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.

项目摘要 成瘾者面临的一个主要问题是，即使是成瘾者， 在长期禁欲之后在“可卡因渴望潜伏期”复发模型中，大鼠自我给药 可卡因使用扩展访问程序，然后经历延长的禁欲期。期间 在戒断期间，大鼠表现出线索诱导的可卡因渴望的进行性强化（潜伏期）。我们有 显示Ca 2+渗透性AMPA受体（CP-AMPAR），仅由GluA 1亚基组成， 在戒断过程中积累在丘脑核核心（NAcc）中，此后需要 暗示诱导的渴望的表达。因此，了解调节CP-AMPAR的机制 维持和去除可能产生减少渴望和延长禁欲的新的治疗靶点。 我们实验室的工作表明，NAcc中CP-AMPAR介导的电流需要活性蛋白质翻译， 因为它们被一般的蛋白质翻译抑制剂阻断。此外，一般蛋白质翻译治疗 在提示诱发的渴望测试之前，抑制剂减少了潜伏的寻求。然而，人们对此知之甚少。 这个关键蛋白质翻译的细节。在某些条件下，一般蛋白质翻译的抑制 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 寡聚蛋白-1（OPHN 1）。在海马中，OPHN 1是eIF 2 α介导的mGluR-LTD所必需的， 移除突触AMPAR。在腹侧被盖区，该通路在双向CP-AMPAR可塑性中起作用， 对腹腔注射可卡因的反应因此，在我们的NAcc研究中，用蛋白翻译抑制剂治疗可能 增加了OPHN 1的翻译，模仿mGluR-LTD并去除突触CP-AMPAR。我 假设是，在可卡因自我给药和长期戒断后，OPHN 1翻译较低， NAcc，允许CP-AMPAR的积累和维持。此外，在孵育大鼠期间， 在寻找试验中，由于eIF 2 α去磷酸化，OPHN 1翻译减少，使CP-AMPAR能够停留在 在测试过程中的突触和介导孵育寻求。目标1将确定OPHN 1翻译是否 使用病毒翻译核糖体亲和纯化（vTRAP）结合 qRT-PCR定量可卡因渴求孵育期间Ophn 1、Gria 1和Gria 2 mRNA。mRNA的变化 将与使用嘌呤霉素标记新生蛋白质的新翻译蛋白质的变化进行比较， 免疫印迹。我还将使用几种技术来本地化关键翻译。目标2将决定 OPHN 1在孵化寻求表达中的作用。在这里，我将使用类似的vTRAP方法， 在孵化的大鼠中检测寻找测试前后主动翻译的mRNA。另外，我将尝试 敲低OPHN 1以测试mGlu 1-LTD介导的CP-AMPAR去除是否必要， 孵化寻找的正常化。当这些研究正在进行时，我将参加一个多方面的 培训计划，以发展所需的非板凳技能，以达到我的目标，成为一个PI在学术环境。