Identifying Dopamine Transporter Regulator Proteins in C. elegans

描述(申请人提供)：这项研究的目标是确定与维持多巴胺(DA)转运体(DAT)在突触前神经末梢的调节活性和定位有关的蛋白质。DA是中枢神经系统兴奋性和抑制性神经传递的调节剂，与包括药物成瘾和精神分裂症在内的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，是精神刺激药物的重要靶点，也是几种神经毒素的通道。因此，DAT的正确表达和活性对于维持大脑中正常的DA稳态是必不可少的。DATS受急性调节，已知与辅助蛋白形成复合体来控制定位和活性，但在体内介导这些过程的机制在很大程度上尚不清楚。此外，尽管许多研究已经确定了与DAT相关的蛋白质，但到目前为止还没有报告表明这些相互作用与功能相关。哺乳动物中枢神经系统的复杂性促使我们的实验室在秀丽隐杆线虫中探索这样的问题。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，对活的DA神经元中线虫DAT的分析可能导致对DAT调节过程和DAT支持的人类药物成瘾的普遍相关性的新见解。在本提案中，我提出了两种鉴定DAT相互作用蛋白的策略：第一，我将使用基于RNA的干扰(RNAi)来干扰已被确定为可能的DAT调节因子的同源基因的表达；第二，使用我已经鉴定和表征的一种新的运动表型，我将实现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，这使得线虫成为鉴定DAT相互作用蛋白的有价值的工具，同时也检查了它们的功能相关性。这项建议中包含的研究将成为我在模型系统遗传学和DA神经传递方面进行培训的极好工具，也是我过渡到研究独立性的理想途径。公众：多巴胺(DA)能神经传递的改变与几种神经精神疾病的病理生理学密切相关，包括帕金森氏病、精神分裂症和药物成瘾。通过突触前DA转运体(DAT)摄取是DA信号在突触终止的主要机制，深入了解调节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。这项研究将确定调节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.