Brianna Marie Lutz

Noncoding RNAs New Players in Chronic Pain

Chronic pain, a common clinical symptom, is often treated inadequately or ineffectively in part due to the incomplete understanding of molecular mechanisms that initiate and maintain this disorder. Newly identified noncoding RNAs govern gene expression. Recent studies have shown that peripheral noxious stimuli drive expressional changes in noncoding RNAs and that these changes are associated with pain hypersensitivity under chronic pain conditions. This review first presents current evidence for the peripheral inflammation/nerve injury–induced change in the expression of two types of noncoding RNAs, microRNAs, and Kcna2 antisense RNA, in pain-related regions, particularly in the dorsal root ganglion. The authors then discuss how peripheral noxious stimuli induce such changes. The authors finally explore potential mechanisms of how expressional changes in dorsal root ganglion microRNAs and Kcna2 antisense RNA contribute to the development and maintenance of chronic pain. An understanding of these mechanisms may propose novel therapeutic strategies for preventing and/or treating chronic pain.

Epigenetic regulation of chronic pain

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 nonsteroidal anti-inflammatory drugs. Inflammation, tissue injury and/or nerve injury-induced changes in gene expression in sensory neurons of the dorsal root ganglion, 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.

Dorsal root ganglion myeloid zinc finger protein 1 contributes to neuropathic pain after peripheral nerve trauma

Abstract 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 (AS) 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 naive 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 AS 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.

DNA methyltransferase DNMT3a contributes to neuropathic pain by repressing Kcna2 in primary afferent neurons

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.

mTOR, a new potential target for chronic pain and opioid-induced tolerance and hyperalgesia

Chronic pain is a major public health problem with limited treatment options. Opioids remain a routine treatment for chronic pain, but extended exposure to opioid therapy can produce opioid tolerance and hyperalgesia. Although the mechanisms underlying chronic pain, opioid-induced tolerance, and opioid-induced hyperalgesia remain to be uncovered, mammalian target of rapamycin (mTOR) is involved in these disorders. The mTOR complex 1 and its triggered protein translation are required for the initiation and maintenance of chronic pain (including cancer pain) and opioid-induced tolerance/hyperalgesia. Given that mTOR inhibitors are FDA-approved drugs and an mTOR inhibitor is approved for the treatment of several cancers, these findings suggest that mTOR inhibitors will likely have multiple clinical benefits, including anticancer, antinociception/anti-cancer pain, and antitolerance/hyperalgesia. This paper compares the role of mTOR complex 1 in chronic pain, opioid-induced tolerance, and opioid-induced hyperalgesia.

G9a participates in nerve injury-induced Kcna2 downregulation in primary sensory neurons

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.

Contribution of the Suppressor of Variegation 3-9 Homolog 1 in Dorsal Root Ganglia and Spinal Cord Dorsal Horn to Nerve Injury-induced Nociceptive Hypersensitivity.

Background:Peripheral nerve injury–induced gene alterations in the dorsal root ganglion (DRG) and spinal cord likely participate in neuropathic pain genesis. Histone methylation gates gene expression. Whether the suppressor of variegation 3-9 homolog 1 (SUV39H1), a histone methyltransferase, contributes to nerve injury–induced nociceptive hypersensitivity is unknown. Methods:Quantitative real-time reverse transcription polymerase chain reaction analysis, Western blot analysis, or immunohistochemistry were carried out to examine the expression of SUV39H1 mRNA and protein in rat DRG and dorsal horn and its colocalization with DRG &mgr;-opioid receptor (MOR). The effects of a SUV39H1 inhibitor (chaetocin) or SUV39H1 siRNA on fifth lumbar spinal nerve ligation (SNL)–induced DRG MOR down-regulation and nociceptive hypersensitivity were examined. Results:SUV39H1 was detected in neuronal nuclei of the DRG and dorsal horn. It was distributed predominantly in small DRG neurons, in which it coexpressed with MOR. The level of SUV39H1 protein in both injured DRG and ipsilateral fifth lumbar dorsal horn was time dependently increased after SNL. SNL also produced an increase in the amount of SUV39H1 mRNA in the injured DRG (n = 6/time point). Intrathecal chaetocin or SUV39H1 siRNA as well as DRG or intraspinal microinjection of SUV39H1 siRNA impaired SNL-induced allodynia and hyperalgesia (n = 5/group/treatment). DRG microinjection of SUV39H1 siRNA also restored SNL-induced DRG MOR down-regulation (n = 6/group). Conclusions:The findings of this study suggest that SUV39H1 contributes to nerve injury–induced allodynia and hyperalgesia through gating MOR expression in the injured DRG. SUV39H1 may be a potential target for the therapeutic treatment of nerve injury–induced nociceptive hypersensitivity.

Intrathecal rapamycin attenuates morphine-induced analgesic tolerance and hyperalgesia in rats with neuropathic pain

Repeated and long-term administration of opioids is often accompanied by the initiation of opioid-induced analgesic tolerance and hyperalgesia in chronic pain patients. Our previous studies showed that repeated intrathecal morphine injection activated the mammalian target of rapamycin complex 1 (mTORC1) in spinal dorsal horn neurons and that blocking this activation prevented the initiation of morphine-induced tolerance and hyperalgesia in healthy rats. However, whether spinal mTORC1 is required for morphine-induced tolerance and hyperalgesia under neuropathic pain conditions remains elusive. We here observed the effect of intrathecal infusion of rapamycin, a specific mTORC1 inhibitor, on morphine-induced tolerance and hyperalgesia in a neuropathic pain model in rats induced by the fifth lumbar spinal nerve ligation (SNL). Continuous intrathecal infusion of morphine for one week starting on day 8 post-SNL led to morphine tolerance demonstrated by morphine-induced reduction in maximal possible analgesic effect (MPAE) to tail heat stimuli and ipsilateral paw withdrawal threshold (PWT) to mechanical stimuli in SNL rats. Such reduction was attenuated by co-infusion of rapamycin. Co-infusion of rapamycin also blocked morphine tolerance demonstrated by attenuation of morphine-induced reduction in MPAE in sham rats and morphine-induced hyperalgesia demonstrated by the reverse of morphine-induced reduction in PWT on both sides of sham rats and on the contralateral side of SNL rats. The results suggest that mTORC1 inhibitors could serve as promising medications for use as adjuvants with opioids in clinical neuropathic pain management.

G9a inhibits CREB-triggered expression of mu opioid receptor in primary sensory neurons following peripheral nerve injury.

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.

Dorsal root ganglion transcriptome analysis following peripheral nerve injury in mice

Background Peripheral 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. Methods The 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. Results Our 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. Conclusion Our 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.