Identifying Dopamine Transporter Regulator Proteins in C. elegans

描述（由申请人提供）：本研究的目的是鉴定参与维持突触前神经末梢多巴胺（DA）转运蛋白（DAT）的调节活性和定位的蛋白质。 DA 是中枢神经系统兴奋性和抑制性神经传递的调节剂，与药物成瘾和精神分裂症等多种神经精神疾病的病理生理学密切相关。 DAT 是突触 DA 失活的主要决定因素，是精神兴奋药物的重要靶标，也是多种神经毒素的门户。因此，DAT 的正确表达和活性对于维持大脑中正常的 DA 稳态至关重要。 DAT 受到急性调节，并且已知与辅助蛋白形成复合物以控制定位和活性，但体内介导这些过程的机制很大程度上未知。此外，尽管大量研究已经鉴定出与 DAT 相关的蛋白质，但迄今为止还没有报告证明这些相互作用的功能相关性。哺乳动物中枢神经系统的复杂性鼓励我们的实验室在秀丽隐杆线虫中探索这些问题。无脊椎动物和脊椎动物神经元在分子水平上的高度保守性表明，对活体 DA 神经元中的线虫 DAT 进行分析可能会得出与人类 DAT 调节过程和 DAT 支持的药物成瘾普遍相关的新见解。在这个提案中，我提出了两种鉴定 DAT 相互作用蛋白的策略：首先，我将采用基于 RNA 的干扰 (RNAi) 来破坏已被鉴定为假定的 DAT 调节因子的直向同源物的表达；其次，使用我已经识别和表征的新型运动表型，我将实施无偏见的正向遗传筛选。与更复杂的脊椎动物模型系统相比，线虫具有相当大的优势，这使得线虫成为识别 DAT 相互作用蛋白的宝贵工具，同时检查其功能相关性。本提案中包含的研究将作为一个很好的工具来推进我在模型系统遗传学和 DA 神经传递方面的培训，并且是我过渡到研究独立性的理想途径。公众：多巴胺能 (DA) 神经传递的改变与多种神经精神疾病的病理生理学密切相关，包括帕金森病、精神分裂症和药物成瘾。通过突触前 DA 转运蛋白 (DAT) 的摄取是终止突触 DA 信号传导的主要机制，彻底了解调节 DAT 活性的过程对于解释 DA 相关疾病的病因和进展至关重要。这项研究将定义调节 DAT 活性的机制，并将有助于开发这些疾病的治疗方法。

DESCRIPTION (provided by applicant): The goal of this study is to identify proteins involved in maintaining the regulated activity and localization of the dopamine (DA) transporter (DAT) at presynaptic nerve terminals. DA is a modulator of both excitatory and inhibitory neurotransmission in the CNS and is critically involved in the pathophysiology of several neuropsychiatric diseases including drug addiction and schizophrenia. DAT is a major determinant of synaptic DA inactivation, an important target for psychostimulant drugs, and a gateway for several neurotoxins. The proper expression and activity of DAT is therefore essential in maintaining normal DA homeostasis in the brain. DATs are subject to acute regulation and are known to form complexes with accessory proteins to control localization and activity, but the mechanisms mediating these processes m vivo are largely unknown. Further, though numerous studies have identified proteins that associate with DAT, no reports to date have demonstrated a functional relevance for these interactions. The intricacy of the mammalian CNS has encouraged our laboratory to pursue such questions in Caenorhabditis elegans. The high degree of conservation between invertebrate and vertebrate neurons at the molecular level suggests that analysis of the C. elegans DAT in living DA neurons could lead to novel insights of general relevance for DAT regulatory processes and DAT-supported drug addiction in man. In this proposal, I present two strategies for the identification of DAT-interacting proteins: first, I will employ RNA-based interference (RNAi) to disrupt the expression of orthologs that have been identified as putative DAT regulators; second, using a novel motor phenotype that I have identified and characterized, I will implement an unbiased, forward genetic screen. C. elegans offers considerable advantages over more complex vertebrate model systems, which make the nematode a valuable tool for identifying DAT-interacting proteins while simultaneously examining their functional relevance. The studies included in this proposal will serve as an excellent vehicle with which to advance my training in model systems genetics and DA neurotransmission and are an ideal avenue for my transition into research independence. Public: Alterations in dopaminergic (DA) neurotransmission are centrally involved in the pathophysiology of several neuropsychiatric diseases, including Parkinson's Disease, Schizophrenia, and drug addiction. Uptake via the presynaptic DA transporter (DAT) is the primary mechanism by which DA signaling at the synapse is terminated, and a thorough understanding of the processes that regulate DAT activity are critical in interpreting the etiology and progression of DA-related diseases. This research will define mechanisms that regulate DAT activity and will aid in the development of treatments for these diseases.