Presynaptic Mechanisms Regulating the Dopamine Transporter

描述（申请人提供）：儿茶酚胺神经递质多巴胺（DA）在神经回路中起作用，调节简单和复杂的行为。在人类中，DA信号调节觉醒、认知、奖励和运动功能。DA信号缺陷与多种神经精神和神经退行性疾病有关，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信号的时空调控是由突触前DA转运蛋白（DAT）控制的。尽管体内许多DAT调节因子的身份和作用尚不明确，但其定位和活动似乎受到高度调控。在秀丽隐杆线虫（Caenorhabditis elegans）中，由于DAT （DAT-1）的药理学或遗传损失而产生的DA突触外溢诱导了一种称为游泳诱发性麻痹（SWIP）的运动表型。因此，缺乏DAT （DAT -1）的蠕虫在小体积的水中游泳几分钟后就会瘫痪，而野生型（N2）蠕虫则会持续游泳。SWIP是由DA的囊泡储存释放引起的，并可通过遗传消除突触后DA受体dop3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前向遗传筛选，选择对利血平敏感的SWIP动物。突变系将根据N2、dat-1和dop3的互补情况进行分组，并对dat-1基因进行测序。在确定互补组之后，将使用SNP作图和全基因组测序来绘制突变位点。RNAi表型和SWIP的转基因救援将用于功能性验证观察到的碱基对变化。为了确定这些基因是否调节DAT，将对交换系进行行为分析，包括游泳的自动分析，对固体表面外源DA的反应，对DAT抑制剂/底物（如丙咪嗪和安非他明）的反应，以及在原代培养和异种系统中评估的野生型DA摄取水平。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dop3依赖性SWIP的突变，不依赖于DAT-1，我们将通过在DA神经元中选择性表达的channelrhodopin -2来评估通过控制DA神经元放电而改变的DA释放的贡献。由于秀丽隐杆线虫表达支配DA生物合成、包装、代谢和再摄取的典型基因，我们基于swip的正向遗传筛选可能揭示线虫中支持DA信号传导的基因，以及在包括人类在内的脊椎动物中保守的基因。

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.