Disrupting DAT Protein-Protein Interactions In Vivo: Implications for Drug Abuse

描述（由申请人提供）：多巴胺（DA）是一种必需的神经递质，在精神兴奋剂（包括可卡因和安非他明）的奖励效应中起主要作用。决定DA在突触处的寿命的DA神经传递的主要成分是通过DA转运体（DAT）将递质再摄取回到神经末梢。重要的是，许多精神兴奋剂主要通过改变DAT的功能产生其强化作用。因此，有必要了解DAT功能相关的调节机制，以评估其在介导精神兴奋剂作用中的作用。最近，我们和其他人已经确定了几个DAT蛋白的相互作用，表明突触分布，功能特性，和DAT的精神兴奋剂的分子作用可以通过相关的蛋白质进行调节。具体来说，我们确定了突触囊泡蛋白synaptogyrin-3（SG 3）作为DAT相互作用蛋白质使用蛋白质组学方法。这些研究已经产生了大量的数据，证明了DAT（DATN）的氨基末端和SG 3在体外的物理和功能之间的相互作用。我们假设DAT/SG 3相互作用促进突触囊泡在DAT附近的质膜处对接，以在再摄取过程中提供具有细胞外多巴胺的囊泡的有效负载。重要的是，这种相互作用可能介导精神兴奋剂的某些作用。迄今为止，蛋白质-蛋白质相互作用对DA稳态的影响或精神兴奋剂的作用尚未在体内进行研究。为了提供一个模式的调查DAT蛋白质-蛋白质相互作用的DA稳态在体内的调节中的作用，我们提出了一个探索性的方法，将联合收割机两个当代的方法。快速扫描循环伏安法（FSCV）已被许多人与电刺激和/或药理学操作结合使用，以获得关于中枢神经系统中儿茶酚胺的时间变化和浓度的信息。我们将采用的第二种当代技术涉及使用TAT缀合的干扰肽来有效地将SG 3的DAT结合结构域递送到大脑。我们的小组已经开始使用这两种方法，目的是辨别破坏DAT/SG 3相互作用是否会影响DA神经传递。事实上，初步数据表明，DAT-N融合到达特的管理中断SG 3和DAT之间的相互作用，并在体内纹状体DA神经传递具有深远的不良后果。我们建议系统地研究破坏DAT/SG 3蛋白质-蛋白质相互作用对DA神经传递的功能后果，并评估这种相互作用对精神兴奋剂反应的影响。本提案中描述的研究有望支持开发药物成瘾新疗法的目标。

DESCRIPTION (provided by applicant): Dopamine (DA) is an essential neurotransmitter and plays a major role in the rewarding effects of psychostimulants, including cocaine and amphetamine. A primary component of DA neurotransmission that determines the lifetime of DA at synapses is the re-uptake of the transmitter back into nerve terminals by the DA transporter (DAT). Importantly, many psychostimulants produce their reinforcing effects primarily by altering the function of DAT. Therefore, it becomes necessary to understand the regulatory mechanisms associated with DAT function in order to assess their role in mediating the effects of pyschostimulants. Recently, we and others have identified several DAT protein interactions, suggesting that synaptic distribution, functional properties, and molecular actions of psychostimulants at DAT can be regulated via associated proteins. Specifically, we identified the synaptic vesicle protein synaptogyrin-3 (SG3) as a DAT interacting protein using a proteomic approach. These studies have produced ample data demonstrating a physical and functional interaction between the amino terminus of DAT (DATN) and SG3 in vitro. We hypothesize that the DAT/SG3 interaction facilitates synaptic vesicle docking at the plasma membrane near DAT to provide efficient loading of the vesicles with extracellular dopamine during the reuptake process. Importantly, this interaction might mediate some actions of psychostimulants. To date, the impact of protein-protein interactions on DA homeostasis or the effects of psychostimulants has not been examined in vivo. In order to provide a mode of investigation for the role of DAT protein-protein interactions in the regulation of DA homeostasis in vivo, we propose an explorative approach that will combine two contemporary methodologies. Fast scan cyclic voltammetry (FSCV), has been used by many in conjunction with electrical stimulation and/or pharmacological manipulation to obtain information about temporal changes and concentration of catecholamines in the central nervous system. The second contemporary technique we will employ involves the use of TAT-conjugated interfering peptides to effectively deliver the DAT binding domain of SG3 to the brain. Our group has begun to use these two approaches together with the goal of discerning whether disrupting the DAT/SG3 interaction impacts DA neurotransmission. Indeed, preliminary data suggests that administration of the DAT-N fused to TAT disrupts the interaction between SG3 and DAT and has profound adverse consequences on in vivo striatal DA neurotransmission. We propose to examine systematically the functional consequences of disrupting the DAT/SG3 protein-protein interaction on DA neurotransmission and to evaluate the effects of this interaction on psychostimulant response. The studies described in this proposal are expected to support the goal of developing novel therapies for drug addiction.