Kappa-Opioid Receptor Mediated Regulation of Dopamine Transport

描述(由申请者提供)：抑郁症和成瘾每年影响数百万人，给社会带来难以估量的代价。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，是临床上使用的抗抑郁药物和几种滥用药物的靶标。？-阿片受体(KOR)丰富于大脑回路，抑制情绪和动机，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情绪失调的发病机制有关，情绪失调是戒除可卡因和其他滥用药物的特征。尽管人们一直在努力开发口服有效的KOR配体来治疗抑郁症和成瘾，但这些药物作用于哪些下游效应器来影响行为和DA传递尚不清楚。KOR激动剂的烦躁和厌恶作用归因于腹侧纹状体(VST)DA释放的减少。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的激活通过依赖于DAT的苏氨酸(Thr)53的ERK1/2磷酸化来增强DAT的活性，而选择性地抑制VST中的ERK1/2可以减弱KOR激动剂的厌恶效应。这些发现确定了KOR配体调节突触前DA传递的新机制，并提示转运蛋白调节失调可能是KOR介导的情绪和情感改变的机制之一。该方案中的研究将验证以下假设：KOR激活通过ERK1/2依赖的磷酸化和DAT的调节来调节DA动力学和VST依赖的行为。具体目的1将通过确定KOR介导的VST中DAT功能和表达的变化是否与Thr53磷酸化有关，以及阻止KOR激动剂引起的VST中ERK1/2激活和DAT磷酸化的操作是否减弱KOR介导的DAT功能变化来确定KOR连锁的DAT调节的细胞机制。具体目标2将通过确定阻止KOR激动剂诱发ERK1/2激活和DAT磷酸化的操作是否改变基础DA动力学来确定这一机制与突触前DA传递的调节的相关性。具体目标3将通过评估在VST中预防KOR-ERK1/2连锁DAT调制是否减弱KOR激动剂和KOR拮抗剂的厌恶和促进抑郁的类似效应，来确定KOR-ERK1/2连锁的DAT调节与KOR激动剂和拮抗剂的行为效应的生理学相关性。KOR-ERK1/2连接的DAT调节在调节KOR激动剂预防可卡因的运动刺激效应中的作用也将被评估。这些研究的结果将加强我们对KOR配体调节情绪和DA传递的神经底物的理解。此外，他们将为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。 与公共健康相关：Kappa-阿片受体在大脑回路中丰富，抑制情绪和动机，并调节位于其中的多巴胺能和5-羟色胺能神经元的基本活动。这项拟议的研究将检验这一假说，即kappa-阿片受体通过激活调节多巴胺转运体功能的激酶级联来影响单胺传递。该提案的结果将有助于确定β-阿片受体调节多巴胺传递的分子靶点，并有助于开发有效的药理药物来治疗成瘾和其他由单胺传递异常引起的疾病状态。

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. PUBLIC HEALTH RELEVANCE: Kappa-opioid receptors are enriched in brain circuits that subserve mood and motivation and regulate the basal activity of both dopaminergic and serotoninergic neurons located therein. The proposed studies will test the hypothesis that kappa-opioid receptors affect monoamine transmission by activating kinase cascades that regulate the function of dopamine transporters. Outcomes from the proposal will enable identification of the molecular targets by which ?-opioid receptors regulate dopamine transmission and aid in the development of effective pharmacological agents for the treatment of addiction and other disease states resulting from aberrant monoamine transmission.