Adult neurogenesis in the subventricular zone: studies on the role of purinergic signalling by the neurosphere assay

In the adult rodent brain, neurogenesis persists in two restricted regions: the subventricular zone (SVZ) of the lateral ventricles and the dentate gyrus of the hippocampus. Within the SVZ, three main cell types are morphologically and functionally distinguished: astrocyte-like stem cells (type B cells) give rise to clusters of transit-amplifying cells (type C cells), which in turn generate migrating neuroblasts (type A cells). These cells can be isolated and grown in vitro as small clonal clusters of proliferating cells, called neurospheres, which can either self-renew and proliferate or undergo differentiation to the three main brain cell types (i.e., astrocytes, neurons and oligodendrocytes) when grown upon specific conditions (Doetsch F, et al., 1999. Cell 97:703-716). The extracellular signaling mechanisms controlling the transition steps among the various cell types described above are still poorly understood; if clearly identified, they could open up new avenues for the pharmacological manipulation of adult neurogenesis which could prove useful to restore brain functions following traumatic and/or ischemic events. Extracellular adenine (i.e., ATP, ADP), uracil (i.e., UTP, UDP) and sugar nucleotides (e.g., UDP-glucose and UDP-galactose) are universal signalling molecules involved in many biological processes acting via specific membrane receptors: the seven ligand-gated purinergic P2X channels and the eight G protein-coupled P2Y receptors. They are involved in the embryonic development of the nervous tissue, and in cell-to-cell communication and in the modulation of cell survival and differentiation in the adult brain (Abbracchio MP et al., 2009. Trends Neurosci 32:19–29). More recently, increasing evidence suggests an important role played by extracellular nucleotides also in controlling adult neurogenesis. On this basis, we investigated the expression of P2Y receptors on neurospheres derived from adult murine SVZ, and we started preliminary pharmacological experiments to characterize the ability of nucleotide derivatives to modulate the self-renewal or differentiative potential of neurospheres. RT-PCR analysis showed that 7-day-old primary neurospheres express the P2Y1,2,6,12,13,14 receptor subtypes but not P2Y4, and also GPR17, P2Y-like receptor responding to both uracil nucleotides (e.g. UDP-glucose) and cysteinyl-leukotrienes (e.g. LTD4 and LTC4), that has been recently demonstrated to be expressed by oligodendrocyte precursor cells (Lecca D et al., 2008. PLoS One 3:e3579). We also studied the differentiative potential of 7-day-old dissociated primary neurospheres, cultured as adherent cells for 7 additional days in the absence of growth factors and in presence of 2% FBS. The majority of the cells (relative to the total of DAPI-labeled nuclei) resulted to be GFAP-positive astrocytes (more then 50%), while only few βIII-tubulin-positive neurons and O4-positive oligodendrocytes were detected (less than 5% and 1% respectively). Moreover, a small proportion of cells with oligodendrocytes morphology resulted to be GPR17+ (less than 1%). Based on these results we tested if exposure of adherent cells to the P2Y1,11,12,13 non-selective agonist ADPβS and to the GPR17 and P2Y14 non-selective agonist UDP-glucose changed the proportion of astrocytes, neurons and oligodendrocytes which are generated upon differentiating conditions. Preliminary results showed no significative differences in the proportion between treated and untreated cells when agonists were added to adherent cells. We are currently performing different protocols of exposure for various time points. One of our future perspectives is to evaluate if and how the tested compounds can affect the proliferation and the differentiative potential of cells grown as free-floating neurospheres.