Regulation and function of P2X7 receptors in the central nervous system under physiological and pathophysiological conditions

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ATP is a universal signalling molecule. Besides its intracellular functions, it acts as an autocrine and paracrine messenger as well as a neurotransmitter. It can be released by various specific mechanisms or non-specifically from damaged tissues and acts on two types of receptors, the G-protein coupled P2Y and the ion channel forming P2X receptors. P2X receptors are homo- or heterotrimeric cation channels with a high Ca2+ permeability. The seven known P2X subunits (P2X1-P2X7) are widely distributed in mammalian tissues and represent important targets for the development of novel drugs. Deletion of the P2X7 subunit gene results in defective cytokine production and abolishes neuropathic and inflammatory pain in animal models. Up-regulation of P2X7 receptor expression is generally observed in neurodegenerative diseases and P2X7 receptor antagonists have been shown to ameliorate tissue damage in models of neurodegeneration. Genetic studies indicate a role of the P2X7 receptor in affective disorders. However, due to the lack of stable and selective ligands and specific antibodies its investigation in native tissue preparations has been challenging. Thus, despite their importance as drug targets, their precise localization, physiological functions, and regulation under pathophysiological conditions remain poorly understood. In particular, the location and function of P2X7 receptors in neurons and different glia cells and their interactions with other proteins remain a matter of ongoing debate. To provide tools that allow a more specific investigation of this receptor, we have generated two bacterial artificial chromosome (BAC) transgenic mouse models. In these mice, a 1) GFP-tagged P2X7 receptor or 2) soluble RFP reporter are over-expressed under the control of P2X7 regulatory elements to allow the visualization of the expression pattern and the localization of these receptors in specific cells. In addition, a BAC construct for the generation of mouse lines in which soluble reporters and/or tagged P2X7 receptors can be conditionally expressed using the loxP/Cre system has been generated and is ready for pronucleus injection. All these BAC constructs can be easily modified to obtain gain of function or loss of function mutants of the P2X7 receptor and provide a modular system that is able to answer major unresolved questions in this field. In this project we aim to1) determine the precise cellular and subcellular localization of P2X7 receptors in the central nervous system2) identify protein-protein interactions of the P2X7 receptor3) investigate the P2X7 receptor expression in two models of demyelinating diseases (neuromyelitis optica and Charcot-Marie-Tooth disease)4) study the physiological and pathophysiological roles of the P2X7 receptors in the CNS by its moderate over-expression and/or over-expression of a gain of function P2X7 mutant