Forward Genetics and the Presynaptic Dopamine Transporter

描述（由申请人提供）：本研究的目的是利用线虫中最近发现的多巴胺（DA）转运体（DAT）依赖表型，称为游泳诱导瘫痪（SWIP），以确定突触前神经末梢可卡因和安非他明敏感DAT蛋白的定位和调节。在脊椎动物中，DA调节涉及运动、认知和奖励的多种行为过程。通过DAT的再摄取是DA信号终止的主要机制，是突触前DA稳态的关键决定因素。人类DAT的多态性与ADHD、精神分裂症和药物成瘾有关，并且DAT是可卡因和安非他明的主要靶点。异源表达研究表明，dat是通过辅助蛋白和激酶连接的信号通路调节的，最终控制转运蛋白的定位和活性。此外，与PKC、CaMKII、PI3K和Akt相关的信号网络与安非他明和可卡因的突触前作用有关，揭示了成瘾风险和治疗发展的许多潜在新靶点。哺乳动物中枢神经系统中DAT调控研究提出的技术挑战促使我们的实验室对秀丽隐杆线虫中DAT相关表型进行表征，在那里可以实现DAT调节剂的正向遗传筛选。之前，我们的实验室发现秀丽隐杆线虫的DAT （DAT-1）与哺乳动物的DAT相同43%，只在DA神经元和终末表达，优先运输DA，并且对可卡因和安非他明都敏感。在博士后NRSA的支持下，我确定了一种表型，称为游泳诱发性麻痹（SWIP），它是功能性DAT-1表达的报告因子。因此，在dat-1敲除或可卡因/安非他明处理的动物中，当DA合成、释放和通过DA受体dop3的突触后信号传导被排除时，SWIP被逆转。基于这些数据，我启动了一项试点前向基因筛选，以鉴定da依赖性SWIP突变体，并确定了两种具有新型dat-1点突变的品系，这些突变在体外和体内引起生物合成、运输和功能缺陷，以及几种突变似乎位于其他基因的品系。在此基础上，我提出了以下具体目标：1)扩大对DA依赖性SWIP表型的筛选，通过多巴胺受体缺陷互补测试，对可卡因/安非他明的反应性和胚胎培养的DA转运试验来验证DAT缺陷；2)使用基于illumina的高通量cDNA测序和转录组分析来鉴定非DAT SWIP产生基因，然后基于生物信息学对人类同源物进行阐明。这些研究为深入了解突触前机制控制dat依赖的DA信号提供了一个强大而独特的机会。公共卫生相关性：多巴胺能（DA）神经传递的改变主要涉及精神兴奋剂反应和成瘾。突触前DA转运体（DAT）是终止DA信号的主要模式，是可卡因和安非他明的直接靶点。本研究利用一个强大的遗传模型系统来阐明调节DA信号和DAT活动的基因，并有机会确定成瘾风险和/或治疗的新靶点。

DESCRIPTION (provided by applicant): The goal of this study is to capitalize on a recently identified dopamine (DA) transporter (DAT)-dependent phenotype in the nematode C. elegans, termed Swimming-Induced Paralysis (SWIP), to identify proteins involved in the localization and regulation of cocaine and amphetamine-sensitive DAT proteins at presynaptic nerve terminals. In vertebrates, DA modulates multiple behavioral processes involved with locomotion, cognition, and reward. Reuptake through DAT is the primary mechanism by which DA signaling is terminated and is a critical determinant of presynaptic DA homeostasis. Polymorphisms in human DAT have been associated with ADHD, schizophrenia, and drug addiction, and DAT is a major target of cocaine and amphetamine. Heterologous expression studies indicate that DATs are regulated via accessory proteins and kinase-linked signaling pathways that ultimately control transporter localization and activity. Moreover, signaling networks linked to PKC, CaMKII, PI3K, and Akt have been linked to the presynaptic actions of amphetamine and cocaine, revealing many potential new targets for addiction risk and therapeutic development. The technical challenges presented by the study of DAT regulation in the mammalian CNS have encouraged our laboratory to pursue a characterization of DAT-associated phenotypes in C. elegans, where forward genetic screens for DAT modulators are attainable. Previously, our laboratory showed that the C. elegans DAT (DAT-1) is 43% identical to mammalian DATs, is expressed exclusively in DA neurons and terminals, preferentially transports DA, and is sensitive to both cocaine and amphetamine. Under the support of a postdoctoral NRSA, I identified a phenotype, termed Swimming Induced Paralysis (SWIP) that is a reporter of functional DAT-1 expression. Thus, SWIP in dat-1 knockout or cocaine/amphetamine treated animals is reversed when DA synthesis, release, and post-synaptic signaling through the DA receptor DOP-3 are precluded. Based on these data, I launched a pilot forward genetic screen to identify DA-dependent SWIP mutants and have identified two lines bearing novel dat-1 point mutations that cause biosynthetic, trafficking and functional defects in vitro and in vivo as well as several lines where mutations appear to lie in other genes. Building on this effort, I propose the following Specific Aims: 1) To expand the screen for DA-dependent SWIP phenotypes, validating DAT deficiency via dopamine receptor deficiency complementation tests, responsivity to cocaine/amphetamine and DA transport assays on embryonic cultures and 2) To identify non-DAT, SWIP-generating genes using Illumina-based, high-throughput cDNA sequencing with transcriptome profiling, followed by a bioinformatics based elucidation of human homologs. These studies provide a powerful and unique opportunity for insight into presynaptic mechanisms controlling DAT-dependent DA signaling. PUBLIC HEALTH RELEVANCE: Alterations in dopaminergic (DA) neurotransmission are centrally involved in psychostimulant response and addiction. The presynaptic DA transporter (DAT) is the primary mode by which DA signaling is terminated and is a direct target for cocaine and amphetamine. This research engages a powerful genetic model system to elucidate genes that regulate DA signaling and DAT activity with opportunities to identify novel targets for addiction risk and/or treatment.