Regulação da neurogénese pelo sensor de nutrientes O-GlcNAc: um enfoque no ASCL1

We know very little on how neurogenesis is regulated at the systemic level, namely by nutrients intake. Our unpublished work (see next section) links a nutrient sensor (O-GlcNAc) with a master regulator of embryonic and adult neurogenesis that is Ascl1. Using Insulin/Glucose signalling as a paradigm, we will investigate how changes in O-GlcNAc impact NSC function (i.e. quiescence/proliferation/differentiation) via regulation of Ascl1 protein levels, addressing the following aims: AIM1. TO CHARACTERIZE THE O-GlcNAc PROTEOME IN NSCs O-GlcNAc is particularly enriched in the brain, where its function has been studied mostly in postmitotic neurons. Recent evidence indicates an important role in neural stem/progenitor cells. Further advances require the characterization of OGT targets in NSCs. We will combine the latest available methods for enriching O-GlcNacylated proteins, with a state-of-the art mass spectrometry pipeline, to characterize the O-GlcNAc proteome in NSCs (Task1). AIM2. TO DETERMINE HOW CHANGES IN O-GlcNAc REGULATE ASCL1 PROTEIN LEVELS Guided by the identification of O-GlcNAc targets in NSCs, we will investigate several non-mutually exclusive mechanisms that could link OGT inhibition with decreased Ascl1 protein levels (Task2). Amongst other possibilities, we will test whether protein O-GlcNAcylation protects Ascl1 from degradation, by: i) promoting heterodimerization of Ascl1 with E-proteins (e.g.E47), an event known to protect Ascl1 from degradation, as result of direct modification of residues in the HLH dimerization domain of either protein; ii) decreasing the interaction of Ascl1 with Huwe1, as result of direct modification of residues in Ascl1; iii) decreasing the interaction of Ascl1 with Huwe1 (a known target of OGT)(14), as result of direct modification of residues in one of Huwe1 regulatory domains. AIM3. TO ASSESS THE FUNCTIONAL IMPACT OF MANIPULATING THE ACTIVITY OF OGT IN NSCs. We will manipulate O-GlcNAc in an in vivo model of Ascl1 driven neurogenesis (the ventral telencephalon), where Ascl1 is expressed in NSCs and intermediate progenitors and its dual function (promoting proliferation and differentiation) can be easily assessed (18,20). To this end, we will combine knock-down and overexpression of OGT by in utero electroporation of mouse embryos, with its pharmacological inhibition in NSC cultures. We will address how changes in O-GlcNAc modulate the behaviour (proliferation, differentiation, migration) of neural stem/progenitor cells, and the importance of Ascl1 regulation in this process (Task3). AIM4. TO ADDRESS HOW INSULIN AND GLUCOSE LEVELS REGULATE THE OGT-ASCL1 AXIS AND NEUROGENESIS Our preliminary data using cultured NSCs show that Insulin promotes O-GlcNAc and increases Ascl1 protein levels with very similar dynamics, suggesting the PTM provides a mechanistic link between metabolism and Ascl1. To test this hypothesis, we will characterize changes in the O-GlcNAc signature in NSCs promoted by Insulin, and investigate them in light of mechanistic findings obtained in Aim2 (Task4). Next, we will use an in vivo model, to investigate how Insulin/Glucose signalling impacts Ascl1 and O-GlcNAc levels, and neurogenesis. We will experiment in the adult neurogenic niche, where robust (i.e. reproducible) systemic interventions are more likely to be informative (compare to the embryo), using fasting protocols established in our team.