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

基本信息

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
项目类别：
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

项目摘要

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，近七分之一的人患有药物滥用障碍，给医疗保健带来沉重负担目前，尚无有效的药物可用于治疗资源、经济和个人生活质量。可卡因使用障碍 (CUD)，很可能是因为该疾病背后的神经生物学很复杂，而且不中脑边缘回路中的多巴胺能神经传递已被证明发挥着重要作用。可卡因抑制多巴胺转运蛋白 (DAT) 的活性以产生其行为。 DAT 是多巴胺信号传导的主要调节因子，其调节主要通过后作用发生。翻译修饰，例如磷酸化，由突触前自动和异源触发这些修饰改变了可卡因与 DAT 的相互作用，导致然而，迄今为止，可卡因的刺激和奖励作用的变化。我们的研究表明，修饰尚未成为 CUD 领域研究的焦点。 DAT 的苏氨酸 53 残基或磷酸化 T53-DAT (PT53-DAT) 在可卡因- 在本提案中，我们使用尖端技术探索这一新颖的发现，包括具有磷酸化缺陷型 DAT-Thr53Ala 突变体 (DAT-A53) 的敲入小鼠模型，这是一种病毒介导的大脑区域特异性阻断 DAT-T53 磷酸化，以及基于光纤光度法的测量使用荧光传感器 dLight 在自由活动的小鼠中检测多巴胺，并定制了这些新模型。生物化学、神经化学和行为研究，当前的提案旨在填补我们在了解 DAT 磷酸化在正常多巴胺神经传递和可卡因的存在，包括在活体动物中的首次存在，作为一个关键机制，我们发现了这一点。 kappa-阿片受体 (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 活性，当 T53 被 A53 取代时，KOR 的厌恶作用在这里，我们将扩展并测试我们的总体假设“可卡因会导致成瘾- 当 KOR 在 T53 处磷酸化 DAT 并改变 DA 动态时，类似的行为”将进行研究。 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。目标 1 还将检查这些 PT53-DAT 依赖性效应是否具有性别和大脑区域特异性。检查 PT53-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 和可卡因将确定 PT53-DAT 是否在 KOR- 和脑切片中发挥作用。拟议研究的结果将为可卡因相关行为提供新的见解。 CUD 机制，为检查 DAT-T53 磷酸化作为基础开辟了新视野强啡肽在 DA 信号传导和可卡因成瘾中的作用机制，并揭示新的分子疗法干预点。