Highlights d Deep profiling of proteome and phosphoproteome in AD progression d Validation of protein alterations in two independent AD cohorts d Identification of Ab-induced protein changes in AD and the 5xFAD mouse model d Prioritization of proteins and pathways in AD by multi-omics

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.

Peripheral nerve injury-induced changes in gene transcription and translation in primary sensory neurons of the dorsal root ganglion (DRG) are considered to contribute to neuropathic pain genesis. Transcription factors control gene expression. Peripheral nerve injury increases the expression of myeloid zinc finger protein 1 (MZF1), a transcription factor, and promotes its binding to the voltage-gated potassium 1.2 (Kv1.2) antisense RNA gene in the injured DRG. However, whether DRG MZF1 participates in neuropathic pain is still unknown. Here, we report that blocking the nerve injury-induced increase of DRG MZF1 through microinjection of MZF1 siRNA into the injured DRG attenuated the initiation and maintenance of mechanical, cold, and thermal pain hypersensitivities in rats with chronic constriction injury (CCI) of the sciatic nerve, without affecting locomotor functions and basal responses to acute mechanical, heat, and cold stimuli. Mimicking the nerve injury-induced increase of DRG MZF1 through microinjection of recombinant adeno-associated virus 5 expressing full-length MZF1 into the DRG produced significant mechanical, cold, and thermal pain hypersensitivities in naïve rats. Mechanistically, MZF1 participated in CCI-induced reductions in Kv1.2 mRNA and protein and total Kv current and the CCI-induced increase in neuronal excitability through MZF1-triggered Kv1.2 antisense RNA expression in the injured DRG neurons. MZF1 is likely an endogenous trigger of neuropathic pain and might serve as a potential target for preventing and treating this disorder.

Chronic pain arising from peripheral inflammation and tissue or nerve injury is a common clinical symptom. Although intensive research on the neurobiological mechanisms of chronic pain has been carried out during previous decades, this disorder is still poorly managed by current drugs such as opioids and non-steroidal anti-inflammatory drugs. Inflammation-, tissue injury-, and/or nerve injury-induced changes in gene expression in sensory neurons of the dorsal root ganglion (DRG), spinal cord dorsal horn, and pain-associated brain regions are thought to participate in chronic pain genesis; however, how these changes occur is still elusive. Epigenetic modifications including DNA methylation and covalent histone modifications control gene expression. Recent studies have shown that peripheral noxious stimulation changes DNA methylation and histone modifications and that these changes may be related to the induction of pain hypersensitivity under chronic pain conditions. This review summarizes the current knowledge and progress in epigenetic research in chronic pain and discusses the potential role of epigenetic modifications as therapeutic antinociceptive targets in this disorder.

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.

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Neuropathic pain, a distressing and debilitating disorder, is still poorly managed in clinic. Opioids, like morphine, remain the mainstay of prescribed medications in the treatment of this disorder, but their analgesic effects are highly unsatisfactory in part due to nerve injury-induced reduction of opioid receptors in the first-order sensory neurons of dorsal root ganglia. G9a is a repressor of gene expression. We found that nerve injury-induced increases in G9a and its catalyzed repressive marker H3K9m2 are responsible for epigenetic silencing of Oprm1, Oprk1, and Oprd1 genes in the injured dorsal root ganglia. Blocking these increases rescued dorsal root ganglia Oprm1, Oprk1, and Oprd1 gene expression and morphine or loperamide analgesia and prevented the development of morphine or loperamide-induced analgesic tolerance under neuropathic pain conditions. Conversely, mimicking these increases reduced the expression of three opioid receptors and promoted the mu opioid receptor-gated release of primary afferent neurotransmitters. Mechanistically, nerve injury-induced increases in the binding activity of G9a and H3K9me2 to the Oprm1 gene were associated with the reduced binding of cyclic AMP response element binding protein to the Oprm1 gene. These findings suggest that G9a participates in the nerve injury-induced reduction of the Oprm1 gene likely through G9a-triggered blockage in the access of cyclic AMP response element binding protein to this gene.