Forward Genetics and the Presynaptic Dopamine Transporter

正向遗传学和突触前多巴胺转运蛋白

• 批准号: 7772187
• 负责人: Randy D. Blakely
• 金额: $ 15.5万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7772187
• 关键词: Alleles; Amphetamines; Animal Model; Animals; Antidepressive Agents; Attention deficit hyperactivity disorder; Behavior; Behavioral; Bioinformatics; Biological Assay; Biological Models; C. elegans genome; Caenorhabditis elegans; Carrier Proteins; Cloning; Cocaine; Cognition; Complementary DNA; Data; Defect; Dopamine; Dopamine Receptor; Drug Addiction; Embryo; Environment; Exhibits; Fellowship; Functional RNA; Future; Gene Expression Profile; Genes; Genetic; Genetic Complementation Test; Genetic Models; Genetic Polymorphism; Genetic Screening; Genome; Genomics; Goals; Haploidy; Homeostasis; Homologous Gene; Human; Imipramine; In Vitro; Knock-out; Laboratories; Lesion; Link; Liquid substance; Locomotion; Maps; Mediating; Methods; Modeling; Molecular; Mutagenesis; Mutation; National Research Service Awards; Nematoda; Nerve; Neurotoxins; Oxidopamine; Paralysed; Phenocopy; Phenotype; Point Mutation; Presynaptic Terminals; Process; Protein Kinase; Proteins; Protocols documentation; Psychotropic Drugs; RNA Interference; Regulation; Regulator Genes; Reporter; Research; Resistance; Rewards; Risk; Schizophrenia; Signal Pathway; Signal Transduction; Single Nucleotide Polymorphism; Structure; Swimming; System; Transgenes; United States National Institutes of Health; Variant; Vertebrates; Work; addiction; base; behavior test; cDNA Library; calmodulin-dependent protein kinase II; design; dopamine transporter; dopaminergic neuron; genetic analysis; genetic regulatory protein; genome sequencing; in vivo; innovation; insight; mutant; neurotransmission; novel; presynaptic; programs; psychostimulant; public health relevance; response; reuptake; therapeutic development; trafficking; uptake; Alleles; Amphetamines; Animal Model; Animals; Antidepressive Agents; Attention deficit hyperactivity disorder; Behavior; Behavioral; Bioinformatics; Biological Assay; Biological Models; C. elegans genome; Caenorhabditis elegans; Carrier Proteins; Cloning; Cocaine; Cognition; Complementary DNA; Data; Defect; Dopamine; Dopamine Receptor; Drug Addiction; Embryo; Environment; Exhibits; Fellowship; Functional RNA; Future; Gene Expression Profile; Genes; Genetic; Genetic Complementation Test; Genetic Models; Genetic Polymorphism; Genetic Screening; Genome; Genomics; Goals; Haploidy; Homeostasis; Homologous Gene; Human; Imipramine; In Vitro; Knock-out; Laboratories; Lesion; Link; Liquid substance; Locomotion; Maps; Mediating; Methods; Modeling; Molecular; Mutagenesis; Mutation; National Research Service Awards; Nematoda; Nerve; Neurotoxins; Oxidopamine; Paralysed; Phenocopy; Phenotype; Point Mutation; Presynaptic Terminals; Process; Protein Kinase; Proteins; Protocols documentation; Psychotropic Drugs; RNA Interference; Regulation; Regulator Genes; Reporter; Research; Resistance; Rewards; Risk; Schizophrenia; Signal Pathway; Signal Transduction; Single Nucleotide Polymorphism; Structure; Swimming; System; Transgenes; United States National Institutes of Health; Variant; Vertebrates; Work; addiction; base; behavior test; cDNA Library; calmodulin-dependent protein kinase II; design; dopamine transporter; dopaminergic neuron; genetic analysis; genetic regulatory protein; genome sequencing; in vivo; innovation; insight; mutant; neurotransmission; novel; presynaptic; programs; psychostimulant; public health relevance; response; reuptake; therapeutic development; trafficking; uptake

