Protein Arginine Methyltransferase 5 Contributes to Paclitaxel-Induced Neuropathic Pain by Activating Transient Receptor Potential Vanilloid 1 Epigenetic Modification in Dorsal Root Ganglion.

BACKGROUND: Paclitaxel (PTX), which is a first-line chemotherapy drug used to treat various types of cancers, exhibits peripheral neuropathy as a common side effect that is difficult to treat. Protein arginine methyltransferase 5 (PRMT 5) is a key regulator of the chemotherapy response, as chemotherapy drugs induce PRMT5 expression. However, little is known about the PRMT5-mediated epigenetic mechanisms involved in PTX-induced neuropathic allodynia. METHODS: Sprague-Dawley rats were intraperitoneally given PTX to induce neuropathic pain. Biochemical analyses were conducted to measure the protein expression levels in the dorsal root ganglion (DRG) of the animals. The von Frey test and hot plate test were used to evaluate nociceptive behaviors. RESULTS: PTX increased the PRMT5 (mean difference [MD]: 0.68, 95% confidence interval [CI], 0.88-0.48; P < .001 for vehicle)-mediated deposition of histone H3R2 dimethyl symmetric (H3R2me2s) at the transient receptor potential vanilloid 1 ( Trpv1 ) promoter in the DRG. PRMT5-induced H3R2me2s recruited WD repeat domain 5 (WDR5) to increase trimethylation of lysine 4 on histone H3 (H3K4me3) at Trpv1 promoters, thus resulting in TRPV1 transcriptional activation (MD: 0.65, 95% CI, 0.82-0.49; P < .001 for vehicle) in DRG in PTX-induced neuropathic pain. Moreover, PTX increased the activity of NADPH oxidase 4 (NOX4) (MD: 0.66, 95% CI, 0.81-0.51; P < .001 for vehicle), PRMT5-induced H3R2me2s, and WDR5-mediated H3K4me3 in the DRG in PTX-induced neuropathic pain. Pharmacological antagonism and the selective knockdown of PRMT5 in DRG neurons completely blocked PRMT5-mediated H3R2me2s, WDR5-mediated H3K4me3, or TRPV1 expression and neuropathic pain development after PTX injection. Remarkably, NOX4 inhibition not only attenuated allodynia behavior and reversed the above-mentioned signaling but also reversed NOX4 upregulation via PTX. CONCLUSIONS: Thus, the NOX4/PRMT5-associated epigenetic mechanism in DRG has a dominant function in the transcriptional activation of TRPV1 in PTX-induced neuropathic pain.

Melatonin Relieves Paclitaxel-Induced Neuropathic Pain by Regulating pNEK2-Dependent Epigenetic Pathways in DRG Neurons.

The neurohormone melatonin (MLT) demonstrates promising potential in ameliorating neuropathic pain induced by paclitaxel (PTX) chemotherapy. However, little is known about its protective effect on dorsal root ganglion (DRG) neurons in neuropathic pain resulting from the chemotherapeutic drug PTX. Here, PTX-treated rats revealed that intrathecal administration of MLT dose-dependently elevated hind paw withdrawal thresholds and latency, indicating that MLT significantly reversed PTX-induced neuropathic pain. Mechanistically, the analgesic effects of MLT were found to be mediated via melatonin receptor 2 (MT2), as pretreatment with an MT2 receptor antagonist inhibited these effects. Moreover, intrathecal MLT injection reversed the pNEK2-dependent epigenetic program induced by PTX. All of the effects caused by MLT were blocked by pretreatment with an MT2 receptor-selective antagonist, 4P-PDOT. Remarkably, multiple MLT administered during PTX treatment (PTX+MLTs) exhibited not only rapid but also lasting reversal of allodynia/hyperalgesia compared to single-bolus MLT administered after PTX treatment (PTX+MLT). In addition, PTX+MLTs exhibited greater efficacy in reversing PTX-induced alterations in pRSK2, pNEK2, JMJD3, H3K27me3, and TRPV1 expression and interaction in DRG neurons than PTX+MLT. These results indicated that MLT administered during PTX treatment reduced the incidence and/or severity of neuropathy and had a better inhibitory effect on the pNEK2-dependent epigenetic program compared to MLT administered after PTX treatment. In conclusion, MLT/MT2 is a promising therapy for the treatment of pNEK2-dependent painful neuropathy resulting from PTX treatment. MLT administered during PTX chemotherapy may be more effective in the prevention or reduction of PTX-induced neuropathy and maintaining quality.

