Role of glia in ethanol tolerance in Drosophila

描述（由申请人提供）：神经胶质细胞对大脑的正常功能至关重要，与神经元广泛沟通，并受到乙醇暴露的影响，但它们在酒精使用障碍发展中的作用知之甚少。本研究的目的是研究胶质细胞对果蝇乙醇行为反应的影响。乙醇暴露基因调控的微阵列研究加上携带乙醇应答基因突变的果蝇基因筛选，鉴定出乙醇耐受所需的新基因。对其中之一的a激酶锚定蛋白Akap200的初步研究表明，它只局限于成年果蝇的神经胶质细胞，并且在神经胶质细胞中需要它的功能来促进乙醇耐受性的发展。AKAP蛋白在空间和时间上协调蛋白激酶A信号通路与其他细胞信号通路，包括蛋白激酶C，我们发现蛋白激酶A和蛋白激酶C，如Akap200，都可以促进胶质细胞的乙醇耐受性。此外，我们发现Akap200显著表达的神经胶质血脑屏障通过改变其形态和功能特性对乙醇暴露做出反应，而Akap200突变体的屏障功能部分受损。我们提出神经胶质细胞通过由Akap200协调的保守信号通路调节乙醇耐受性。我们提出的研究有三个主要目标。首先，我们计划通过确定乙醇对神经胶质结构/功能的时间和剂量依赖性影响，以及神经胶质细胞过程在乙醇行为反应发展中所起的作用，来表征每一类中枢神经胶质细胞对乙醇暴露的反应。其次，我们计划表征Akap200和Akap200相互作用蛋白在乙醇耐受性和胶质细胞结构/功能发展中的作用，以建立胶质细胞调节乙醇行为反应的机制。第三，我们计划对调节乙醇行为反应的新型神经胶质基因进行基因筛选。最近，针对特定神经胶质类型的基因靶向工具的可用性，限制了成人的基因操作，以及评估神经胶质结构和功能的方法，再加上我们发现的神经胶质蛋白激酶a信号通路对乙醇耐受性的影响，使我们有机会在理解神经胶质对乙醇行为反应的贡献方面取得快速进展。蛋白激酶A信号和许多神经胶质特性的进化保护表明，在果蝇身上学到的东西可以转移到哺乳动物系统中，并可能影响人类酒精使用障碍的治疗。

DESCRIPTION (provided by applicant): Glial cells are crucial for the normal functioning of the brain, communicate extensively with neurons, and are affected by ethanol exposure, yet their role in the development of alcohol use disorders is poorly understood. The goal of this proposal is to study the contribution of glia to ethanol behavioral responses in the fruit fly Drosophila. A microarray study of gene regulation by ethanol exposure coupled with a genetic screen of flies carrying mutations in ethanol-responsive genes led to the identification of novel genes required for ethanol tolerance. Preliminary studies of one of these, the A-kinase anchoring protein Akap200, indicate that it is exclusively localized to glia in adult flies and that its function is required in glia to promote the development of ethanol tolerance. AKAP proteins spatially and temporally coordinate protein kinase A signaling with other cell signaling pathways, including protein kinase C, and we found that both protein kinase A and protein kinase C, like Akap200, can promote ethanol tolerance in glia. Additionally, we found that the glial blood-brain barrier, where Akap200 is prominently expressed, responds to ethanol exposure by changing its morphological and functional properties, and that barrier function is partially compromised in Akap200 mutants. We propose that glia regulate ethanol tolerance through conserved signaling pathways that are coordinated by Akap200. There are three main goals of our proposed research. First, we plan to characterize the response of each class of central nervous system glia to ethanol exposure by determining the time and dose-dependent effects of ethanol on glial structure/function, and the role played by glial cellular processes in the development of ethanol behavioral responses. Second, we plan to characterize the role of Akap200 and Akap200-interacting proteins in the development of ethanol tolerance and glial structure/function in order to establish the mechanisms by which glia regulate ethanol behavioral responses. Third, we plan to carry out a genetic screen for novel glial genes that regulate ethanol behavioral responses. The recent availability of tools for genetically targeting specific glial types, for limiting genetic manipulations to adults, and methodology for assessing glial structure and function, coupled with our discovery of a glial protein kinase A signaling pathway for ethanol tolerance, has given us an opportunity to make rapid progress in understanding the contributions of glia to ethanol behavioral responses. Evolutionary conservation of protein kinase A signaling and many glial properties suggests that what is learned in Drosophila can transfer to mammalian systems and may impact the treatment of alcohol use disorders in humans. PUBLIC HEALTH RELEVANCE: Glial cells are critically important for the normal functioning of the brain and they show complex and dynamic responses to ethanol, but their role in the development of alcohol use disorders remains to be characterized. We found that glia in the fruit fly Drosophila alter their shape and function in response to ethanol, and that evolutionarily conserved signal transduction pathways act in fly glia to regulate ethanol tolerance. Sophisticated genetic tools, a high level of molecular conservation, and similar glial properties make Drosophila an ideal system for uncovering molecular mechanisms that can provide novel insights in vertebrates, including humans.