Regulation of Alcohol Intake by Purinergic P2X4 Receptors

我们已经鉴定了P2 X4受体（P2 X4 Rs）作为药物开发的靶点，以预防和/或治疗 酒精使用障碍（AUD）这一假设来自于令人信服的系统;遗传学，药理学 和行为证据报告乙醇（EtOH）摄入量和P2 X4 R活性之间的反比关系。 支持这一假设，我们发现p2 rx 4敲除小鼠（P2 X4 KO）消耗的EtOH显著增加， 与野生型（WT）同窝对照相比。我们和其他人发现伊维菌素（IVM），一种正变构调节剂， 在P2 X4 Rs的受体，显著拮抗EtOH对ATP门控P2 X4 Rs的抑制，并减少EtOH的摄入。 小鼠和大鼠。我们已经组建了一个多学科团队，他们将共同使用基因，分子， 电生理学、化学和行为技术，系统地探索中脑边缘系统中的靶点， 多巴胺（DA）系统，将适合药物开发。拟议的研究将转化为 实验室研究结果转化为发现和开发新的预防和治疗方法的机会 治疗AUD。目的1研究将检验DA奖赏系统中的P2 X4 Rs调节 乙醇摄入量。我们将使用：用途：结合P2 X4 R调节的体内微透析（研究1）和慢病毒- 介导的短发夹RNA（shRNA）敲低P2 X4 R在核内的表达，和/或 腹侧被盖区（VTA）（研究2和3），以深入了解P2 X4 R在脑内的具体贡献。 中脑边缘DA系统与乙醇的关系雄性和雌性P2 X4 KO和/或WT小鼠将使用两种 饮用模式：（1）24小时访问，两瓶选择（自由选择）和（2）在黑暗中饮用（DID;狂欢 EtOH饮用）。目标2研究将使用脑切片电生理学和敲除/敲除的组合， 向下技术来测试相关的假设，我们预测，在P2 X4 R 减少，P2 X介导的DA神经元放电抑制和EtOH调节的嘌呤能抑制将被 减少或消除。这项工作将提供关于P2 X4 Rs和EtOH相互作用的关键信息， 以及它如何影响中脑边缘DA神经元的嘌呤能和EtOH反应。Aim 3研究将测试 假设IVM可以用作鉴定和开发新化合物的平台， 调节P2 X4 Rs并减少EtOH摄入。这一目标将通过设计和合成一种 一系列半合成阿维菌素和米利倍霉素类似物，然后进行体外和体内评价。 通过这种结合合成和生物学评价的迭代过程，我们将鉴定出 具有最大的抗酒精作用同时使IVM样毒性最小化（即，增加治疗指数）， 产生用于治疗AUD的进一步评估的候选者。总之，这些研究将进一步推动我们长期的- 长期目标是确定关键大脑奖励区域内的神经化学机制， 乙醇摄入量。此外，这项工作将为未来的翻译研究奠定基础， 药物治疗AUD。

We have identified P2X4 receptors (P2X4Rs) as a target for the development of drugs to prevent and/or treat alcohol use disorder (AUD). This hypothesis is derived from compelling systems; genetic, pharmacological and behavioral evidence reporting an inverse relationship between ethanol (EtOH) intake and P2X4R activity. Supporting this hypothesis we found that p2rx4 knock-out mice (P2X4 KO) consumed significantly more EtOH than wildtype (WT) littermate controls. We and others found that ivermectin (IVM), a positive allosteric modula- tor of P2X4Rs, significantly antagonized EtOH inhibition of ATP-gated P2X4Rs and reduced EtOH intake in mice and rats. We have assembled a multidisciplinary team that together will use genetic, molecular, electrophysiological, chemical and behavioral techniques to systematically explore 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 AUD. Aim 1 studies will test the hypothesis that P2X4Rs within the DA reward system regulate EtOH intake. We will use: in vivo microdialysis coupled with P2X4R modulation (Study 1) and lentiviral- mediated short hairpin RNA (shRNA) knockdown P2X4R expression in the nucleus accumbens (NAc) and/or ventral tegmental area (VTA) (Studies 2 & 3) to gain insights into specific contributions of P2X4Rs in the mesolimbic DA system to EtOH drinking. Male and female P2X4KO and/or WT mice will be tested using two drinking paradigms: (1) 24 hr access, two-bottle choice (free choice) and (2) drinking in the dark (DID; binge EtOH drinking). Aim 2 studies will use a combination of brain slice electrophysiological and knock-out/knock- down technologies to test interrelated hypotheses where we predict that under conditions in which P2X4Rs are reduced, P2X-mediated inhibition of DA neuron firing and EtOH regulation of purinergic inhibition will be reduced or eliminated. This work will provide key information regarding the interaction of P2X4Rs and EtOH, and how it may affect both purinergic and EtOH responses of mesolimbic DA neurons. Aim 3 studies will test the hypothesis that IVM can be used as a platform to identify and develop new compounds that positively modulate P2X4Rs and decrease EtOH-intake. This Aim will be accomplished by designing and synthesizing a series of semisynthetic avermectin and milibemycin analogs followed by their in vitro and in vivo evaluation. Through this iterative process combining synthetic and biological evaluation, we will identify molecules that have maximal anti-alcohol effects while minimizing the IVM-like toxicities (i.e., increased therapeutic index) to yield candidates for further assessment for treating AUD. Taken together, these studies will further our long- term goal of identifying neurochemical mechanisms within key brain reward regions important for regulating EtOH intake. Moreover, this work will set the stage for future translational studies to develop novel pharmacotherapies for AUD.