Phosphorylated Upstream Frameshift 1-dependent Nonsense-mediated µ-Opioid Receptor mRNA Decay in the Spinal Cord Contributes to the Development of Neuropathic Allodynia-like Behavior in Rats.

BACKGROUND: Nonsense-mediated messenger RNA (mRNA) decay increases targeted mRNA degradation and has been implicated in the regulation of gene expression in neurons. The authors hypothesized that nonsense-mediated µ-opioid receptor mRNA decay in the spinal cord is involved in the development of neuropathic allodynia-like behavior in rats. METHODS: Adult Sprague-Dawley rats of both sexes received spinal nerve ligation to induce neuropathic allodynia-like behavior. The mRNA and protein expression contents in the dorsal horn of animals were measured by biochemical analyses. Nociceptive behaviors were evaluated by the von Frey test and the burrow test. RESULTS: On Day 7, spinal nerve ligation significantly increased phosphorylated upstream frameshift 1 (UPF1) expression in the dorsal horn (mean ± SD; 0.34 ± 0.19 in the sham ipsilateral group vs. 0.88 ± 0.15 in the nerve ligation ipsilateral group; P < 0.001; data in arbitrary units) and drove allodynia-like behaviors in rats (10.58 ± 1.72 g in the sham ipsilateral group vs. 1.19 ± 0.31 g in the nerve ligation ipsilateral group, P < 0.001). No sex-based differences were found in either Western blotting or behavior tests in rats. Eukaryotic translation initiation factor 4A3 (eIF4A3) triggered SMG1 kinase (0.06 ± 0.02 in the sham group vs. 0.20 ± 0.08 in the nerve ligation group, P = 0.005, data in arbitrary units)-mediated UPF1 phosphorylation, leading to increased nonsense-mediated mRNA decay factor SMG7 binding and µ-opioid receptor mRNA degradation (0.87 ± 0.11-fold in the sham group vs. 0.50 ± 0.11-fold in the nerve ligation group, P = 0.002) in the dorsal horn of the spinal cord after spinal nerve ligation. Pharmacologic or genetic inhibition of this signaling pathway in vivo ameliorated allodynia-like behaviors after spinal nerve ligation. CONCLUSIONS: This study suggests that phosphorylated UPF1-dependent nonsense-mediated µ-opioid receptor mRNA decay is involved in the pathogenesis of neuropathic pain.

Phosphate NIMA-Related Kinase 2-Dependent Epigenetic Pathways in Dorsal Root Ganglion Neurons Mediates Paclitaxel-Induced Neuropathic Pain.

BACKGROUND: The microtubule-stabilizing drug paclitaxel (PTX) is an important chemotherapeutic agent for cancer treatment and causes peripheral neuropathy as a common side effect that substantially impacts the functional status and quality of life of patients. The mechanistic role for NIMA-related kinase 2 (NEK2) in the progression of PTX-induced neuropathic pain has not been established. METHODS: Adult male Sprague-Dawley rats intraperitoneally received PTX to induce neuropathic pain. The protein expression levels in the dorsal root ganglion (DRG) of animals were measured by biochemical analyses. Nociceptive behaviors were evaluated by von Frey tests and hot plate tests. RESULTS: PTX increased phosphorylation of the important microtubule dynamics regulator NEK2 in DRG neurons and induced profound neuropathic allodynia. PTX-activated phosphorylated NEK2 (pNEK2) increased jumonji domain-containing 3 (JMJD3) protein, a histone demethylase protein, to specifically catalyze the demethylation of the repressive histone mark H3 lysine 27 trimethylation (H3K27me3) at the Trpv1 gene, thereby enhancing transient receptor potential vanilloid subtype-1 (TRPV1) expression in DRG neurons. Moreover, the pNEK2-dependent PTX response program is regulated by enhancing p90 ribosomal S6 kinase 2 (RSK2) phosphorylation. Conversely, intrathecal injections of kaempferol (a selective RSK2 activation antagonist), NCL 00017509 (a selective NEK2 inhibitor), NEK2-targeted siRNA, GSK-J4 (a selective JMJD3 inhibitor), or capsazepine (an antagonist of TRPV1 receptor) into PTX-treated rats reversed neuropathic allodynia and restored silencing of the Trpv1 gene, suggesting the hierarchy and interaction among phosphorylated RSK2 (pRSK2), pNEK2, JMJD3, H3K27me3, and TRPV1 in the DRG neurons in PTX-induced neuropathic pain. CONCLUSIONS: pRSK2/JMJD3/H3K27me3/TRPV1 signaling in the DRG neurons plays as a key regulator for PTX therapeutic approaches.

