Presynaptic Mechanisms Regulating the Dopamine Transporter

调节多巴胺转运蛋白的突触前机制

• 批准号: 8387035
• 负责人: James Andrew Hardaway
• 金额: $ 2.21万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-12-01 至 2013-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8387035
• 关键词: Alleles; Amphetamines; Anabolism; Animals; Arousal; Attention deficit hyperactivity disorder; Base Pairing; Behavior; Behavioral; Behavioral Assay; Biological Assay; Biotinylation; Bipolar Disorder; Brain; Caenorhabditis elegans; Carrier Proteins; Catecholamines; Cell surface; Cells; Chromosomes; Cloning; Cocaine; Cognition; Complex; Computer Simulation; Computers; DNA Resequencing; Data; Dopamine; Dopamine Receptor; Ensure; Exhibits; Functional disorder; Genes; Genetic; Genetic Screening; Genome; Haploidy; Hawaiian population; Homologous Gene; Human; Imipramine; Individual; Induced Mutation; Laboratories; Lesion; Light; Link; Maps; Mediating; Metabolism; Modeling; Molecular; Motor; Mutation; Nematoda; Nerve; Neurodegenerative Disorders; Neurotransmitters; Organism; Paralysed; Parkinson Disease; Phenocopy; Phenotype; Phylogeny; Physiological; Physiology; Play; Protein Kinase; Psychotropic Drugs; RNA Interference; Regulation; Reserpine; Rewards; Role; Schizophrenia; Signal Transduction; Single Nucleotide Polymorphism Map; Site; Solid; Specificity; Speed; Structure; Surface; Swimming; Synapses; System; Testing; Transgenes; Transgenic Organisms; Vertebrates; Water; Work; base; behavior test; dopamine transporter; dopaminergic neuron; gain of function; gain of function mutation; genome sequencing; improved; in vivo; inhibitor/antagonist; insight; loss of function; mutant; neural circuit; neuropsychiatry; novel; overexpression; presynaptic; promoter; public health relevance; relating to nervous system; response; reuptake; selective expression; trafficking; transmission process; uptake

DESCRIPTION (provided by applicant): The catecholamine neurotransmitter dopamine (DA) functions in neural circuits across phylogeny to modulate both simple and complex behaviors. In humans, DA signaling modulates arousal, cognition, reward, and motor function. Deficits in DA signaling are associated with multiple neuropsychiatric and neurodegenerative disorders including schizophrenia, attention-deficit hyperactivity disorder (ADHD), and Parkinson's disease. Temporal and spatial regulation of synaptic DA signaling is controlled by the presynaptic DA transporter (DAT). DAT localization and activity appear to be highly regulated, though the identity of, and roles played by, many DAT regulators in vivo is ill-defined. In the nematode Caenorhabditis elegans, synaptic spillover of DA produced by pharmacological or genetic loss of DAT (DAT-1) induces a locomotory phenotype called Swimming Induced Paralysis (SWIP). Thus, worms lacking DAT (dat-1) paralyze in a few minutes after swimming in a small volume of water whereas wild-type (N2) worms swim continuously. SWIP is induced by the release of vesicular stores of DA and can be suppressed by genetic elimination of the post-synaptic DA receptor DOP-3. To reveal genes necessary for regulating synaptic DA levels, we propose to carry out a forward genetic screen in the nematode, selecting for animals that exhibit reserpine-sensitive SWIP. Mutant lines will be grouped based on their complementation by N2, dat-1 and dop-3, and after sequencing of the DAT-1 gene. Following the identification of complementation groups, SNP mapping and whole genome sequencing will be employed to map the sites of mutations. RNAi phenocopy and transgenic rescue of SWIP will be used to functionally validate observed base-pair changes. To determine if these genes regulate DAT, swip lines will be subjected to behavioral analysis, including automated analysis of swimming, response to exogenous DA on solid surface, response to DAT inhibitors/substrates such as imipramine and amphetamine, and the presence of wild-type levels of DA uptake as assessed in primary cultures and in heterologous systems. Transgenic expression of GFP-DAT-1 will be used to evaluate the effect of mutations on DAT trafficking in vivo, and to demonstrate the specificity of these genes for DAT trafficking. For mutations that induce reserpine and DOP-3 dependent SWIP independent of DAT-1, we will assess the contribution of altered DA release through the controlled firing of DA neurons using Channelrhodopsin-2 selectively expressed in DA neurons. As C elegans express the canonical genes that dictate DA biosynthesis, packaging, metabolism and reuptake, our SWIP-based forward genetic screen may reveal genes that both support DA signaling in the nematode and that are conserved in vertebrates, including humans.

