Mechanisms of ATP Release from Glial Progenitors

描述（由申请人提供）：神经胶质细胞的核苷酸释放在CNS和PNS中起重要的自分泌和旁分泌信号传导作用。最近的研究表明，神经胶质细胞的非溶解性ATP释放可能涉及调节的胞外分泌或通过核苷渗透通道流出。细胞外ATP作用于质乳嗜离子性P2X和代谢性P2Y受体，可调节神经元活动，诱导星形胶质细胞中的钙瞬变，并产生关键的细胞内第二信使，例如涉及脑外伤后胶质瘤的发展和神经干细胞的增殖和迁移。最近，我们已经获得证据表明，神经祖细胞显示自发钙振荡，高度依赖于P2Rs的激活。大量应用嘌呤能受体拮抗剂或ATP降解酶apyrase可防止自发钙波动的发生。此外，阻断P2Rs可降低这些祖细胞的增殖和迁移速率。这些结果表明，神经祖细胞具有释放ATP的能力，ATP在早期中枢神经系统发育过程中起着至关重要的自分泌和旁分泌信号作用。虽然已经提出了成熟星形胶质细胞释放递质的途径，但对ATP从前体释放的机制的发育方面尚不清楚。因此，本资助申请的目标是评估星形胶质细胞发育的三个可能的递质释放途径（胞外分泌/分泌和通过嗜离子P2XRs和连接蛋白半通道的扩散）是否以及在哪个阶段是功能正常的，并确定它们对钙振荡和细胞迁移的贡献。免疫细胞化学、生化和药理学方法结合发光和荧光成像将用于识别ATP释放途径。这一鉴定对于理解中枢神经系统发育和修复过程中的信号机制至关重要。

DESCRIPTION (provided by applicant): Nucleotide release from glial cells plays important autocrine and paracrine signaling roles in the CNS and PNS. Recent studies suggest that the non-lytic release of ATP from glial cells can either involve regulated exocytosis or efflux through nucleotide-permeable channels. Extracellular ATP acting on plasmalemmal ionotropic P2X and metabotropic P2Y receptors modulates neuronal activity, induces calcium transients in astrocytes and generates critical intracellular second messengers implicated for instance in the development of gliosis following brain trauma and in the proliferation and migration of neural stem cells. Recently, we have obtained evidence that neural progenitor cells display spontaneous calcium oscillations that are highly dependent on activation of P2Rs. Bath application of purinergic receptor antagonists or of the ATP degrading enzyme apyrase prevented the occurrence of spontaneous calcium fluctuations. Moreover, blockade of P2Rs reduced the proliferation and the migration rates of these progenitors. These results suggest that neural progenitors have the ability to release ATP, which then plays a crucial autocrine, paracrine signaling role during early CNS development. Although identification of pathways involved on transmitter release from glial cells have been suggested for mature astrocytes, nothing is known about the developmental aspects of the mechanism(s) by which ATP is released from precursors. Therefore, the goal of this grant application is to evaluate whether and at which stage of astrocyte development three of the putative pathways for transmitter release are functionally competent (exocytosis/secretion and diffusion through ionotropic P2XRs and connexin hemichannels) and to determine their contribution to calcium oscillations and cell migration. Immunocytochemical, biochemical and pharmacological approaches combined with luminescence and fluorescence imaging will be used to identify ATP release pathways. This identification is fundamental for the understanding of the signaling mechanisms ongoing during CNS development and repair.