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）通过主动重摄取突触释放的 DA 来控制 DA 稳态和神经传递。 DAT 是导致安非他明 (AMPH) 和可卡因的有益特性和滥用潜力的主要分子靶点。 AMPH 作为 DAT 底物，通过一种尚未完全了解的机制，促进 DA 流出（逆转 DAT 的 DA 转运）进入细胞外空间。细胞外 DA 水平的增加对于 AMPH 的精神运动兴奋特性至关重要。 DAT 的 N 末端是一个结构域，对于 AMPH 引起 DA 流出的能力至关重要。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 Syntaxin1 (Stx1) 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 Stx1 在 Ser14 处被酪蛋白激酶 2 (CK2)1 磷酸化，我们假设 AMPH 会促进这一事件，并促进 DAT-Stx1 关联，从而支持 DA 外流。我们的分子假设是 AMPH 诱导 Stx1 磷酸化，导致 Stx1 与 DAT N 末端结合，从而触发 DAT 介导的 DA 外流。我们建议通过以下具体目标来检验我们的分子假设：1）确定 CK2 和 Stx1 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，我们已经确定运动是 DAT 调节的行为，并受到 AMPH 的刺激。 DA 神经元中果蝇 DAT (dDAT) 的缺失会抑制 AMPH 诱导果蝇运动的能力。 AMPH 诱导的运动通过 dDAT 缺陷的 DA 神经元中人类 DAT (hDAT) 的表达来恢复。通过这种策略，我们将把我们的分子观察转化为体内模型，从而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 CK2 介导的 Stx1 磷酸化。因此，我们的第二个具体目标是：2) 确定 CK2 和 Stx1 磷酸化在 AMPH 诱导行为中的作用。 这项研究的长期目标是了解如何选择性地操纵 DAT 运输周期的不同方面来损害 DA 外流和 AMPH 行为。根据我们的初步数据，我们假设抑制 CK2 功能将特异性损害 DAT 介导的 DA 流出，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“可成药”靶点（CK2），用于治疗 AMPH 滥用。