Modulation of proliferation and differentiative potential of adult brain subventricular zone cells by purinergic signaling in vitro and in vivo: contribution of reactive astrocytes

The subventricular zone (SVZ) of the lateral ventricles is one of the two neurogenic regions persisting in the adult brain. Here, GFAP+ precursors (Type B cells) give raise to transit-amplifying Mash1+ Type C cells, which eventually further differentiate to doublecortin+ neuroblasts (Type A cells). It is now emerging that brain injuries boost neurogenesis in the adult SVZ, also through action on surrounding parenchyma or niche cells. Nevertheless, very few newborn neurons survive and integrate in the damaged areas, suggesting a non-permissive environment. Thus, understanding the pro- or anti-neurogenic activity of the various molecules composing the extracellular milieu of the neurogenic niche would greatly help designing appropriate pharmacological approaches to promote neurogenesis while reducing inhibitory signals. In pathological conditions, the concentrations of extracellular nucleotides (eNTs) raise several folds, and contribute to reactive astrogliosis [Abbracchio & Ceruti, PUSI 2:595, 2006]. Among their multiple functions in brain parenchima, astrocytes are also key components of the neurogenic niche, and regulate neural stem cells (NSC) proliferation and differentiation. Moreover, also eNTs could directly modulate SVZ cells, possibly through the G protein-coupled P2Y1 nucleotide receptor [Mishra et al., Development, 33:675, 2006; Grimm et al., J Cell Sci 122:2524, 2009], although very few data are available especially in vivo. In the present study, we tested the ability of the stable P2Y1 agonist ADPβS to control adult NSC activities, with a focus on the possible effects exerted by reactive astrocytes. ADPβS administration in the lateral ventricle of adult mice caused both reactive astrogliosis in the brain parenchyma and activation of SVZ progenitors. Indeed, proliferation of GFAP+ NSCs increased, leading to a significant expansion of the population of transit-amplifying Mash1+ progenitors and doublecortin+ neuroblasts in the SVZ. Lineage tracing experiments further demonstrated that ADPβS promoted GLAST+ progenitor proliferation, and sustained their progression towards the generation of rapidly dividing progenitors. Data were fully confirmed in vitro by the neurosphere assay, where ADPβS stimulated the differentiation of undissociated NS towards GFAP+ astrocytes, and β-IIITub+ neurons. To test whether ADPβS was acting directly on NSCs only or whether reactive astrocytes were involved, we grew NS in the conditioned media derived from Control astrocytic cultures or from astrocytes cultured in presence of ADPβS. Both astrocyte-conditioned medium reduced the number and size of primary NS with respect to control neurosphere medium. Notably, a significant enhancement in SVZ progenitor proliferation was observed when SVZ cells, initially grown in the supernatant of astrocytes exposed to ADPβS, were then shifted to normal medium. This suggests that ADPβS stimulates the release of yet-to-be identified mediator(s) whose removal boosted proliferation of SVZ cells. Our preliminary results from an ELISA assay suggest that IL-10 could likely play a role in this effect. Taken together our results strengthen evidence that purinergic system crucially regulates SVZ progenitors, both directly and through the involvement of reactive astrocytes. 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.