Protein arginine methyltransferase-6 regulates heterogeneous nuclear ribonucleoprotein-F expression and is a potential target for the treatment of neuropathic pain

Abstract

Protein arginine methyltransferase-6 participates in a range of biological functions, particularly RNA processing, transcription, chromatin remodeling, and endosomal trafficking. However, it remains unclear whether protein arginine methyltransferase-6 modifies neuropathic pain and, if so, what the mechanisms of this effect. In this study, protein arginine methyltransferase-6 expression levels and its effect on neuropathic pain were investigated in the spared nerve injury model, chronic constriction injury model and bone cancer pain model, using immunohistochemistry, western blotting, immunoprecipitation, and label-free proteomic analysis. The results showed that protein arginine methyltransferase-6 mostly co-localized with β-tubulin III in the dorsal root ganglion, and that its expression decreased following spared nerve injury, chronic constriction injury and bone cancer pain. In addition, PRMT6 knockout (Prmt6^–/–) mice exhibited pain hypersensitivity. Furthermore, the development of spared nerve injury–induced hypersensitivity to mechanical pain was attenuated by blocking the decrease in protein arginine methyltransferase-6 expression. Moreover, when protein arginine methyltransferase-6 expression was downregulated in the dorsal root ganglion in mice without spared nerve injury, increased levels of phosphorylated extracellular signal-regulated kinases were observed in the ipsilateral dorsal horn, and the response to mechanical stimuli was enhanced. Mechanistically, protein arginine methyltransferase-6 appeared to contribute to spared nerve injury–induced neuropathic pain by regulating the expression of heterogeneous nuclear ribonucleoprotein-F. Additionally, protein arginine methyltransferase-6-mediated modulation of heterogeneous nuclear ribonucleoprotein-F expression required amino acids 319 to 388, but not classical H3R2 methylation. These findings indicated that protein arginine methyltransferase-6 is a potential therapeutic target for the treatment of peripheral neuropathic pain.

Introduction

Neuropathic pain that develops after nerve injury is usually intractable and is characterized by symptoms such as spontaneous pain, hyperalgesia, and paresthesia (Fitzcharles et al., 2021; Mbrah et al., 2022; Cheng et al., 2023; Wang and Jia, 2023). In clinical practice, neuropathic pain is primarily treated with tricyclic antidepressants and selective norepinephrine reuptake inhibitors, but these conventional analgesics have side effects, and their efficacy is inadequate (Salvemini and Doyle, 2023). Several mechanisms of neuropathic pain have been reported (Zeng et al., 2023; Zhang et al., 2024), but many patients’ pain remains unresolved, suggesting that our understanding of the origin of neuropathic pain remains incomplete (Saloman et al., 2022). Therefore, elucidating causal mechanisms of neuropathic pain is essential for identifying new therapeutic targets and developing novel effective analgesics.

Previous studies have suggested that ectopic firing and abnormal excitability of dorsal root ganglion (DRG) neurons after peripheral nerve injury leads to neuropathic pain (Zhang et al., 2021; Graham et al., 2022; Estivill-Torrús et al., 2024; Margiotta et al., 2024). The persistent excitability of DRG neurons is associated with altered DRG gene transcription and protein translation. Notably, altered transcription of genes encoding voltage-gated ion channels and receptors results in abnormal and spontaneous DRG neuron activation, and consequently increased secretion of neurotransmitters from primary afferents (Zhang et al., 2022, 2023a). Nerve injury can induce downregulation of μ opioid receptor (MOR) expression in the DRG, causing neuropathic pain and drug tolerance (Wu et al., 2019). The inappropriate transcriptional changes are associated with modified protein methylation (Ghosh and Pan, 2022).

