Identifying the Distinct Intracellular Pathways That Mediate Dopamine-Driven Behaviors

项目概要/摘要 多巴胺（DA），一种与学习和动机有关的神经递质，也是学习和动机的关键因素。 精神兴奋剂作用机制和药物滥用病理学。多巴胺结合五种受体（D1-D2）。 D5 R），其中D2 R具有特殊的相关性，因为它在不同的大脑区域中发挥各种功能作用 是所有抗精神病药物的目标。多巴胺受体是G蛋白偶联的 GPCR是激活细胞内G蛋白及其下游信号级联的GPCR。该信号 在抑制蛋白与受体的胞内结构域结合时终止。最近，逮捕 已经发现，通过将各种蛋白质支架在细胞表面， 受体激活这些不同的细胞内途径的功能作用尚不清楚。 D2 R在丘脑核的纹状体-苍白球中型棘神经元（spMSN）上表达， 它的功能是调节许多精神兴奋剂诱导的行为。先前对老鼠的研究表明， 在这些细胞中，arrestin通路能够介导运动，但不能介导动机，因此表明， 某些行为可由抑制蛋白独立于G蛋白信号传导来驱动。GSK-3β是一种激酶， 在抑制蛋白的下游被激活，并且对于某些可卡因诱导的行为的表达是必需的。 此外，在激活时，纹状体D2 R与NMDA受体NR 2B亚基二聚化，这是一种机制， 可能是GSK-3β依赖性的，似乎介导可卡因的条件性位置偏爱（CPP），但不是 食物在精神兴奋剂依赖者的死后纹状体切片中，D2 R/NMDAR的比例 尽管D2 R表达显著降低，但异聚体的表达比对照高约3倍。是 不清楚是什么介导了D2 R下游这些异聚体的形成。 我推测，在spMSNs中，D2 R驱动的arrestin和下游GSK-3β信号传导介导可卡因 而不是通过促进D2 R-NR 2B异聚化和转录组学变化的食物奖励加工。我会 通过以下具体目标解决这一假设：目标1将确定D2 R驱动的 可卡因与食物奖励加工中的arrestin和GSK-3β信号传导。我会用CRISPR敲除β- 在成年小鼠的spMSNs中的抑制蛋白2或GSK-3β，并测定与 愿意为食物和可卡因而工作。Aim 2将使用相同的淘汰策略进行调查 慢性精神兴奋剂作用下β arr 2和GSK-3β介导的分子和转录组学变化 exposure.我将分析离体NAc组织的NR 2B-D2 R异聚化，以及GSK-3β和NR 2B 磷酸化，并在慢性可卡因暴露后对spMSNs进行转录组学分析， RNAseq。总之，拟议的实验将提供深入了解D2 R驱动的分子基础。 行为，并确定开发多巴胺相关疾病药物的新途径。

PROJECT SUMMARY/ABSTRACT Dopamine (DA), a neurotransmitter implicated in learning and motivation, is also a critical player in the mechanisms of psychostimulant action and the pathology of drug abuse. Dopamine binds five receptors (D1- D5R), of which D2R carries particular relevance, as it takes on various functional roles in different brain regions and cells types and is a target of all antipsychotic medications. Dopamine receptors are G protein-coupled receptors (GPCRs) that activate intracellular G proteins and their downstream signaling cascades. This signal is terminated upon arrestin binding to the intracellular domain of the receptor. More recently, arrestins have been found to initiate their own G protein-independent signaling cascades by scaffolding various proteins upon receptor activation. The functional roles of these distinct intracellular pathways are unclear. D2R is expressed on striato-pallidal medium spiny neurons (spMSNs) of the nucleus accumbens, where it functions to mediate many psychostimulant-induced behaviors. Previous research in mice has shown that in these cells the arrestin pathway is able to mediate locomotion but not motivation, thus indicating that certain behaviors may be driven by arrestin independently of G protein signaling. GSK-3β, a kinase, is activated downstream of arrestin and is essential for the expression of certain cocaine-induced behaviors. Additionally, upon activation, striatal D2Rs dimerize with the NMDA receptor NR2B subunit, a mechanism that may be GSK-3β-dependent and seems to mediate conditioned place preference (CPP) for cocaine but not food. In post-mortem striatal sections from psychostimulant dependent humans, the proportion of D2R/NMDAR heteromers was ~3-fold higher compared to controls despite a substantial decrease of D2R expression. It is not clear what mediates the formation of these heteromers downstream of D2R. I hypothesize that, in spMSNs, D2R-driven arrestin and downstream GSK-3β signaling mediate cocaine but not food reward processing by facilitating D2R-NR2B heteromerization and transcriptomic changes. I will address this hypothesis through the following Specific Aims: Aim 1 will determine the roles of D2R-driven arrestin and GSK-3β signaling in cocaine versus food reward processing. I will use CRISPR to knockout β- arrestin 2 or GSK-3β in spMSNs of adult mice and assay preference for locations associated with, and willingness to work for, a food reward and cocaine. Aim 2 will use the same knockout strategy to investigate the molecular and transcriptomic changes mediated by βarr2 and GSK-3β upon chronic psychostimulant exposure. I will analyze ex vivo NAc tissue for NR2B-D2R heteromerization, as well as GSK-3β and NR2B phosphorylation, and perform transcriptomic analysis on spMSNs after chronic cocaine exposure using nuclear RNAseq. Together, the proposed experiments will provide insight into the molecular basis for D2R-driven behaviors and identify novel avenues for developing drugs for dopamine-related diseases.