Synthesis and Degradation of Synaptic Proteins During Cocaine Withdrawal

可卡因戒断过程中突触蛋白的合成和降解

• 批准号: 8842876
• 负责人: Craig Thomas Werner
• 金额: $ 2.68万
• 依托单位: ROSALIND FRANKLIN UNIV OF MEDICINE & SCI
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-20 至 2015-09-07
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8842876
• 关键词: AMPA Receptors; Abstinence; Accounting; Adult; Animal Model; Biological Assay; Cell surface; Co-Immunoprecipitations; Cocaine; Cocaine Dependence; Cocaine Users; Coupled; Cues; Detection; Drug usage; Genetic Translation; Glutamates; Goals; Incubated; Individual; Injection of therapeutic agent; Lead; Learning; Measures; Mediating; Memory; Mentors; Messenger RNA; Methods; Minority; Mission; Modeling; Motor; Neurons; Nucleus Accumbens; Permeability; Pharmaceutical Preparations; Polyribosomes; Protein Biosynthesis; Proteins; Rattus; Relapse; Research; Retrieval; Reverse Transcriptase Polymerase Chain Reaction; Role; Saline; Self Administration; Self-Administered; Subcellular Fractions; Surface; Synapses; Synaptic plasticity; Synaptosomes; System; Techniques; Testing; The Sun; Time; Tissues; Training; Translations; Ubiquitin; Withdrawal; Wolves; Work; addiction; burden of illness; craving; density; disorder later incidence prevention; drug withdrawal; innovation; interest; memory retrieval; multicatalytic endopeptidase complex; new therapeutic target; novel; postsynaptic; preference; prevent; protein degradation; public health relevance; receptor; research study; response; skills; therapeutic target; AMPA Receptors; Abstinence; Accounting; Adult; Animal Model; Biological Assay; Cell surface; Co-Immunoprecipitations; Cocaine; Cocaine Dependence; Cocaine Users; Coupled; Cues; Detection; Drug usage; Genetic Translation; Glutamates; Goals; Incubated; Individual; Injection of therapeutic agent; Lead; Learning; Measures; Mediating; Memory; Mentors; Messenger RNA; Methods; Minority; Mission; Modeling; Motor; Neurons; Nucleus Accumbens; Permeability; Pharmaceutical Preparations; Polyribosomes; Protein Biosynthesis; Proteins; Rattus; Relapse; Research; Retrieval; Reverse Transcriptase Polymerase Chain Reaction; Role; Saline; Self Administration; Self-Administered; Subcellular Fractions; Surface; Synapses; Synaptic plasticity; Synaptosomes; System; Techniques; Testing; The Sun; Time; Tissues; Training; Translations; Ubiquitin; Withdrawal; Wolves; Work; addiction; burden of illness; craving; density; disorder later incidence prevention; drug withdrawal; innovation; interest; memory retrieval; multicatalytic endopeptidase complex; new therapeutic target; novel; postsynaptic; preference; prevent; protein degradation; public health relevance; receptor; research study; response; skills; therapeutic target

