Identifying the Distinct Intracellular Pathways That Mediate Dopamine-Driven Behaviors

项目摘要/摘要 多巴胺(DA)是一种与学习和动机有关的神经递质，也是 精神刺激作用的机制和药物滥用的病理学。多巴胺与五种受体结合(d1- D5R)，其中D2R具有特别的相关性，因为它在不同的大脑区域扮演着不同的功能角色 和细胞类型，是所有抗精神病药物的靶点。多巴胺受体是G蛋白偶联的 激活细胞内G蛋白及其下游信号级联的受体(GPCRs)。这个信号 在arrestin与受体的胞内结构域结合时终止。最近，逮捕行动已经 被发现通过将不同的蛋白支架在G蛋白上启动他们自己的G蛋白不依赖的信号级联 受体激活。这些不同的细胞内通路的功能作用尚不清楚。 D2R在伏核的纹状-苍白球中棘神经元(SpMSN)上表达， 在那里它起着调节许多精神刺激剂诱导的行为的作用。先前对小鼠的研究表明 在这些细胞中，arrestin途径能够调节运动，但不能调节动机，因此表明 某些行为可能是由arrestin驱动的，而不是G蛋白信号。葛兰素史克-3β，一种激酶，是 在Arrestin下游被激活，对某些可卡因诱导的行为的表达是必不可少的。 此外，一旦激活，纹状体D2Rs与NMDA受体NR2B亚单位二聚化，这一机制 可能依赖葛兰素史克-3β，似乎介导了对可卡因的条件性位置偏爱，但不是 食物。在精神刺激依赖者的死后纹状体切片中，D2R/NMDAR的比例 尽管D2R的表达显著降低，但异构体的表达比对照组高出约3倍。它是 尚不清楚是什么中介了D2R下游这些异构体的形成。 我推测，在SPMSN中，D2R驱动的arrestin和下游的GSK-3β信号介导可卡因 但不是通过促进D2R-NR2B异构化和转录变化来进行食物奖励处理。这就做 通过以下具体目标解决这一假设：目标1将确定D2R驱动的角色 可卡因与食物奖赏过程中的arrestin和gsk-3β信号传递。我会用CRISPR击倒β- 成年小鼠SPMSN中Arrestin 2或GSK-3β的表达以及与 愿意为之工作，食物奖励和可卡因。目标2将使用相同的击倒策略来调查 βARR2和GSK-3β对慢性精神刺激剂分子和转录的影响 曝光。我将分析体外NAC组织中NR2B-D2R的异构化，以及GSK-3β和NR2B 磷酸化，并对慢性可卡因暴露后的spMSN进行转录分析 RNAseq.总之，拟议的实验将为D2R驱动的分子基础提供洞察力 并确定开发治疗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.