OPHN1 translation and AMPA receptor plasticity during incubation of craving

渴望孵化过程中 OPHN1 翻译和 AMPA 受体可塑性

• 批准号: 9911677
• 负责人: Alexander Borg Kawa
• 金额: $ 6.49万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-02 至 2022-03-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9911677
• 关键词: ADORA2A gene; AMPA Receptors; Abstinence; Acute; Affinity Chromatography; Anisomycin; Automobile Driving; Bathing; Biological Assay; Brain; Cocaine; Cocaine Users; Coupled; Cues; Dendrites; Drug usage; Electrophysiology (science); Enterobacteria phage P1 Cre recombinase; Eukaryotic Initiation Factors; Excision; Exhibits; Exposure to; Female; GRM1 gene; Genetic Translation; Goals; Hippocampus (Brain); Hour; Immunoblotting; Incidence; Incubated; Individual; Infusion procedures; Injections; Label; Ligation; Maintenance; Measures; Mediating; Messenger RNA; Methods; Mission; Modeling; Neurons; Nucleus Accumbens; Open Reading Frames; Pathway interactions; Permeability; Pharmaceutical Preparations; Pharmacology; Phosphorylation; Physiology; Play; Procedures; Protein Dephosphorylation; Proteins; Puromycin; Quantitative Reverse Transcriptase PCR; Rattus; Relapse; Research; Ribosomes; Role; Saline; Self Administration; Signal Pathway; Signal Transduction; Slice; Surface; Synapses; Techniques; Testing; Therapeutic Intervention; Time; Training; Translating; Translations; United States National Institutes of Health; Viral; Withdrawal; Work; addiction; cocaine exposure; cocaine use; craving; experience; inhibitor/antagonist; knock-down; male; metabotropic glutamate receptor type 1; neuropsychiatric disorder; new therapeutic target; novel; positive allosteric modulator; prevent; receptor; response; skills

Project Summary A major problem for individuals suffering from addiction is the persistent vulnerability to relapse, even after long periods of abstinence. In the `incubation of cocaine craving' model of relapse, rats self-administer cocaine using an extended access procedure, and then experience a prolonged abstinence period. During abstinence, rats exhibit a progressive intensification (incubation) of cue-induced cocaine craving. We have shown that Ca2+-permeable AMPA receptors (CP-AMPAR), comprised exclusively of the GluA1 subunit, accumulate in the nucleus accumbens core (NAcc) during abstinence and thereafter are required for the expression of incubated cue-induced craving. Thus, understanding the mechanisms regulating CP-AMPAR maintenance and removal may yield novel therapeutic targets for reducing craving and prolonging abstinence. Work from our lab has shown that CP-AMPAR mediated currents in the NAcc require active protein translation, as they are blocked by general protein translation inhibitors. Also, treatment with a general protein translation inhibitor just before the cue-induced craving test reduces incubated seeking. However, little is known about the specifics of this critical protein translation. Under some conditions, inhibition of general protein translation actually increases the translation of a subset of mRNA with 5' upstream open reading frames, such as Oligophrenin-1 (OPHN1). In the hippocampus, OPHN1 is necessary for eIF2α-mediated mGluR-LTD and the removal of synaptic AMPARs. In the VTA, this pathway plays a role in bidirectional CP-AMPAR plasticity in response to i.p. cocaine exposure. Thus, in our NAcc studies, treatment with protein translation inhibitors may have increased translation of OPHN1, mimicking mGluR-LTD and removing synaptic CP-AMPARs. My hypothesis is that, following cocaine self-administration and prolonged abstinence, OPHN1 translation is low in the NAcc, permitting the accumulation and maintenance of CP-AMPARs. In addition, in incubated rats during the seeking test, OPHN1 translation is reduced due to eIF2α-dephosphorylation, enabling CP-AMPARs to stay in synapses during the test and mediate incubated seeking. Aim 1 will determine if OPHN1 translation is dysregulated in incubated rats using viral Translating Ribosome Affinity Purification (vTRAP) coupled with qRT-PCR to quantify Ophn1, Gria1 and Gria2 mRNA during incubation of cocaine craving. Changes in mRNA will be compared to changes in newly translated proteins using puromycin-labeling of nascent proteins and immunoblotting. I will also use several techniques to localize the critical translation. Aim 2 will determine the role of OPHN1 in the expression of incubated seeking. Here I will use a similar vTRAP approach to examine actively translated mRNAs before and after a seeking test in incubated rats. Also, I will experimentally knockdown OPHN1 to test if it is necessary for mGlu1-LTD-mediated removal of CP-AMPARs and normalization of incubated seeking. While these studies are underway, I will participate in a multi-faceted training plan to develop the non-bench skills needed to reach my goal of becoming a PI in an academic setting.

