Kappa Opioid Receptors and Phospho-Dopamine Transporters Drive Cocaine Reward

Kappa 阿片受体和磷酸多巴胺转运蛋白驱动可卡因奖励

• 批准号: 10467723
• 负责人: LANKUPALLE D JAYANTHI
• 金额: $ 65.73万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-15 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10467723
• 关键词: Affect; Affinity; Animals; Attenuated; Behavior; Behavioral; Biochemical; Brain; Brain region; Chronic; Cocaine; Cocaine Dependence; Cocaine use disorder; Complex; Corpus striatum structure; Disease; Dopamine; Dose; Dynorphins; Etiology; Fiber; Gene Delivery; Gene Expression; Goals; Healthcare; Individual; Intake; Interruption; Investigation; Knock-in Mouse; Knowledge; Ligands; Link; Measurement; Measures; Mediating; Microdialysis; Modeling; Modification; Molecular; Mus; Mutant Strains Mice; Neurobiology; Neurons; Nucleus Accumbens; Opioid Antagonist; Opioid agonist; Outcome; Pathogenesis; Periodicity; Persons; Pharmaceutical Preparations; Pharmacology; Phosphorylation; Photometry; Play; Post-Translational Protein Processing; Pre-Clinical Model; Proteins; Quality of life; Receptor Activation; Receptor Signaling; Regulation; Relapse; Reporting; Resources; Retroviral Vector; Rewards; Role; Scanning; Signal Transduction; Site; Slice; Societies; Stress; Substance Use Disorder; Surface; Swimming; Synaptosomes; System; Techniques; Testing; Therapeutic; Therapeutic Intervention; Threonine; Time; Up-Regulation; Viral; Wild Type Mouse; addiction; base; behavioral study; cocaine exposure; cocaine relapse; conditioned place preference; dopamine transporter; dopaminergic neuron; drug seeking behavior; extracellular; in vivo; insight; kappa opioid receptors; mouse model; mutant; negative affect; neurochemistry; neurotransmission; novel; optogenetics; presynaptic; protein protein interaction; receptor; regional difference; response; sensor; sex; syntaxin 1A; therapeutically effective; trafficking; transmission process; uptake

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.

概括 在美国，药物使用障碍涉及几乎七分之一的人，对医疗保健施加了沉重的烧伤 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），很可能是因为该疾病的神经生物学很复杂，而不是 完全理解。在中脑链纤维电路中的多巴胺能神经传递已显示出一种 在CUD中的关键作用。可卡因抑制多巴胺转运蛋白（DAT）的活性产生其行为 效果。 DAT的调节是多巴胺信号传导的主要调节剂，是通过后发生的 转化修饰，例如磷酸化，这些磷酸化是由突触前的自动和异质触发的 受体连接的信号级联。这些修改改变了可卡因与DAT的相互作用， 改变可卡因的刺激和奖励作用。但是，迄今为止，翻译后 修改并不是CUD领域调查的重点。我们的研究表明， DAT或磷酸-T53-DAT（PT53-DAT）的苏氨酸-53居住在调节可卡因方面起着关键作用 定向行为。在此提案中，我们使用尖端技术探索了这一小说发现，包括 用磷酸化缺陷DAT-THR53ALA突变体（DAT-A53）敲入小鼠模型，病毒介导 DAT-T53辐射的大脑区域特异性阻断，以及基于光纤光度法的测量值 使用荧光传感器Dlight的自由移动小鼠中的多巴胺。这些新型号和量身定制 生化，神经化学和行为研究，目前的提议旨在填补我们的关键空白 了解DAT磷酸化在正常多巴胺神经传递中的核心作用以及 可卡因的存在，包括第一次在活动物中。作为关键机制，我们发现 Kappa-Apoid受体（KOR）通过PT53-DAT调节DAT。我们的研究还表明，kor激活 通过PT53-DAT增加DAT活动，当T53用A53取代时，Kor的厌恶效应 激动剂被减弱。在这里，我们将扩展和检验我们的总体假设“可卡因诱导成瘾 - 就像行为一样，kor磷酸化在T53处磷酸化并改变了DA动力学”。AIM 1将研究 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。 AIM 1还将检查这些PT53-DAT依赖性作用是否为性别和大脑区域。 AIM 2意志 检查PT53-DAT对细胞外DA动力学和DA释放以及清除调制的影响 kor和可卡因在体内以及脑切片中。 AIM 3将确定PT53-DAT是否在KOR和KOR-和 可卡因相关的行为。拟议研究的结果将为您提供新的见解 CUD的机制，开放的新视野，用于检查Dat-T53的磷酸化作为基础 DA信号和可卡因成瘾中的动酚作用机制，并暴露了新的分子疗法 干预点。