Arginine methylation plays an essential role in the central nervous system (Chang et al., 2023). To date, genes encoding nine protein arginine methyltransferases (PRMTs), which catalyze arginine methylation, have been identified in mammalian genomes (Xu and Richard, 2021). Ubiquitous PRMT expression has been reported in various mammalian cells, and these proteins play essential roles in several cellular functions, such as RNA splicing and transcription (Li et al., 2021). PRMT dysregulation has been linked to the development and onset of different cancer types (Guccione et al., 2021). Moreover, coactivator-associated arginine methyltransferase-1 has been associated with pain hypersensitivity that arises after peripheral nerve injury (Mo et al., 2018). Furthermore, PRMT7 modulates DRG neuron excitability by regulating NaV1.9 currents, causing ectopic firing and hyperexcitability (Ma et al., 2022). Although accumulating evidence indicates that PRMTs are involved in various cellular processes and disorders (Lee et al., 2022), their role in neuropathic pain requires further elucidation.

PRMT6 catalyzes methylation of the histone protein H3R2 to repress gene expression (Bouchard et al., 2018). In addition, it plays an essential role in tumor progression and promotes transcription by acting as a coactivator for nuclear factor kappa B (NF-κB) (Di Lorenzo et al., 2014). In contrast, the role of PRMT6 in neuropathic pain remains unclear. Here, we evaluated PRMT6 distribution and expression in the DRG after inducing neuropathic pain in an animal model of spared nerve injury (SNI). We then asked whether altering PRMT6 expression in the DRG would change pain behavior in mice and investigated its mechanism in neuropathic pain.

Methods

Experimental animals

All animal experiments were performed in accordance with the International Association for the Study of Pain and National Institutes of Health policies, including the Guide for the Care and Use of Laboratory Animals (8^th ed., National Research Council, 2011), and were approved by the Laboratory Animal Ethics Committee of Shanghai General Hospital, School of Medicine, Shanghai Jiao Tong University on April 20, 2021 (approval No. 2021AW072). This study was reported in accordance with the ARRIVE 2.0 guidelines (Animal Research: Reporting of In Vivo Experiments) (Percie du Sert et al., 2020). PRMT6 knockout (Prmt6^–/–) mice were provided by the Naval Medical University of Shanghai, China (Zhang et al., 2019). The mice were viable and fertile and did not display any overt phenotypes. A total of 160 healthy male C57BL/6 mice (8-week-old, specific-pathogen–free grade, treatment-naive) were purchased from JOINN Laboratories (Suzhou, China, license No. SCXK (Su) 2018-0006). The mice were housed four to a cage under a 12/12-hour dark/light cycle and allowed free access to water and food. The animal room was maintained at a constant temperature of 24°C with 50%–60% humidity. The animals were randomly assigned to different experimental groups. Before initiating behavioral testing, the mice were acclimated to the laboratory conditions for 1 week. During behavioral testing, the researchers were blinded to the treatment conditions. Genomic DNA was isolated from tail biopsies and analyzed by polymerase chain reaction (PCR) amplification using the following primers: 5′-AGT CCA TGC TGA GCT CCG T-3′ and 5′-TCC ATG CAG CTC ATA TCC A-3′ for the PRMT6 WT allele; and 5′-AAG GTC ACT GGA AGA AGG-3′ and 5′-ACT CTC AGA ATT GCC TAG-3′ for the PRMT6 knockout allele.

Model establishment

The murine SNI-induced neuropathic pain model was established as described previously (Matsuoka et al., 2019). First, C57BL/6 mice were anesthetized with 2%–3% sevoflurane by inhalation. The biceps femoris muscle was separated, and then the sciatic nerve and its branches were exposed. A silk thread was used to ligate the common peroneal nerve and branches of the tibial nerve. Denervation was performed by removing around 1 mm of the axons to prevent retraction of the nerve section. C57BL/6 mice in the sham group underwent the same surgical procedure, but without nerve ligation.

The murine chronic constriction injury (CCI) neuropathic pain model was established as described previously (Sun et al., 2022). Sevoflurane (2%–3%) was used to anesthetize C57BL/6 mice. Following sciatic nerve exposure, at the proximal end of the sciatic trigeminal nerve was loosely ligated in three places, about 1-mm apart, with a 7-0 silk thread. C57BL/6 mice in the sham group underwent the same surgical procedure, but without nerve ligation.

