Characterization of a novel presynaptic target for ethanol action

描述(申请人提供)：酗酒和酗酒影响4.5%的美国人口，造成约1840亿美元/年的经济负担。开发新的药物疗法来帮助对抗酒精依赖和复发的能力，需要了解酒精对神经系统的作用机制。确定乙醇结合的靶点尤其重要，因为这些靶点可能会带来最完整的治疗效果。虽然酒精对突触前功能有明显而深远的影响，但这种巨大影响背后的机制基本上是未知的。这项提议的长期目标是确定酒精对突触前功能发挥作用的分子机制。这一探索性应用的目的是研究Munc13.1蛋白和乙醇之间的生理和行为相互作用。Munc13.1是一种突触前活动区蛋白，对脑内神经递质的释放至关重要。在秀丽隐杆线虫中，同源的Unc13蛋白负责对挥发性麻醉药的敏感性。Munc13蛋白家族的C1或二酰甘油结合域在结构上与蛋白激酶C相似，蛋白激酶C调节酒精的行为效应，并具有酒精结合位点(S)。初步数据表明，乙醇也会与Munc13.1中的这个C1结构域结合。有待检验的中心假设是，乙醇对神经系统功能的显著影响是由于乙醇与Munc13.1C1域的结合。这项提议的第一个目的将检验这样的假设，即乙醇与Munc13.1的C1域结合会改变这一突触前蛋白的活性。这将通过光标记和质谱学来鉴定酒精结合残基，并通过在膜转位分析中阐明这种结合对Munc13.1活性的影响来完成。第二个目的是确定Dunc13活性的降低如何改变果蝇对酒精的行为和生理反应。这个无脊椎动物模型系统之所以被选中，是因为它能够快速、经济地改变Dunc13的水平，并监测其功能后果。乙醇-Dunc13相互作用的这些后果将通过测量野生型和Dunc13功能动物的乙醇偏好、刺激和镇静来揭示。此外，将使用突触-PHluorin传感器来检测这种相互作用的影响，以成像醉酒和清醒的果蝇特定的GABA能神经元亚群的囊泡释放。这些GABA能神经元是乙醇对果蝇的刺激和镇静作用的关键调节器。这种方法是创新的，因为它首次应用了超敏感的生物化学和强大的神经遗传学技术来剖析乙醇-受体相互作用的功能。这一建议意义重大，因为它有望揭示乙醇对突触前功能影响的新的主要机制。最终，这种理解可能会导致旨在破坏乙醇-unc13.1相互作用的新药，为争取戒酒提供宝贵的武器。 与公共健康相关：尽管与酒精有关的死亡人数巨大(美国每年约75,000-100,000人)，社会成本(美国每年1,840亿美元)，但治疗酒精滥用和成瘾的药物很少。为了开发新的药物和有效的干预，有必要在分子水平上确定酒精作用的靶点和机制。本研究的目的是确定突触前蛋白在调节脑内酒精活动中的作用，本研究的意义在于，它可能允许基于该靶蛋白的新疗法的开发，以对抗酒精成瘾。

DESCRIPTION (provided by applicant): Alcohol abuse and alcoholism affect 4.5% of the United States population causing an economic burden of approximately 184 billion dollars/year. The ability to develop new pharmacotherapies to help fight the descent into alcohol dependence and recidivism requires an understanding of mechanisms of alcohol actions on the nervous system. It is particularly important to define the targets of ethanol binding as these may bring about the most complete therapeutic effect. Although alcohol is known to have distinct and profound effects on presynaptic function, the mechanisms underlying this large impact are essentially unknown. The long-term goal of this proposal is to define the molecular mechanism by which alcohol exerts its action on presynaptic function. The objective of this exploratory application is to examine the physiological and behavioral interactions between Munc13.1 protein and ethanol. Munc13.1 is a presynaptic active zone protein essential for neurotransmitter release in brain. In Caenorhabditis elegans, the homologous Unc13 protein is responsible for the sensitivity to volatile anesthetics. The C1 or diacylglycerol binding domain of the Munc13 family of proteins is structurally similar to that of protein kinase C (PKC) which regulates behavioral effects of alcohol and has alcohol binding site(s). Preliminary data demonstrate that ethanol will also bind to this C1 domain in Munc13.1. The central hypothesis to be tested is that a significant effect of ethanol on nervous system function is due to the binding of ethanol to the Munc13.1 C1 domain. The first aim of this proposal will examine the hypothesis that ethanol binding to the C1 domain of the Munc13.1 modifies the activity of this presynaptic protein. This will be accomplished by photolabeling and mass spectrometry to identify alcohol binding residues, and by elucidating the effects of this binding on Munc13.1 activity in membrane translocation assays. The second aim is to determine how a reduction in Dunc13 activity changes the behavioral and physiological responses to ethanol in Drosophila melanogaster. This invertebrate model system was chosen for its ability to rapidly and economically alter Dunc13 levels and to monitor the functional consequences. These consequences of the ethanol-Dunc13 interaction will be revealed by measuring ethanol preference, stimulation, and sedation in wild type and Dunc13 reduction of function animals. Moreover, the effect of the interaction will be examined using the synapto-pHluorin sensor to image vesicle release from a specific subset of GABAergic neurons in intoxicated and sober flies. These GABAergic neurons are critical modulators of the stimulatory and sedative effects of ethanol in Drosophila. The approach is innovative as it applies the strengths of ultrasensitive biochemistry and powerful neurogenetic techniques for the first time to dissect the function of an ethanol-receptor interaction. This proposal is significant as it is expected to uncover a novel primary mechanism for an effect of ethanol on presynaptic function. Ultimately, this understanding may lead to new drugs designed to disrupt the ethanol-unc13.1 interaction providing a valuable weapon in the fight for sobriety. PUBLIC HEALTH RELEVANCE: Despite the huge number of alcohol related deaths (annually ~75,000-100,000 in the US) and cost to society ($184 billion/yr in the US) very few medications are available for treating alcohol abuse and addiction. To develop new medications and effective intervention it is necessary to define the target and mechanism of alcohol action at the molecular level. The goal of the present study is to establish the role of a presynaptic protein in regulating alcohol actions within the brain and the significance of this study is that it may allow the development of new therapeutics based on this target protein to combat alcohol addiction.