Nerve injury induces changes in gene transcription in dorsal root ganglion (DRG) neurons, which may contribute to nerve injury-induced neuropathic pain. DNA methylation represses gene expression. Here, we report that peripheral nerve injury increases expression of the DNA methyltransferase DNMT3a in the injured DRG neurons via the activation of the transcription factor octamer transcription factor 1. Blocking this increase prevents nerve injury-induced methylation of the voltage-dependent potassium (Kv) channel subunit Kcna2 promoter region and rescues Kcna2 expression in the injured DRG and attenuates neuropathic pain. Conversely, in the absence of nerve injury, mimicking this increase reduces the Kcna2 promoter activity, diminishes Kcna2 expression, decreases Kv current, increases excitability in DRG neurons and leads to spinal cord central sensitization and neuropathic pain symptoms. These findings suggest that DNMT3a may contribute to neuropathic pain by repressing Kcna2 expression in the DRG.

Nerve injury-induced downregulation of voltage-gated potassium channel subunit Kcna2 in the dorsal root ganglion (DRG) is critical for DRG neuronal excitability and neuropathic pain genesis. However, how nerve injury causes this downregulation is still elusive. Euchromatic histone-lysine N-methyltransferase 2, also known as G9a, methylates histone H3 on lysine residue 9 to predominantly produce a dynamic histone dimethylation, resulting in condensed chromatin and gene transcriptional repression. We showed here that blocking nerve injury-induced increase in G9a rescued Kcna2 mRNA and protein expression in the axotomized DRG and attenuated the development of nerve injury-induced pain hypersensitivity. Mimicking this increase decreased Kcna2 mRNA and protein expression, reduced Kv current, and increased excitability in the DRG neurons and led to spinal cord central sensitization and neuropathic pain-like symptoms. G9a mRNA is co-localized with Kcna2 mRNA in the DRG neurons. These findings indicate that G9a contributes to neuropathic pain development through epigenetic silencing of Kcna2 in the axotomized DRG.

Nerve injury‐induced change in gene expression in primary sensory neurons of dorsal root ganglion (DRG) is critical for neuropathic pain genesis. N 6 ‐methyladenosine (m 6 A) modification of RNA represents an additional layer of gene regulation. Here, it is reported that peripheral nerve injury increases the expression of the m 6 A demethylase fat‐mass and obesity‐associated proteins (FTO) in the injured DRG via the activation of Runx1, a transcription factor that binds to the Fto gene promoter. Mimicking this increase erases m 6 A in euchromatic histone lysine methyltransferase 2 ( Ehmt2 ) mRNA (encoding the histone methyltransferase G9a) and elevates the level of G9a in DRG and leads to neuropathic pain symptoms. Conversely, blocking this increase reverses a loss of m 6 A sites in Ehmt2 mRNA and destabilizes the nerve injury‐induced G9a upregulation in the injured DRG and alleviates nerve injury‐associated pain hypersensitivities. FTO contributes to neuropathic pain likely through stabilizing nerve injury‐induced upregulation of G9a, a neuropathic pain initiator, in primary sensory neurons.

Opioids are the gold standard for pharmacological treatment of neuropathic pain, but their analgesic effects are unsatisfactory in part due to nerve injury-induced downregulation of opioid receptors in dorsal root ganglia (DRG) neurons. How nerve injury drives such downregulation remains elusive. DNA methyltransferase-(DNMT-) triggered DNA methylation represses gene expression. We show here that blocking the nerve injury-induced increase in DRG DNMT3a (a de novo DNMT) rescued the expression of Oprm1 and Oprk1 mRNAs and their respective encoding mu opioid receptor (MOR) and kappa opioid receptor (KOR) proteins in the injured DRG. Blocking this increase also prevented the nerve injury-induced increase in DNA methylation in the promoter and 5′-untranslated region of the Oprm1 gene in the injured DRG, restored morphine or loperamide (a peripheral acting MOR preferring agonist) analgesic effects, and attenuated the development of their analgesic tolerance under neuropathic pain conditions. Mimicking this increase reduced the expression of Oprm1 and Oprk1 mRNAs and their coding MOR and KOR in DRG and augmented MOR-gated neurotransmitter release from the primary afferents. Mechanistically, DNMT3a regulation of Oprm1 gene expression required the methyl-CpG-binding protein 1, MBD1, as MBD1 knockout resulted in the decreased binding of DNMT3a to the Oprm1 gene promoter and blocked the DNMT3a-triggered repression of Oprm1 gene expression in DRG neurons. These data suggest that DNMT3a is required for nerve injury-induced and MBD1-mediated epigenetic silencing of the MOR and KOR in the injured DRG. DNMT3a inhibition may serve as a promising adjuvant therapy for opioid use in neuropathic pain management.

BackgroundPeripheral nerve injury leads to changes in gene expression in primary sensory neurons of the injured dorsal root ganglia. These changes are believed to be involved in neuropathic pain genesis. Previously, these changes have been identified using gene microarrays or next generation RNA sequencing with poly-A tail selection, but these approaches cannot provide a more thorough analysis of gene expression alterations after nerve injury.MethodsThe present study chose to eliminate mRNA poly-A tail selection and perform strand-specific next generation RNA sequencing to analyze whole transcriptomes in the injured dorsal root ganglia following spinal nerve ligation. Quantitative real-time reverse transcriptase polymerase chain reaction assay was carried out to verify the changes of some differentially expressed RNAs in the injured dorsal root ganglia after spinal nerve ligation.ResultsOur results showed that more than 50 million (M) paired mapped sequences with strand information were yielded in each group (51.87 M–56.12 M in sham vs. 51.08 M–57.99 M in spinal nerve ligation). Six days after spinal nerve ligation, expression levels of 11,163 out of a total of 27,463 identified genes in the injured dorsal root ganglia significantly changed, of which 52.14% were upregulated and 47.86% downregulated. The largest transcriptional changes were observed in protein-coding genes (91.5%) followed by noncoding RNAs. Within 944 differentially expressed noncoding RNAs, the most significant changes were seen in long interspersed noncoding RNAs followed by antisense RNAs, processed transcripts, and pseudogenes. We observed a notable proportion of reads aligning to intronic regions in both groups (44.0% in sham vs. 49.6% in spinal nerve ligation). Using quantitative real-time polymerase chain reaction, we confirmed consistent differential expression of selected genes including Kcna2, Oprm1 as well as lncRNAs Gm21781 and 4732491K20Rik following spinal nerve ligation.ConclusionOur findings suggest that next generation RNA sequencing can be used as a promising approach to analyze the changes of whole transcriptomes in dorsal root ganglia following nerve injury and to possibly identify new targets for prevention and treatment of neuropathic pain.