The murine bone cancer pain (BCP) model was established as described previously (Zhao et al., 2017). C57BL/6 male mice were anesthetized with 2%–3% sevoflurane, the leg was shaved, the right condyle of the distal femur was exposed, and an arthrotomy was performed. A suspension of boiled tumor cells or live tumor cells (5 µL, 1 × 10⁵ cells) was injected into the intramedullary cavity of the mouse femur using a 30-gauge needle. Bone wax was used to seal the injection site to prevent tumor cell leakage. The maximum tumor size allowed by the Laboratory Animal Ethics Committee was 1.0 cm. When the maximum tumor size exceeds 1.0 cm, the animal is excluded from the experiment and euthanized.

Behavioral function tests

Behavioral tests were conducted following published protocols (Zhao et al., 2017) by investigators who were blinded to the group assignments. The intervals between tests were at least 1 hour.

Two calibrated von Frey filaments (weighing 0.4 g or 0.07 g, Stoelting Co., Wood Dale, IL, USA) were applied to measure mechanical stimulus–mediated paw withdrawal frequency. One von Frey filament was used to stimulate the hind paw for approximately 1 second. The same stimulation was repeated 10 times for both hind paws at 5-minute intervals. Rapid paw withdrawal was considered a positive response. The paw withdrawal frequency (%) was calculated as follows: (number of paw withdrawals/10 trials) × 100.

The Hargreaves thermal test was performed using a Model 336 Analgesic Meter (IITC Inc., Woodland Hills, CA, USA), as previously described (Wang et al., 2021). Each mouse was placed on a glass plate, and a radiant heat source underneath the glass plate was used to expose the median plantar area of the hind paw to heat. Immediate lifting of the hindlimb in response to the thermal stimulus was taken as a cue to turn off the light. The paw withdrawal latency was defined as the time that elapsed before the mouse lifted its hindlimb. Five tests were performed on each side every 5 minutes. A cutoff time of 20 seconds per stimulus was set to avoid tissue damage.

Cold responses were tested by measuring paw withdrawal latency on a 0°C metal plate (Wang et al., 2021). The time between when the mouse were placed on the plate and when it jumped was recorded. Each test was repeated three times at 15-minute intervals, and a 20-second cutoff was used to prevent potential thermal injury.

Conditioned place preference (CPP) tests were conducted to assess spontaneous continuous pain following a published protocol (Zhao et al., 2017). The test apparatus comprised two Plexiglas chambers–one with a smooth wall and floor with vertical black and white stripes, and one with a rough wall and floor with horizontal black and white stripes–connected by an inner door. The time spent in each chamber and the movements of the mice were detected by a photobeam detector mounted on the chamber wall. The detector can simultaneously monitor two chambers. MED-PC IV CPP software (Med Associates Inc, St. Albans, VT, USA) was used to evaluate the time spent in each and movement of mice between the chambers. 0.9% saline and lidocaine was intrathecally injected into mice, which was paired with each conditioned chamber. A more detailed description of this experiment is provided in Additional file 1 ^{(111KB, pdf)} .

Functional motor activity was assessed using the placement, righting reflex, and grasp tests, as previously described (Zhao et al., 2017). For the reflexive placement test, the hindlimb of the mouse was held relatively lower than the forelimb, the back of the hindlimb was brought into contact with the edge of the table, and the examiner noted whether the mouse reflexively placed its hind paw on the table surface. For the righting reflex test, mice were placed on their backs and observed to see if they immediately returned to an upright position. For the grasp reflex test, the mice were placed on a wire grid, and the experimenter observed whether the hind paw made contact with the wire and grasped it. Each test was performed five times in succession. Each reflex was scored by counting the number of times that the normal reflex was observed.

