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

描述(申请人提供)：多巴胺(DA)是一种基本的神经递质，在包括可卡因和苯丙胺在内的精神刺激剂的奖赏效应中发挥重要作用。DA神经传递的一个主要组成部分决定了DA在突触上的寿命，它是DA转运体(DAT)将递质重新摄取回神经末梢的过程。重要的是，许多心理刺激剂主要通过改变DAT的功能来产生增强作用。因此，有必要了解与DAT功能相关的调节机制，以评估它们在介导双刺激物质效应中的作用。最近，我们和其他人发现了几种DAT蛋白的相互作用，表明DAT中的突触分布、功能特性和精神刺激剂的分子作用可以通过相关蛋白来调节。具体地说，我们用蛋白质组学的方法鉴定了突触小泡蛋白Synaptogyrin-3(SG3)是一种DAT相互作用蛋白。这些研究已经产生了大量的数据，证明在体外DAT的氨基末端(DATN)和SG3之间存在物理和功能上的相互作用。我们假设DAT/SG3的相互作用促进了突触小泡在DAT附近的质膜上的对接，从而在重摄取过程中为小泡提供了有效的细胞外多巴胺负荷。重要的是，这种相互作用可能会调节精神刺激剂的某些行为。到目前为止，蛋白质-蛋白质相互作用对DA动态平衡的影响或精神刺激剂的影响还没有在体内进行检验。为了提供一种研究DAT蛋白-蛋白相互作用在体内DA动态平衡调节中的作用的模式，我们提出了一种结合两种当代方法的探索性方法。快速扫描循环伏安法(FSCV)已被许多人结合电刺激和/或药物操作来获得中枢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-共轭干扰肽来有效地将SG3的DAT结合结构域传递到大脑。我们的团队已经开始将这两种方法结合使用，目的是确定干扰DAT/SG3相互作用是否会影响DA神经传递。事实上，初步数据表明，给药融合到TAT的DAT-N扰乱了SG3和DAT之间的相互作用，并对体内纹状体DA神经传递产生了深远的不良后果。我们建议系统地研究破坏DAT/SG3蛋白-蛋白相互作用对DA神经传递的功能后果，并评估这种相互作用对心理刺激反应的影响。这项提案中描述的研究有望支持开发治疗药物成瘾的新疗法的目标。

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.