The Role of the Dopamine Transporter in Psychostimulant Abuse

项目概要/摘要：安非他明（AMPH）是一种精神兴奋剂，通常用于 神经精神疾病的治疗。它们也被滥用，造成毁灭性的后果。滥用可能性 AMPH的作用与其引起细胞质多巴胺（DA）动员的能力有关， 细胞外DA水平的增加。这种增加是介导的，至少部分是通过逆转DA 多巴胺转运蛋白（DAT）功能，其引起非囊泡DA释放（DA流出）。这种DA外排被认为是 对AMPH的精神兴奋特性至关重要，这一概念得到了证据的支持， DA流出的抑制削弱了AMPH引起运动行为的能力。迄今为止，没有药物治疗 可用于治疗AMPH滥用。因此，有必要了解：a）分子 由AMPH靶向以促进DA流出的机制; B）DA流出是否破坏脑中的DA功能，以及 这些干扰是否支持AMPH诱导的行为;以及c）我们如何将这些机制靶向于 损害DA外排以调节AMPH行为。 以前，使用生物化学，电生理学以及行为测定的组合，我们和 其他人已经表明DAT N-末端（NT）是AMPH促进细胞增殖的能力的关键结构域。 具体行为。这是因为AMPH刺激DAT NT磷酸化，这对于AMPH的能力至关重要。 引起DA外排。此外，我们提供了人类DAT（hDAT）NT直接参与 与可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体蛋白syntaxin 1的相关性 （Stx1），这种相互作用，除了hDAT磷酸化，调节AMPH诱导的DA流出， 行为。这是第一次证明质膜蛋白与Stx1的相互作用是 对精神刺激行为至关重要值得注意的是，我们的数据表明，这种相互作用的强度 由AMPH诱导的Stx1磷酸化调节。 我们假设hDAT NT磷酸化的功能和行为作用是由其磷酸化水平决定的。 与质膜蛋白Stx1相互作用，这是一个由Stx1磷酸化状态调节的过程。 我们建议通过以下具体目标来验证这一假设：1）确定hDAT如何相互作用 与Stx 1; 2）以确定Stx 1在AMPH诱导的DA流出中的参与。分子发现 S.A. #1和S。#2将在神经元中，在分离的果蝇大脑中进行评估，并通过使用 果蝇作为研究AMPH作用的动物模型。我们现在能够通过表达hDAT来“人性化”苍蝇 在缺乏内源性DAT（KO）的果蝇DA神经元中。在这个系统中，我们已经建立了AMPH- 相关的行为，如运动和梳理，是DAT依赖性的。在这个动物模型中，我们 现在可以确定对AMPH的偏好（奖励）和回避。因此，具体目标3）是确定是否 AMPH诱导的行为需要hDAT NT-Stx1相互作用。

Project Summary/Abstract: Amphetamines (AMPHs) are psychostimulants commonly used for the treatment of neuropsychiatric disorders. They are also abused, with devastating outcomes. The abuse potential of AMPHs has been associated with their ability to cause mobilization of cytoplasmic dopamine (DA), leading to an increase in extracellular DA levels. This increase is mediated, at least in part, by the reversal of the DA transporter (DAT) function, which causes non-vesicular DA release (DA efflux). This DA efflux is thought to be essential for the psychomotor stimulant properties of AMPHs, a notion supported by evidence that specific inhibition of DA efflux impairs the ability of AMPH to cause locomotor behaviors. To date, no pharmacotherapies are available for the treatment of AMPHs abuse. Therefore, it is essential to understand: a) the molecular mechanisms targeted by AMPH to promote DA efflux; b) whether DA efflux disrupts DA functions in brain, and whether these disruptions support AMPH-induced behaviors; and c) how we can target these mechanisms to impair DA efflux to regulate AMPH behaviors. Previously, using a combination of biochemistry, electrophysiology, as well as behavioral assays, we and others have shown that the DAT N-Terminus (NT) is a critical structural domain for the ability of AMPH to promote specific behaviors. This is because AMPH stimulates DAT NT phosphorylation, which is vital for its ability to cause DA efflux. Furthermore, we provided the first evidence that the human DAT (hDAT) NT engages in direct associations with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein, syntaxin 1 (Stx1), and that this interaction, in addition to hDAT phosphorylation, regulates AMPH-induced DA efflux and behaviors. This was the first demonstration that the interaction of a plasma membrane protein with Stx1 is essential for psychostimulant behaviors. Noteworthy is that our data suggest that the strength of this interaction is regulated by AMPH-induced phosphorylation of Stx1. We hypothesize that the functional and behavioral role of the hDAT NT phosphorylation is dictated by its interactions with the plasma membrane protein, Stx1, a process regulated by the phosphorylation status of Stx1. We propose to test this hypothesis through the following specific aims: 1) to determine how hDAT interacts with Stx1; 2) to determine the involvement of Stx1 in AMPH-induced DA efflux. The molecular discoveries of S.A. #1 and S.A. #2 will be evaluated in neurons, in isolated Drosophila brains, and behaviorally, by using Drosophila as an animal model to study AMPH actions. We are now able to “humanize” flies by expressing hDAT in DA neurons of Drosophila lacking the endogenous DAT (KO). In this system, we have established that AMPH- associated behaviors, such as locomotion and grooming, are DAT-dependent. Also, in this animal model, we can now determine preference (reward) and avoidance for AMPH. Thus, specific aim 3) is to determine whether hDAT NT-Stx1 interactions are required for AMPH-induced behaviors.