Understanding the Role of Dopamine Efflux in Amphetamine-induced Behaviors

了解多巴胺流出在安非他明诱导行为中的作用

• 批准号: 8835506
• 负责人: Andrea Nicole Belovich
• 金额: $ 2.78万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-03-01 至 2018-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8835506
• 关键词: Amphetamine Abuse; Amphetamines; Animal Model; Behavior; Behavioral; Behavioral Model; Behavioral Paradigm; Cell membrane; Cocaine; Data; Disease; Dopamine; Drosophila genus; Drosophila melanogaster; Event; Extracellular Space; Goals; Health; Homeostasis; Human; Investigation; Learning; Locomotion; Mediating; Modeling; Molecular; Molecular Target; Movement; N-terminal; Neurons; Organism; Pharmacological Treatment; Phosphorylation; Phosphotransferases; Property; Research; Rewards; Role; SNAP receptor; System; Testing; Time; Translating; behavior observation; behavior test; casein kinase II; dopamine transporter; dopaminergic neuron; extracellular; fly; in vivo; in vivo Model; knockout gene; neurotransmission; public health relevance; receptor; research study; reuptake; social; stimulant abuse; uptake

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.

描述（由申请人提供）：多巴胺（DA）转运蛋白（DAT）通过突触释放的DA的主动再摄取来控制DA稳态和神经传递。 DAT是负责苯丙胺（AMPH）和可卡因的奖励性质和滥用潜力的主要分子靶标。 AMPH充当DAT底物，并且通过未完全理解的机制，可以促进DA（DAT转运DA的运输）向细胞外空间的外流。细胞外DA水平的增加是对AMPHS的精神运动刺激特性的重要性。 DAT的N末端是一个对AMPH引起DA外流能力至关重要的结构域。我们已经表明，可溶性的N-乙基甲基酰亚胺敏感因子附着蛋白受体（SNARE）蛋白语法（STX1）与DAT N末端相互作用，这种相互作用支持AMPH引起DA外排的能力。此外，已知STX1被酪蛋白激酶2（CK2）1在Ser14上磷酸化，我们假设的事件由AMPH促进，并促进DATX1关联的支持，因此，DA Efflux。我们的分子假设是AMPH诱导STX1磷酸化，从而导致STX1与DAT N末端的关联，从而触发DAT介导的DA FEFFLUX。我们建议通过以下特定目的检验分子假设：1）确定CK2和STX1磷酸化在AMPH诱导的DA外排的作用。 接下来，我们将在体内测试我们的分子发现。我们已经开发了果蝇，作为测试AMPH行为效应的模型。在此系统中，我们已经确定运动是一种受调节的行为，并被AMPH刺激。 DA神经元中果蝇DAT（DDAT）的缺失抑制了AMPH诱导苍蝇运动的能力。 AMPH诱导的运动通过DDAT缺乏DA神经元中人DAT（HDAT）的表达恢复。通过这种策略，我们将把分子观测转换为体内模型，从而使我们能够测试行为有效性。我们的体内假设是，对诱导的行为（例如，运动）取决于CK2介导的STX1磷酸化。因此，我们的第二个特定目的是：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选择性地操纵DAT运输周期的不同方面，以损害DA FEFFLUX和AMPH行为。在我们的初步数据的支持下，我们假设抑制CK2功能会特别损害DAT介导的DA外排，但不会损害DAT介导的DA摄取。这项拟议的研究将发现一个新的“可药”目标（CK2）来治疗AMPH滥用。