Amphetamine Regulation of DAT Function in c.elegans

安非他明对线虫 DAT 功能的调节

• 批准号: 7589238
• 负责人: Lucia Carvelli
• 金额: $ 23.01万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-15 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7589238
• 关键词: Acute; Address; Affect; Amphetamines; Animal Behavior; Animals; Behavior; Behavioral; Binding Sites; Biochemical; Biochemical Genetics; Caenorhabditis elegans; Cell membrane; Chromosome Pairing; Cleaved cell; Cocaine; Cognitive; Cultured Cells; Disease; Dopamine; Drug Addiction; Emotional; Environment; Future; GABA Transporter 1; Goals; Homologous Gene; In Vitro; Life; Link; Manuscripts; Measures; Mediating; Membrane Proteins; Methodology; Modeling; Molecular; Molecular Target; N-terminal; Neurons; Norepinephrine; Numbers; Parkinson Disease; Pathway interactions; Preparation; Process; Property; Proteins; Proteomics; Psychotropic Drugs; Public Health; Regulation; Reporting; Research; Rewards; Role; SNAP receptor; Schizophrenia; Serotonin; Signal Transduction; Synapses; System; Testing; Transgenic Organisms; UNC-64 protein; addiction; base; calmodulin-dependent protein kinase II; cellular imaging; dopamine system; dopamine transporter; dopaminergic neuron; extracellular; gamma-Aminobutyric Acid; in vivo; mutant; nervous system disorder; novel; protein protein interaction; psychostimulant; reuptake; syntaxin 1A; tool

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.

描述（由申请人提供）：该项目的长期目标是在体外和体内研究安非他明（AMPH）对多巴胺（DA）转运体（DAT）功能的急性调节。中枢神经系统突触中的DA信号调节着多种认知、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，包括吸毒成瘾、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功能的努力主要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。尽管这些努力揭示了AMPH作用的许多新方面，但很难建立一个单一的系统来操纵神经元环境中AMPH对DAT功能的调节。本项目将结合分子、生化和生物物理方法来表征神经元制剂和体内AMPH对DAT功能的调节。在本提案中，我们将利用秀丽隐杆线虫提供的转基因和实验工具创建一个新的实验平台来研究DAT蛋白-蛋白相互作用。需要解决的关键问题包括DAT蛋白与SNARE蛋白UNC-64 （syntaxin 1A的线虫同源物）的关联如何对amph诱导的DA外排和amph诱导的行为至关重要。我们的实验计划将amph诱导的DAT功能调节与动物行为的刺激联系起来。提出的研究有以下具体目的：1)创建DAT/UNC-64相互作用受损的转基因蠕虫系。2)在体内和体外研究DAT/UNC-64相互作用是否调节DAT-1功能和DAT-1介导的AMPH效应。目标是建立一个基于实验的模型，促进我们对体外和体内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中DA信号中断的新途径和分子。公共卫生相关性：多巴胺转运蛋白（DAT）是在质膜上进行多巴胺再摄取过程的蛋白质，是包括安非他明（AMPH）和可卡因在内的几种精神活性药物的主要分子靶点。多巴胺能系统的异常与许多精神和神经疾病有关，包括吸毒成瘾、精神分裂症和帕金森病。本研究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外溢的影响，旨在阐明这些蛋白如何支持活体动物中amph诱导的行为。