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，在整个发育过程中用于调节控制运动、注意力、奖励和认知的回路。人类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信号传导受到突触前DA转运蛋白（DAT）的严格控制，DAT是成瘾性精神兴奋剂（如可卡因和苯丙胺（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鉴定出人类DAT的多个功能等位基因，这为我们破译控制DA释放和失活的突触前机制的努力增加了翻译意义。到目前为止，这些机制主要是通过药理学和遗传操作的基因二十年前或更长时间前确定的研究。在当前的应用中，我们利用一个强大的，向前的遗传学方法来识别和表征新的突触前调节DA信号使用强大的模型系统秀丽隐杆线虫。在过去的15年里，布莱克利实验室发展了C。elegans模型，重点是DAT和DA信号转导，由C.线虫DAT基因（T23G5.5，dat-1）。目前的努力源于我们发现了一种简单的DA和dat-1依赖性表型，称为游泳诱导的瘫痪（SWIP）。野生型动物在水中以约1 Hz的频率甩动长达30分钟，而dat-1（ok 157）（DAT缺陷）动物在3-5分钟内瘫痪。这种表型依赖于DA合成、囊泡DA包装和DA释放，并被DAT活性克服。我们请求支持，以推进我们的屏幕，以确定和表征基因突触前调节DA的释放和再摄取，本地化其模式的DA信号的贡献，评估其对AMPH的行动的影响，并启动保守的脊椎动物同源物的分析。我们的共同努力提供了一个机会，以确定新的和保守的调节DA信号，这将是非常难以阐明的脊椎动物模型。

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.