Cell specific CRF-PACAP effects in mice (Ressler)

小鼠中细胞特异性 CRF-PACAP 效应 (Ressler)

• 批准号: 10116477
• 负责人: KERRY J. RESSLER
• 金额: $ 48.86万
• 依托单位: MCLEAN HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2024-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10116477
• 关键词: Acute; Amygdaloid structure; Anxiety; Behavior; Behavioral; Blood; Brain; Brain region; Cell Nucleus; Cells; Chronic; Complement; Corticotropin-Releasing Hormone; Cre driver; DNA; DNMT3a; Data; Diagnosis; Disease; Education; Emotions; Epigenetic Process; Estrogens; Fright; Genes; Genetic; Genetic Transcription; Hospitals; Human; Immobilization; Individual; Knock-out; Knockout Mice; Knowledge; Literature; Measures; Medial; Mediating; Mediator of activation protein; Messenger RNA; Methylation; MicroRNAs; Modeling; Molecular; Motivation; Mus; Neurons; Nuclear; Pathway Analysis; Pathway interactions; Pattern; Peptides; Phenotype; Physiological; Physiology; Population; Post-Traumatic Stress Disorders; Prefrontal Cortex; Process; Reaction; Receptor Gene; Regulation; Research; Response Elements; Ribosomes; Role; Signal Transduction; Sleep; Sleep Deprivation; Sorting - Cell Movement; Stress; Structure of terminal stria nuclei of preoptic region; Symptoms; System; Testing; The Sun; Trauma; Woman; Work; acute stress; anxiety-related behavior; base; biological adaptation to stress; cell type; conditioned fear; design; emotional behavior; experimental study; genome-wide analysis; knock-down; mRNA Expression; novel; overexpression; parabrachial nucleus; pituitary adenylate cyclase activating polypeptide; program dissemination; psychological trauma; receptor; sex; stress related disorder; whole genome

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.

摘要：项目1 促肾上腺皮质激素释放因子（CRF）和腺苷酸环化酶激活多肽（PACAP）是 与物种间的应激反应有关。我们已经证明，PACAP及其受体（PAC 1 R） 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。在受创伤的受试者中，PACAP血液水平与定量恐惧指标相关， 以及创伤后应激障碍的诊断和症状我们发现了一种性别特异性的关联，暗示着雌激素 PAC 1 R基因中的反应元件，预测女性的恐惧和PTSD症状。在小鼠中，PAC 1 R mRNA在外周和杏仁核中被恐惧和雌激素叠加诱导，最近的敲除数据 表明PACAP对正常CRF依赖性应激信号传导是关键的。尽管这些多行 支持这些肽系统的重要性的证据，文献目前缺乏明确的机制， 解释它们如何在分子水平上在已知的压力，创伤和 情绪行为现在很清楚的是，通用报告格式和泛太平洋会计准则体系是相互交织的，必须加以研究 一起了解他们在压力反应中的个人和互动作用。项目1旨在 在深层次的机制水平上运作，以了解细胞类型特异性CRF和PACAP表达的作用， 中央杏仁核（CeA）、终纹床核（BNST）、前额叶皮质内的神经元群 (PFC)臂旁核（PBn）。我们的假设是，miRNA、mRNA和 DNA甲基化，在CRF和PACAP神经元的CeA，BNST，PFC，和PBn将确定新的目标， 与压力有关的疾病在目标1中，我们将进行mRNA和miRNA谱分析，以检查 应力我们将使用CRF-和PACAP-Cre驱动系与含有TRAP-核糖体（mRNA）的小鼠杂交， 和太阳标记的标记物（核分选/miRNA分析）来检查恐惧条件反射的差异效应， 固定应激和睡眠限制应激对CeA内这些神经元中miRNA/mRNA表达的影响， 在目标2中，我们将进行DNA甲基化（mDNA）分析，以检查差异效应 压力我们将使用与Aim 1相同的操作，以及核分选和全基因组测序。 甲基化阵列来检查应激对特定细胞/区域中的mDNA的差异影响。在目标3中，我们 进行RNA表达和DNA甲基化的细胞类型特异性操作以减轻压力。我们将瞄准 一组限制性基因（对于Aim 3a：FKBP 5;对于Aim 3b：新鉴定的靶标），使用过表达 和mRNA/miRNA的敲低，以及使用dCas 9-泰特和-Dnmt 3a的靶向DNA（去）甲基化， CRF和PACAP表达细胞。这一目标将探讨这些表观遗传调控的因果关系， CRF/PACAP系统在调节压力和情绪相关行为中的作用。项目1 实验是根据现有知识设计的，但正如Conte中心所预期的那样，我们将使用新的 所有项目的发现，通过行政核心完善和优化我们的目标。