Peripheral Receptor Mechanisms in Orofacial Muscle Pain

项目总结 DNA甲基化是一种表观遗传因子，在调控基因表达和基因改变中发挥重要作用。 DNA甲基化是一种与许多人类疾病相关的特征。炎症与环境 心理物理应激等因素诱导伤害性感受基因去甲基化导致其异常 表情。此更新应用程序的目标是研究肌肉炎症如何远程 调节三叉神经节(TG)中多个伤害性感受基因的DNA甲基化 牵涉到疼痛和痛觉过敏。我们的中心假设是咬肌炎症导致 甘油三酯中促伤害性基因的甲基化减少导致其异常表达，这有助于 疼痛和机械性痛觉过敏的发展。我们进一步假设心理物理压力 通过在甘油三酯中过量产生活性氧物种(ROS)来加强这些影响，这调节了 DNA甲基化。在目标1中，我们将确定DNA甲基化在炎性疼痛反应中的作用。 具体地说，我们将检查是否通过Dnmts在启动子区域增加DNA甲基化 单独的致痛基因，防止炎症性疼痛和痛觉过敏。我们已经证实， TRPV1、TRPA1、P2X3和Piezo2等伤害感受基因含有结合Dnmts和Piezo2的CG岛。 抑制DNMT活性可增加其在TG中的表达。为了确定DNA的作用 在单个基因的甲基化，我们设计并验证了一种新型的DNMT融合蛋白复合体，它以 特定基因的启动子区域，使用CRISPR-dCAS9技术。我们期待的是 TG内的融合蛋白将阻止靶基因的上调，并揭示其相对贡献 在肌炎条件下疼痛和痛敏的特定基因的DNA甲基化的研究。在目标2中，我们将 探讨神经节内ROS在伤害性基因DNA甲基化中的作用。我们将研究 ROS是否调节TG中TRPV1和TRPA1基因的DNA甲基化以及应激是否升高 神经节内ROS，维持促伤害性基因甲基化水平的降低。我们的 初步数据表明，ROS是参与两种伤害性感受性物质DNA甲基化的关键上游因素 基因。我们预测，阻断ROS在TG中的积累或DNA启动子的靶向甲基化将 防止应激介导的痛觉过敏增强以及TRPV1和TRPA1的上调。成功 该项目的完成将揭开涉及DNA甲基化和神经节内的新机制 氧化代谢产物对多个伤害性基因的功能调控，为其提供机制基础 炎症和压力如何使感觉神经节引起持久的肌肉疼痛。这个 预期的结果应该对治疗的发展具有广泛的翻译意义 靶向调控特定伤害性感受器DNA甲基化的转录机制 基因或具有相似表观遗传机制的基因簇。

PROJECT SUMMARY DNA methylation, an epigenetic factor, plays an important role in regulating gene expression and alterations in DNA methylation is a feature associated with a number of human diseases. Inflammation and environmental factors such as psychophysical stress induces demethylation of pro-nociceptive genes leading to their aberrant expression. The objective of this renewal application is to investigate how muscle inflammation remotely regulates DNA methylation of multiple pro-nociceptive genes in trigeminal ganglia (TG) that have been implicated in pain and hyperalgesia. Our central hypothesis is that masseter muscle inflammation results in reduced methylation of pro-nociceptive genes in TG leading to their aberrant expression, which contributes to the development of pain and mechanical hyperalgesia. We further hypothesize that psychophysical stress potentiates these effects via the excess production of reactive oxygen species (ROS) within TG, which regulate DNA methylation. In Aim 1, we will determine the role of DNA methylation in inflammatory pain responses. Specifically, we will examine whether increased DNA methylation via DNMTs at the promoter region of individual pro-nociceptive genes, prevents inflammatory pain and hyperalgesia. We have confirmed that pro-nociceptive genes such as TRPV1, TRPA1, P2X3 and PIEZO2 contain CG islands that bind DNMTs and that the inhibition of DNMT activities increased their expression in TG. In order to determine the role of DNA methylation in individual genes, we designed and validated a novel DNMT fusion protein complex that targets the promoter region of a specific gene, using the CRISPR-dCAS9 technology. We expect that the expression of the fusion protein within TG will prevent the upregulation of the target gene and reveal the relative contribution of DNA methylation for a specific gene in pain and hyperalgesia under a myositis condition. In Aim 2, we will investigate the role of intraganglionic ROS in DNA methylation of pro-nociceptive genes. We will examine whether ROS regulates DNA methylation of TRPV1 and TRPA1 genes in TG and whether stress elevates intraganglionic ROS, which maintains the reduced level of methylation of the pro-nociceptive genes. Our preliminary data suggest ROS as a key upstream factor involved in DNA methylation of the two pro-nociceptive genes. We predict that the blockade of ROS accumulation in TG or targeted methylation of DNA promoters will prevent stress-mediated potentiation of hyperalgesia and the upregulation of TRPV1 and TRPA1. Successful achievement of this project should unravel novel mechanisms involving DNA methylation and intraganglionic oxidative metabolites on functional regulation of multiple pro-nociceptive genes, providing a mechanistic basis for how inflammation and stress engage sensory ganglia to induce prolonged persistent muscle pain. The anticipated outcomes should have broad translational implications for the development of therapeutic approaches targeting transcriptional machineries that regulate DNA methylation of a specific pro-nociceptive gene or a cluster of genes sharing similar epigenetic mechanisms.