A genetically-encoded sensor for imaging extracellular ATP onto astrocytes and other cells

项目描述 星形胶质细胞遍布哺乳动物大脑，并在空间和空间上相互作用 与神经元、血管和其他神经胶质细胞发挥功能。它们服务于多种稳态 功能并参与突触的形成、去除和调节。一个长期存在和 主要的悬而未决的问题涉及星形胶质细胞如何与神经元等其他细胞进行通信， 小胶质细胞和星形胶质细胞。从这个角度来看，很多注意力都集中在细胞外 ATP，由神经元、星形胶质细胞和多种其他细胞释放 机制。一旦释放，ATP 就会激活一系列离子型和代谢型 ATP 受体，其降解产物激活腺苷受体。然而，事实证明 直接测量细胞外 ATP 水平和我们的大部分知识极具挑战性 大脑中的 ATP 信号传导基于药理学和遗传干预 靶向 ATP 受体。因此，ATP 的释放、浓度、动态和扩散 活体脑组织几乎未被探索过，尽管事实上它广泛涉及 星形胶质细胞-神经胶质细胞和星形胶质细胞-神经元相互作用。得益于设计的重大进步 并使用基因编码的谷氨酸传感器，我们着手设计和表征 细胞外 ATP 的基因编码传感器。在此应用程序的初步数据中，我们 报告基于强度的 ATP 感应荧光报告基因 (iATPSnFR)。 iATPSnFR 是 表达于细胞表面，并对预期的细胞外 ATP 浓度做出反应 适当连接的循环排列超级文件夹的荧光强度增加 GFP（cpSFGFP）。 iATPSnFR 可以通过基因靶向特定细胞类型并使用 标准落射荧光和共焦显微镜。 iATPsNFR 的使用将揭示 细胞释放 ATP 并揭示星形胶质细胞在 生理和病理生理过程。我们预计 iATPSnFR 将允许 首次直接测量和跟踪细胞外 ATP 动态。虽然我们 专注于星形胶质细胞，iATPsNFR 可应用于任何细胞类型。在这个提案中我们有两个 我们寻求制定新方法并提供重要的、新的、 星形胶质细胞和 ATP 信号传导研究广泛适用且急需的资源。

Project description Astrocytes are found throughout the mammalian brain and interact spatially and functionally with neurons, blood vessels and other glia. They serve multiple homeostatic functions and are involved in synapse formation, removal and regulation. One long standing and major open question concerns how astrocytes communicate with other cells such as neurons, microglia and astrocytes. From this perspective, much attention has focussed on extracellular ATP, which is released from neurons, astrocytes and multiple other cells by several mechanisms. Once released, ATP activates a family of ionotropic and metabotropic ATP receptors, and its degradation product activates adenosine receptors. However, it has proven extremely challenging to measure extracellular ATP levels directly and much of our knowledge about ATP signaling in the brain is based on pharmacological and genetic interventions targeting ATP receptors. Hence, the release, concentration, dynamics and spread of ATP in living brain tissue has hardly been explored, despite the fact that it is implicated widely in astrocyte-glial and astrocyte-neuron interactions. Buoyed by significant advances in the design and use of genetically-encoded glutamate sensors, we set out to design and characterise a genetically-encoded sensor for extracellular ATP. In the preliminary data of this application we report an intensity-based ATP-sensing fluorescent reporter (iATPSnFR). iATPSnFR is expressed on cell surfaces and responds to expected extracellular ATP concentrations with an increase in fluorescence intensity of an appropriately attached circularly permuted super folder GFP (cpSFGFP). iATPSnFR can be genetically targeted to specific cell types and imaged with standard epifluoresence and confocal microscopes. The use of iATPsNFR will shed light on the cells releasing ATP and reveal when, where and how astrocytes receive ATP signals during physiological and pathophysiological processes. We expect that iATPSnFR will permit the measurement and tracking of extracellular ATP dynamics directly for the first time. Although we focus on astrocytes, iATPsNFR can be applied to any cell type. In this proposal we have two specific aims with which we seek to develop our new approach and provide important, new, broadly applicable and much needed resources for astrocyte and ATP signalling research.