Cellular mechanisms underlying central sensitization in a mouse model of chronic muscle pain.

Chronic pain disorders are often associated with negative emotions, including anxiety and depression. The central nucleus of the amygdala (CeA) has emerged as an integrative hub for nociceptive and affective components during central pain development. Prior adverse injuries are precipitating factors thought to transform nociceptors into a primed state for chronic pain. However, the cellular basis underlying the primed state and the subsequent development of chronic pain remains unknown. Here, we investigated the cellular and synaptic alterations of the CeA in a mouse model of chronic muscle pain. In these mice, local infusion of pregabalin, a clinically approved drug for fibromyalgia and other chronic pain disorders, into the CeA or chemogenetic inactivation of the somatostatin-expressing CeA (CeA-SST) neurons during the priming phase prevented the chronification of pain. Further, electrophysiological recording revealed that the CeA-SST neurons had increased excitatory synaptic drive and enhanced neuronal excitability in the chronic pain states. Finally, either chemogenetic inactivation of the CeA-SST neurons or pharmacological suppression of the nociceptive afferents from the brainstem to the CeA-SST neurons alleviated chronic pain and anxio-depressive symptoms. These data raise the possibility of targeting treatments to CeA-SST neurons to prevent central pain sensitization.

The Anesthetic Strategy for Patients with Mucopolysaccharidoses: A Retrospective Cohort Study.

Anesthesia for patients with mucopolysaccharidoses (MPS) is quite challenging due to vital systemic dysfunction following progressive accumulation of lysosomal glycosaminoglycans. Previous studies focused on perioperative difficult airway management under general anesthesia but rarely depicted the concern of choosing the size of the endotracheal tube (ETT) as well as neuraxial anesthesia. This study aimed to analyze the overall anesthetic management and related complications for a thorough anesthetic strategy. Within the study period from 2002 to 2021, each record of the anesthetic and perioperative quality assurance/improvement system for patients with a diagnosis of MPS at MacKay Memorial Hospital was retrospectively reviewed. A total of 51 individuals with 151 anesthesia for 163 interventions were cohort studied, and there were 136 general anesthesia and 15 neuraxial anesthesia. We found that the most common interventions for MPS patients were otolaryngological surgeries (49.6%). Additionally, a secured airway played a marked preference for the most general anesthesia (87.1%). The incidence of difficult intubation was 12.5%. In view of ETT size, a smaller than estimated size was used in MPS type II, III, IV, and VI patients and also in patients who received intubation with multiple attempts. However, a larger than estimated size of ETT was adopted whilst choosing cuffed ones. For neuraxial anesthesia, two failed spinal anesthesia procedures were converted to general anesthesia and 73 percent of the patients received perioperative sedation. In conclusion, through the individualized anesthetic strategy and build-up of an experienced team for airway management, high-quality anesthesia can be ensured in each patient.

MicroRNA-489-3p attenuates neuropathic allodynia by regulating oncoprotein DEK/TET1-dependent epigenetic modification in the dorsal horn.

Originally characterized as an oncoprotein overexpressed in many forms of cancer that participates in numerous cellular pathways, DEK has since been well described regarding the regulation of epigenetic markers and transcription factors in neurons. However, its role in neuropathic allodynia processes remain elusive and intriguingly complex. Here, we show that DEK, which is induced in spinal dorsal horn neurons after spinal nerve ligation (SNL), is regulated by miR-489-3p. Moreover, SNL-induced decrease in miR-489-3p expression increased the expression of DEK, which recruited TET1 to the promoter fragments of the Bdnf, Grm5, and Stat3 genes, thereby enhancing their transcription in the dorsal horn. Remarkably, these effects were also induced by intrathecally administering naïve animals with miR-489-3p inhibitor, which could be inhibited by knockdown of TET1 siRNA or DEK siRNA. Conversely, delivery of intrathecal miR-489-3p-mimic into SNL rats attenuated allodynia behavior and reversed protein expression coupled to the promoter segments in the dorsal horn. Thus, a spinal miR-489-3p/DEK/TET1 transcriptional axis may contribute to neuropathic allodynia. These results may provide a new target for treating neuropathic allodynia.

