A Neurogenomic Model for Dopamine Transporter Regulation

描述：(申请人提供) 突触前多巴胺(DA)转运体(DAT)构成主要机制 使大脑中的多巴胺失活。DAT蛋白是高亲和力靶点 重要的成瘾和治疗药物，包括可卡因、苯丙胺和 哌醋甲酯(利他林)。DATS受到严格的监管 但支持DAT调控的分子机制尚不清楚。 最近的研究表明，DAT受到协调的强烈调控 涉及激酶激活、转运体磷酸化和改变的机制 膜转运/稳定，尽管到目前为止与此相关的基因 监管规定仍有待确定。我们已经建立了一个模型系统 允许在本地神经元环境中进行DAT评估，利用强大的 秀丽隐杆线虫的转基因和基因组工具。在这 系统中，我们具有选择引入或使基因失活的独特能力 在活的DA神经元中，允许我们测试关于DAT的特定假说 结构/功能、药物调节与药物和激酶触发的DAT 监管。此外，还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和光学分析 对已识别的DA神经元的调节，并且可以扩展以支持 新的DAT调控基因的鉴定。在我们的CEBRA提案中，我们寻求 1)分析支持线虫DAT的功能和调控途径 (CeDAT)在培养的DA神经元中的表达；2)建立一种系统，其中 转基因和蛋白质组学方法的结合可以应用于允许 对假想的和新的DAT调节器的评估。

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.