Nerve injury inhibits Oprd1 and Cnr1 transcription through REST in primary sensory neurons

Abstract

The transcription repressor REST in the dorsal root ganglion (DRG) is upregulated by peripheral nerve injury and promotes the development of chronic pain. However, the genes targeted by REST in neuropathic pain development remain unclear. The expression levels of four opioid receptor genes (Oprm1, Oprd1, Oprl1 and Oprk1) and the cannabinoid CB1 receptor (Cnr1) gene in the DRG regulate nociception. In this study, we determined the role of REST in controlling their expression in the DRG induced by spared nerve injury (SNI). SNI induced chronic pain hypersensitivity in wild-type mice and was accompanied by increased levels of Rest transcript and protein. Transcriptomic analyses of wild-type mouse DRGs suggested that SNI upregulates the expression of Rest transcripts and downregulates the transcripts of all four opioid receptor genes and the Cnr1 gene. Quantitative reverse transcription polymerase chain reaction analyses of these tissues validated these results. Analysis of publicly available bioinformatic data suggested that REST binds to the promoter regions of Oprm1 and Cnr1. Chromatin immunoprecipitation analyses indicated the presence of REST at these promoters. Full-length Rest conditional knockout in primary sensory neurons reduced SNI-induced pain hypersensitivity and rescued the SNI-induced reduction in the expression of Oprd1 and Cnr1 in mouse DRG. Our results suggest that nerve injury represses the transcription of at least the Oprd1 and Cnr1 genes via REST in primary sensory neurons and that REST is a potential therapeutic target for neuropathic pain. Thus, inhibiting REST activity could potentially reduce chronic neuropathic pain and augment opioid/cannabinoid analgesic actions by increasing the transcription of Oprd1 and Cnr1 genes in DRG neurons.

Keywords: Cannabinoid CB1 receptor; Chronic pain; Dorsal root ganglion; Opioid receptors; REST; Therapeutic target.

Fig. 1

(a) Peripheral nerve injury induces chronic neuropathic pain hypersensitivities in wild-type mice. Either spared nerve injury (SNI) or sham surgery (Sham) was performed on the left hind limbs of male wild-type mice. Pain symptoms, in terms of ipsilateral hindpaw withdrawal thresholds (withdrawal latency), were determined in response to a noxious pressure stimulus (mechanical hyperalgesia), noxious radiant heat (thermal hyperalgesia), and von Frey filaments (tactile allodynia) over 27 days (Days post-surgery). N = 6 per group. A Student’s t-test was used. P-values are shown (* <0.05; **<0.01; ***<0.001; ****<0.0001) compared with sham group. Data are presented as mean ± SEM. (b–d) SNI results in increased mRNA and protein levels of the REST gene in the injured DRG of wild-type mice, but it does not cause significant changes in the rotarod assays. (b) mRNA samples from the L3/L4 DRG tissues of mice, characterized in Fig. 1a, were used for RT-qPCR analysis of Rest transcripts. Gapdh was used as an internal control. A Student’s t-test was used to measure the p-value. N = 5 mice per group. Data are presented as mean ± SEM. (c) Western blotting assays from the L3/L4 DRG tissues indicated a corresponding increase in the REST protein. M = Molecular weight ladder. Approximate molecular weights in kilodalton are also shown. (d) Rotarod assays of wild-type mice indicate no significant difference between sham and SNI treatment.

Fig. 2

SNI leads to transcriptomic activation of Rest and repression of opioid receptor and cannabinoid CB1 receptor genes in the injured DRG of wild-type mice. mRNA samples from the L3/L4 DRG tissues of 3 individual male mice were examined by RNA-Seq analyses. (a) Hierarchical clustering of differentially expressed genes between SNI and Sham surgery groups. (b) Volcano plot showing differentially expressed genes. (c) Differential expression analysis of Rest, Oprd1, Oprk1, Oprl1, Oprm1, and Cnr1 between SNI and Sham surgery groups. The size of the circle is -log10(FDR). (d) Gene Set Enrichment Analysis shows downregulation of the opioid signaling pathway. Experiments were performed in triplicate. NES represents Normalized Enrichment Score.

Fig. 3

(a) SNI results in decreased mRNA levels of opioid receptor and cannabinoid CB1 receptor genes in the injured DRG of wild-type mice. mRNA samples from the L3/L4 DRG tissues of mice, characterized in Fig. 1, were used for RT-qPCR analysis of Oprm1, Oprd1, Oprk1, Oprl1, and Cnr1 transcripts. Gapdh was used as an internal control. A Student’s t-test was used to measure the p-value. N = 5 mice per group. Data are presented as mean ± SEM. (b) REST binds to the Oprm1 and Cnr1 promoters in the injured DRG of wild-type mice. REST binding to Oprm1 and Cnr1 promoters was measured by qChIP assay in sham and SNI in mice. N = 3 mice per group. A Student’s t-test was used to measure the p-value. Data are presented as mean ± SEM.

Fig. 4

(a) DRG neuron-specific Rest cKO attenuates nerve injury-induced pain hypersensitivity in mice. Nerve injury-induced pain hypersensitivity was assessed in Rest cKO mice as described in Fig. 1. The mouse groups were: Rest cKO (-Cre) + sham, Rest cKO (-Cre) + SNI, and Rest cKO (+ Cre) + SNI. N = 6 per group. A two-way ANOVA followed by Tukey’s multiple comparison test was used. P-values are shown compared with Rest cKO (-Cre) + SNI group. Data are presented as mean ± SEM. (b–d) SNI results in decreased mRNA and protein levels of the Rest gene in the injured DRG of Rest cKO mice compared with their wild type littermates, but it does not cause significant changes in the rotarod assays. (b) mRNA samples from the L3/L4 DRG tissues of mice, as characterized in Fig. 4a, were analyzed. The mouse groups were: Rest cKO (-Cre) + sham, Rest cKO (-Cre) + SNI, and Rest cKO (+ Cre) + SNI. RT-qPCR analysis was used to measure the levels of Rest transcripts. Gapdh was used as an internal control. A two-way ANOVA followed by Tukey’s multiple comparison test was used. Data are presented as mean ± SEM. N = 5 mice per group. P-values are shown. (c) Western blotting assays from the L3/L4 DRG tissues to detect REST protein. GAPDH was used as an internal control. Approximate molecular weights in kilodalton are also shown. (d) Rotarod assays of Rest cKO mice indicated no significant difference between sham and SNI treatment.

Fig. 5

DRG neuron-specific Rest cKO rescues the SNI-induced reduction in the expression of Oprd1 and Cnr1 in the DRG of mice. mRNA samples from the L3/L4 DRG tissues of mice, characterized in Fig. 4, were used for RT-qPCR analysis of Oprm1, Oprd1, Oprk1, Oprl1, and Cnr1 transcripts. Gapdh was used as an internal control. Data from Rest cKO (-Cre) + sham, Rest cKO (-Cre) + SNI, and Rest cKO (+ Cre) + SNI are shown. A two-way ANOVA followed by Tukey’s multiple comparison test was used. Data are presented as mean ± SEM. P-values are shown. N = 5 mice per group.