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.
To understand astrocyte-neuron interactions and their impact on brain disorders we need to record the activity of chemicals that allow these cells to communicate in a manner that is specific to each cell type. Here, we propose to develop generally applicable reagents to exploit a genetically-encoded sensor of extracellular cell surface ATP that we have made, which will allow us to image defined cells in mice and thus explore ATP signaling in a non-invasive and cell specific manner. We will openly share this tool with the neuroscience community. The tool will have a major impact on brain disorder research, especially related to astrocytes.
