UC San Diego

Extracellular nucleotides signaling through P2 nucleotide receptors expressed throughout the nervous system regulate a variety of neuronal functions. The characterization of nucleotide effects on neuronal differentiation, growth and survival are potentially important targets that may facilitate regeneration and survival of neurons after injury, disease, or age-related loss of function. Work in this thesis tested the hypothesis that extracellular nucleotides, acting through P2 receptor(s), alter neuronal differentiation, growth and survival. I characterized morphological changes that occur with nerve growth factor (NGF) differentiation and corresponding changes in ATP release. Target tissues, such as vascular endothelial and cardiac cells, were identified as sources of ATP release and promoters of differentiation of PC12 cells independent of NGF. The altered expression of P2X and P2Y receptors in differentiated versus non-differentiated PC12 cells, combined with the enhancement of NGF-stimulated differentiation by ATP and UTP led to the identification of P2Y2 receptors as mediating nucleotide-promoted neuronal differentiation and growth in primary cultured neurons and PC12 cells. Such effects occurred via increased NGF sensitivity and enhanced activation of the NGF receptor, TrkA. Endogenous co-localization and co- immunoprecipitation of P2Y2 and TrkA receptors requiring activated Src as a physical intermediate. In vivo treatment with ATP?S treatment enhanced a marker of neuronal growth. P2Y2 receptor activation, independent of NGF/TrkA, also mediated inhibition of neuronal apoptosis by ATP/UTP. In addition to effects on differentiation, P2Y2 inhibition of apoptosis occurred through Src-mediated activation of ERK and Akt. In other experiments I found that undifferentiated, compared to NGF-differentiated, PC12 cells had lower, briefer ATP?S-stimulated norepinephrine release and reuptake; only NGF- differentiated PC12 cells were capable of desensitization to ATP?S. These results identify a role for P2Y2 receptors in neurotrophin-dependent differentiation as well as neurotrophin-independent inhibition of neuronal apoptosis. P2Y2 receptors thus regulate neuronal differentiation, survival, and function in part via interaction with neurotrophin/receptor tyrosine kinase receptors. As such, P2Y2 receptors provide a new target for therapeutic intervention in neuropathological conditions