Chronic Stress and Abdominal Pain: Novel Mechanisms

描述(申请人提供)：慢性应激与腹痛：新机制慢性应激激活下丘脑-垂体-肾上腺(HPA)轴，是腹痛的已知诱因。最近的报道表明，慢性心理应激导致与记忆和情绪相关的中枢神经系统区域基因功能的表观遗传调节发生变化。表观遗传学是指通过DNA甲基化、组蛋白修饰和染色质重塑，在不改变DNA序列的情况下，基因功能发生稳定和/或可遗传的变化。表观遗传学领域在过去十年中迅速崛起，其基础是开创性研究证明了原代细胞和人类细胞系中甲基化和乙酰化位点在全基因组范围内的分布。目前尚不清楚慢性应激是否通过表观遗传机制调节外周疼痛通路。内源性瞬时受体电位(TRP)通路在痛觉传递中起着关键作用，而TRPV1受体受作用于CB1受体的内源性大麻素(CB)的调节，从而抑制了TRPV1的功能。慢性应激下调CB通路的功能，导致TRPV1受体功能上调和腹痛。我们将验证这一假说，即慢性应激通过对支配盆腔器官的背根节(DRG)神经元中CB1和TRPV1受体的表观遗传调节而不是对下肢的躯体感觉分布进行区域和细胞特异性的调节来诱导内脏疼痛。具体地说，我们的初步数据支持两个高度新颖的假说：1.慢性间歇性应激促进DNMT1介导的糖皮质激素受体(GR)启动子位置的甲基化，导致GR表达下降，这与抗伤害性内源性大麻素CB1受体的水平和功能降低有关，并增强组蛋白乙酰化，与背根节(DRG)神经元中伤害性内源性TRPV1受体的表达和功能增加有关；2.慢性应激诱导的皮质酮(Cort)介导的表观遗传变化是区域和细胞特有的，并与支配GI(结肠)和躯体感觉(坐骨神经)的伤害性DRG“硬连接”。在慢性间歇性应激的背景下，这种硬连接的表达模式使支配胃肠道的伤害性神经元对结直肠扩张的反应产生痛敏反应。这些研究将包括应用切割边缘方法来确定应激反应基因启动子上可能的调控CpG甲基化位点，以及相关组蛋白修饰靶点的染色质免疫沉淀(ChIP)分析。伤害性感受神经元的亚群将使用逆行标记的免疫组织化学标记和激光捕获显微镜结合相关靶点的定量单细胞聚合酶链式反应来获取不同的DRG神经元亚群。对于特定受体和信号转导通路在CB和Trp通路中观察到的变化的作用的确认，将通过原位靶向传递基因沉默(si-RNA)试剂以及这些干预与行为的相关性来确认，例如，对结直肠扩张的内脏运动反应。我们还将利用FRET/TIRF显微镜和电生理记录来研究CB1-TRPV1受体复合体的形成是否在DRG神经元和转基因细胞的应激相关内脏痛敏中发挥作用。阐明慢性应激性内脏疼痛的表观遗传调控机制将对我们理解疼痛通路是如何调控的，并可能对影响胃肠道的功能性疼痛障碍的管理产生重大影响。

DESCRIPTION (provided by applicant): Chronic Stress and Abdominal Pain: Novel Mechanisms Chronic stress activates the Hypothalamic-Pituitary-Adrenal (HPA) axis and is a known inducer of abdominal pain. Recent reports indicate that chronic psychological stress causes changes in epigenetic regulation of gene function in CNS regions associated with memory and mood. Epigenetics refers to stable and/or heritable changes in gene function without changes in the DNA sequence via DNA methylation, histone modification and chromatin remodeling. The field of epigenetics has emerged rapidly in the past decade based on seminal studies demonstrating genome-wide distribution of methylation and acetylation sites in primary cells and human cell lines. It is unknown whether chronic stress regulates peripheral pain pathways via epigenetic mechanisms. The endovanilloid transient receptor potential (TRP) pathway plays a pivotal role in pain transmission and the TRPV1 receptor is known to be regulated by endocannabinoids (CB) acting on CB1 receptors which inhibit TRPV1 function. Chronic stress down-regulates the function of the CB pathway resulting in up-regulation of TRPV1 receptor function and abdominal pain. We will examine the hypothesis that chronic stress induces visceral pain via epigenetic regulation of CB1 and TRPV1 receptors in a region- and cel- specific manner in dorsal root ganglion (DRG) neurons innervating pelvic organs but not the somatosensory distribution to the lower extremities. Specifically, our preliminary data support two highly novel hypotheses: 1. Chronic intermittent stress promotes DNMT1-mediated methylation of glucocorticoid receptor (GR) promoter sites resulting in decreased GR expression that is linked to reduced levels and function of the anti-nociceptive endocannabinoid CB1 receptor, and enhances histone acetylation linked to increased expression and function of the pro-nociceptive endovanilloid TRPV1 receptor in dorsal root ganglion (DRG) neurons; and 2. Chronic stress-induced, corticosterone (CORT)-mediated epigenetic changes are region- and cell-specific, and "hardwired" to nociceptive DRGs innervating the GI tract (colon) vs. somatosensory (sciatic nerve) distribution. The hardwired expression pattern predisposes nociceptive neurons innervating the GI tract to hyperalgesia in response to colorectal distension in the setting of chronic intermittent stress. These studies will include the application of cuttin-edge methods to identify putative regulatory CpG methylation sites at the promoters of stress response genes and chromatin immunoprecipitation (ChIP) analysis of relevant histone modification targets. The subpopulation of nociceptive neurons will be identified using both immunohistochemical markers with retrograde labeling and laser capture microscopy to harvest distinct populations of DRG neurons in conjunction with quantitative single-cell PCR of relevant targets. Confirmation of the role of specific receptors and signal transduction pathways to the changes observed in CB and TRP pathways will be confirmed using targeted delivery of gene silencing (si-RNA) reagents in situ and correlation of these interventions with behavior, e.g. visceral motor response to colorectal distension. We will also examine whether the formation of CB1-TRPV1 receptor complexes plays a role in stress-associated visceral hyperalgesia in DRG neurons and transfected cells using FRET/TIRF microscopy and electrophysiological recordings. Clarifying the mechanisms underlying epigenetic regulation of chronic stress-induced visceral pain will have a significant impact on our understanding of how pain pathways are regulated and, likely, the management of functional pain disorders affecting the GI tract.