PURINERGIC SIGNALING AND NEUROGENESIS: MODULATION OF ADULT BRAIN SUBVENTRICULAR ZONE CELL FUNCTIONS AND PARENCHIMAL PROGENITORS MULTIPOTENCY.

Despite previous beliefs, the generation of new neurons and new glia in the central nervous system (CNS) continues throughout life. Adult neurogenesis occurs in both classical neurogenic niches (e.g., the subventricular zone, SVZ, of the lateral ventricles) as well as in the entire brain's parenchyma, which is full of quiescent neural progenitors that are activated after injury. In particular, NG2-positive polydendrocytes, that usually differentiate to mature oligodendrocytes participating to re-myelination after injury, retain some multipotency and, under some conditions, can also generate neurons and astrocytes (Nishiyama et al., 2009). Among various neurotransmitters and growth factors, extracellular nucleotides (ATP, UTP, their break-down products and sugar nucleotides), which are released at high amounts at the sites of CNS damage (Ulrich et al., 2012) are key actors in regulating reparative responses via purinergic P2 receptors (Abbracchio et al., 2009). Previous studies from our laboratory have identified the purinergic receptor GPR17 as a new marker of early stages of NG2 cell differentiation, showing that GPR17 activation accelerates NG2 cells’ oligodendrocyte fate (Fumagalli et al., 2011; Ceruti et al., 2011; Boda et al., 2011). Interestingly, GPR17 is also one of the key genes expressed by human adult neural stem cells (Maisel et al., 2007), suggesting a possible role in cell fate determination. Based on these premises, the aim of my PhD project was to investigate the role of purinergic signaling in regulating stem cell proprieties of adult brain subventricular zone and of NG2+ parenchymal progenitors. In the first part of my PhD thesis, by using a conditional GLAST::CreERT2 Rosa YFP mouse model and an in vitro neurosphere assay, we have demonstrated that the P2Y receptor agonist ADPβS promotes the proliferation of SVZ neural progenitors and sustains their progression towards the generation of neuroblasts, either directly or through the activation of parenchymal astrocytes. In the second part of my PhD, OPCs have been then cultured accordingly to two published protocols (Kondo & Raff, 2000; Liu et al., 2007) both able to unveil their stem cell properties. In both protocols, we observed an increase in the percentage of cells expressing the neuronal marker β-tubulinIII (βtubIII) upon treatment with the non-selective GPR17 antagonist Cangrelor and VPA with respect to control. Interestingly GPR17, which is normally expressed only by NG2+ cells, was surprisingly detected in a subset of βtubIII+ cells already under control differentiative condition, suggesting its potential involvement in neurogenesis. The appearance of this cell population was further incremented by the exposure to VPA and Cangrelor. Taken together, our results suggest that antagonizing GPR17 functions can address the fate of NG2 cells towards the generation of new neurons, as observed with epigenetic modulators like VPA. In conclusion, our results strengthen the evidence that the purinergic system crucially regulates neuronal progenitors, either in a classical neurogenic niche or in the brain parenchyma. The pharmacological modulation of the purinergic system could therefore represent a promising and innovative approach to exploit the intrinsic ability of the adult brain to regenerate in acute and chronic neurodegenerative disorders.