OPHN1 translation and AMPA receptor plasticity during incubation of craving

项目摘要 对于患有毒瘾的人来说，一个主要的问题是持续的易复发，甚至 在长期禁欲之后。在“潜伏的可卡因渴求”复吸模型中，大鼠自我给药 使用延长准入程序的可卡因，然后经历延长的戒断期。在.期间 戒断后，大鼠表现出对线索诱导的可卡因渴求的逐渐加剧(孵化)。我们有 研究表明，钙离子通透性AMPA受体(CP-AMPAR)完全由GluA1亚基组成， 在戒断期间积聚在伏核核心(NAcc)，此后是 潜伏期线索诱导的渴望的表达。因此，了解CP-AMPAR的调控机制 维持和移除可能产生新的治疗目标，以减少渴望和延长禁欲。 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， 因为它们被普通的蛋白质翻译抑制剂所阻断。此外，使用普通蛋白质翻译进行治疗 在线索诱导的渴求测试之前的抑制会减少潜伏的寻觅。然而，人们对此知之甚少 这一关键蛋白质翻译的细节。在某些条件下，抑制一般蛋白质的翻译 实际上增加了带有5‘上游开放阅读框的mRNA子集的翻译，例如 寡聚体-1(OPHN1)。在海马区，OPHN1是eIF2α介导的mGluR-LTD所必需的，并且 移除突触AMPAR。在VTA中，这一通路在CP-AMPAR双向可塑性中起作用 对IP的回应。接触可卡因。因此，在我们的NAcc研究中，用蛋白质翻译抑制剂治疗可能 增加了OPHN1的翻译，模仿mGluR-LTD并移除突触CP-AMPAR。我的 假说是，在可卡因自我管理和长期戒断之后，OPHN1的翻译水平较低 NAcc，允许CP-AMPAR的积累和维护。此外，在孵化的大鼠中， SEEING测试，OPHN1翻译由于eIF2α去磷酸化而减少，使CP-AMPAR能够保持 在突触测试和中介孵化寻找过程中。目标1将确定OPHN1翻译是否 病毒翻译核糖体亲和纯化(VTRAP)联合应用对孵化大鼠的调节失调 定量逆转录聚合酶链式反应检测Ophn1、Gria1和Gria2基因在可卡因渴求培养过程中的表达。基因表达的变化 将与使用新生蛋白的嘌呤霉素标记的新翻译蛋白的变化进行比较 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 OPHN1在孵化寻觅表达中的作用在这里，我将使用类似的vTRAP方法 在孵化的大鼠进行寻找试验前后，检查主动翻译的mRNAs。此外，我还将在实验中 敲除OPHN1以测试它是否对mGlu1-Ltd介导的CP-AMPAR和 孵化寻找的常态化。在这些研究进行的同时，我将参加一个多方面的 培训计划，以发展所需的非替补技能，以达到我在学术环境中成为一名PI的目标。

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.