Epigenetic gene silencing underlies C-fiber dysfunctions in neuropathic pain

Abstract

Peripheral nerve injury causes neuropathic pain, which is characterized by the paradoxical sensations of positive and negative symptoms. Clinically, negative signs are frequently observed; however, their underlying molecular mechanisms are largely unknown. Dysfunction of C-fibers is assumed to underlie negative symptoms and is accompanied by long-lasting downregulation of Na(v)1.8 sodium channel and mu-opioid receptor (MOP) in the dorsal root ganglion (DRG). In the present study, we found that nerve injury upregulates neuron-restrictive silencer factor (NRSF) expression in the DRG neurons mediated through epigenetic mechanisms. In addition, chromatin immunoprecipitation analysis revealed that nerve injury promotes NRSF binding to the neuron-restrictive silencer element within MOP and Na(v)1.8 genes, thereby causing epigenetic silencing. Furthermore, NRSF knockdown significantly blocked nerve injury-induced downregulations of MOP and Na(v)1.8 gene expressions, C-fiber hypoesthesia, and the losses of peripheral morphine analgesia and Na(v)1.8-selective blocker-induced hypoesthesia. Together, these data suggest that NRSF causes pathological and pharmacological dysfunction of C-fibers, which underlies the negative symptoms in neuropathic pain.

Figures

Figure 1.

Downregulation of NRSE-containing MOP and Nav1.8 genes after nerve injury. A, Time course of MOP and Nav1.8 mRNA expressions in the DRG after nerve injury. The mRNA expression levels were assessed using quantitative real-time PCR and normalized to that of GAPDH mRNA. Data are calculated as percentages of day 0 and expressed as the means ± SEM from at least three mice. *p < 0.05 versus day 0. B, Schematic diagram indicating the locations of NRSE sequences within MOP and Nav1.8 genes. Coding exons are shown as black boxes, the noncoding exons as open boxes, and the NRSE sequences as gray boxes. The black arrows indicate the translation initiation sites, and the lower open arrows indicate the orientation of the NRSE sequences. C, Deviations of MOP- and Nav1.8-NRSEs from the consensus NRSE. The capital letters are conserved among functional NRSE sequences, and the bold capital letters are important for NRSF binding. The scores were obtained from the TFSEARCH program.

Figure 2.

Epigenetic upregulation of NRSF gene expression. A, B, Time course of total (A) and exon-specific (B) NRSF mRNA expressions in the DRG after nerve injury. The mRNA expression levels were assessed using quantitative real-time PCR and normalized to that of GAPDH mRNA. Data are calculated as percentages of day 0. *p < 0.05 versus day 0. EI, EII, EIII, Exons I, II, III, respectively. C, Acetylation of histone H3 (AcH3) and H4 (AcH4) at NRSF promoter II (PII) at day 7 after injury, assessed using ChIP assay. Quantitative analysis was performed using real-time PCR, and the data were normalized to the corresponding input. D, NRSF protein expression at day 7 postinjury, assessed using Western blot analysis. Results are normalized to the histone H3 protein expression level. For C and D, data are calculated as percentages of sham-operated group. *p < 0.05 versus sham-operated group. Data are expressed as the means ± SEM from at least three mice. E, Immunohistochemical double labeling between NRSF (red) and NeuN (green), a neuronal marker, in the DRG of sham-operated and nerve-injured mice. Scale bars, 50 μm.

Figure 3.

Epigenetic silencing of MOP and Nav1.8 genes through NRSF binding. ChIP assay was performed at day 7 postinjury. A, B, ChIP assays using anti-NRSF antibody. A, Gel images show the representative data. B, Quantification of NRSF-binding at MOP-NRSE and at Nav1.8-NRSE-2 (Nav1.8–2). C, D, Scanning ChIP analysis of acetylation levels of histone H3 (AcH3) and H4 (AcH4) in the genomic regions spanning NRSE sequences within MOP (C) and Nav1.8 (D) genes. The schematics at the top show the PCR-targeted regions. Quantitative analysis was performed using real-time PCR, and the data were normalized to the corresponding input. Results are calculated as percentages of sham-operated group and expressed as means ± SEM from at least three mice. *p < 0.05 versus sham-operated group.

Figure 4.

Blockade of nerve injury-induced MOP and Nav1.8 downregulations by NRSF knockdown. Mice were intrathecally pretreated with vehicle (Veh), AS-ODN (AS) against NRSF, or the corresponding MS-ODN (MS). A, AS-ODN-induced reduction of NRSF protein expression in the DRG, assessed by Western blot analysis. Data are normalized to histone H3 protein expression levels and then expressed as percentages of the levels in the Veh-treated group. *p < 0.05 versus Veh-treated group. Data represent the means ± SEM from three mice. B, C, The effects of NRSF AS-ODN on nerve injury-induced downregulations of MOP and Nav1.8 (B) and TRPM8, TRPA1, and CGRP (C) in the DRG at day 7 postinjury. The mRNA expression levels were quantified by real-time PCR, and normalized to that of GAPDH mRNA. Results are calculated as percentages of Veh-treated and sham-operated groups and expressed as means ± SEM from at least three mice. *p < 0.05 versus Veh-treated and sham-operated groups and #p < 0.05 versus MS-ODN-treated and nerve-injured groups.

Figure 5.

Blockade of nerve injury-induced C-fiber hypoesthesia and loss of peripheral A-803467 hypoesthesia by NRSF knockdown. A–C, Blockade of nerve injury-induced hypoesthesia of C-fiber (5 Hz) (A), but not hypersensitization of Aδ-fiber (250 Hz) (B) and Aβ-fiber (2000 Hz) (C) by NRSF AS-ODN. Paw withdrawal thresholds to electrical stimulation (μA) were measured using the EPW test. *p < 0.05 versus vehicle (Veh)-treated and sham-operated groups and #p < 0.05 versus Veh-treated and nerve-injured groups. D, Recovery of nerve injury-induced loss of peripheral A-803467 hypoesthesia by AS-ODN. The C-fiber responses were assessed 30 min after intraplantar injection of control (Cont) or A-803467 (30 nmol). *p < 0.05 versus Veh-treated, sham-operated, and Cont-treated groups. #p < 0.05 versus Veh-treated, nerve-injured, and Cont-treated groups. +p < 0.05 versus AS-ODN-treated, nerve-injured, and Cont-treated groups. Data represent the means ± SEM from at least three mice.

Figure 6.

Blockade of nerve injury-induced loss of peripheral morphine analgesia by NRSF knockdown. Thermal pain threshold was assessed at day 7 postinjury by using a thermal paw withdrawal test. A, Time course of thermal paw withdrawal latencies (PWL, in seconds) after morphine (30 nmol, intraplantar) injection. *p, #p, or +p < 0.05 versus corresponding 0 min, respectively. B, Comparison of morphine analgesia by area under the curve (AUC). *p < 0.05 versus vehicle (Veh)-treated and sham-operated groups and #p < 0.05 versus Veh-treated and nerve-injured groups. Data are expressed as the means ± SEM from at least four mice.