Role of dorsal root ganglion FTO, a RNA demethylase, in neuropathic pain

背根神经节 FTO（一种 RNA 去甲基酶）在神经性疼痛中的作用

• 批准号: 9978158
• 负责人: Yuan-Xiang Tao
• 金额: $ 50.99万
• 依托单位: RBHS-NEW JERSEY MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-15 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9978158
• 关键词: Absence of pain sensation; Affect; Animals; Attenuated; Binding; Cell Nucleus; Chronic; Code; Data; Development; Disease; Down-Regulation; Epigenetic Process; Fatty acid glycerol esters; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Transcription; Histones; Homologous Gene; Human; Hypersensitivity; Injury; Lead; Ligation; Link; Maintenance; Malignant Neoplasms; Messenger RNA; Modification; Morphine; Neurons; Neuropathy; Obesity; Opioid Analgesics; Pain; Peripheral nerve injury; Pharmacology; Pilot Projects; Prevention strategy; Proteins; Public Health; RNA; RNA Degradation; RNA metabolism; Rattus; Regulator Genes; Role; Spinal Anesthesia; Spinal Ganglia; Spinal nerve structure; Symptoms; Testing; Time; Transcription Coactivator; Transcriptional Activation; Up-Regulation; WT1 gene; central sensitization; chronic pain; dorsal horn; histone methyltransferase; human disease; injured; knock-down; mechanical allodynia; morphine tolerance; mu opioid receptors; nerve injury; neurotransmitter release; novel; novel therapeutics; opiate tolerance; overexpression; pain reduction; pain symptom; painful neuropathy; promoter; transcription factor; treatment strategy

PROJECT SUMMARY: RNA modifications were recently rediscovered as essential regulators of gene expression. N6-methyladenosine (m6A) is identified as the most prevalent internal modification of eukaryotic RNA. The m6A modification controls RNA metabolism including RNA degradation and has been linked to human diseases such as obesity and cancer. However, its role in chronic pain including neuropathic pain is unknown. Our preliminary data suggest that a nerve injury-induced increase in dorsal root ganglion (DRG) FTO (fat mass and obesity-associated protein), a well-characterized RNA demethylase, may participate in neuropathic pain by reducing the level of m6A on Ehmt2 mRNA (encoding G9a, a histone methyltransferase), stabilizing nerve injury-induced G9a upregulation, and, consequently, silencing the expression of mu opioid receptor (MOR) in the injured DRG. Given that the transcriptional and translational changes in DRG gene expression following peripheral nerve injury participate in the development and maintenance of neuropathic pain and that G9a as a repressor of gene expression is a key endogenous contributor to neuropathic pain genesis, we propose that the increased FTO in the injured DRG contributes to neuropathic pain. In Aim 1, we will first determine whether pharmacological inhibition or genetic knockdown of DRG FTO attenuates nerve injury-induced pain hypersensitivity during the development and maintenance periods, and whether mimicking nerve injury-induced increase in DRG FTO leads to major symptoms of neuropathic pain in naive rats. In Aim 2, we will examine whether and how peripheral nerve injury upregulates the expression of FTO in the DRG. Time-dependent changes in the expression of Fto and its transcriptional activator Runx1 mRNAs and their proteins and in the level of m6A on specific RNAs in the DRG after peripheral nerve injury will be examined. We will also define whether nerve injury-induced up-regulation of DRG FTO is attributed to an increase in Runx1 in the injured DRG. In Aim 3, we will examine how DRG FTO participates in neuropathic pain. We will first observe whether FTO binds to Ehmt2 mRNA and whether this binding activity is increased in the injured DRG neurons after the fifth lumbar spinal nerve ligation (SNL). We will then define whether FTO contributes to the SNL-induced upregulation of Ehmt1 mRNA/G9a and the G9a-controlled downregulation of MOR by stabilizing the increased G9a expression in the injured DRG. Finally, we will determine whether blocking the SNL-induced increase in DRG FTO reduces the MOR-controlled primary afferent neurotransmitter release, restores the decrease of opioid analgesia, and attenuates opioid tolerance development. These studies will not only advance our understanding of posttranscriptional mechanisms of neuropathic pain, but will also open a door to develop a new strategy for the prevention and treatment of this disorder.

项目概要： 最近，RNA 修饰被重新发现为基因表达的重要调节因子。 N6-甲基腺苷 (m6A) 被认为是真核 RNA 最普遍的内部修饰。 m6A 修改控件 RNA 代谢（包括 RNA 降解）与肥胖等人类疾病有关 癌症。然而，其在慢性疼痛（包括神经性疼痛）中的作用尚不清楚。我们的初步数据表明 神经损伤引起的背根神经节 (DRG) FTO（脂肪量和肥胖相关 蛋白），一种充分表征的 RNA 去甲基化酶，可能通过降低 Ehmt2 mRNA（编码 G9a，一种组蛋白甲基转移酶）上的 m6A，稳定神经损伤诱导的 G9a 上调，从而沉默受损 DRG 中 mu 阿片受体 (MOR) 的表达。 鉴于周围神经后 DRG 基因表达的转录和翻译变化 损伤参与神经病理性疼痛的发生和维持，G9a 作为基因的阻遏物 表达是神经性疼痛发生的关键内源性因素，我们认为 FTO 的增加 受损的 DRG 会导致神经性疼痛。在目标 1 中，我们将首先确定药理学是否 抑制或基因敲低 DRG FTO 可减轻神经损伤引起的疼痛超敏反应 发育和维持期，以及是否模仿神经损伤引起的 DRG FTO 增加 导致幼鼠出现神经性疼痛的主要症状。在目标 2 中，我们将研究是否以及如何 周围神经损伤上调 DRG 中 FTO 的表达。随时间变化的 Fto 及其转录激活剂 Runx1 mRNA 及其蛋白的表达以及 m6A 水平 将检查周围神经损伤后 DRG 中的特定 RNA。我们还将定义是否神经 损伤引起的 DRG FTO 上调归因于受伤 DRG 中 Runx1 的增加。在目标 3 中，我们 将检查 DRG FTO 如何参与神经性疼痛。我们首先观察FTO是否与Ehmt2结合 第五腰椎损伤后损伤的 DRG 神经元中的 mRNA 以及这种结合活性是否增加 神经结扎（SNL）。然后我们将定义 FTO 是否有助于 SNL 诱导的 Ehmt1 上调 mRNA/G9a 和 G9a 通过稳定增加的 G9a 表达来控制 MOR 的下调 受伤的DRG。最后，我们将确定阻止 SNL 诱导的 DRG FTO 增加是否会减少 MOR 控制的初级传入神经递质释放，恢复阿片类镇痛的减少，以及 减弱阿片类药物耐受性的发展。这些研究不仅会加深我们对 神经性疼痛的转录后机制，但也将为开发新策略打开一扇大门 预防和治疗这种疾病。