Presynaptic Regulation of C.elegans Dopamine Transporter

描述（由申请人提供）：多巴胺（DA）是一种神经递质，在整个系统发育过程中用于调节控制运动，注意力，奖励和认知的回路。男性DA信号的改变与帕金森病、注意力缺陷多动障碍（ADHD）、成瘾和精神分裂症有关。DA信号是由突触前DA转运蛋白（DAT）严格控制的，它是上瘾性精神兴奋剂（如可卡因和安非他明（AMPH））以及用于治疗ADHD的药物的主要靶点。我们最近在ADHD和双相情感障碍受试者中发现了人类DAT的多个功能性等位基因，这为我们破译控制DA释放和失活的突触前机制增加了翻译意义。迄今为止，这些机制在很大程度上是通过药理学和基因操作来研究的，这些基因是在20年前或更早以前发现的。在目前的应用中，我们利用强大的秀丽隐杆线虫模型系统，利用强大的遗传方法来识别和表征DA信号的新型突触前调节因子。在过去的15年里，Blakely实验室开发了秀丽隐杆线虫模型的技能，重点关注DAT和DA信号，开始于秀丽隐杆线虫DAT基因（T23G5.5, DAT -1）的鉴定。目前的努力源于我们发现了一种简单的DA和dat-1依赖表型，称为游泳诱导瘫痪（SWIP）。野生型动物在~1赫兹的水中捶打长达30分钟，而dat-1 (ok157) （dat-缺陷）动物在3-5分钟内瘫痪。这种表型依赖于DA合成、囊状DA包装和DA释放，并被DAT活性所克服。我们要求支持我们的筛选，以确定和表征突触前调节DA释放和再摄取的基因，定位它们对DA信号传导的贡献模式，评估它们对AMPH作用的影响，并启动保守的脊椎动物同源物的分析。总之，我们的努力提供了一个机会来识别新的和保守的DA信号调节因子，这在脊椎动物模型中是非常难以阐明的。

DESCRIPTION (provided by applicant): Dopamine (DA) is a neurotransmitter used throughout phylogeny to modulate circuits controlling movement, attention, reward and cognition. Alterations in DA signaling in man have been implicated in Parkinson's Disease, Attention-Deficit Hyperactivity Disorder (ADHD), addiction and schizophrenia. DA signaling is tightly controlled by a presynaptic, DA transporter (DAT) that is a major target for addictive psychostimulants such as cocaine and amphetamine (AMPH), as well as agents used in the treatment of ADHD. Our recent identification of multiple, functional alleles of human DAT in ADHD and Bipolar disorder subjects adds translational significance to our efforts to decipher the presynaptic mechanisms that control DA release and inactivation. To date, these mechanisms have largely been studies through pharmacological and genetic manipulation of genes identified two decades or more ago. In the current application, we capitalize on a robust, forward genetic approach to identify and characterize novel presynaptic regulators of DA signaling using the powerful model system Caenorhabditis elegans. Over the past 15 years, the Blakely lab has developed skills in the C. elegans model with a focus on DAT and DA signaling, initiated by the identification of the C. elegans DAT gene (T23G5.5, dat-1). The present effort arises from our discovery of a simple DA and dat-1 dependent phenotype termed Swimming-Induced Paralysis (SWIP). Whereas wildtype animals thrash in water at ~1 Hz for up to 30 minutes, dat-1 (ok157) (DAT-deficient) animals paralyze in 3-5 minutes. This phenotype is dependent on DA synthesis, vesicular DA packaging, and DA release and is overcome by DAT activity. We request support to advance our screen to identify and characterize genes acting presynaptically to regulate DA release and reuptake, localize their mode of contribution to DA signaling, assess their impact on the actions of AMPH, and initiate an analysis of conserved vertebrate homologs. Together our efforts provide an opportunity to identify novel and conserved regulators of DA signaling that would be extremely difficult to elucidate in vertebrate models. PUBLIC HEALTH RELEVANCE: DAT proteins are major presynaptic regulators of DA signaling and are targets of therapeutic and addictive psychostimulants. To identify novel presynaptic mechanisms that control DA release and reuptake, we implement a novel, forward genetic approach in the model system C. elegans. As all known regulators of DA signaling in mammals are conserved in C. elegans, our efforts have a high probability of yielding fundamental insights that underlie potential contributors to human, DA-dependent disorders as well as novel targets for medication development.