Bio-Scaffolds as Cell or Exosome Carriers for Nerve Injury Repair.

Central and peripheral nerve injuries can lead to permanent paralysis and organ dysfunction. In recent years, many cell and exosome implantation techniques have been developed in an attempt to restore function after nerve injury with promising but generally unsatisfactory clinical results. Clinical outcome may be enhanced by bio-scaffolds specifically fabricated to provide the appropriate three-dimensional (3D) conduit, growth-permissive substrate, and trophic factor support required for cell survival and regeneration. In rodents, these scaffolds have been shown to promote axonal regrowth and restore limb motor function following experimental spinal cord or sciatic nerve injury. Combining the appropriate cell/exosome and scaffold type may thus achieve tissue repair and regeneration with safety and efficacy sufficient for routine clinical application. In this review, we describe the efficacies of bio-scaffolds composed of various natural polysaccharides (alginate, chitin, chitosan, and hyaluronic acid), protein polymers (gelatin, collagen, silk fibroin, fibrin, and keratin), and self-assembling peptides for repair of nerve injury. In addition, we review the capacities of these constructs for supporting in vitro cell-adhesion, mechano-transduction, proliferation, and differentiation as well as the in vivo properties critical for a successful clinical outcome, including controlled degradation and re-absorption. Finally, we describe recent advances in 3D bio-printing for nerve regeneration.

Elevation of hilar mossy cell activity suppresses hippocampal excitability and avoidance behavior.

Modulation of hippocampal dentate gyrus (DG) excitability regulates anxiety. In the DG, glutamatergic mossy cells (MCs) receive the excitatory drive from principal granule cells (GCs) and mediate the feedback excitation and inhibition of GCs. However, the circuit mechanism by which MCs regulate anxiety-related information routing through hippocampal circuits remains unclear. Moreover, the correlation between MC activity and anxiety states is unclear. In this study, we first demonstrate, by means of calcium fiber photometry, that MC activity in the ventral hippocampus (vHPC) of mice increases while they explore anxiogenic environments. Next, juxtacellular recordings reveal that optogenetic activation of MCs preferentially recruits GABAergic neurons, thereby suppressing GCs and ventral CA1 neurons. Finally, chemogenetic excitation of MCs in the vHPC reduces avoidance behaviors in both healthy and anxious mice. These results not only indicate an anxiolytic role of MCs but also suggest that MCs may be a potential therapeutic target for anxiety disorders.

Blocking the Spinal Fbxo3/CARM1/K+ Channel Epigenetic Silencing Pathway as a Strategy for Neuropathic Pain Relief.

Many epigenetic regulators are involved in pain-associated spinal plasticity. Coactivator-associated arginine methyltransferase 1 (CARM1), an epigenetic regulator of histone arginine methylation, is a highly interesting target in neuroplasticity. However, its potential contribution to spinal plasticity-associated neuropathic pain development remains poorly explored. Here, we report that nerve injury decreased the expression of spinal CARM1 and induced allodynia. Moreover, decreasing spinal CARM1 expression by Fbxo3-mediated CARM1 ubiquitination promoted H3R17me2 decrement at the K+ channel promoter, thereby causing K+ channel epigenetic silencing and the development of neuropathic pain. Remarkably, in naïve rats, decreasing spinal CARM1 using CARM1 siRNA or a CARM1 inhibitor resulted in similar epigenetic signaling and allodynia. Furthermore, intrathecal administration of BC-1215 (a novel Fbxo3 inhibitor) prevented CARM1 ubiquitination to block K+ channel gene silencing and ameliorate allodynia after nerve injury. Collectively, the results reveal that this newly identified spinal Fbxo3-CARM1-K+ channel gene functional axis promotes neuropathic pain. These findings provide essential insights that will aid in the development of more efficient and specific therapies against neuropathic pain.

Locally Applied Stem Cell Exosome-Scaffold Attenuates Nerve Injury-Induced Pain in Rats.

