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，奖励寻求和执行功能。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，并在空间和时间上限制DA的作用。因此，被破坏的DA信号被认为会增加多种脑部疾病的风险，包括精神分裂症、注意力缺陷多动障碍（ADHD）、成瘾和帕金森病。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方法很难在哺乳动物中实现。我们发现，秀丽隐杆线虫dat-1基因的去除会导致线虫在水中出现高渗透、依赖DA信号的瘫痪表型，我们将这种表型称为游泳诱发性瘫痪（Swip）。通过与携带猫-1（哺乳动物囊泡单胺转运蛋白（VMATs）的同源基因）和猫-2（编码酪氨酸羟化酶）功能等位基因缺失的动物杂交，以及用VMAT抑制剂利血平治疗动物，可以挽救猫-1动物的Swip。当dat-1动物与缺乏特定的突触后d2型多巴胺受体（dop3）的动物杂交时，也可以看到Swip行为的恢复。最近，我们的实验室实施了一项前向遗传筛选，以确定可以通过利血平以及与其他DA信号突变体的杂交来拯救的Swip系。从这个屏幕上，我已经帮助确定了携带新的，失去功能的dat-1等位基因的动物，以及映射到已知的DA信号通路的位点的突变体。其中一种突变体swip13 （vt32）显示DA水平降低，与dat-1敲除动物相似，并表现出对神经毒素6-OHDA的抗性，这种神经毒素需要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swip13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Blakely实验室和合作研究来研究这个swip13如何调节秀丽隐杆线虫的DA信号，研究相对未被研究的swip13同源ERK 7/8。

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.