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

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 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.