PURPOSE: Nerve injury-induced pain is difficult to treat. In this study, we developed an alginate scaffold with human umbilical cord mesenchymal stem cell exosomes (EX-SC) to treat nerve injury-induced pain. MATERIALS AND METHODS: The scaffold was prepared and characterized for its physical traits and biocompatibility. In vitro studies of PC12 and HEK293 cells were used to evaluate the neuroprotective and neurotrophic effects of exosomes. Right L5/6 spinal nerve ligation (SNL) was performed in Sprague-Dawley rats to induce mechanical allodynia and thermal hyperalgesia, evaluated by von Frey hair and radiant heat tests. The EX-SC was wrapped around ligated L5/6 spinal nerves for treatment. Western blotting and immunofluorescence staining were used to evaluate neuron/glial activation, cytokines and neurotrophic factor of affected dorsal root ganglion (DRG). RESULTS: In cell culture assay, the exosomes induce neurite outgrowth of PC12 cells and protect PC12 and HEK293 cells against formaldehyde acid treatment. On post-ligation day 21, rats receiving EX-SC had significantly higher median (interquartile range) withdrawal threshold and latency [14.1 (13.7-15.5) g, 14.2 (13.7-15.3) s] than saline-SC-treated rats [2.1 (1.7-3.0) g, 2.0 (1.8-2.4) s, P=0.02 and 0.002]. The EX-SC also attenuated SNL-induced up-regulation of c-Fos, GFAP, Iba1, TNF-α and IL-1ß, while enhancing the level of IL-10 and GDNF, in the ipsilateral L5/6 DRG. After implantation for 21 days, the EX-SC enhanced the expression of myelin basic protein and IL-10 in injured L5/6 axons. CONCLUSION: We demonstrate the EX-SC possesses antinociceptive, anti-inflammation and pro-neurotrophic effects in the SNL pain model. It could be a promising therapeutic alternative for nerve injury-induced pain.

Inhibiting MLL1-WDR5 interaction ameliorates neuropathic allodynia by attenuating histone H3 lysine 4 trimethylation-dependent spinal mGluR5 transcription.

ABSTRACT: Mixed lineage leukemia 1 (MLL1)-mediated histone H3 lysine 4 trimethylation (H3K4me3) of a subset of genes has been linked to the transcriptional activation critical for synaptic plasticity, but its potential contribution to neuropathic allodynia development remains poorly explored. Here, we show that MLL1, which is induced in dorsal horn neuron after spinal nerve ligation (SNL), is responsible for mechanical allodynia and increased H3K4me3 at metabotropic glutamate receptor subtype 5 (mGluR5) promoter. Moreover, SNL induced WD (Trp-Asp) repeat domain 5 subunit (WDR5) expression as well as the MLL1-WDR5 interaction accompany with H3K4me3 enrichment and transcription of mGluR5 gene in the dorsal horn in neuropathic allodynia progression. Conversely, WDR5-0103, a novel inhibitor of the MLL1-WDR5 interaction, reversed SNL-induced allodynia and inhibited SNL-enhanced mGluR5 transcription/expression as well as MLL1, WDR5, and H3K4me3 at the mGluR5 promoter in the dorsal horn. Furthermore, disrupting the expression of MLL1 or WDR5 using small interfering RNA attenuated mechanical allodynia and reversed protein transcription/expression and complex localizing at mGluR5 promoter in the dorsal horn induced by SNL. This finding revealed that MLL1-WDR5 complex integrity regulates MLL1 and WDR5 recruitment to H3K4me3 enrichment at mGluR5 promoter in the dorsal horn underlying neuropathic allodynia. Collectively, our findings indicated that SNL enhances the MLL1-WDR5 complex, which facilitates MLL1 and WDR5 recruitment to H3K4me3 enrichment at mGluR5 promoter in spinal plasticity contributing to neuropathic allodynia pathogenesis.

Transcription Repressor Hes1 Contributes to Neuropathic Pain Development by Modifying CDK9/RNAPII-Dependent Spinal mGluR5 Transcription.

