Role of glia in ethanol tolerance in Drosophila

描述(申请人提供)：神经胶质细胞对大脑的正常功能至关重要，与神经元进行广泛的交流，并受到酒精暴露的影响，但它们在酒精使用障碍发展中的作用尚不清楚。这项建议的目的是研究果蝇体内神经胶质细胞对酒精行为反应的贡献。一项通过酒精暴露进行基因调控的微阵列研究，以及对携带乙醇反应基因突变的果蝇的遗传筛查，导致了对酒精耐受性所需的新基因的鉴定。对其中之一的A-激酶锚定蛋白Akap200的初步研究表明，它只定位于成年果蝇的神经胶质细胞，它的功能是促进神经胶质细胞对乙醇耐受的发展所必需的。AKAP蛋白在空间和时间上协调蛋白激酶A和其他细胞信号通路，包括蛋白激酶C，我们发现蛋白激酶A和蛋白激酶C，如Akap200，都可以促进神经胶质细胞对乙醇的耐受。此外，我们发现Akap200突出表达的胶质血脑屏障通过改变其形态和功能属性来响应乙醇暴露，并且该屏障功能在Akap200突变体中部分受损。我们认为，神经胶质细胞通过Akap200协调的保守信号通路来调节乙醇耐受性。我们提出的研究有三个主要目标。首先，我们计划通过确定酒精对神经胶质细胞结构/功能的时间和剂量依赖性的影响，以及神经胶质细胞过程在酒精行为反应发展中所起的作用，来表征每类中枢神经系统神经胶质细胞对酒精暴露的反应。其次，我们计划研究Akap200和Akap200相互作用蛋白在酒精耐受性和神经胶质细胞结构/功能发展中的作用，以建立神经胶质细胞调节酒精行为反应的机制。第三，我们计划对调节酒精行为反应的新的神经胶质基因进行遗传筛选。最近针对特定神经胶质细胞类型的基因定位工具的出现，将遗传操作限制在成人中，以及评估神经胶质细胞结构和功能的方法学的出现，再加上我们发现了酒精耐受的神经胶质蛋白激酶A信号通路，使我们有机会在理解神经胶质细胞对酒精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守表明，在果蝇中学到的东西可以转移到哺乳动物系统，并可能影响人类酒精使用障碍的治疗。 公共卫生相关性：神经胶质细胞对大脑的正常运作至关重要，它们对乙醇表现出复杂和动态的反应，但它们在酒精使用障碍发展中的作用仍有待确定。我们发现果蝇体内的神经胶质细胞对乙醇的反应改变了其形态和功能，进化上保守的信号转导通路在果蝇神经胶质细胞中起着调节酒精耐受性的作用。复杂的遗传工具、高度的分子保守和相似的神经胶质特性使果蝇成为揭示分子机制的理想系统，可以为包括人类在内的脊椎动物提供新的见解。

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.