Presynaptic Mechanisms Regulating the Dopamine Transporter

描述（由申请人提供）：儿茶酚胺神经递质多巴胺（DA）在整个发育过程中的神经回路中起作用，以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信号传导的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。突触前DA转运体（DAT）控制着突触DA信号的时空调节。DAT的定位和活性似乎是高度调节的，虽然身份，并发挥作用，许多DAT调节剂在体内是不明确的。在线虫秀丽隐杆线虫中，由DAT（DAT-1）的药理学或遗传学损失产生的DA的突触溢出诱导称为游泳诱导的麻痹（SWIP）的运动表型。因此，缺乏DAT（dat-1）的蠕虫在少量水中游泳后几分钟内就会瘫痪，而野生型（N2）蠕虫则会持续游泳。SWIP由DA囊泡储存的释放诱导，并且可以通过突触后DA受体DOP-3的遗传消除来抑制。为了揭示调节突触DA水平所必需的基因，我们建议在线虫中进行正向遗传筛选，选择表现出利血平敏感SWIP的动物。在DAT-1基因测序后，根据N2、dat-1和dop-3的互补性对突变株系进行分组。在鉴定互补组之后，将采用SNP作图和全基因组测序来定位突变位点。RNAi表型复制和SWIP的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swip系进行行为分析，包括游泳的自动分析、对固体表面上的外源DA的响应、对DAT抑制剂/底物如丙咪嗪和安非他明的响应，以及在原代培养物和异源系统中评估的DA摄取的野生型水平的存在。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。对于诱导不依赖于DAT-1的利血平和DOP-3依赖性SWIP的突变，我们将使用在DA神经元中选择性表达的视紫红质-2评估通过DA神经元的受控放电改变DA释放的贡献。由于线虫表达的经典基因，决定DA的生物合成，包装，代谢和再摄取，我们的SWIP为基础的正向遗传筛选可能会揭示基因，既支持DA信号在线虫和保守的脊椎动物，包括人类。 公共卫生相关性：人脑中多巴胺信号的改变与许多神经精神和神经变性疾病相关，包括精神分裂症、双相情感障碍、注意力缺陷多动障碍和帕金森病。使用线虫秀丽隐杆线虫和多巴胺介导的运动行为称为游泳诱导的麻痹或SWIP，我们的目标是确定负责调节多巴胺信号的基因。C.线虫的DA神经元中表达许多与高级生物相同的基因，因此我们相信我们的筛选可能揭示调节人类DA水平的保守基因。

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. PUBLIC HEALTH RELEVANCE: Alterations in dopamine signaling in the human brain are associated with numerous neuropsychiatric and neurodegenerative disorders including schizophrenia, bipolar disorder, attention-deficit hyperactivity disorder and Parkinson's disease. Using the nematode Caenorhabditis elegans and a dopamine-mediated locomotory behavior known as Swimming Induced Paralysis or SWIP, we aim to identify genes responsible for regulating dopamine signaling. C. elegans express many of the same genes within their DA neurons as higher order organisms, so we believe our screen may reveal conserved genes that regulate DA levels in humans.