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

基本信息

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
项目类别：
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

项目摘要

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.

项目摘要/摘要多巴胺（DA）是一种在学习和动机中实施的神经递质，也是关键参与者精神刺激作用的机制和药物滥用的病理。多巴胺结合五个受体（D1- D5R），其中D2R具有特殊的相关性，因为它在不同的大脑区域扮演了各种功能角色和细胞类型，是所有抗精神病药的靶标。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）。这个信号终止于抑制蛋白与接收器的细胞内结构域结合。最近，逮捕了发现它们可以通过在受体激活。这些不同细胞内途径的功能作用尚不清楚。 D2R在伏隔核的纹状体 - 胶合培养基神经元（SPMSN）上表达它在介导许多精神刺激诱导的行为的功能中起作用。先前在小鼠中的研究表明在这些细胞中，阻止素途径能够介导运动，但不能动机，因此表明某些行为可能是由与G蛋白信号传导无关的阻止驱动的。 GSK-3β，一种激酶，是被抑制蛋白的下游激活，对于表达某些可卡因诱导的行为至关重要。此外，激活后，纹状体D2Rs用NMDA受体NR2B亚基二聚（一种机制）可能是GSK-3β依赖性的，似乎可以介导可卡因的条件位置偏好（CPP），但不是食物。在来自精神刺激依赖人类的验尸后纹状体部分中，D2R/NMDAR的比例与对照组的目的地相比，杂体高约3倍，D2R表达大大降低。这是不清楚是什么介导了D2R下游的这些异构体的形成。我假设在SPMSN中，D2R驱动的阻止蛋白和下游GSK-3β信号介导可卡因但是，不是通过促进D2R-NR2B异构化和转录组变化来进行食物奖励处理。我会通过以下特定目的解决这一假设：AIM 1将确定D2R驱动的作用可卡因与食品奖励处理中的逮捕蛋白和GSK-3β信号传导。我将使用CRISPR敲除β- 在成年小鼠的SPMSN中阻止蛋白2或GSK-3β，并分析与与之相关的位置的偏好愿意工作的意愿，食物奖励和可卡因。 AIM 2将使用相同的淘汰赛策略进行调查慢性精神刺激剂对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异构化，以及GSK-3β和NR2B 磷酸化并在慢性可卡因暴露后对SPMSN进行转录组分析 rnaseq。一起，提出的实验将提供有关D2R驱动的分子基础的洞察力行为并确定用于开发与多巴胺相关疾病的药物的新途径。