|dc.description.abstract||Adult hippocampal neurogenesis plays an essential role in the formation and consolidation of new memories, spatial processing and some forms of learning. Identifying the molecular mechanisms that regulate hippocampal neural progenitor cells as they proliferate, differentiate, and are selected for either survival or cell death will provide a fundamental understanding of how this neurogenic niche coordinates these activities. Here, the roles of P2X7 receptors are examined for their influence over neural progenitor cell biology, particularly cell death, proliferation, and phagocytosis of apoptotic progenitors that have undergone programmed cell death. As a purinergic cation channel, P2X7 receptors are exceptionally versatile; their primary role is as ATP-gated calcium channels, and they have notable roles in the immune system, where they regulate cytokine release and form large transmembrane pores resulting in cell death. By acting as scavenger receptors, they can also mediate phagocytosis. These diverse roles were investigated in neural progenitor cells of the adult murine hippocampal neurogenic niche.
Primary cultures of hippocampal neural progenitor cells were derived from adult female C57BL/6 mice and characterised using multimarker immunocytochemistry as P2X7 receptor positive type 2 neural progenitor cells, as defined by Sox2pos, nestinpos, BLBPpos, Mash1pos/neg, vimentinpos, Pax6pos, Prox1pos, DCXneg, GFAPneg staining patterns. For some experiments, cultures derived from P2X7 knock out mice (Pfizer) were also used. Calcium influx assays using the indicator dye Fluo-8-AM demonstrated functional activity of P2X7 receptors with the general agonist ATP (1 mM) and the more specific agonist BzATP (100 μM). Ethidium bromide uptake demonstrated that P2X7 receptors were able to form large transmembrane pores, a canonical function unique to this receptor, and confirmed the presence of a full length protein, as opposed to various splice variants. Live cell confocal microscopy revealed hippocampal neural progenitors are capable of phagocytosing fluorescent latex beads, and flow cytometry in conjunction with specific inhibitors demonstrated that P2X7 receptors are capable of facilitating this phagocytosis.
The effects of purinergic signalling on neural progenitor proliferation were assessed using the thymidine analogue EdU. P2X7 receptors activated with either extracellular ATP or BzATP showed a significant dose-dependent decrease in proliferation. Cell death was not observed under these conditions and proliferation could be rescued upon exchange of medium. P2X7 receptor inhibition reduced the effects of extracellular ATP on proliferation, and use of neural progenitor cultures derived from genetically null mice corroborated this observation. Convergence with growth factor signalling pathways was also explored.
The data presented here provides good evidence that P2X7 receptors function as scavenger receptors in the absence of ATP, allowing neural progenitor cells to phagocytose their apoptotic peers during target-independent programmed cell death, as well as governing rates of proliferation in the presence of ATP, possibly by regulating calcium dependent downstream signalling. Effector molecules of calcium signalling pathways were investigated following P2X7 receptor activation to determine some of the downstream mechanisms involved in P2X7 receptor mediated decreases in proliferation. Live cell calcium imaging identified the instigation of secondary calcium oscillations following extracellular ATP application; it was hypothesised that the decrease in proliferation was due to calcium dependent signalling cascades, involving calcium release from internal stores. Using confocal microscopy, calcium dependent transcription factors NFκB and NFAT1 were evaluated for their potential to translocate to the nucleus following purinergic stimulation. Extracellular ATP did not cause translocation of NFκB or NFAT1. A possible convergence with growth factor signalling pathways was investigated as the growth factors present in culture conditions exert powerful regulation over the cells and also utilise calcium and endoplasmic reticulum signalling to exert their effects. Inhibition of proteins involved in endoplasmic reticulum signalling caused a decrease in proliferation, as did growth factor withdrawal. Transcription factor analysis revealed that withdrawal of both EGF and bFGF caused NFAT1, but not NFκB, to translocate to the nucleus, a novel finding in these cells.
The data presented here is among the first to examine the dichotomous signalling roles of P2X7 receptors in adult hippocampal neural progenitor cells. In mature neurons, P2X7 receptors have been implicated in various pathologies, and may present a therapeutic target for a number of neurological disorders. Understanding how these receptors regulate the physiology of stem and progenitor cells is an important first step in developing any regenerative therapies. Given the crucial role neurogenesis plays in both memory formation and hippocampal function, understanding these biological mechanisms is essential to addressing significant questions regarding neurogenesis and regeneration.||