A Neurogenomic Model for Dopamine Transporter Regulation

描述：（申请人提供） 突触前多巴胺（DA）转运蛋白（DAT）构成了 大脑中DA的失活DAT蛋白是高亲和力的靶点 重要的成瘾和治疗药物，包括可卡因、安非他明和 哌甲酯（利他林）。DAT受到重要的监管 调节，但支持DAT调节的分子机制是未知的。 最近的研究表明，DAT是由协调的 涉及激酶活化、转运蛋白磷酸化和改变的 膜运输/稳定化，尽管迄今为止负责这一点的基因 监管仍有待确定。我们建立了一个模型系统 允许在天然神经元背景下进行DAT评估，利用强大的 转基因和基因组工具提供的秀丽隐杆线虫。在这 系统，我们有独特的能力，选择性地导入或转移基因， 在活的DA神经元中，使我们能够测试关于DAT的特定假设， 结构/功能、药物调节以及药物和激酶触发的DAT 调控此外，已经建立了一种新的细胞培养方法， 允许对DAT功能进行详细的电生理学和光学分析， 在已识别的DA神经元中进行调节，并且可以缩放以支持 新DAT调节基因的鉴定。在我们的CEBRA提案中，我们寻求 1)分析支持C.秀丽隐翅虫DAT （CeDAT）在培养的DA神经元中的表达和2）开发一个系统， 可以应用转基因和蛋白质组学方法的组合， 评估假设的和新的DAT调节剂。

DESCRIPTION: (provided by the applicant) Presynaptic dopamine (DA) transporters (DATs) constitute the primary mechanism for inactivation of DA in the brain. DAT proteins are high-affinity targets for important addictive and therapeutic drugs including cocaine, amphetamines and methylphenidate (Ritalin). DATs are subject to significant regulatory modulation but molecular mechanisms supporting DAT regulation are unknown. Recent studies indicate that DATs are acutely regulated by coordinated mechanisms involving kinase activation, transporter phosphorylation and altered membrane trafficking/stabilization, though as yet genes responsible for this regulation remain to be identified. We have established a model system permitting DAT evaluation in a native neuronal context, exploiting the powerful transgenic and genomic tools afforded in Caenorhabditis elegans. In this system, we have the unique ability to selectivelv introduce or inactivate genes in living DA neurons, allowing us to test specific hypotheses regarding DAT structure/function, drug modulation and drug and kinase triggered DAT regulation. Moreover, a novel cell culture approach has been established that permits a detailed electrophysiologic and optical analysis of DAT function and regulation in identified DA neurons and that can be scaled to support the identification of novel DAT regulatory genes. In our CEBRA proposal, we seek to 1) analyze the functional and regulatory pathways supporting C. elegans DAT (CeDAT) expression in cultured DA neurons and 2) to develop a system where a combination of transgenic and proteomic approaches can be applied to permit the evaluation of hypothesized and novel DAT regulators.