The Molecular Mechanisms of the GDNF-mediated Decrease in Alcohol Consumption

描述（由申请人提供）：强迫性饮酒是一种影响其他健康个体的疾病状态。GDNF信号通路在酒精消耗的调节中起作用。这项研究有两个主要目的：1）确定GDNF这种作用的下游效应物和分子机制，2）确定GDNF信号通路是否是酒精消耗负调节的稳态机制的一部分。首先，GDNF信号传导对两个候选蛋白Kv4.3和Synapsin I的磷酸化的作用将被研究。使用体外模型系统，包括永生化细胞系和原代神经元培养物，将用于生化测定蛋白磷酸化的程度。由于神经递质多巴胺在酒精消耗中起着重要作用，因此将研究GDNF以及GDNF介导的这些蛋白质靶点的磷酸化对多巴胺释放的影响。Kv4.3和/或突触蛋白I蛋白的siRNA敲低，沿着高效液相色谱（HPLC）定量多巴胺释放，将用于确定候选效应子是否在GDNF介导的多巴胺释放中起作用。其次，乙醇对GDNF的上调和随后的其信号通路的激活的影响将在体外和体内模型中进一步表征。RT-PCR和生物化学技术将用于支持这一目标。了解酒精消耗对GDNF信号传导的影响，以及GDNF对酒精消耗的影响，将有助于理解酒精成瘾的神经生物学，以及开发有效的治疗方法来缓解酒精的疾病状态。相关性：酗酒是一个广泛而昂贵的社会问题。确定新的分子靶点，可用于开发药物治疗，可以调节酒精消费是一个值得追求。这项研究将有助于了解和治疗这种和其他药物成瘾的现有知识基础。

DESCRIPTION (provided by applicant): Compulsive consumption of alcohol is a disease state that affects otherwise healthy individuals. The GDNF signaling pathway plays a role in the regulation of alcohol consumption. The proposed research has two broad aims: 1) to determine the downstream effectors and molecular mechanisms that underlie this action of GDNF, and 2) to determine whether the GDNF signaling pathway is part of a homeostatic mechanism by which alcohol consumption is negatively regulated. Firstly, the actions of GDNF signaling on the phosphorylation of two candidate proteins, Kv4.3 and Synapsin I, will be investigated. Use of in vitro model systems, including an immortalized cell line and primary neuron cultures, will be used to biochemically determine the extent of protein phosphorylation. Because the neurotransmitter dopamine plays a major role in the consumption of alcohol, the effects of GDNF, as well as the GDNF-mediated phosphorylation of these protein targets, on dopamine release will be investigated. siRNA knockdown of the Kv4.3 and/or Synapsin I proteins, along with high-performance liquid chromatography (HPLC) to quantify dopamine release, will be used to determine whether either candidate effector functions in GDNF-mediated dopamine release. Secondly, the effect of ethanol on the upregulation of GDNF and subsequent activation of its signaling pathway will be further characterized in both in vitro and in vivo models. RT-PCR and biochemical techniques will be used to support this aim. Understanding the effect of alcohol consumption on GDNF signaling, as well as the effects of GDNF on alcohol consumption, will contribute to the understanding of the neurobiology of alcohol addiction, and the development of efficacious therapies to alleviate the alcoholic's disease state. Relevance: Alcoholism is a widespread and costly societal problem. Identifying novel molecular targets that may be used to develop drug therapies that can regulate alcohol consumption is a worthwhile pursuit. This research will contribute to the current knowledge base for the understanding and treatment of this and other drug addictions.