描述（由申请人提供）：儿茶酚胺神经递质多巴胺 (DA) 在整个系统发育的神经回路中发挥作用，调节简单和复杂的行为。在人类中，DA 信号调节唤醒、认知、奖励和运动功能。 DA 信号传导缺陷与多种神经精神和神经退行性疾病相关，包括精神分裂症、注意力缺陷多动障碍 (ADHD) 和帕金森病。突触 DA 信号传导的时间和空间调节由突触前 DA 转运蛋白 (DAT) 控制。尽管体内许多 DAT 调节因子的身份和作用尚不明确，但 DAT 的定位和活性似乎受到高度调节。在线虫秀丽隐杆线虫中，DAT (DAT-1) 的药理或遗传缺失产生的 DA 突触溢出会诱导一种称为游泳诱发麻痹 (SWIP) 的运动表型。因此，缺乏 DAT (dat-1) 的线虫在少量水中游泳后几分钟内就会瘫痪，而野生型 (N2) 线虫则可以连续游泳。 SWIP 是由 DA 囊泡储存的释放诱导的，并且可以通过突触后 DA 受体 DOP-3 的基因消除来抑制。为了揭示调节突触 DA 水平所需的基因，我们建议在线虫中进行正向遗传筛选，选择表现出对利血平敏感的 SWIP 的动物。突变系将根据 N2、dat-1 和 dop-3 的互补性以及 DAT-1 基因测序后进行分组。识别互补组后，将采用 SNP 作图和全基因组测序来绘制突变位点图谱。 RNAi 表型复制和 SWIP 转基因拯救将用于功能验证观察到的碱基对变化。为了确定这些基因是否调节 DAT，将对游泳线进行行为分析，包括游泳的自动分析、对固体表面上的外源 DA 的反应、对 DAT 抑制剂/底物（如丙咪嗪和安非他明）的反应以及野生型的存在在原代培养物和异源系统中评估的 DA 吸收水平。 GFP-DAT-1的转基因表达将用于评估突变对体内DAT运输的影响，并证明这些基因对DAT运输的特异性。对于诱导利血平和独立于 DAT-1 的 DOP-3 依赖性 SWIP 的突变，我们将通过使用在 DA 神经元中选择性表达的通道视紫红质 2 控制 DA 神经元的放电来评估改变 DA 释放的贡献。由于线虫表达决定 DA 生物合成、包装、代谢和再摄取的规范基因，我们基于 SWIP 的正向遗传筛选可能会揭示既支持线虫中 DA 信号传导又在包括人类在内的脊椎动物中保守的基因。

项目成果

Metallo-β-lactamase Domain-Containing Protein 1 (MBLAC1) Is a Specific, High-Affinity Target for the Glutamate Transporter Inducer Ceftriaxone.

金属-β-内酰胺酶结构域含有蛋白 1 (MBLAC1) 是谷氨酸转运蛋白诱导剂头孢曲松的特异性、高亲和力靶标。

• 发表时间: 2017-10-18
• 影响因子: 5
• 作者: Retzlaff, Cassandra L;Kussrow, Amanda;Schorkopf, Tim;Saetear, Phoonthawee;Bornhop, Darryl J;Hardaway, J Andrew;Sturgeon, Sarah M;Wright, Jane;Blakely, Randy D
• 通讯作者: Blakely, Randy D