Understanding the Role of Dopamine Efflux in Amphetamine-induced Behaviors

描述(申请人提供)：多巴胺(DA)转运体(DAT)通过主动重新摄取突触释放的DA来控制DA的动态平衡和神经传递。DAT是苯丙胺(AMPH)和可卡因的有益特性和滥用潜力的主要分子靶标。AMPH作为DAT底物，通过一种尚未完全了解的机制，促进DA(DAT转运DA的逆转)外流到细胞外空间。这种细胞外多巴胺水平的增加对于AMPH的精神运动刺激特性是一个重要的事件。DAT的N末端是一个结构域，对Amph导致DA外流的能力至关重要。我们已经证明，可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体(SNARE)蛋白Synaxin1(STX1)与DAT的N-末端相互作用，这种相互作用支持AMPH导致DA外流的能力。此外，已知STX1在Ser14处被酪蛋白激酶2(CK2)1磷酸化，我们推测这一事件是由AMPH促进的，并促进DAT-STX1关联，从而支持DA外流。我们的分子假说是AMPH诱导STX1的磷酸化，导致STX1与DAT的N端结合，从而触发DAT介导的DA外流。我们建议通过以下特定目的来验证我们的分子假说：1)确定CK2和STX1磷酸化在AMPH诱导的DA外流中的作用。接下来，我们将在体内测试我们的分子发现。我们已经开发了黑腹果蝇作为模型来测试amph的行为效应。在这个系统中，我们已经确定了运动是一种DAT调节的行为，并受到AMPH的刺激。果蝇DAT(DDAT)在DA神经元中的缺失抑制了Amph诱导果蝇运动的能力。在dDAT缺陷的DA神经元中，人DAT(HDAT)的表达可恢复AMPH诱导的运动。有了这个策略，我们将把我们的分子观察转化为活体模型，从而使我们能够测试行为的有效性。我们的体内假说是AMPH诱导的行为(例如，运动)依赖于CK2介导的STX1磷酸化。因此，我们的第二个特定目标是：2)确定CK2和STX1磷酸化在AMPH诱导的行为中的作用。这项研究的长期目标是学习如何有选择地操纵DAT运输周期的不同方面，以损害DA外流和AMPH行为。在我们的初步数据支持下，我们假设抑制CK2功能将特异性地损害DAT介导的DA外流，但不会损害DAT介导的DA摄取。这项拟议的研究将揭示一种新的治疗AMPH滥用的“可用药”靶点(CK2)。

DESCRIPTION (provided by applicant): The dopamine (DA) transporter (DAT) controls DA homeostasis and neurotransmission by the active reuptake of synaptically released DA. The DAT is the major molecular target responsible for the rewarding properties and abuse potential of amphetamine (AMPH) and cocaine. AMPH acts as a DAT substrate, and, through a mechanism not fully understood, promotes the efflux of DA (reversal of DAT's transport of DA) into the extracellular space. This increase of extracellular DA levels is an event of importance for the psychomotor stimulant properties of AMPHs. The N-terminus of the DAT is a structural domain critical to AMPH's ability to cause DA efflux. We have shown that the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein syntaxin1 (Stx1) interacts with the DAT N-terminus and this interaction supports the ability of AMPH to cause DA efflux. Additionally, Stx1 is known to be phosphorylated at Ser14 by casein kinase 2 (CK2)1, an event we hypothesize is promoted by AMPH and promotes DAT-Stx1 association supporting, therefore, DA efflux. Our molecular hypothesis is that AMPH induces Stx1 phosphorylation, leading to the association of Stx1 with the DAT N-terminus and thereby triggering DAT-mediated DA efflux. We propose to test our molecular hypothesis through the following specific aim: 1) To determine the roles of CK2 and Stx1 phosphorylation in AMPH- induced DA efflux. Next, we will test our molecular discoveries in vivo. We have developed Drosophila melanogaster as a model to test the behavioral effects of AMPH. In this system, we have established that locomotion is a DAT- regulated behavior and is stimulated by AMPH. Deletion of Drosophila DAT (dDAT) in DA neurons inhibits AMPH's ability to induce locomotion in flies. AMPH-induced locomotion is restored by the expression of the human DAT (hDAT) in dDAT-deficient DA neurons. With this strategy, we will translate our molecular observations to an in vivo model, allowing us to test behavioral validity. Our in vivo hypothesis is that AMPH- induced behaviors (e.g., locomotion) are dependent on CK2-mediated Stx1 phosphorylation. Thus, our second specific aim is: 2) To determine the role of CK2 and Stx1 phosphorylation in AMPH-induced behaviors. The long term goal of this research is to learn how to selectively manipulate different aspects of the DAT transport cycle to impair DA efflux and AMPH behaviors. Supported by our preliminary data, we hypothesize that inhibiting CK2 function will specifically impair DAT-mediated DA efflux, but not DAT-mediated DA uptake. This proposed research will uncover a new "druggable" target (CK2) for the treatment of AMPH abuse.