Mechanisms of ATP Release from Glial Progenitors

描述（由申请方提供）：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，从胶质细胞的ATP的非裂解释放可以涉及调节胞吐或流出通过核苷酸渗透通道。作用于质膜亲离子型P2 X和亲代谢型P2 Y受体的细胞外ATP调节神经元活性，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，其涉及例如脑创伤后神经胶质增生的发展以及神经干细胞的增殖和迁移。最近，我们已经获得的证据表明，神经祖细胞显示自发的钙振荡是高度依赖于激活的P2受体。浴应用嘌呤能受体拮抗剂或ATP降解酶腺苷三磷酸双磷酸酶防止自发钙波动的发生。此外，阻断P2 Rs降低了这些祖细胞的增殖和迁移率。这些结果表明，神经祖细胞有能力释放ATP，然后在早期CNS发育过程中发挥重要的自分泌，旁分泌信号作用。虽然已经提出了成熟星形胶质细胞中神经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一无所知。因此，该资助申请的目标是评估星形胶质细胞发育的三种推定的递质释放途径是否以及在哪个阶段具有功能能力（胞吐/分泌和通过离子型P2 XR和连接蛋白半通道的扩散），并确定它们对钙振荡和细胞迁移的贡献。免疫细胞化学，生物化学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CNS发育和修复过程中持续的信号传导机制至关重要。

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.