Kappa Opioid Receptors and Phospho-Dopamine Transporters Drive Cocaine Reward

总结 物质使用障碍困扰着美国近七分之一的人，给医疗保健带来沉重负担 资源、经济和个人生活质量。目前，没有有效的药物可用于 可卡因使用障碍（CUD），很可能是因为这种疾病的神经生物学基础是复杂的， 完全理解中脑边缘回路中的多巴胺能神经传递已被证明在神经系统中起着重要作用。 在CUD中的重要作用。皮质醇抑制多巴胺转运蛋白（DAT）的活性，以产生其行为 方面的影响. DAT是多巴胺信号传导的主要调节器，其调节主要通过后 翻译修饰，如磷酸化，这是由突触前的自体和异种激活的。 受体相关的信号级联。这些修饰改变了可卡因与DAT的相互作用， 可卡因的刺激和奖励作用的变化。然而，到目前为止，翻译后 在CUD领域中，修饰不是研究的焦点。我们的研究表明， DAT的Thr-53残基，或磷酸-T53-DAT（PT 53-DAT），在调节可卡因- 定向行为。在这项提案中，我们使用尖端技术探索这一新发现，包括 用磷酸化缺陷型DAT-Thr 53 Ala突变体（DAT-A53）敲入小鼠模型， DAT-T53磷酸化的脑区域特异性阻断，以及基于纤维光度计的 多巴胺在自由活动的小鼠使用荧光传感器，dLight。有了这些新型号和量身定制的 生物化学，神经化学和行为研究，目前的建议旨在填补我们的关键空白， 理解DAT磷酸化在正常多巴胺神经传递和多巴胺神经传递中的中心作用。 可卡因的存在，包括第一次在活动物体内。作为一个关键机制，我们发现， κ-阿片受体（KOR）通过PT 53-DAT调节DAT。我们的研究还表明，KOR激活 通过PT 53-DAT增加DAT活性，并且当T53被A53取代时，KOR的厌恶作用 激动剂是减弱的。在这里，我们将扩展和测试我们的总体假设“可卡因诱导成瘾- 类似于当KOR在T53磷酸化DAT并改变DA动力学时的行为”。目标1将调查 T53磷酸化对KOR介导的DAT上调、运输和蛋白质-蛋白质相互作用的影响。 目的1还将检查这些PT 53-DAT依赖性效应是否具有性别和脑区域特异性。目标2将 检查PT 53-DAT对细胞外DA动力学以及DA释放和清除调节的影响， 体内和脑切片中的KOR和可卡因。目标3将确定PT 53-DAT是否在KOR中发挥作用， 可卡因相关的行为从拟议的研究结果将提供新的见解， CUD的机制，打开了新的视野，检查磷酸化的DAT-T53作为一个潜在的 强啡肽在多巴胺信号传导和可卡因成瘾中的作用机制及新的分子治疗药物 干预点。

SUMMARY Substance use disorder afflicts nearly one in seven people in the US, placing a heavy burden on healthcare resources, the economy, and individual quality of life. Currently, there is no effective medication available for cocaine use disorder (CUD), most likely because the neurobiology underlying the disease is complex and not completely understood. Dopaminergic neurotransmission in the mesolimbic circuit has been shown to play a critical role in CUD. Cocaine inhibits the activity of the dopamine transporter (DAT) to produce its behavioral effects. Regulation of the DAT, a principal regulator of dopamine signaling, occurs primarily through post- translational modifications, such as phosphorylation, which are triggered by presynaptic auto and hetero- receptor-linked signaling cascades. These modifications alter the interaction of cocaine with the DAT, leading to changes in the stimulating and rewarding effects of cocaine. However, to date, post-translational modifications have not been a focus of investigation in the CUD field. Our studies show that phosphorylation of the Threonine-53 residue of DAT, or phospho-T53-DAT (PT53-DAT), plays a pivotal role in regulating cocaine- directed behaviors. In this proposal, we explore this novel finding using cutting-edge techniques, including a knock-in mouse model with a phosphorylation-defective DAT-Thr53Ala mutant (DAT-A53), a viral-mediated brain region-specific blockade of DAT-T53 phosphorylation, and fiber photometry-based measurements of dopamine in freely moving mice using the fluorescent sensor, dLight. With these new models and tailored biochemical, neurochemical, and behavioral studies, the current proposal aims to fill a critical gap in our understanding of the central role of DAT phosphorylation in normal dopamine neurotransmission and in the presence of cocaine, including in live animals for the first time. As a key mechanism, we have discovered that the kappa-opioid receptor (KOR) regulates DAT via PT53-DAT. Our studies also show that KOR activation increases DAT activity through PT53-DAT, and when T53 is substituted with A53, the aversive effects of a KOR agonist are attenuated. Here, we will expand and test our overarching hypothesis “Cocaine induces addiction- like behaviors when the KOR phosphorylates DAT at T53 and alters DA dynamics”. Aim 1 will investigate the effect of T53 phosphorylation on KOR-mediated DAT upregulation, trafficking and protein-protein interactions. Aim 1 will also examine if these PT53-DAT-dependent effects are sex-, and brain-region specific. Aim 2 will examine the impact of PT53-DAT on extracellular DA dynamics and DA release and clearance modulation by KOR and cocaine in vivo and in brain slices. Aim 3 will determine whether PT53-DAT plays a role in KOR- and cocaine- associated behaviors. Outcomes from the proposed studies will provide novel insights into the mechanisms of CUD, open new horizons for examining phosphorylation of DAT-T53 as an underlying mechanism of dynorphin actions in DA-signaling and cocaine addiction and expose new molecular therapeutic intervention points.