Kappa-Opioid Receptor Mediated Regulation of Dopamine Transport

描述（由申请人提供）：抑郁症和成瘾每年影响数百万人，给社会带来难以估量的成本。多巴胺转运蛋白（DAT）是一种清除释放到细胞外空间的多巴胺（DA）的膜蛋白，是临床使用的抗抑郁药和一些滥用药物的靶标。？-阿片受体（-opioid receptor， KOR）在大脑回路中富集，为情绪和动机提供服务，并调节位于其中的DA神经元的基础活动。KOR系统的上调与抑郁症和心境失调的发病机制有关，心境失调是可卡因和其他滥用药物戒断的特征。尽管正在努力开发口服有效的KOR配体来治疗抑郁症和成瘾，但这些药物影响行为和DA传递的下游效应物尚不清楚。KOR激动剂的不安和厌恶作用归因于腹侧纹状体（VST） DA释放减少。然而，重要的是，VST中的KOR与多巴胺转运体相反。我们的研究表明，KOR激活通过ERK1/2依赖于DAT的苏氨酸（Thr）53的磷酸化来增加DAT活性，而在VST中选择性抑制ERK1/2可减弱KOR激动剂的不良作用。这些发现确定了KOR配体调节突触前DA传递的新机制，并表明转运蛋白失调可能是KOR介导的情绪和情感改变的一种机制。本研究将验证以下假设：KOR激活通过erk1 /2依赖性磷酸化和DAT调控来调节DA动力学和vst依赖性行为。特异性目的1将通过确定kor介导的DAT功能和VST中表达的变化是否与Thr53磷酸化有关，以及阻止kor激动剂引起的ERK1/2激活和VST中DAT磷酸化是否会减弱kor介导的DAT功能变化，从而确定kor相关DAT调节的细胞机制。特异性目的2将通过确定阻止kor激动剂的操作是否诱发ERK1/2激活和DAT磷酸化改变基础DA动力学来确定该机制与突触前DA传递调节的相关性。特异性目的3将通过评估预防VST中KOR- erk1 /2相关的DAT调节是否会减弱KOR激动剂的厌恶和促抑郁作用，以及KOR拮抗剂的抗抑郁作用，来确定KOR- erk1 /2相关的DAT调节与KOR激动剂和拮抗剂的行为作用的生理相关性。还将评估KOR- erk1 /2相关的DAT调节在介导KOR激动剂预防可卡因运动刺激作用中的作用。这些研究的发现将增强我们对KOR配体调节情绪和DA传递的神经基质的理解。此外，它们将为DAT磷酸化在调节突触DA清除和行为中的作用提供新的见解。

DESCRIPTION (provided by applicant): Depression and addiction affect millions of individuals each year exerting untold costs on society. The dopamine transporter (DAT), a membrane protein that clears dopamine (DA) released into the extracellular space is a target of clinically used antidepressants and several drugs of abuse. ?-opioid receptors (KOR) are enriched in brain circuits that subserve mood and motivation and regulate the basal activity of DA neurons located therein. Upregulation of KOR systems has been implicated in the pathogenesis of depression and the mood dysregulation that characterizes withdrawal from cocaine and other drugs of abuse. Despite on-going efforts to develop orally effective KOR ligands for the treatment of depression and addiction, the downstream effectors upon which these agents act to affect behavior and DA transmission are unknown. The dysphoric and aversive effects of KOR agonists have been attributed to decreased DA release in the ventral striatum (VST). Importantly, however, KOR in VST are apposed to the dopamine transporter. Our studies show that KOR activation increases DAT activity through ERK1/2 dependent phosphorylation of threonine (Thr)53 of DAT and selective inhibition of ERK1/2 in the VST attenuates the aversive effects of KOR agonists. These findings identify a novel mechanism by which KOR ligands regulate presynaptic DA transmission and suggest that transporter dysregulation may be one mechanism underlying KOR-mediated alterations in mood and affect. The studies in this proposal will test the hypotheses that: KOR activation modulates DA dynamics and VST-dependent behaviors via ERK1/2-dependent phosphorylation and regulation of DAT. Specific Aim 1 will identify the cellular mechanisms of KOR-linked DAT modulation by determining whether KOR-mediated changes in DAT function and expression in the VST are associated with Thr53 phosphorylation and whether manipulations that prevent KOR-agonist evoked ERK1/2 activation and DAT phosphorylation in the VST attenuate KOR-mediated changes in DAT function. Specific Aim 2 will establish the relevance of this mechanism to the regulation of presynaptic DA transmission by determining whether manipulations that prevent KOR-agonist evoked ERK1/2 activation and DAT phosphorylation alter basal DA dynamics. Specific Aim 3 will determine the physiological relevance of KOR-ERK1/2 linked DAT modulation to the behavioral effects of KOR agonists and antagonists by assessing whether prevention of KOR-ERK1/2 linked DAT modulation in the VST attenuates the aversive and pro-depressive like effects of KOR agonists as well as the antidepressant-like effects of KOR antagonists. The role of KOR-ERK1/2 linked DAT modulation in mediating the efficacy of KOR agonists in preventing the locomotor stimulant effects of cocaine will also be assessed. Findings from these studies will enhance our understanding of the neural substrates upon which KOR ligands act to regulate mood and DA transmission. Furthermore, they will provide new insights as to the role of DAT phosphorylation in regulating synaptic DA clearance and behavior.