The Role of the Dopamine Transporter in Psychostimulant Abuse

项目摘要/摘要：苯丙胺(AMPH)是一种精神刺激剂，通常用于 神经精神障碍的治疗。他们也被滥用，造成了毁灭性的后果。滥用的可能性 AMPHs的活性与它们引起细胞质多巴胺(DA)动员的能力有关，导致 细胞外多巴胺水平升高。这种增加至少在一定程度上是由DA的逆转所调节的 转运体(DAT)功能，导致非囊泡性DA释放(DA外流)。这种DA外流被认为是 对于AMPHs的精神运动刺激特性是必不可少的，这一概念得到了证据的支持 抑制DA外流会削弱AMPH引起运动行为的能力。到目前为止，还没有药物疗法 可用于治疗AMPHs滥用。因此，有必要了解：a)分子 AMPH靶向促进DA外流的机制；b)DA外流是否扰乱大脑中的DA功能，以及 这些干扰是否支持AMPH诱导的行为；以及c)我们如何针对这些机制 损害DA外流以调节AMH行为。 此前，结合生物化学、电生理学和行为分析，我们和 另一些研究表明，DAT N-末端(NT)是AMPH促进能力的关键结构域 特定的行为。这是因为Amph刺激DAT NT的磷酸化，这对它的能力至关重要。 导致DA外流。此外，我们首次提供了人类DAT(HDAT)NT参与直接 可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体蛋白--突触素1的相关性 (STX1)，这种相互作用，除了HDAT磷酸化外，还调节Amph诱导的DA外流和 行为。这是第一次证明质膜蛋白与STX1的相互作用是 对于精神刺激行为来说是必不可少的。值得注意的是，我们的数据表明，这种相互作用的强度 是由Amph诱导的STX1的磷酸化调节的。 我们假设HDAT NT磷酸化的功能和行为作用是由其决定的 与质膜蛋白STX1的相互作用，这是一个受STX1磷酸化状态调控的过程。 我们建议通过以下具体目标来检验这一假设：1)确定HDAT如何相互作用 2)确定STX1在Amph诱导的DA外流中的作用。人类的分子发现 S.A.#1和S.A.#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 以果蝇为动物模型来研究amph的作用。我们现在能够通过表达HDAT来使苍蝇“人性化” 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，是依赖DAT的。此外，在这个动物模型中，我们 现在可以决定AMPH的偏好(奖励)和回避。因此，具体目标3)是确定 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.