Immunofluorescence staining and imaging

Mice were deeply anesthetized with sevoflurane, subjected to intracardial perfusion with phosphate-buffered saline (PBS, 0.01 M, pH 7.4), and fixed by perfusion with paraformaldehyde (PFA, 4% in 0.01 M PBS). Then, the L3–L5 DRG, L3–L5 spinal cord segment, sciatic nerve, and mouse plantar hindlimb skin were harvested and postfixed overnight in PFA (4%). Cryopreserved tissues were dehydrated using a sucrose gradient in 0.01 M PBS at 4°C and then sectioned into 10-µm-thick slices. The sections were blocked overnight in 5% BSA and 1% Triton X-100 in PBS and then incubated with primary antibodies against PRMT6 (rabbit, 1:200; Novus Biologicals, Littleton, CO, USA, Cat# NB100-56642, RRID: AB_838734), heterogeneous nuclear ribonucleoprotein F (hnRNP-F) (mouse,1:200; Thermo Fisher Scientific, Waltham, MA, USA, Cat# MA5-18024, RRID: AB_2539408), calcitonin gene-related peptide (CGRP; mouse, 1:200, Abcam, Cambridge, MA, USA, Cat# ab81887, RRID: AB_1658411), isolectin B4 (IB4; 1:200, Vector Laboratories, Burlingame, CA, USA, Cat# FL-1201, RRID: AB_2314663), neurofilament-200 (NF200; mouse, 1:200, Sigma, St. Louis, MO, USA, Cat# N5389, RRID: AB_260781), glutamine synthetase (GS; mouse,1:200; Abcam, Cat# ab64613, RRID: AB_1140869), and β-tubulin III (mouse, 1:200; Abcam, Cat# ab78078, RRID: AB_2256751) for 1 hour at room temperature. Next, the sections were then rinsed several times and incubated with fluorescent dye–conjugated secondary antibodies for 2 hours at room temperature, followed by staining with 4′,6-diamidino-2-phenylindole (DAPI, 1:1500, Invitrogen, Waltham, MA, USA, Cat# D1306) for 2 minutes at room temperature. The fluorescent dye–conjugated secondary antibodies are as follow: Cy3 goat anti-rabbit (1:200; Abclonal, Wuhan, China, Cat# AS007, RRID: AB_2769089) and 488-conjugated goat anti-mouse (1:200; Abclonal, Cat# AS037, RRID: AB_2768319). The double-stained sections were imaged using a fluorescence microscope (DMI4000, Leica, Wetzlar, Germany) equipped with a digital camera (DFC365FX, Leica). ImageJ 1.80 software (NIH, Bethesda, MD, USA; Schneider et al., 2012) was utilized to perform quantitative analysis of the images (n = 3 mice per experimental group, n = 4–6 sections per animal). The average percentage of PRMT6-positive neurons out of the total number of neurons in the different tissue slices from each animal was calculated, and the average ± SE for each group of animals was determined.

Drug delivery and intrathecal injection

A selective small-molecule PRMT6 inhibitor was administered via intrathecal injection to determine whether PRMT6 inhibition in the DRG induced hyperalgesia. The details of this experimental method are described in Additional file 1 ^{(111KB, pdf)} .

Quantitative reverse transcription-PCR assay

Total RNA was extracted using RNA-easy Isolation Reagent (Vazyme, Nanjing, China, Cat# R701-01) according to the manufacturer’s instructions. HiScript II Q Reverse Transcriptase SuperMix (Vazyme, Cat# R222-01) and oligo (dT) or random primers (Sangon Biotech, Shanghai, China) were used to perform reverse transcription. The reverse-transcribed template cDNA (1 µL) was then PCR-amplified using the specific primers listed in Table 1 and a Bio Rad CFX96 real-time PCR system. Each sample was prepared in triplicate in a 20-µL reaction volume containing 10 µL AceQ qPCR SYBR Green Master Mix (Vazyme, Cat# Q111-02), 20 ng cDNA, and 250 nM forward and reverse primers. The PCR program, consisted of 39 cycles of 30 seconds at 95°C, 30 seconds at 60°C, and 30 seconds at 72°C, followed by a final extension of 5 minutes at 72°C. mRNA expression levels were calculated using the ΔCt method (2^–ΔΔCt) after normalization to the corresponding Tuba1a.

Table 1.

