Purinergic signaling modulates adult neurogenesis in the subventricular zone: role of parenchymal astrocytes

Background and Purpose - The subventricular zone (SVZ) of the lateral ventricles is one of the two neurogenic regions persisting in the adult brain [1]. Evidence is accumulating that neurogenesis in the SVZ is boosted following trauma or ischemia, also through the interaction with surrounding parenchyma or niche cells. Astrocytes are key components of the neurogenic niche, and play a vital role in regulating neural stem cells (NSC) proliferation and differentiation. However, the exact molecular mechanisms by which astrocytes modulate NSC functions have not been identified. Besides significantly contributing to reactive astrogliosis, increasing evidence suggests that extracellular nucleotides play a role in controlling adult neurogenesis; these functions become prominent especially under pathological conditions where nucleotides concentrations raise several folds. From the few data published so far, a primary role for the P2Y1 G protein-coupled receptor subtype is clearly emerging in controlling the proliferation and differentiative potential of SVZ cells [2]. Therefore, we tested the ability of ADP-beta-S, a stable P2Y1 receptor agonist, to modulate stem cell properties in the adult brain in vitro and in vivo, with a particular focus on the possible modulatory effects exerted by reactive astrocytes Methods and Results – In vitro results: neurospheres (NS; floating aggregates of SVZ precursor cells, maintaining the ability to proliferate and self-renew in culture) were obtained by mouse SVZ [3] and grown in the absence or presence of ADPβS. When cells derived from the dissociation of SVZ were plated in the presence of ADPβS, an increased number of NS was generated with respect to cultures grown under control conditions. Moreover, ADPβS stimulated the differentiation of undissociated NS towards GFAP+ astrocytes, and β-IIITub+ neurons. Interestingly, a significant enhancement in secondary NS generation was detected when SVZ cells were initially grown as primary NS in the supernatant of astrocytic culture exposed to ADP-beta-S, and then shifted to normal medium. This suggests that extracellular nucleotide stimulate the release of yet-to-be identified astrocytic mediator(s) whose removal from the culture medium boosted the self-renewal capability of SVZ cells. Preliminary results reveal that ADPβS influence the release from astrocytes of several pro- and anti-inflammatory cytokines that could likely play a role in this effect. In vivo results: a 7-day long intracerebroventricular (i.c.v.) administration of 100 µM ADPβS stimulated reactive astrogliosis in the brain parenchima surrounding the SVZ, and induced a massive reaction of GFAP-expressing precursors and astrocytes in the SVZ, which became hypertrophic. Moreover, ADPβS promoted BrdU incorporation, indicating a proliferative effect. Confirming in vitro data, ADPβS administration induced also a significant expansion of the population of Mash1+ transit-amplifying cells and of doublecortin+ neuroblasts. By taking advantage of a conditional GLAST::CreERT2 Rosa YFP mouse model, we also demonstrated that ADPβS promoted the proliferation of GLAST-expressing progenitors in the neurogenic niche, and sustained their progression towards the generation of rapidly dividing transit-amplifying cells. Conclusions - Taken together, our data suggest that nucleotides can be used to increase the pool of NSCs and their differentiation towards neuroblasts, either directly or through the activation of parenchymal astrocytes. This effect could be exploited to restore brain functions following acute and chronic neurodegenerative disorders, by stimulating the self-repair intrinsic ability of the brain. References [1] Doetsch F, Garcia-Verdugo JM, Alvarez-Buylla A (1997) Cellular composition and three-dimensional organization of the subventricular germinal zone in the adult mammalian brain. J Neurosci 17:5046-5061. [2] Suyama S, Sunabori T, Kanki H, Sawamoto K , Gachet C, Koizumi S, Okano H (2012) Purinergic signaling promotes proliferation of adult mouse subventricular zone cells. J Neurosci 32:9238-9247. [2] Johansson C.B. et al., 1999. Exp.Cell Res. 253:733-736.