Cell specific CRF-PACAP effects in mice (Ressler)

摘要：项目1 促肾上腺皮质激素释放因子(CRF)和垂体腺苷环化酶激活多肽(PACAP)是 与跨物种的应激反应有关。我们已经证明PACAP及其受体(PAC1R)是 心理创伤后异常过程的关键介质，包括创伤后应激障碍 (PTSD)，在人类身上。在受创伤的受试者中，PACAP的血液水平与量化的恐惧措施有关，如 以及创伤后应激障碍的诊断和症状。我们发现了一种特定性别的联系，这与雌激素有关 PAC1R基因中的反应元件，预测女性的恐惧和创伤后应激障碍症状。在小鼠身上，PAC1R 外周和杏仁核中的恐惧和雌激素以及最近的基因敲除数据都额外地诱导了信使核糖核酸 提示PACAP在正常的CRF依赖的应激信号中起关键作用。尽管有这多条线路 支持这些多肽系统重要性的证据，目前文献缺乏明确的机制 解释它们如何在分子水平上相互作用，在已知的应激、创伤和 情绪化行为。现在很明显，CRF和PACAP系统是交织在一起的，必须进行研究 共同了解他们在压力反应中的个人和交互作用。项目1旨在 在深层机制水平上操作，以了解细胞类型特异性CRF和PACAP表达的作用 杏仁中央核(CEA)、终纹床核(BNST)、前额叶皮质内的神经元群 (PFC)和臂旁核(PBN)。我们的假设是miRNA、mRNA和 CEA、BNST、PFC和PBN的CRF和PACAP神经元中的DNA甲基化将识别新的靶点 与压力相关的障碍。在目标1中，我们将进行信使核糖核酸和微核糖核糖核酸分析，以检查不同的影响 压力。我们将使用CRF-和PACAP-Cre驱动系与含有TRAP-核糖体(MRNA)的小鼠杂交 和Sun标记的标记物(核分类/miRNA图谱)来检查恐惧条件作用的不同影响， 束缚应激和睡眠限制应激对CEA内这些神经元miRNA/mRNA表达的影响， BNST、PBN和PFC。在目标2中，我们将进行DNA甲基化(MDNA)分析以检查差异效应 压力的影响。我们将使用与目标1相同的操作以及核分类和全基因组 甲基化阵列，以检测压力对特定细胞/区域的mDNA的差异影响。在《目标3》中，我们将 对RNA表达和DNA甲基化进行细胞类型的特定操作，以缓解压力。我们的目标是 一组受限的基因(对于目标3a：FKBP5；对于目标3b：新识别的靶点)，使用过度表达 和mRNA/miRNA的敲除，以及使用dCas9-Tet和-DNMT3A的靶向DNA(去)甲基化，在 表达CRF和PACAP的细胞。这一目标将探索这些表观遗传调控的因果关系 CRF/PACAP系统中调节应激和情绪相关行为的过程。项目1 实验是基于当前知识设计的，但正如Conte Center所预期的那样，我们将使用新的 从所有项目中发现，通过管理核心来改进和优化我们的目标。

SUMMARY: PROJECT 1 Corticotrophin Releasing Factor (CRF) and Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) are implicated in the stress response across species. We have shown that PACAP and its receptor (PAC1R) are critical mediators of abnormal processes following psychological trauma, including posttraumatic stress disorder (PTSD), in humans. In traumatized subjects, PACAP blood levels associate with quantitative fear measures as well as with PTSD diagnosis and symptoms. We have found a sex-specific association, implicating an estrogen response element in the PAC1R gene, that predicts fear and PTSD symptoms in women. In mice, PAC1R mRNA is induced additively by fear and estrogen in both periphery and the amygdala, and recent knockout data suggest that PACAP is critical for normal CRF-dependent stress signaling. Despite these multiple lines of evidence supporting the importance of these peptide systems, the literature currently lacks clear mechanistic explanations of how they interact at a molecular level within pathways known to underlie stress, trauma, and emotional behaviors. It is now clear that the CRF and PACAP systems are interwoven and must be studied together to understand their individual and interactive roles in stress responsiveness. Project 1 is designed to operate at a deep mechanistic level to understand the role of cell-type specific CRF- and PACAP-expressing neuron populations within central amygdala (CeA), bed nucleus of the stria terminalis (BNST), prefrontal cortex (PFC), and parabrachial nucleus (PBn). Our hypothesis is that differential regulation of miRNA, mRNA, and DNA methylation, in CRF and PACAP neurons of the CeA, BNST, PFC, and PBn will identify new targets for stress-related disorders. In Aim 1, we will conduct mRNA and miRNA profiling to examine differential effects of stress. We will use CRF- and PACAP-Cre driver lines crossed with mice containing TRAP-ribosome (mRNA) and Sun-tagged markers (nuclear sorting/miRNA profiling) to examine differential effects of fear conditioning, immobilization stress, and sleep restriction stress, on miRNA/mRNA expression in these neurons within CeA, BNST, PBn, and PFC. In Aim 2, we will conduct DNA Methylation (mDNA) profiling to examine differential effects of stress. We will use the same manipulations as in Aim 1 together with nuclear sorting and whole-genome methylation arrays to examine differential effects of stress on mDNA in specific cells/regions. In Aim 3, we will perform cell-type specific manipulation of RNA expression and DNA methylation to mitigate stress. We will target a circumscribed set of genes (for Aim 3a: FKBP5; for Aim 3b: newly identified targets), using over-expression and knockdown of mRNA/miRNA, and targeted DNA (de)methylation using dCas9-TET and -Dnmt3a, within CRF and PACAP-expressing cells. This aim will explore causal relationships of these epigenetic regulatory processes within CRF/PACAP systems in mediating stress- and emotion-related behaviors. Project 1 experiments are designed based on current knowledge, but as expected with Conte Centers, we will use new discoveries from all projects to refine and optimize our objectives via the Administrative Core.