Identifying the Distinct Intracellular Pathways That Mediate Dopamine-Driven Behaviors

确定介导多巴胺驱动行为的独特细胞内途径

• 批准号: 10477942
• 负责人: CAROLINE RAUFFENBART
• 金额: $ 4.68万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10477942
• 关键词: Address; Adult; Antipsychotic Agents; Arrestins; Automobile Driving; Autopsy; Behavior; Behavioral; Binding; Biological Assay; Brain; Brain region; Categories; Cells; Chronic; Clustered Regularly Interspaced Short Palindromic Repeats; Cocaine; Collaborations; Complex; Corpus striatum structure; DRD2 gene; Data; Development; Disease; Dopamine; Dopamine Receptor; Dopaminergic Agents; Drug abuse; Engineering; Exhibits; Food; Future; G-Protein-Coupled Receptors; GTP-Binding Proteins; Gene Expression; Genes; Human; Investigation; Knock-out; Knockout Mice; Learning; Location; Locomotion; Mediating; Mediator of activation protein; Molecular; Motivation; Mus; NR2B NMDA receptor; Neurons; Neurotransmitters; Nuclear; Nucleus Accumbens; Pathology; Pathway interactions; Pharmaceutical Preparations; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Process; Protein Phosphatase 2A Regulatory Subunit PR53; Proteins; RNA Interference; Receptor Activation; Recording of previous events; Research; Rewards; Role; Signal Pathway; Signal Transduction; Signaling Protein; Surface; Tissues; Treatment Efficacy; Up-Regulation; Work; arrestin 2; beta-arrestin; brain cell; cell type; cocaine exposure; cocaine self-administration; conditioned place preference; design; differential expression; dimer; experimental study; glycogen synthase kinase 3 beta; human subject; insight; network models; novel; preference; prevent; psychostimulant; putamen; receptor; recruit; reward processing; scaffold; side effect; stimulant dependence; stimulant exposure; transcriptome; transcriptome sequencing; transcriptomics; willingness

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.

项目概要/摘要 多巴胺 (DA) 是一种与学习和动机有关的神经递质，也是 精神兴奋作用机制和药物滥用病理学。多巴胺结合五种受体（D1- D5R），其中 D2R 具有特殊的相关性，因为它在不同的大脑区域中发挥着不同的功能作用 和细胞类型，是所有抗精神病药物的目标。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 (GPCR)。这个信号 当抑制蛋白与受体的胞内结构域结合时终止。最近，抑制素已 已发现通过在其上搭建各种蛋白质来启动其自己的不依赖于 G 蛋白的信号级联 受体激活。这些不同的细胞内途径的功能作用尚不清楚。 D2R 在伏隔核的纹状体苍白球中型多棘神经元 (spMSN) 上表达， 它的作用是调节许多精神兴奋剂引起的行为。先前对小鼠的研究表明 在这些细胞中，抑制蛋白途径能够介导运动，但不能介导动机，因此表明 某些行为可能是由抑制蛋白驱动的，与 G 蛋白信号传导无关。 GSK-3β 是一种激酶 抑制蛋白下游被激活，对于某些可卡因诱导行为的表达至关重要。 此外，激活后，纹状体 D2R 与 NMDA 受体 NR2B 亚基形成二聚体，这一机制 可能是 GSK-3β 依赖性的，似乎介导可卡因的条件性位置偏好 (CPP)，但不是 食物。在精神兴奋剂依赖性人类的死后纹状体切片中，D2R/NMDAR 的比例 尽管 D2R 表达大幅下降，但异聚体的含量比对照高约 3 倍。这是 尚不清楚是什么介导了 D2R 下游这些异聚体的形成。 我假设，在 spMSN 中，D2R 驱动的抑制蛋白和下游 GSK-3β 信号传导可卡因 但不是通过促进 D2R-NR2B 异聚化和转录组变化来进行食物奖励处理。我会 通过以下具体目标来解决这一假设： 目标 1 将确定 D2R 驱动的角色 可卡因与食物奖励处理中的抑制蛋白和 GSK-3β 信号传导。我将使用 CRISPR 敲除 β- 成年小鼠 spMSN 中的抑制蛋白 2 或 GS​​K-3β 以及相关位置的测定偏好，以及 愿意为之工作、食物奖励和可卡因。目标2将使用相同的淘汰赛策略进行调查 慢性精神兴奋剂βarr2和GSK-3β介导的分子和转录组变化 接触。我将分析离体 NAc 组织的 NR2B-D2R 异聚化，以及 GSK-3β 和 NR2B 磷酸化，并在慢性可卡因暴露后使用核对 spMSN 进行转录组分析 RNA测序。总之，所提出的实验将深入了解 D2R 驱动的分子基础 行为并确定开发治疗多巴胺相关疾病药物的新途径。