DESCRIPTION (provided by applicant): The goal of this study is to capitalize on a recently identified dopamine (DA) transporter (DAT)-dependent phenotype in the nematode C. elegans, termed Swimming-Induced Paralysis (SWIP), to identify proteins involved in the localization and regulation of cocaine and amphetamine-sensitive DAT proteins at presynaptic nerve terminals. In vertebrates, DA modulates multiple behavioral processes involved with locomotion, cognition, and reward. Reuptake through DAT is the primary mechanism by which DA signaling is terminated and is a critical determinant of presynaptic DA homeostasis. Polymorphisms in human DAT have been associated with ADHD, schizophrenia, and drug addiction, and DAT is a major target of cocaine and amphetamine. Heterologous expression studies indicate that DATs are regulated via accessory proteins and kinase-linked signaling pathways that ultimately control transporter localization and activity. Moreover, signaling networks linked to PKC, CaMKII, PI3K, and Akt have been linked to the presynaptic actions of amphetamine and cocaine, revealing many potential new targets for addiction risk and therapeutic development. The technical challenges presented by the study of DAT regulation in the mammalian CNS have encouraged our laboratory to pursue a characterization of DAT-associated phenotypes in C. elegans, where forward genetic screens for DAT modulators are attainable. Previously, our laboratory showed that the C. elegans DAT (DAT-1) is 43% identical to mammalian DATs, is expressed exclusively in DA neurons and terminals, preferentially transports DA, and is sensitive to both cocaine and amphetamine. Under the support of a postdoctoral NRSA, I identified a phenotype, termed Swimming Induced Paralysis (SWIP) that is a reporter of functional DAT-1 expression. Thus, SWIP in dat-1 knockout or cocaine/amphetamine treated animals is reversed when DA synthesis, release, and post-synaptic signaling through the DA receptor DOP-3 are precluded. Based on these data, I launched a pilot forward genetic screen to identify DA-dependent SWIP mutants and have identified two lines bearing novel dat-1 point mutations that cause biosynthetic, trafficking and functional defects in vitro and in vivo as well as several lines where mutations appear to lie in other genes. Building on this effort, I propose the following Specific Aims: 1) To expand the screen for DA-dependent SWIP phenotypes, validating DAT deficiency via dopamine receptor deficiency complementation tests, responsivity to cocaine/amphetamine and DA transport assays on embryonic cultures and 2) To identify non-DAT, SWIP-generating genes using Illumina-based, high-throughput cDNA sequencing with transcriptome profiling, followed by a bioinformatics based elucidation of human homologs. These studies provide a powerful and unique opportunity for insight into presynaptic mechanisms controlling DAT-dependent DA signaling. PUBLIC HEALTH RELEVANCE: Alterations in dopaminergic (DA) neurotransmission are centrally involved in psychostimulant response and addiction. The presynaptic DA transporter (DAT) is the primary mode by which DA signaling is terminated and is a direct target for cocaine and amphetamine. This research engages a powerful genetic model system to elucidate genes that regulate DA signaling and DAT activity with opportunities to identify novel targets for addiction risk and/or treatment.

描述（由申请人提供）：这项研究的目的是利用最近确定的多巴胺（DA）转运蛋白（DAT）依赖性表型在线虫C.秀素中，称为游泳诱导的瘫痪（SWIP），以鉴定参与cocaine和Amphetamine sensence proteApters的蛋白质，以识别蛋白质的蛋白质。在脊椎动物中，DA调节了与运动，认知和奖励有关的多个行为过程。通过DAT进行的重新摄取是DA信号终止的主要机制，是突触前DA稳态的关键决定因素。人DAT中的多态性与ADHD，精神分裂症和药物成瘾有关，DAT是可卡因和苯丙胺的主要靶标。异源表达研究表明，DAT是通过辅助蛋白和激酶连接的信号通路来调节DAT的，这些信号通路最终控制转运蛋白的定位和活性。此外，与PKC，CAMKII，PI3K和AKT相关的信号网络与苯丙胺和可卡因的突触前作用有关，揭示了许多潜在的成瘾风险和治疗性发展的新目标。哺乳动物中枢神经系统中DAT调节的研究提出的技术挑战鼓励我们的实验室在秀丽隐杆线虫中追求与DAT相关的表型的表征，在那里可以实现DAT调节剂的正向基因筛选。以前，我们的实验室表明，秀丽隐杆线虫DAT（DAT-1）与哺乳动物DATS相同43％，仅在DA神经元和末端表达，优先运输DA，并且对可卡因和苯丙胺均敏感。在博士后NRSA的支持下，我确定了一种表型，称为游泳诱发瘫痪（SWIP），该表型是功能性dat-1表达的记者。因此，当不排除DA合成，释放和通过DA受体DOP-3的DA合成，释放和突触后信号传导时，将dat-1基因敲除或可卡因/苯丙胺处理的动物进行逆转。基于这些数据，我启动了一个飞行员前向遗传筛选，以识别DA依赖性的SWIP突变体，并鉴定了两条带有新型DAT-1点突变的线，它们在体外和体内引起了生物合成，运输和功能缺陷，以及突变似乎位于其他基因的几行。 Building on this effort, I propose the following Specific Aims: 1) To expand the screen for DA-dependent SWIP phenotypes, validating DAT deficiency via dopamine receptor deficiency complementation tests, responsivity to cocaine/amphetamine and DA transport assays on embryonic cultures and 2) To identify non-DAT, SWIP-generating genes using Illumina-based, high-throughput cDNA sequencing with transcriptome profiling, followed by基于生物信息学的人类同源物的阐明。这些研究为洞悉控制DAT依赖性DA信号传导的突触前机制提供了强大而独特的机会。公共卫生相关性：多巴胺能（DA）神经传递的改变与心理刺激反应和成瘾有关。突触前DA转运蛋白（DAT）是DA信号终止的主要模式，是可卡因和苯丙胺的直接目标。这项研究使一个强大的遗传模型系统阐明了调节DA信号传导和DAT活动的基因，并有机会识别成瘾风险和/或治疗的新目标。