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

三磷酸腺苷是一种普遍存在的信号分子。除了细胞内功能外，它还充当自分泌和旁分泌信使以及神经递质。它可以通过各种特定的机制或从受损的组织中非特异性地释放出来，作用于两种类型的受体，即G蛋白偶联的P2Y受体和形成P2X受体的离子通道。P2X受体是一种同源或异源三聚体阳离子通道，具有很高的钙通透性。已知的7个P2X亚基(P2X1-P2X7)广泛存在于哺乳动物组织中，是新药开发的重要靶点。在动物模型中，P2X7亚单位基因的缺失会导致细胞因子产生缺陷，并消除神经病理性和炎症性疼痛。在神经退行性疾病中，通常可以观察到P2X7受体表达上调，而在神经退行性疾病模型中，P2X7受体拮抗剂已被证明可以改善组织损伤。遗传学研究表明，P2X7受体在情感障碍中发挥了作用。然而，由于缺乏稳定和选择性的配体和特异性抗体，其在天然组织制剂中的研究一直具有挑战性。因此，尽管它们作为药物靶点很重要，但它们的精确定位、生理功能和在病理生理条件下的调节仍然知之甚少。特别是，P2X7受体在神经元和不同胶质细胞中的位置和功能以及它们与其他蛋白质的相互作用仍然是一个持续争论的问题。为了提供工具，允许对这种受体进行更具体的研究，我们建立了两个细菌人工染色体(BAC)转基因小鼠模型。在这些小鼠中，1)GFP标记的P2X7受体或2)可溶性RFP报告在P2X7调控元件的控制下过表达，以允许可视化表达模式和这些受体在特定细胞中的定位。此外，用于产生可利用loxP/Cre系统有条件地表达可溶性报告基因和/或标记的P2X7受体的小鼠株系的BAC构建已经产生，并准备用于原核注射。所有这些BAC结构都可以很容易地进行修饰，以获得P2X7受体的功能突变或功能缺失，并提供一个能够回答该领域主要悬而未决的问题的模块化系统。在这个项目中，我们的目标是：1)确定P2X7受体在中枢神经系统中的精确细胞和亚细胞定位；2)确定P2X7受体的蛋白质-蛋白质相互作用；3)研究两种脱髓鞘疾病模型(视神经脊髓炎和夏科-玛丽-牙病)中P2X7受体的表达；4)通过适度过度表达和/或过度表达功能突变体，研究P2X7受体在中枢神经系统中的生理和病理生理作用。

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