Diverse transcriptional controls in the dorsal horn have been observed in pain hypersensitivity. However, the understanding of the exact causes and mechanisms of neuropathic pain development is still fragmentary. Here, the results demonstrated nerve injury decreased the expression of spinal hairy and enhancer of split 1 (Hes1), a transcriptional repressor, and enhanced metabotropic glutamate receptor subtype 5 (mGluR5) transcription/expression, which was accompanied with behavioral allodynia. Moreover, nerve injury decreased Hes1 levels and reciprocally increased cyclin dependent kinase-9 (CDK9) levels and recruited CDK9 to phosphorylate RNA polymerase II (RNAPII) in the promoter fragments of mGluR5, thereby enhancing mGluR5 transcription/expression in the dorsal horn. These effects were also induced by intrathecally administering naïve rats with Hes1 small interfering RNA (siRNA). Conversely, Hes1 overexpression using intrathecal lentiviral vectors in nerve injury rats produced reversal of pain behavior and reversed protein expressions, phosphorylation, and coupling to the promoter segments in the dorsal horn. Collectively, the results in this study indicated nerve injury diminishes spinal Hes1-dependent suppression of CDK9-dependent RNAPII phosphorylation on the mGluR5 promoter that possibly enhances mGluR5 transcription/expression for neuropathic pain development.

Mesenchymal stem cell exosomes as a cell-free therapy for nerve injury-induced pain in rats.

Nerve injury-induced neuropathic pain is difficult to treat. In this study, we used exosomes derived from human umbilical cord mesenchymal stem cell (UCMSC) as a cell-free therapy for nerve injury-induced pain in rats. Isolated UCMSC exosomes range in size from 30 to 160 nm and contain CD63, HSP60, and CD81 exosome markers. After L5/6 spinal nerve ligation surgery, single intrathecal injection of exosomes reversed nerve ligation-induced mechanical and thermal hypersensitivities of right hindpaw of rats at initial and well-developed pain stages. Moreover, continuous intrathecal infusion of exosomes achieved excellent preventive and reversal effects for nerve ligation-induced pain. In immunofluorescent study, lots of Exo-green-labelled exosomes could be found majorly in the ipsilateral L5 spinal dorsal horn, dorsal root ganglion, and peripheral axons, suggesting the homing ability of UCMSC exosomes. They also appeared in the central terminals or cell bodies of IB4, CGRP, and NF200 sensory neurons. In addition, exosome treatment suppressed nerve ligation-induced upregulation of c-Fos, CNPase, GFAP, and Iba1. All these data suggest that the analgesic effects of exosomes may involve their actions on neuron and glial cells. Exosomes also inhibited the level of TNF-α and IL-1ß, while enhanced the level of IL-10, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor in the ipsilateral L5/6 dorsal root ganglion of nerve-ligated rats, indicating anti-inflammatory and proneurotrophic abilities. Protein analysis revealed the content of vascular endothelial growth factor C, angiopoietin-2, and fibroblast growth factor-2 in the exosomes. In summary, intrathecal infusion of exosomes from UCMSCs may be considered as a novel therapeutic approach for nerve injury-induced pain.

Spinal RNF20-Mediated Histone H2B Monoubiquitylation Regulates mGluR5 Transcription for Neuropathic Allodynia.

To date, histone H2B monoubiquitination (H2Bub), a mark associated with transcriptional elongation and ongoing transcription, has not been linked to the development or maintenance of neuropathic pain states. Here, using male Sprague Dawley rats, we demonstrated spinal nerve ligation (SNL) induced behavioral allodynia and provoked ring finger protein 20 (RNF20)-dependent H2Bub in dorsal horn. Moreover, SNL provoked RNF20-mediated H2Bub phosphorylated RNA polymerase II (RNAPII) in the promoter fragments of mGluR5, thereby enhancing mGluR5 transcription/expression in the dorsal horn. Conversely, focal knockdown of spinal RNF20 expression reversed not only SNL-induced allodynia but also RNF20/H2Bub/RNAPII phosphorylation-associated spinal mGluR5 transcription/expression. Notably, TNF-α injection into naive rats and specific neutralizing antibody injection into SNL-induced allodynia rats revealed that TNF-α-associated allodynia involves the RNF20/H2Bub/RNAPII transcriptional axis to upregulate mGluR5 expression in the dorsal horn. Collectively, our findings indicated TNF-α induces RNF20-drived H2B monoubiquitination, which facilitates phosphorylated RNAPII-dependent mGluR5 transcription in the dorsal horn for the development of neuropathic allodynia.SIGNIFICANCE STATEMENT Histone H2B monoubiquitination (H2Bub), an epigenetic post-translational modification, positively correlated with gene expression. Here, TNF-α participated in neuropathic pain development by enhancing RNF20-mediated H2Bub, which facilitates phosphorylated RNAPII-dependent mGluR5 transcription in dorsal horn. Our finding potentially identified neuropathic allodynia pathophysiological processes underpinning abnormal nociception processing and opens a new avenue for the development of novel analgesics.

