Epigenetic regulation of neuropathic pain: Role of DRG histone methyltransferase G9a

 描述（由申请人提供）：目前对神经损伤诱导的神经性疼痛（最常见的临床综合征之一）的治疗是有限的。了解与这种疾病相关的病理变化可能有助于将治疗策略从症状缓解转移到神经性疼痛特异性新疗法。周围神经损伤引起的背根神经节（DRG）中受体、酶和电压依赖性离子通道表达的变化有助于神经病理性疼痛的发生。然而，神经损伤如何引起这些变化的分子机制仍然是难以捉摸的。最近的研究表明，基因调控的机制涉及表观遗传修饰，如组蛋白甲基化。G9 a是一种组蛋白甲基转移酶，主要催化组蛋白H3赖氨酸9单甲基化（H3 K9 me 1）和二甲基化（H3 K9 me 2），是基因表达的关键调节因子。我们的初步数据表明，神经损伤诱导的DRG G9 a的增加可能是负责通过管理DRG μ阿片受体（莫尔）和κ阿片受体（KOR）基因的表达神经病理性疼痛。该提案将确定DRG G9 a是否以及如何在外周神经损伤后上调，以及G9 a是否以及如何在神经性疼痛条件下促进疼痛超敏反应。在目的1中，我们将确定是否药物抑制或基因敲低DRG G9 a减弱神经损伤引起的疼痛超敏反应的发展和维持期间，是否过度表达DRG G9 a导致神经性疼痛的主要症状。目的二：观察周围神经损伤后DRG中G9 a、C/EBPβ的表达及H3 K9 me 1、H3 K9 me 2的表达。我们还将研究神经损伤诱导的DRG G9 a上调是否归因于神经病理性疼痛条件下C/EBPβ与DRG中G9 a基因启动子的结合活性增加。在目的3中，我们将检查G9 a是否以及如何参与受损DRG中神经损伤诱导的莫尔和KOR的下调。我们将首先观察G9 a是否与莫尔和KOR基因的启动子和5 '端调控区结合，以及这些结合活性在第四脊神经损伤（SNL）后受损的DRG神经元中是否增加。然后，我们将确定G9 a是否有助于SNL诱导的DRG莫尔和KOR的下调，通过阻断转录因子环AMP反应元件结合蛋白进入这两个基因内的结合基序。最后，我们将确定阻断SNL诱导的DRG G9 a增加是否挽救受损DRG中莫尔和KOR的下调，减少初级传入神经递质释放，恢复阿片类镇痛的减少，并减弱阿片类耐受性的发展。这些研究不仅将促进我们对神经性疼痛的表观遗传机制的理解，而且还将为开发预防和治疗这种疾病的新策略打开大门。

 DESCRIPTION (provided by applicant): Current treatment for nerve injury-induced neuropathic pain, one of the most common clinical syndromes, is limited. Understanding pathological changes related to this disorder may help shift treatment strategies from symptomatic relief to neuropathic pain-specific novel therapies. Peripheral nerve injury-induced changes in the expression of receptors, enzymes, and voltage-dependent ion channels in the dorsal root ganglion (DRG) contribute to neuropathic pain genesis. However, the molecular mechanisms of how nerve injury causes these changes are still elusive. Recent studies suggest that the mechanism for gene regulation involves epigenetic modification, such as histone methylation. G9a, a histone methyltransferase, primarily catalyzes histone H3 lysine 9 monomethylation (H3K9me1) and dimethylation (H3K9me2) and is a key regulator of gene expression. Our preliminary data indicate that a nerve injury-induced increase in DRG G9a may be responsible for neuropathic pain by governing the expression of DRG mu opioid receptor (MOR) and kappa opioid receptor (KOR) genes. This proposal will determine whether and how DRG G9a is up-regulated following peripheral nerve injury and whether and how G9a contributes to pain hypersensitivity under neuropathic pain conditions. In Aim 1, we will determine whether pharmacological inhibition or genetic knockdown of DRG G9a attenuates nerve injury-induced pain hypersensitivity during the development and maintenance periods and whether overexpression of DRG G9a leads to major symptoms of neuropathic pain. In Aim 2, we will observe the expression of G9a and C/EBPβ and the levels of H3K9me1 and H3K9me2 in the DRG after peripheral nerve injury. We will also examine whether nerve injury-induced up-regulation of DRG G9a is attributed to an increase in the binding activity of C/EBPβ to the G9a gene promoter in the DRG under neuropathic pain conditions. In Aim 3, we will examine whether and how G9a participates in the nerve injury- induced down-regulation of MOR and KOR in the injured DRG. We will first observe whether G9a binds to the promoter and 5'-end regulatory regions of MOR and KOR genes and whether these binding activities are increased in the injured DRG neurons after the fourth spinal nerve injury (SNL). We will then define whether G9a contributes to the SNL-induced down-regulation of DRG MOR and KOR by blocking the access of the transcriptional factor cyclic AMP response element binding protein to its binding motifs within these two genes. Finally, we will determine whether blocking the SNL-induced increase in DRG G9a rescues the downregulation of MOR and KOR in the injured DRG, reduces primary afferent neurotransmitter release, restores the decrease of opioid analgesia, and attenuates opioid tolerance development. These studies will not only advance our understanding of the epigenetic mechanisms of neuropathic pain but will also open a door to develop a new strategy for the prevention and treatment of this disorder.