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的大脑，在空间上相互作用， 与神经元、血管和其他神经胶质细胞有功能联系。它们提供多种稳态 参与突触的形成、移除和调节。一个长期存在的， 主要的未决问题涉及星形胶质细胞如何与其他细胞如神经元通信， 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是由神经元、星形胶质细胞和多种其他细胞释放的， 机制等一旦释放，ATP激活亲离子型和亲代谢型ATP家族 受体，其降解产物激活腺苷受体。但已被证实 直接测量细胞外ATP水平极具挑战性， 关于ATP信号在大脑中的作用是基于药理学和遗传学的干预 靶向ATP受体。因此，ATP的释放，浓度，动力学和传播， 活的脑组织几乎没有被探索过，尽管事实上它广泛地牵涉到 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。设计上的重大进步 和使用基因编码的谷氨酸传感器，我们开始设计和制造一种 细胞外ATP的基因编码传感器在本申请的初步数据中， 报道了基于强度的ATP传感荧光报告基因（iATPSnFR）。iATPSnFR是 在细胞表面表达，并响应预期的细胞外ATP浓度， 适当连接的圆形排列的超级折叠体的荧光强度增加 GFP（cpSFGFP）。iATPSnFR可以在遗传上靶向特定的细胞类型， 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何时，何地以及如何接收ATP信号， 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动力学。虽然我们 iATPsNFR专注于星形胶质细胞，可应用于任何细胞类型。在这个提案中，我们有两个 具体目标，我们寻求发展我们的新方法，并提供重要的，新的， 广泛适用和急需的资源星形胶质细胞和ATP信号研究。

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.