Threshold Inhibition of Methyltransferase G9a/Glp Exacerbates Neuropathic Hypersensitivity through Mediating GRIN2B Methylation

Background In this study, we investigated whether G9a related DNA methylation and histone modification is involved in the transcription alteration of NR2B following peripheral nerve injury and subsequently contributes to pain facilitation of G9a inhibition. Methods After approval by the institutional ethical committee on pain research in conscious animals, C57BL/6 mice were used to induce neuropathic pain with spared nerve injury (SNI). G9a/Glp expression, GRIN2B gene 5’regulatory region CpG sites methylation profile, as well as NR2B expression in the spinal dorsal horn following SNI was detected with immunofluorescence, bisulfite sequencing, and western blot, respectively. For mechanism study, threshold doses of G9a/Glp inhibitors BIX01294/UNC0638 or direct DNA demethylation agent 5-Aza was intrathecal injected through the pre-buried catheter daily in bolus for 3 days, G9a/Glp and its enzymatic substrate H3K9me2/H3K9me3 expression, GRIN2B gene methylation alteration, as well as NR2B expression were observed. Nociceptive behavior was depicted in response to von Frey filaments following SNI with or without intrathecal BIX/UNC and 5-Aza treatments. Results Ipsilateral mechanical withdrawal threshold rather than thermal withdrawal latency prominently decreased and peaked at day 7 to 14 post SNI. Besides, nerve injury consistently increased G9a/Glp, H3K9me2 expression, GRIN2B gene 5’-regulatory region CpG sites methylation, as well as NR2B expression in the spinal dorsal horn at day 7 post SNI. Furthermore, either G9a/Glp inhibition by BIX/UNC or H3K9me2 blockade by 5-Aza independently reversed GRIN2B gene high methylation, followed with disinhibition of NR2B transcription inhibition. Consistent with molecular changes, either BIX/UNC or 5-Aza further worsens nerve injuryinduced allodynia. Conclusion G9a/Glp contributes to the pathogenesis of neuropathic pain via methylating GRIN2B gene affecting NR2B transcription. G9a/Glp at an elevated setpoint may prevent the over-sensitization following peripheral nerve injury.■