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)在整个系统发育过程中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信号调节觉醒、认知、奖励和运动功能。DA信号的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。突触前DA转运体(DAT)控制突触DA信号的时空调控。DAT的定位和活性似乎受到高度调控，尽管体内许多DAT调控因子的身份和作用尚不明确。在线虫中，药物或遗传的DAT-1缺失(DAT-1)产生的DA的突触溢出导致一种运动表型，称为游泳诱导瘫痪(SWIP)。因此，缺乏DAT(dat-1)的蠕虫在少量水中游泳后几分钟内瘫痪，而野生型(N2)蠕虫则持续游泳。SWIP由DA囊泡库的释放诱导，并可通过突触后DA受体DOP-3的遗传消除而抑制。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的SWIP的动物。突变株系将根据它们与N2、dat-1和dop-3的互补性以及DAT-1基因的测序进行分组。在确定互补群后，将使用SNP作图和全基因组测序来定位突变位置。RNAi表型复制和SWIP的转基因挽救将被用于从功能上验证观察到的碱基对变化。为了确定这些基因是否调节DAT，Swip系将接受行为分析，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(如丙咪嗪和苯丙胺)的反应，以及在原代培养和异源系统中评估是否存在野生型的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。对于导致利血平和DOP-3依赖的SWIP而不依赖DAT-1的突变，我们将利用在DA神经元中选择性表达的通道视紫红质-2来评估DA释放的变化对DA神经元的控制放电的贡献。由于线虫表达决定DA生物合成、包装、代谢和重吸收的规范基因，我们基于SWIP的正向遗传筛查可能揭示既支持线虫中的DA信号又在包括人类在内的脊椎动物中保守的基因。 公共卫生相关性：人脑中多巴胺信号的变化与许多神经精神和神经退行性疾病有关，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氏症。利用线虫秀丽线虫和多巴胺介导的运动行为，即游泳诱导瘫痪或SWIP，我们的目标是识别负责调节多巴胺信号的基因。线虫在其DA神经元中表达许多与更高级生物体相同的基因，因此我们相信我们的筛查可能揭示调节人类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.