项目概要 对于患有成瘾的人来说，一个主要问题是持续容易复发，即使 在长期戒断后的“可卡因渴望孵化”模型中，老鼠自我给药。 使用延长获取程序的可卡因，然后经历长时间的戒断期。 戒断后，老鼠表现出对线索诱导的可卡因渴望的逐渐增强（潜伏期）。 研究表明，Ca2+ 通透性 AMPA 受体 (CP-AMPAR) 仅由 GluA1 亚基组成， 禁欲期间在伏隔核 (NAcc) 中积累，此后为 因此，了解调节 CP-AMPAR 的机制。 维持和去除可能会产生减少渴望和延长戒断时间的新治疗目标。 我们实验室的工作表明 CP-AMPAR 介导的 NAcc 电流需要活性蛋白质翻译， 因为它们被一般蛋白质翻译抑制剂阻断。另外，用一般蛋白质翻译治疗。 在提示诱发的渴望测试之前使用抑制剂会减少培养性寻求。然而，人们对此知之甚少。 这种关键蛋白质翻译的细节在某些条件下，抑制一般蛋白质翻译。 实际上增加了具有 5' 上游开放阅读框的 mRNA 子集的翻译，例如 Oligophrenin-1 (OPHN1) 在海马中，OPHN1 对于 eIF2α 介导的 mGluR-LTD 和 在 VTA 中，该通路在双向 CP-AMPAR 可塑性中发挥作用。 因此，在我们的 NAcc 研究中，使用蛋白质翻译抑制剂进行治疗可能会降低对可卡因暴露的反应。 增加了 OPHN1 的翻译，模仿 mGluR-LTD 并去除突触 CP-AMPAR。 假设是，在自我服用可卡因和长期戒断后，OPHN1 翻译水平较低 NAcc，允许 CP-AMPAR 的积累和维持。此外，在孵化期间的大鼠中。 在寻找测试中，OPHN1 翻译由于 eIF2α 去磷酸化而减少，从而使 CP-AMPAR 能够保留 测试期间在突触中进行调节并介导孵化寻找。目标 1 将确定 OPHN1 翻译是否正确。 使用病毒翻译核糖体亲和纯化（vTRAP）与 qRT-PCR 可量化可卡因渴望孵化过程中 Ophn1、Gria1 和 Gria2 mRNA 的变化。 将使用新生蛋白质的嘌呤霉素标记与新翻译的蛋白质的变化进行比较 我还将使用多种技术来定位目标 2 将确定的关键翻译。 OPHN1 在孵化寻找表达中的作用，这里我将使用类似的 vTRAP 方法来进行。 在孵化大鼠中进行寻找测试之前和之后检查活跃翻译的 mRNA。 另外，我将进行实验。 敲低 OPHN1 以测试 mGlu1-LTD 介导的 CP-AMPAR 去除是否有必要 当这些研究正在进行时，我将参与多方面的研究。 培训计划旨在培养实现我在学术环境中成为 PI 的目标所需的非替补技能。