The role of sex- and cell-type specific protein degradation increases in the amygdala in fear memory formation

项目总结/摘要 这项计划的主要目标是了解恐惧记忆是如何形成和储存在大脑中的。后 创伤性应激障碍（PTSD）影响了近5%的世界人口，然而，目前的治疗方法 逆转这种疾病症状的疗效有限。此外，女性比男性更有可能 发展PTSD，尽管控制这种性别依赖倾向的机制仍然不明确。 最近，我们小组和其他人的工作涉及由泛素介导的蛋白质降解， 蛋白酶体系统（UPS）在杏仁核恐惧记忆形成中的作用，杏仁核是参与恐惧记忆形成的主要大脑区域。 情绪处理然而，蛋白质降解在恐惧记忆形成中的功能作用还没有得到证实。 被识别。此外，以前关于UPS介导的蛋白质降解的研究仅集中在 因此，关于是否类似的UPS机制控制着恐惧记忆的形成， 女性在我们的初步研究中，我们发现在雄性中，蛋白质降解定位于细胞核， 杏仁核细胞的恐惧学习，这表明在转录控制的潜在作用，虽然这已经 从未被直接测试过。令人惊讶的是，女性在UPS介导的蛋白质中没有表现出任何变化， 但是，恐惧学习后杏仁核的降解确实使基线泛素蛋白酶体升高， 活性与男性相比，这与DNA 5-羟甲基化（5-hmc）的增加有关。 uba 52是泛素编码基因之一。这表明，女性基线UPS活性升高， 通过改变Uba 52的DNA甲基化来调节，可能足以让他们获得恐惧记忆。 本提案中的工作旨在回答这些关于性别依赖性差异的重要问题， 核蛋白降解在恐惧记忆形成过程中转录调控中的作用。使用 药理学和蛋白酶体活性的复杂CRISPR-dCas 9操作的组合， 结合质谱和全基因组下一代RNA-seq技术，Aim 1将测试 核蛋白降解是否参与恐惧记忆形成过程中的转录控制， 男性和女性之间是否存在差异。Aim 2将使用尖端的CRISPR-dCas 9技术， 控制Uba 52的甲基化状态，并将测试这如何影响泛素- 蛋白酶体活性的男性和女性的杏仁核。最后，在目标3中，我们将使用CRISPR-dCas 9 一项技术来测试女性杏仁核中基线蛋白质降解的增加是否会使她们 获得恐惧记忆总的来说，这项研究将回答有关性别依赖的重要问题， 蛋白质降解在恐惧记忆形成中的作用差异。该项目取得的成果 可能对理解恐惧记忆中性别相关差异的病因学有重要意义 形成并导致新的治疗策略的发展，以治疗主要的精神疾病。

Project Summary/Abstract The broad goal of this proposal is to understand how fear memories are formed and stored in the brain. Post- traumatic stress disorder (PTSD) affects nearly 5% of the world population, however, current treatments have limited efficacy in reversing the symptoms of this disorder. Furthermore, females are more likely than males to develop PTSD, though the mechanisms controlling this sex-dependent predisposition remain equivocal. Recently, work from our group and others have implicated protein degradation mediated by the ubiquitin- proteasome system (UPS) in fear memory formation in the amygdala, the primary brain region involved in emotional processing. However, the functional role of protein degradation in fear memory formation has yet to be identified. Furthermore, previous studies on UPS-mediated protein degradation have focused exclusively on males, so little is known about whether similar UPS mechanisms control the formation of fear memories in females. In our preliminary studies, we found that in males protein degradation was localized to the nucleus of amygdala cells following fear learning, suggesting a potential role in transcriptional control, though this has never been directed tested. Surprisingly, females did not show any changes in UPS-mediated protein degradation in the amygdala following fear learning though did have elevated baseline ubiquitin-proteasome activity in comparison with males, which was associated with increased DNA 5-hydroxymethylation (5-hmc) of Uba52, one of the ubiquitin coding genes. This suggests that elevated baseline UPS activity in females could be regulated by altered DNA methylation of Uba52 and may be sufficient for them to acquire fear memories. The work in this proposal is designed to answer these important questions about sex-dependent differences in the role of nuclear protein degradation in transcriptional control during fear memory formation. Using a combination of pharmacology and sophisticated CRISPR-dCas9 manipulations of proteasome activity in combination with mass spectrometry and whole genome next generation RNA-seq technology, Aim 1 will test whether nuclear protein degradation is involved in transcriptional control during fear memory formation and whether this varies between males and females. Aim 2 will use cutting-edge CRISPR-dCas9 technology to control the methylation state of Uba52 and will test how this effects baseline differences in ubiquitin- proteasome activity in the amygdala of males and females. Finally, in Aim 3 we will using CRISPR-dCas9 technology to test whether increased baseline protein degradation in the amygdala of females primes them to acquire fear memories. Collectively, this study will answer important questions about sex-dependent differences in the role of protein degradation in fear memory formation. The results obtained from this project could have important implications for understanding the etiology of sex-related differences in fear memory formation and lead to the development of novel therapeutic strategies to treat major psychiatric disorders.