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: 9024345
• 负责人: Andrea Nicole Belovich
• 金额: $ 2.59万
• 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 2017-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9024345
• 关键词: Amphetamine Abuse; Amphetamines; Animal Model; Behavior; Behavioral; Behavioral Model; Behavioral Paradigm; Carrier Proteins; 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）和可卡因的奖励特性和滥用潜力的主要分子靶标。AMPH作为DAT底物，并通过尚未完全理解的机制促进DA流出（DAT转运DA的逆转）进入细胞外间隙。这种细胞外DA水平的增加对于AMPH的精神兴奋特性是重要的事件。 DAT的N-末端是AMPH引起DA外排能力的关键结构域。我们已经表明，可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体（SNARE）蛋白syntaxin 1（Stx 1）与DAT N-末端相互作用，这种相互作用支持AMPH引起DA流出的能力。此外，已知Stx 1在Ser 14被酪蛋白激酶2（CK 2）1磷酸化，我们假设这一事件是由AMPH促进的，并促进DAT-Stx 1缔合，因此支持DA外排。我们的分子假设是AMPH诱导Stx 1磷酸化，导致Stx 1与DAT N-末端的关联，从而触发DAT介导的DA流出。我们拟通过以下具体目标来验证我们的分子假设：1）确定CK 2和Stx 1磷酸化在AMPH诱导的DA流出中的作用。 接下来，我们将在体内测试我们的分子发现。我们已经开发了果蝇作为模型来测试AMPH的行为影响。在这个系统中，我们已经建立了运动是DAT调节的行为，并由AMPH刺激。DA神经元中果蝇DAT（dDAT）的缺失抑制AMPH诱导果蝇运动的能力。AMPH诱导的运动通过dDAT缺陷DA神经元中人DAT（hDAT）的表达而恢复。通过这种策略，我们将把我们的分子观察转化为体内模型，使我们能够测试行为的有效性。我们的体内假设是AMPH诱导的行为（例如，运动）依赖于CK 2介导的Stx 1磷酸化。因此，我们的第二个具体目标是：2）确定CK 2和Stx 1磷酸化在AMPH诱导的行为中的作用。 本研究的长期目标是了解如何选择性地操纵DAT转运周期的不同方面，以损害DA流出和AMPH行为。我们的初步数据支持，我们假设，抑制CK 2功能将专门损害DAT介导的DA流出，但不是DAT介导的DA摄取。这项拟议的研究将揭示一个新的“可药物化”目标（CK 2），用于治疗AMPH滥用。