Amphetamine Regulation of DAT Function in c.elegans

描述（由申请人提供）：本项目的长期目标是了解安非他明（AMPH）在体外和体内对多巴胺（DA）转运蛋白（DAT）功能的急性调节。中枢神经系统突触上的DA信号调节多种认知、情感和行为功能。DA系统的异常与许多精神和神经系统疾病有关，包括药物成瘾、精神分裂症和帕金森病。AMPH诱导DA的反向转运，从而增加细胞外DA水平并导致其精神兴奋作用。迄今为止，研究AMPH调节DAT功能的努力在很大程度上依赖于传统的生物化学和/或生物物理方法，通常在异源表达系统中。虽然这些努力已经揭示了许多新的方面AMPH的行动，它一直难以建立一个单一的系统，其中操纵的AMPH调节DAT功能在神经元环境中。本项目将联合收割机分子，生物化学和生物物理学的方法来表征AMPH调节DAT功能在神经元制剂和在体内。在这个建议中，我们将创建一个新的实验平台，研究DAT蛋白质-蛋白质相互作用使用的转基因和实验工具提供的秀丽隐杆线虫。DAT蛋白是如何与SNARE蛋白结合的，这是有待解决的关键问题。elegans syntaxin 1A的同源物）对于AMPH诱导的DA流出和AMPH诱导的行为是必需的。我们的实验计划将AMPH诱导的DAT功能调节与刺激动物行为联系起来。提出的研究解决了以下具体目的：1）创建具有受损的DAT/DAT-64相互作用的转基因蠕虫品系。2)确定DAT/AMPH-64相互作用是否在体内和体外调节DAT-1功能和DAT-1介导的AMPH效应。我们的目标是生成一个基于实验的模型，推进我们在体外和体内对AMPH作用的理解，并发现可能有助于在成瘾和帕金森病等疾病状态中破坏DA信号传导的新途径和分子。公共卫生相关性：多巴胺转运蛋白（DAT）是在质膜上进行DA再摄取过程的蛋白质，是几种精神活性药物（包括安非他明（AMPH）和可卡因）的主要分子靶点。多巴胺能系统中的抑制与许多精神和神经疾病有关，包括药物成瘾、精神分裂症和帕金森病。本提案的主要目标是建立一个体内表达系统，研究DAT相互作用蛋白及其对AMPH诱导的DA外排的影响，目的是阐明这些蛋白如何支持AMPH诱导的动物行为。

DESCRIPTION (provided by applicant): The long-term objective of this project is to develop an understanding of the acute modulation of dopamine (DA) transporter (DAT) function by amphetamine (AMPH) in vitro and in vivo. DA signaling at CNS synapses regulates a variety of cognitive, emotional and behavioral functions. Abnormalities in the DA system have been implicated in a number of psychiatric and neurological disorders, including drug addiction, schizophrenia and Parkinson's disease. AMPH induces reverse transport of DA, thereby increasing extracellular DA levels and leading to its psychostimulant effects. To date, efforts to investigate AMPH regulation of DAT function have largely relied on traditional biochemical and/or biophysical approaches, often in heterologous expression systems. Although these efforts have revealed many novel aspects of AMPH actions, it has been difficult to establish a single system in which to manipulate the AMPH regulation of DAT function within a neuronal environment This project will combine molecular, biochemical and biophysical approaches to characterize AMPH regulation of DAT function both in neuronal preparations and in vivo. In this proposal, we will create a novel experimental platform to study DAT protein-protein interactions using the transgenic and experimental tools offered by Caenorhabditis elegans. The key issues to resolve include how DAT protein associations with the SNARE protein UNC-64 (the C. elegans homolog of syntaxin 1A) are essential for AMPH-induced DA efflux and AMPH-induced behaviors. Our experimental plan links the AMPH-induced functional regulation of DAT to stimulation of animal behaviors. The proposed studies address the following Specific Aims: 1) To create transgenic worm lines with impaired DAT/UNC-64 interaction. 2) To determine whether DAT/UNC-64 interactions regulate DAT-1 function and DAT-1- mediated AMPH effects in vivo and in vitro. The goal is to generate an experimentally-based model advancing our understanding in vitro and in vivo of the AMPH actions and to discover novel pathways and molecules that may contribute to disrupted DA signaling in disease states such as addiction and Parkinson's disease. PUBLIC HEALTH RELEVANCE: The dopamine transporter (DAT), the protein which carries out the DA reuptake process at the plasma membrane, is the major molecular target of several psychoactive drugs, including amphetamine (AMPH) and cocaine. Abnormalities in the dopaminergic system have been implicated in a number of psychiatric and neurological disorders, including drug addiction, schizophrenia and Parkinson's disease. The major goal of this proposal is to create an in vivo expression system to study DAT-interacting proteins and their effects on AMPH-induced DA efflux with the intent of elucidating how these proteins support AMPH-induced behaviors in living animals.