Regulation of alcohol intake by purinergic P2X4 receptors

描述（由申请人提供）：酒精使用障碍（AUDs）具有惊人的社会经济影响，但目前的治疗策略在很大程度上是不充分的，部分原因是缺乏关于乙醇作用的分子靶点的信息。目前的提案验证了P2X4受体（P2X4Rs）代表开发预防和/或治疗AUDs药物的新靶点的假设。这个目标还没有被探索过。我们已经组建了一个多学科的研究团队，他们将共同使用最先进的基因、分子、化学和行为技术，系统地测试中脑边缘多巴胺（DA）系统中的靶点，这些靶点将适用于药物开发。拟议的研究将把实验室发现转化为发现和开发预防和治疗AUDs的新疗法的机会。Aim 1研究将检验大脑多巴胺奖励系统中的P2X4Rs调节乙醇摄入的假设。研究1，我们将评估p2rx4基因组成敲除（P2X4KO）对酒精摄入量的影响（24小时接触和夜间饮酒[DID]测试）。研究2，利用慢病毒介导的短发夹RNA （shRNA）靶向递送，我们将敲低伏隔核（NAc）和/或腹侧被盖区（VTA）中的P2X4R表达，以深入了解中边缘DA系统对P2X4R减少/阻断的短期影响的具体贡献。这项工作还将有助于阐明P2X4Rs在引起或调节乙醇效应中所起的作用，而不会引起可能伴随构成性KOs的潜在发育改变。我们将使用一种新的选择性P2X4R拮抗剂5-BDBD并开发新的相关探针，作为确定P2X4Rs在中毒中的作用的第三种方法。成功的药理学操作P2X4Rs将为P2X4Rs作为未来AUDs治疗的可行靶点的重要性提供新的见解。研究3：利用分子生物学、脑切片电生理学和快速扫描循环伏安法研究P2X4Rs在中脑边缘DA系统NAc和VTA中的作用。这项工作将提供关于P2X4Rs在中脑边缘DA系统中的机制见解，以及它们如何相互作用来调节小鼠的乙醇摄入和行为（研究1和2）。目的2研究将使用P2X4Rs阳性调节剂伊维菌素（IVM）与P2X4KO小鼠联合使用，以验证刺激P2X4Rs会减少乙醇摄入量的假设。此外，我们将设计、合成和测试对P2X4R具有阳性、阴性或无活性的新化学实体，这些实体可以作为药理学探针来询问P2X4R系统。总之，这项工作将进一步推动我们确定神经化学机制和控制酒精摄入的关键大脑区域的长期目标。此外，这项工作将确定新的靶点，并将开始开发新的化合物，用于治疗药物的转化开发，以预防或治疗AUDs。

DESCRIPTION (provided by applicant): Alcohol use disorders (AUDs) have a staggering socio-economic impact, yet current therapeutic strategies are largely inadequate, due, in part, to a lack of information regarding the molecular target(s) on which ethanol acts. The current proposal tests the hypothesis that P2X4 receptors (P2X4Rs) represent a novel target for the development of drugs to prevent and/or treat AUDs. This target has not yet been explored. We have assembled a multidisciplinary team of investigators that together will use state of the art genetic, molecular, chemical and behavioral techniques to systematically test for targets in the mesolimbic dopamine (DA) system that will be amenable for drug development. The proposed studies will translate laboratory findings into opportunities to discover and develop novel therapeutics for the prevention and treatment of AUDs. Aim 1 studies will test the hypothesis that P2X4Rs within the brain dopamine reward systems regulate ethanol intake. Study 1, we will evaluate the effects of constitutive knockout of the p2rx4 gene (P2X4KO) on alcohol intake (24 hr access and drinking in the dark [DID] tests). Study 2, using targeted delivery of lentiviral-mediated short hairpin RNA (shRNA), we will knockdown P2X4R expression in the nucleus accumbens (NAc) and/or ventral tegmental area (VTA) to gain insights into specific contributions of the mesolimbic DA system regarding short- term effects of P2X4R reduction/blockade. This work will also help clarify the role of P2X4Rs in causing or modulating the effects of ethanol without the potential developmental alterations that may accompany constitutive KOs. We will use a new, selective P2X4R antagonist, 5-BDBD and develop new related probes, as a third method to determine P2X4Rs role in intoxication. Successful pharmacological manipulation of P2X4Rs would provide new insights into the importance of P2X4Rs as a viable target for future therapies for AUDs. Study 3, using molecular biological, brain slice electrophysiology and fast scan cyclic voltammetry we will begin to investigate the role of P2X4Rs in the NAc and VTA of the mesolimbic DA system. This work will provide mechanistic insights regarding P2X4Rs in the mesolimbic DA system and how they interact to modulate ethanol intake and behavior in mice (Studies 1 and 2). Aim 2 studies will use ivermectin (IVM), a positive modulator of P2X4Rs, in combination with P2X4KO mice, to test the hypothesis that stimulation of P2X4Rs will decrease ethanol intake. In addition, we will design, synthesize and test new chemical entities with positive, negative or null activity on P2X4R that can serve as pharmacologic probes to interrogate the P2X4R system. Taken together, the work will further our long term goal of identifying neurochemical mechanisms and key brain regions that control alcohol intake. Moreover, this work will identify new targets and will begin to develo new compounds for the translational development of therapeutic agents to prevent and or treat AUDs.