Kappa Opioid Receptors and Phospho-Dopamine Transporters Drive Cocaine Reward

摘要 在美国，近七分之一的人患有药物使用障碍，给医疗保健带来了沉重的负担 资源、经济和个人生活质量。目前，还没有有效的药物来治疗 可卡因使用障碍(CUD)，很可能是因为这种疾病背后的神经生物学很复杂，而不是 完全理解。中脑边缘环路中的多巴胺能神经传递已被证明在 在CUD中的关键作用。可卡因抑制多巴胺转运体(DAT)的活性，使其产生行为 效果。作为多巴胺信号的主要调节者，DAT的调节主要通过后 翻译修饰，如由突触前自身和异源基因触发的磷酸化。 受体连接的信号传递级联。这些修饰改变了可卡因与DAT的相互作用，导致 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，修改并不是调查的重点。我们的研究表明，蛋白的磷酸化 DAT的苏氨酸-53残基，或磷酸-T53-DAT(PT53-DAT)，在调节可卡因的生长发育过程中起着关键作用。 定向行为。在这个提案中，我们使用尖端技术来探索这一新发现，包括 病毒介导的磷酸化缺陷DAT-Thr53Ala突变体(DAT-A53)的敲入小鼠模型 脑区特异性阻断DAT-T53磷酸化，以及基于纤维光度法的测量 使用荧光传感器dLight的自由活动小鼠体内的多巴胺。有了这些新型号和量身定做的 生化、神经化学和行为研究，目前的建议旨在填补我们在 了解DAT磷酸化在正常的多巴胺神经传递和在 可卡因的存在，包括第一次在活体动物中。作为一个关键的机制，我们已经发现 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的厌恶效应 激动剂被减弱。在这里，我们将扩展和测试我们的首要假设：可卡因导致上瘾-- 当KOR在T53处使DAT磷酸化并改变DA动态时，类似的行为“。目标1将调查 T53磷酸化对KOR介导的DAT上调、转运和蛋白质相互作用的影响。 目标1还将检查这些PT53-DAT依赖的效应是否具有性别和大脑区域特异性。目标2将 检测PT53-DAT对细胞外DA动力学及DA释放和清除调节的影响 KOR和可卡因在体内和脑片中。目标3将确定PT53-DAT是否在KOR-AND中发挥作用 与可卡因相关的行为。拟议研究的结果将为我们提供对 CUD的机制为研究DAT-T53的磷酸化开辟了新的视野 强啡肽在DA信号转导和可卡因成瘾中的作用机制及新的分子治疗 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.