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）：该项目的长期目的是在体外和体内对多巴胺（DA）转运蛋白（DAT）功能的急性调节（DA）功能的急性调节。中枢神经系统的DA信号传导调节多种认知，情感和行为功能。 DA系统中的异常已与许多精神病和神经系统疾病有关，包括吸毒成瘾，精神分裂症和帕金森氏病。 AMPH诱导了DA的反向运输，从而提高了细胞外DA水平并导致其精神刺激作用。迄今为止，研究AMPH调节DAT功能的努力在很大程度上取决于传统的生化和/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动作的许多新方面，但很难建立一个单个系统，在该系统中，在神经元环境中操纵DAT功能的AMPH调节该项目将结合分子，生化和生物物理方法来表征DAT功能的AMPH功能在神经元制剂中的AMPH调节。在此提案中，我们将创建一个新型的实验平台，以使用秀丽隐杆线虫提供的转基因和实验工具来研究DAT蛋白 - 蛋白质相互作用。解决的关键问题包括DAT蛋白与SNARE蛋白UNC-64（C. elegrans syntaxin 1a的同源物）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功能调节与刺激动物行为联系起来。拟议的研究解决了以下特定目的：1）创建具有DAT/UNC-64相互作用受损的转基因蠕虫线。 2）确定DAT/UNC-64相互作用是否调节DAT-1功能和Dat-1-介导的AMPH效果在体内和体外。目的是生成一个基于实验的模型，以推动我们在体外和体内对AMPH作用的理解，并发现可能有助于疾病状态（如成瘾和帕金森氏病）中破坏DA信号的新颖途径和分子。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的主要分子靶标，包括苯丙胺（AMPH）和可卡因。多巴胺能系统的异常已与许多精神病和神经系统疾病有关，包括吸毒成瘾，精神分裂症和帕金森氏病。该提案的主要目的是创建一个体内表达系统，以研究研究蛋白质及其对AMPH诱导的DA外排的影响，以阐明这些蛋白质如何支持活动物中的AMPH诱导的行为。