Amphetamine Regulation of DAT Function in c.elegans

描述(由申请人提供)：该项目的长期目标是了解苯丙胺(AMPH)在体外和体内对多巴胺(DA)转运体(DAT)功能的急性调节。DA信号在中枢神经系统突触中调节多种认知、情绪和行为功能。DA系统的异常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氏症。AMPH诱导DA的反向运输，从而增加细胞外DA的水平，从而导致其精神刺激作用。到目前为止，研究amph对DAT功能的调控在很大程度上依赖于传统的生化和/或生物物理方法，通常是在异源表达系统中。尽管这些工作揭示了amph作用的许多新方面，但很难建立一个单一的系统来在神经元环境中操纵DAT功能的amph调节。本项目将结合分子、生化和生物物理方法来表征神经元制备和体内DAT功能的amph调节。在这个计划中，我们将创建一个新的实验平台，利用秀丽线虫提供的转基因和实验工具来研究DAT蛋白-蛋白质之间的相互作用。需要解决的关键问题包括DAT蛋白如何与SNARE蛋白UNC-(线虫Synaxin 1A的同源物)结合在Amph诱导的DA外流和Amph诱导的行为中是必不可少的。我们的实验计划将Amph诱导的DAT的功能调节与刺激动物行为联系起来。本研究的具体目标如下：1)建立DAT/UNC-相互作用受损的转基因蠕虫系。2)研究体内、外DAT/UNC-相互作用对DAT-1功能及DAT-1介导的Amph效应的影响。我们的目标是建立一个基于实验的模型，促进我们在体外和体内对AMPH作用的理解，并发现可能导致成瘾和帕金森病等疾病状态下DA信号中断的新途径和分子。与公共健康相关：多巴胺转运体(DAT)是在质膜上执行DA再摄取过程的蛋白质，是包括苯丙胺(AMPH)和可卡因在内的几种精神活性药物的主要分子靶标。多巴胺能系统的异常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氏症。这项建议的主要目的是建立一个体内表达系统来研究DAT相互作用的蛋白及其对AMPH诱导的DA外流的影响，目的是阐明这些蛋白如何支持AMPH诱导的活体动物行为。

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.