DESCRIPTION (provided by applicant): Cocaine addicts have a long-term vulnerability to relapse during abstinence, which may be triggered by drug-associated cues. To identify therapeutic targets for prevention of relapse, the synaptic plasticity that occurs during withdrawal must be better understood. In the incubation animal model of addiction, rats show a progressive increase in cue-induced craving after withdrawal from extended access self-administration, a phenomenon termed 'incubation'. Previous work in my lab revealed that high conductance Ca2+-permeable (CP) AMPA receptors (AMPARs) accumulate at synapses in the nucleus accumbens (NAc) during prolonged withdrawal and mediate the expression of 'incubated' cue-induced craving. The accumulation of CP-AMPARs in the NAc during incubation coincided with an increase in total and cell surface levels of GluA1, but not GluA2; furthermore, co-immunoprecipitation studies indicated an increase in GluA1 homomers. These findings suggest that an increase in GluA1 protein mediates formation of CP-AMPARs during incubation, and that most of the new CP-AMPARs are GluA1 homomers. The mechanism(s) that underlie this increase in GluA1 protein levels are unknown, but major candidates include persistent changes in protein synthesis and protein degradation during withdrawal. My central hypothesis is that incubation is associated with increased GluA1 synthesis coupled with altered ubiquitin-proteasome system (UPS) dependent protein degradation. This will be tested through three aims. The first two aims will be conducted in rats after 45-55 days of withdrawal from extended access cocaine or saline self-administration, when synaptic CP-AMPAR levels are elevated in the NAc of rats that self-administered cocaine. Aim 1 will determine if enhanced GluA1 mRNA translation and/or an increase in dendritic GluA1 mRNA levels occurs in the NAc during incubation. I will use polysome profile analysis to measure GluA1 mRNA translation rate and prepare synaptoneurosomes from NAc tissue to quantify dendritic GluA1 mRNA. These are two techniques that have not been utilized in studying cocaine- induced plasticity. Aim 2 will determine whether persistent changes in proteasome activity occur in the NAc during incubation. There have been no prior studies on the role of the UPS in plasticity elicited by cocaine self- administration and withdrawal. I will quantify polyubiquitinated proteins targeted fo proteasomal degradation using GST pull-downs and measure proteasome activity using fluorogenic assays. In Aim 3, I will determine whether UPS activity in the NAc is altered during the expression of 'incubated' cue-induced cocaine seeking, in other words, after memory retrieval, using the same methods as in Aim 2. This proposal will better our understanding of the plasticity that underlies enhancement of craving during withdrawal and the plasticity involved in retrieval of a memory that elicits cocaine craving. While these studies are underway, I will participate in a Training Plan that employs coursework, individual mentoring, and collaborative interactions to develop the non-bench skills needed to reach my goal of becoming a PI in an academic setting.

描述（由申请人提供）：可卡因成瘾者在禁欲期间具有长期脆弱性，可能是由药物相关的提示触发的。为了确定预防复发的治疗靶标，必须更好地理解戒断期间发生的突触可塑性。在成瘾的孵化动物模型中，大鼠在退出扩展访问自我给药后提示引起的渴望逐渐增加，这是一种称为“孵化”的现象。我实验室中的先前工作表明，高电导率Ca2+可渗透（CP）AMPA受体（AMPAR）在长时间戒断期间积聚在伏伏核（NAC）的突触处，并介导“孵育”提示诱导的渴望的表达。在孵育过程中，NAC中CP-Ampar的积累与GLUA1的总和细胞表面水平的升高相吻合，而不是GLUA2。此外，共免疫沉淀研究表明GLUA1同源物的增加。这些发现表明，GLUA1蛋白在孵育过程中介导了CP-AMPAR的形成，并且大多数新的CP-Ampars都是GLUA1同源物。 GLUA1蛋白水平增加的基础的机制尚不清楚，但主要候选者包括戒断过程中蛋白质合成和蛋白质降解的持续变化。我的中心假设是，孵育与GLUA1合成增加以及泛素蛋白 - 蛋白酶体系统（UPS）依赖性蛋白质降解相关。这将通过三个目标进行测试。在从扩展的可卡因或盐水自我管理中退出45-55天后，将在大鼠中进行前两个目标，当突触CP-Ampar水平升高到自我管理可卡因的大鼠的NAC中。 AIM 1将确定在孵育过程中NAC中发生了增强的GLUA1 mRNA翻译和/或树突状GLUA1 mRNA水平的增加。我将使用多聚体谱分析来测量GLUA1 mRNA翻译速率并制备从NAC组织的突触式介体以量化树突状GLUA1 mRNA。这是研究可卡因诱导的可塑性的两种技术。 AIM 2将确定蛋白酶体活动的持续变化在孵育过程中是否发生在NAC中。先前没有关于可卡因自我给药和戒断引起的UPS在可塑性中的作用的研究。我将使用GST下拉降低靶向FO蛋白酶体降解并使用荧光测定来测量蛋白酶体活动。在AIM 3中，我将确定使用与AIM 2中相同的方法在“孵化”提示引起的可卡因寻求可卡因期间是否改变了NAC中的UPS活性。该提议将更好地理解我们对在撤回和恢复cocain cocain cocain cocain cocain cocain cocain cocain cocain cocain cocain cocain cocain cocain cocain cocain cocain cocain cocain cocain的理解。尽管正在进行这些研究，但我将参加一项培训计划，该计划采用课程工作，个人指导和协作互动来发展在学术环境中成为PI的目标所需的非基础技能。