Abstract
Type 1 cannabinoid receptors (CB1Rs) are expressed in the dorsal root ganglion (DRG) and contribute to the analgesic effect of cannabinoids. However, the epigenetic mechanism regulating the expression of CB1Rs in neuropathic pain is unknown. G9a (encoded by the Ehmt2 gene), a histone 3 at lysine 9 methyltransferase, is a key chromatin regulator responsible for gene silencing. In this study, we determined G9a's role in regulating CB1R expression in the DRG and in CB1R-mediated analgesic effects in an animal model of neuropathic pain. We show that nerve injury profoundly reduced mRNA levels of CB1Rs but increased the expression of CB2 receptors in the rat DRG. ChIP results indicated increased enrichment of histone 3 at lysine 9 dimethylation, a G9a-catalyzed repressive histone mark, at the promoter regions of the CB1R genes. G9a inhibition in nerve-injured rats not only up-regulated the CB1R expression level in the DRG but also potentiated the analgesic effect of a CB1R agonist on nerve injury-induced pain hypersensitivity. Furthermore, in mice lacking Ehmt2 in DRG neurons, nerve injury failed to reduce CB1R expression in the DRG and to decrease the analgesic effect of the CB1R agonist. Moreover, nerve injury diminished the inhibitory effect of the CB1R agonist on synaptic glutamate release from primary afferent nerves to spinal cord dorsal horn neurons in WT mice but not in mice lacking Ehmt2 in DRG neurons. Our findings reveal that nerve injury diminishes the analgesic effect of CB1R agonists through G9a-mediated CB1R down-regulation in primary sensory neurons.
Highlights
Type 1 cannabinoid receptors (CB1Rs) are expressed in the dorsal root ganglion (DRG) and contribute to the analgesic effect of cannabinoids
Our study demonstrates that nerve injury induced long-lasting CB1R down-regulation in the DRG and diminished the analgesic effect of the CB1R agonist on neuropathic pain
In both rats and mice, nerve injury caused by spinal nerve ligation (SNL) and Spared nerve injury (SNI), respectively, consistently reduced the expression of CB1Rs in the DRG
Summary
Nerve injury differentially alters the expression level of CB1Rs and CB2Rs in the DRG. Intraperitoneal injection of ACEA at both 1 mg/kg and 5 mg/kg significantly increased the mechanical and thermal withdrawal thresholds in Ehmt2-cKO mice 3 weeks after SNI (n ϭ 8 mice, Fig. 6) These data provide unambiguous evidence that G9a in DRG neurons is indispensable for nerve injury– induced down-regulation of CB1Rs and the diminished analgesic effect of the CB1R agonist on neuropathic pain. The inhibitory effect of ACEA on the amplitude of evoked EPSCs in lamina II neurons did not differ significantly between SNI Ehmt2-cKO mice (n ϭ 12 neurons) and sham-treated Ehmt2-cKO mice (n ϭ 10 neurons) These findings clearly indicate that G9a in DRG neurons is responsible for nerve injury–induced reduction in CB1R-mediated inhibition of primary afferent input to spinal dorsal horn neurons. In WT mice subjected to SNI, bath application of up to 200 nM ACEA had no significant inhibitory effect
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