BMP-2 induces angiogenesis by provoking integrin α6 expression in human endothelial progenitor cells.

Bone morphogenetic protein-2 (BMP-2) is a multifunctional cytokine, capable of governing several cellular functions, including proliferation, motility, differentiation, and angiogenesis. Circulating endothelial progenitor cells (EPCs) have been shown to facilitate tissue repair, postnatal neovascularization, and tumor associated angiogenesis. Nevertheless, the impact of BMP-2 on angiogenesis of human EPCs has largely remained a mystery. In this study, we found that BMP-2 promoted cell migration and tube formation of EPCs in a concentration-dependent manner, indicating BMP-2 induced in vitro angiogenesis in human EPCs. Furthermore, BMP-2 significantly increased microvessel formation in Matrigel plug assay, and BMP-2 antagonist noggin prevented BMP-2-induced in vivo angiogenesis. Mechanistic investigations showed BMP-2 profoundly induced the expression of Id-1 and integrin α6 as well as EPCs angiogenesis by activating PI3K/Akt and MEK/ERK signaling pathways. Moreover, knockdown of Id-1 and integrin α6 by siRNA transfection obviously attenuated BMP-2-indueced tube formation of EPCs. These results suggest that BMP-2 promotes angiogenesis in human EPCs through the activation of PI3K/Akt, MEK/ERK, and Id-1/integrin α6 signaling cascades. This is the first demonstration that BMP-2 exhibits the angiogenesis property on human EPCs. BMP-2 might serve as the potential therapeutic target for treatment of angiogenesis-related diseases.

Bromodomain-containing Protein 4 Activates Voltage-gated Sodium Channel 1.7 Transcription in Dorsal Root Ganglia Neurons to Mediate Thermal Hyperalgesia in Rats.

BACKGROUND: Bromodomain-containing protein 4 binds acetylated promoter histones and promotes transcription; however, the role of bromodomain-containing protein 4 in inflammatory hyperalgesia remains unclear. METHODS: Male Sprague-Dawley rats received hind paw injections of complete Freund's adjuvant to induce hyperalgesia. The dorsal root ganglia were examined to detect changes in bromodomain-containing protein 4 expression and the activation of genes involved in the expression of voltage-gated sodium channel 1.7, which is a key pain-related ion channel. RESULTS: The intraplantar complete Freund's adjuvant injections resulted in thermal hyperalgesia (4.0 ± 1.5 s; n = 7). The immunohistochemistry and immunoblotting results demonstrated an increase in the bromodomain-containing protein 4-expressing dorsal root ganglia neurons (3.78 ± 0.38 fold; n = 7) and bromodomain-containing protein 4 protein levels (2.62 ± 0.39 fold; n = 6). After the complete Freund's adjuvant injection, histone H3 protein acetylation was enhanced in the voltage-gated sodium channel 1.7 promoter, and cyclin-dependent kinase 9 and phosphorylation of RNA polymerase II were recruited to this area. Furthermore, the voltage-gated sodium channel 1.7-mediated currents were enhanced in neurons of the complete Freund's adjuvant rats (55 ± 11 vs. 19 ± 9 pA/pF; n = 4 to 6 neurons). Using bromodomain-containing protein 4-targeted antisense small interfering RNA to the complete Freund's adjuvant-treated rats, the authors demonstrated a reduction in the expression of bromodomain-containing protein 4 (0.68 ± 0.16 fold; n = 7), a reduction in thermal hyperalgesia (7.5 ± 1.5 s; n = 7), and a reduction in the increased voltage-gated sodium channel 1.7 currents (21 ± 4 pA/pF; n = 4 to 6 neurons). CONCLUSIONS: Complete Freund's adjuvant triggers enhanced bromodomain-containing protein 4 expression, ultimately leading to the enhanced excitability of nociceptive neurons and thermal hyperalgesia. This effect is likely mediated by the enhanced expression of voltage-gated sodium channel 1.7.

Continuous Intrathecal Infusion of Cannabinoid Receptor Agonists Attenuates Nerve Ligation-Induced Pain in Rats.

