Fine-tuning neurogenesis : microRNA regulatory networks

Tese de doutoramento, Farmácia (Biologia Celular e Molecular), Universidade de Lisboa, Faculdade de Farmácia, 2015Neurogenesis, the process of producing neurons from neural stem cells (NSCs) occurs in discrete areas of the adult mammalian brain. However, NSCs produce very few neurons following injury or pathological conditions. Thus, the identification of novel players that improve neurogenesis may have huge impact in the development of strategies for neural repair. Recent discoveries have revealed that microRNAs (miRNAs or miRs) have important roles in stem cell biology, controlling stem cell fate and behavior. Strikingly, additional evidence suggests the involvement of apoptosis-associated miRNAs in stem cell differentiation. Thus, our studies were conducted with the purpose of further characterizing the molecular mechanisms by which specific apoptosis-associated miRNAs regulate NSC differentiation to ultimately improve neurogenesis as an alternative to cell loss. In initial studies we evaluated the expression of specific apoptosis-associated miRNAs during the differentiation process of mouse NSCs. Validation by qRT-PCR revealed that pro-apoptotic miR-16, let-7a, let-7b, miR-34 family members, and miR- 143/145 cluster, as well as anti-apoptotic miR-21 are differentially expressed throughout NSC differentiation. We further investigated the role of two of the most modulated miRNAs. First, we assessed the impact of miR-34a during neuronal differentiation. We showed that miR- 34a is significantly downregulated during NSC differentiation and that miR-34a overexpression significantly impairs neurogenesis progression. Additionally, we demonstrated that miR-34a downregulation is crucial to allow upregulation of synaptotagmin 1 (Syt1) and autophagy-related 9a (Atg9a) proteins, essential for differentiation to progress. Finally, we explored the role of miR-145 in NSC differentiation. We discovered that neuronal differentiation was associated with a marked increase in miR-145 levels and reported that forced miR-145 downregulation negatively affects this process. Additional experiments, in turn, indicated that miR-145 regulates differentiation of mouse NSCs through the sex-determining region Y-box 2 (Sox2)– Lin28/let-7 pathway. These results demonstrate that miR-34a downregulation and miR- 145 upregulation are required for proper neuronal differentiation. In conclusion, our findings clarify the role of miR-34a and miR-145 during neuronal differentiation and may prove useful in the development of novel therapeutic strategies to improve long-term survival and differentiation of endogenous and/or transplanted stem cells.Fundo Europeu de Desenvolvimento Regional (FEDER