Lingli Liang

A long noncoding RNA contributes to neuropathic pain by silencing Kcna2 in primary afferent neurons

Neuropathic pain is a refractory disease characterized by maladaptive changes in gene transcription and translation in the sensory pathway. Long noncoding RNAs (lncRNAs) are emerging as new players in gene regulation, but how lncRNAs operate in the development of neuropathic pain is unclear. Here we identify a conserved lncRNA, named Kcna2 antisense RNA, for a voltage-dependent potassium channel mRNA, Kcna2, in first-order sensory neurons of rat dorsal root ganglion (DRG). Peripheral nerve injury increased Kcna2 antisense RNA expression in injured DRG through activation of myeloid zinc finger protein 1, a transcription factor that binds to the Kcna2 antisense RNA gene promoter. Mimicking this increase downregulated Kcna2, reduced total voltage-gated potassium current, increased excitability in DRG neurons and produced neuropathic pain symptoms. Blocking this increase reversed nerve injury–induced downregulation of DRG Kcna2 and attenuated development and maintenance of neuropathic pain. These findings suggest endogenous Kcna2 antisense RNA as a therapeutic target for the treatment of neuropathic pain.

Opioid receptor–triggered spinal mTORC1 activation contributes to morphine tolerance and hyperalgesia

The development of opioid-induced analgesic tolerance and hyperalgesia is a clinical challenge for managing chronic pain. Adaptive changes in protein translation in the nervous system are thought to promote opioid tolerance and hyperalgesia; however, how opioids drive such changes remains elusive. Here, we report that mammalian target of rapamycin (mTOR), which governs most protein translation, was activated in rat spinal dorsal horn neurons after repeated intrathecal morphine injections. Activation was triggered through μ opioid receptor and mediated by intracellular PI3K/Akt. Spinal mTOR inhibition blocked both induction and maintenance of morphine tolerance and hyperalgesia, without affecting basal pain perception or locomotor functions. These effects were attributed to the attenuation of morphine-induced increases in translation initiation activity, nascent protein synthesis, and expression of some known key tolerance-associated proteins, including neuronal NOS (nNOS), in dorsal horn. Moreover, elevating spinal mTOR activity by knocking down the mTOR-negative regulator TSC2 reduced morphine analgesia, produced pain hypersensitivity, and increased spinal nNOS expression. Our findings implicate the μ opioid receptor-triggered PI3K/Akt/mTOR pathway in promoting morphine-induced spinal protein translation changes and associated morphine tolerance and hyperalgesia. These data suggest that mTOR inhibitors could be explored for prevention and/or reduction of opioid tolerance in chronic pain management.

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.

mTOR and its downstream pathway are activated in the dorsal root ganglion and spinal cord after peripheral inflammation, but not after nerve injury ☆

Protein translation controlled through activation of mammalian target of rapamycin (mTOR) participates in many physiological and pathological processes. However, whether such activation is required for chronic pain is still unknown. Here, we examined activation of the mTOR signaling pathway during complete Freunds adjuvant (CFA)-induced chronic inflammatory pain and L5 spinal nerve ligation (SNL)-induced neuropathic pain in rats. Western blot analysis showed significantly increased levels of phosphorylated mTOR (p-mTOR) and phosphorylated p70S6 kinase 1 (p-S6K1, a downstream effector of mTOR) in the ipsilateral L4/5 spinal cord 2h, 1 day, 3 days, and 7 days after intraplantar CFA injection and in the ipsilateral L4/5 dorsal root ganglions (DRGs) 1 and 3 days after CFA injection. Immunohistochemistry also demonstrated increases in number of p-mTOR-labeled neurons in the ipsilateral L4/5 DRGs and in density of p-mTOR-labeled immunoreactivity in the ipsilateral L4/5 superficial dorsal horn 1 day after CFA injection. Moreover, intrathecal administration of rapamycin, a selective inhibitor of mTOR, significantly blocked CFA-induced mechanical allodynia and thermal hyperalgesia 1 day post-CFA injection. Interestingly, expression of neither p-mTOR nor p-S6K1 was markedly altered on days 3, 7, or 14 after L5 SNL in L5 spinal cord or DRG. These findings indicate that in DRG and spinal cord, mTOR and S6K1 are activated during chronic inflammatory pain, but not during neuropathic pain. Our results strongly suggest that mTOR and its downstream pathway contribute to the development of chronic inflammatory pain.

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.

Nerve injury–induced epigenetic silencing of opioid receptors controlled by Dnmt3a in primary afferent neurons

Abstract 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.

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.

Erratum: Identification of Early RET+ Deep Dorsal Spinal Cord Interneurons in Gating Pain (Neuron (2016) 91(5) (1137–1153) (S0896627316304238) (10.1016/j.neuron.2016.07.038))

(Neuron 91, 1137–1153; September 7, 2016) In the originally published version of the article and Supplemental Information, the units used for electrical stimulation were ‘‘pA,’’ but should have been ‘‘mA.’’ Additionally, the current number in the legend to Figure 6B was written as 25, but should have been 30. Finally, the authors did not acknowledge Dr. D. Ginty for sharing Split mice. These errors have been corrected both online and in print. The authors regret the error.