BACKGROUND AND OBJECTIVES: Cannabinoid receptors (CB1R/CB2R) are known to play important roles in pain transmission. In this study, we investigated the effects of continuous intrathecal infusion of CB1/2R agonists in the L5/6 spinal nerve ligation pain model. METHODS: Under isoflurane anesthesia, rats received nerve ligation and intrathecal catheter connected to an infusion pump. After surgery, saline (1 µL/h), CB1/2R agonist WIN55,212-2, CB1R agonist ACEA, or CB2R agonist AM1241 (1 µmol/h) was given intrathecally for 7 days. The mechanical and thermal sensitivities of rat hindpaw were determined by von Frey hair and radiant heat tests. The expression of CB1/2R and protein levels of CB1/2R, Iba1, glial fibrillary acidic protein, and tumor necrosis factor α were examined by immunofluorescence study and Western blotting. RESULTS: On postligation day 7, rats that received WIN55,212-2, ACEA or AM1241 had significantly higher mean withdrawal thresholds (6.8, 8.4, and 10.2 g) and latencies (6.3, 7.3, and 9.1 seconds) than did saline-treated rats (1.7 g, 2.2 seconds). Cannabinoid receptors were expressed not only in IB4 (isolectin B4) and CGRP (calcitonin gene-related peptide) dorsal root ganglion neurons, their central terminals, and peripheral axons, but also in neurons, microglia, and astrocytes in spinal cord. Cannabinoid receptor agonists enhanced nerve ligation-induced up-regulation of cannabinoid receptor in spinal cord and dorsal root ganglion. Treatment with WIN55,212-2 or AM1241, but not ACEA, markedly reduced nerve ligation-induced up-regulation of Iba1, glial fibrillary acidic protein, and tumor necrosis factor α in spinal cord. CONCLUSIONS: Continuous intrathecal infusion of CB1/2R agonists elicits antinociception in the pain model. The mechanisms might involve their actions on neurons and glial cells. CB2R, but not CB1R, seems to play an important role in the regulation of nerve injury-induced neuroinflammation.

K+ Channel Modulatory Subunits KChIP and DPP Participate in Kv4-Mediated Mechanical Pain Control.

The K+ channel pore-forming subunit Kv4.3 is expressed in a subset of nonpeptidergic nociceptors within the dorsal root ganglion (DRG), and knockdown of Kv4.3 selectively induces mechanical hypersensitivity, a major symptom of neuropathic pain. K+ channel modulatory subunits KChIP1, KChIP2, and DPP10 are coexpressed in Kv4.3+ DRG neurons, but whether they participate in Kv4.3-mediated pain control is unknown. Here, we show the existence of a Kv4.3/KChIP1/KChIP2/DPP10 complex (abbreviated as the Kv4 complex) in the endoplasmic reticulum and cell surface of DRG neurons. After intrathecal injection of a gene-specific antisense oligodeoxynucleotide to knock down the expression of each component in the Kv4 complex, mechanical hypersensitivity develops in the hindlimbs of rats in parallel with a reduction in all components in the lumbar DRGs. Electrophysiological data further indicate that the excitability of nonpeptidergic nociceptors is enhanced. The expression of all Kv4 complex components in DRG neurons is downregulated following spinal nerve ligation (SNL). To rescue Kv4 complex downregulation, cDNA constructs encoding Kv4.3, KChIP1, and DPP10 were transfected into the injured DRGs (defined as DRGs with injured spinal nerves) of living SNL rats. SNL-evoked mechanical hypersensitivity was attenuated, accompanied by a partial recovery of Kv4.3, KChIP1, and DPP10 surface levels in the injured DRGs. By showing an interdependent regulation among components in the Kv4 complex, this study demonstrates that K+ channel modulatory subunits KChIP1, KChIP2, and DPP10 participate in Kv4.3-mediated mechanical pain control. Thus, these modulatory subunits could be potential drug targets for neuropathic pain.SIGNIFICANCE STATEMENT Neuropathic pain, a type of moderate to severe chronic pain resulting from nerve injury or disorder, affects 6.9%-10% of the global population. However, less than half of patients report satisfactory pain relief from current treatments. K+ channels, which act to reduce nociceptor activity, have been suggested to be novel drug targets for neuropathic pain. This study is the first to show that K+ channel modulatory subunits KChIP1, KChIP2, and DPP10 are potential drug targets for neuropathic pain because they form a channel complex with the K+ channel pore-forming subunit Kv4.3 in a subset of nociceptors to selectively inhibit mechanical hypersensitivity, a major symptom of neuropathic pain.