Identification and Characterization of a Novel Regulator of Dopamine Signaling

描述(由申请人提供)：多巴胺(DA)信号调节多种行为，包括模式运动活动，奖赏寻求和执行功能。可卡因敏感的DA转运体(DAT)是突触前DA稳态的关键决定因素，也是限制DA在空间和时间上的活动的关键决定因素。因此，DA信号的中断被认为是多种大脑疾病的风险因素，包括精神分裂症、注意力缺陷多动障碍(ADHD)、成瘾和帕金森氏症。不幸的是，能够阐明影响DA信号和DAT控制的分子网络的正向遗传学方法在哺乳动物中很难实现。我们发现，当线虫被放置在水中时，切割线虫dat-1基因会导致一种高渗透性的、依赖DA信号的麻痹表型，我们称之为游泳诱导麻痹(SWIP)。DAT-1动物的SWIP可以通过与具有CAT-1、哺乳动物囊泡单胺转运体(VMATs)的同源基因、编码酪氨酸羟基酶的CAT-2功能等位基因缺失的动物杂交以及VMAT抑制剂利血平的治疗来拯救。当dat-1动物与缺乏特定突触后D2型多巴胺受体(dop-3)的动物杂交时，也可以看到拯救Swip行为的方法。最近，我们的实验室实施了一种正向遗传筛选，以确定利血平以及与其他DA信号突变体杂交可以挽救的Swip系。从这个屏幕上，我帮助识别了携带新的、功能缺失的dat-1等位基因的动物，以及映射到缺失已知的DA信号贡献者的基因座的突变。其中一个突变体swp13(Vt32)表现出与DAT-1基因敲除动物相似的DA水平降低，并对神经毒素6-OHDA表现出抗性，而神经毒素6-OHDA需要DAT-1活性才能导入DA神经元。由于SWIP是一种高多巴胺能表型，SWIP13(Vt32)动物DA含量的降低和6-OHDA敏感性的丧失表明SWIP13蛋白可能调节DAT的表达、运输或活性。我最近确定了swp13表型的基因，并发现它在哺乳动物中高度保守。在这里，我建议通过布莱克利实验室和合作研究相对未被研究的swp13直系同源ERK 7/8来研究swp13如何调节线虫中的DA信号。

DESCRIPTION (provided by applicant): Dopamine (DA) signaling modulates multiple behaviors including patterned motor activity, reward seeking and executive function. The cocaine-sensitive DA transporter (DAT) is a critical determinant of presynaptic DA homeostasis as well as in limiting the actions of DA in space and time. Consequentially, disrupted DA signaling is thought to contribute to risk for multiple brain disorders including schizophrenia, attention-deficit hyperactivity disorder (ADHD), addiction and Parkinson's disease. Unfortunately, forward genetic approaches that can elucidate the molecular networks impinging on the control of DA signaling and DAT are difficult to implement in mammals. We have found that ablation of the C. elegans dat-1 gene results in a highly-penetrant, DA signaling-dependent, paralysis phenotype when worms are placed in water, a phenotype we term Swimming- Induced Paralysis (Swip). The Swip of dat-1 animals can be rescued by crosses to animals bearing loss of function alleles of cat-1, ortholog of mammalian vesicular monoamine transporters (VMATs), cat-2, which encodes tyrosine hydroxylase, as well as by treatment of animals with the VMAT inhibitor reserpine. Rescue of Swip behavior is also seen when dat-1 animals are crossed to animals lacking a specific, postsynaptic D2-type dopamine receptor (dop-3). Recently, our lab implemented a forward genetic screen to identify Swip lines that can be rescued by reserpine as well as by crosses to other DA signaling mutants. From this screen, I have helped identify animals bearing novel, loss of function dat-1 alleles, as well as mutants that map to loci that lac known contributors to DA signaling. One such mutant, swip13 (vt32), displays reduced DA levels, similar to dat-1 knockout animals, and exhibits resistance to the neurotoxin 6-OHDA, which requires DAT-1 activity for DA neuron import. As Swip is a hyperdopaminergic phenotype, the reduced DA content of swip13 (vt32) animals and loss of 6-OHDA sensitivity suggest that the SWIP13 protein may regulate DAT expression, trafficking or activity. I have recently identified the gene accounting for the swip13 phenotype, and found it to be highly conserved in mammals. Here, I propose to investigate how this swip13 regulates DA signaling in C. elegans with Blakely lab and collaborative studies examining the relatively unstudied swip13 ortholog ERK 7/8.