cells in vivo will be performed by in situ hybridization. The expression of RUNX proteins will be examined by Western blotting. Our preliminary results show that neural stem cells isolated from adult mice SVZ express the three forms of Runx. It will also be assessed if there are changes in the expression of Runx after induction of neural stem cell differentiation. For this objective, differentiation of SVZ neural stem cells into neurons will be induced, followed by RNA extraction with subsequent qPCR to evaluate the expression of Runx family members and differentiation markers. Differentiation also will be evaluated by immunocytochemistry using antibodies for specific cell types and for the three forms of RUNX followed by fluorescence microscopy. To assess whether Runx controls the proliferation and differentiation of neural stem cells, we will silence the three isoforms of Runx by RNAi and will evaluate proliferation and differentiation of neural stem cells in vitro. Neurogenesis and differentiantion of neuronal precursors present in the central nervous system (CNS) of adult mammals are physiological events for the maintenance of nervous tissue and attempts to regenerate after injury and neurodegeneration. Thus, it is possible to hypothesize that members of the Runx family could also play an important role in controlling proliferation and differentiation of neural stem cells present in the neurogenic niche of adult mammalian CNS.
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