Mechanisms of ATP Release from Glial Progenitors

描述(申请人提供)：神经胶质细胞的核苷酸释放在中枢神经系统和三叉神经节中起着重要的自分泌和旁分泌信号作用。最近的研究表明，神经胶质细胞对ATP的非裂解释放可能涉及调控的胞吐作用，也可能是通过核苷酸通透通道的外流。细胞外ATP作用于质膜亲离子型和代谢型P2X受体，调节神经元活性，诱导星形胶质细胞内钙瞬变，并产生关键的细胞内第二信使，例如参与脑创伤后胶质细胞的形成和神经干细胞的增殖和迁移。最近，我们获得了神经前体细胞表现出自发的钙振荡的证据，这种振荡高度依赖于P2Rs的激活。沐浴应用嘌呤能受体拮抗剂或ATP降解酶apyrase可防止自发性钙波动的发生。此外，阻断P2Rs降低了这些祖细胞的增殖和迁移率。这些结果表明，神经前体细胞具有释放三磷酸腺苷的能力，三磷酸腺苷在中枢神经系统发育早期起着关键的自分泌、旁分泌信号作用。虽然成熟的星形胶质细胞已经发现了神经胶质细胞释放递质的途径，但对前体细胞释放三磷酸腺苷的机制(S)的发育方面还知之甚少。因此，这项拨款申请的目的是评估星形胶质细胞发育的三个可能的递质释放途径是否以及在哪个阶段具有功能(胞吐/分泌和通过离子型P2XRs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。将使用免疫细胞化学、生化和药理学方法结合发光和荧光成像来识别ATP释放途径。这种识别对于理解中枢神经系统发育和修复过程中正在进行的信号机制是至关重要的。

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.