All primers used in this study

Names	Sequences (5'–3')
qRT-PCR
Prmt6	F: ATT CAG ACC AGC AAT GTA ACC AA
	R: CAC AGA GCA ACC AGC CAC T
Tuba1a	F: GTG CAT CTC CAT CCA TGT TG
	R: GTG GGT TCC AGG TCT ACG AA
Plasmid construction
PRMT6	Sense siRNA: GAG CAC UCU AAU CUA AUA ATT
	Antisense siRNA: UUA UUA GAU UAG AGU GCU CTT
hnRNP-F	Sense siRNA: GGA GGU ACA UUG GCA UUG UTT
	Antisense siRNA: ACA AUG CCA AUG UAC CUC CTT
Plasmid construction
PRMT6	F: AGA GCT AGC GAA TTC GCC ACC ATG TCG CTG AGC AAG
	R: CGC GGC CGC GGA TCC TCA GTC CTC CAT GGC AAA GTC
PRMT6Δ1-88	F: ACG GTG CTG GAC GTG GGC GCG GGC ACC GGC AT
	R: CAT GAA TTC TGC AGA TAT CCA GCA CAG TGG CG
PRMT6Δ89-188	F: CCA GCT TCC GCG GAG CTC TTC GTG GCC CCG AT
	R: CTT GCC TCG CAG CGC GGC CCA GTT CTT CAG GAT
PRMT6Δ189-318	F: ACC CAC TGG AAG CAG GCG CTC CTC TACT T
	R: CAG GAG GAG ACC GCC CTC CTT CAG CCA TTT
PRMT6Δ319-388	F: TCG GTA CCA AGC TTT GGT AAG CCT ATC CCT A
	R: GGC CGG GTG AAA AGG CGA GGT GGA CAG CA
PRMT6(dead)	F: ACG GTA GGT ACC ATG GCA AGT ATA GTT TTC CGT ACA AGT CC
	R: GCT ACT GGA TCC CTT TGA TCG ACT TCG CCA GCG GAC

F: Forward; hnRNP-F: heterogeneous nuclear ribonucleoprotein F; PRMT6: protein arginine methyltransferase-6; qRT-PCR: quantitative reverse transcription-polymerase chain reaction; R: reverse.

Western blotting

Western blotting was performed as described previously (Zhao et al., 2017). Mice were anesthetized with sevoflurane and then sacrificed. The L3/4 DRG tissues were collected from two mice, pooled, and frozen in liquid nitrogen. Neuro-2a and HEK293T cells or DRG tissues were then homogenized in cold RIPA lysis buffer (Beyotime Biotechnology, Beijing, China, Cat# P0013C) containing a cocktail of phosphatase and protease inhibitors (Beyotime Biotechnology, Cat# P1046). Equal amounts of protein samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (10%) and transferred to polyvinylidene difluoride membranes. After blocking with fat-free milk (5%), the membranes were incubated overnight at 4°C with the following primary antibodies: rabbit anti-PRMT6 (1:1000; Novus Biologicals, Cat# NB100-56642, RRID: AB_838734), mouse anti-hnRNP-F (1:1000; Thermo Fisher Scientific, Cat# MA5-18024, RRID: AB_2539408), rabbit anti-MOR (1:1000; Abclonal, Cat# A16939, RRID: AB_2770729), rabbit anti-asymmetric dimethylation of histone H3 arginine 2 (H3R2me2a; 1:1000; Abclonal, Cat# A3155, RRID: AB_2764949), rabbit anti-ERK (1:1000; Cell Signaling Technology, Danvers, MA, USA, Cat# 4695S, RRID: AB_390779), rabbit anti-phospho-ERK (1:1000; Cell Signaling Technology, Cat# 4377, RRID: AB_331775), and rabbit anti-H3 (1:2000; Cell Signaling Technology, Cat# 4499, RRID: AB_10544537). Finally, the membranes were cinubatee with an anti-rabbit secondary antibody (1:5000) conjugated with horseradish peroxidase (BBI, Nashville, TN, USA, Cat# D110058) and an anti-mouse secondary antibody (1:5000; BBI, Cat# D110085) for 2 hours at room temperature. The protein bands were detected and quantified using a Chemidoc XRS imager and laboratory imaging software (Bio-Rad, Hercules, CA, USA). Total protein values were normalized to histone H3 nucleoprotein (rabbit, 1:2000; Cell Signaling Technology, Cat# 4499, RRID: AB_10544537) and Tubulin (rabbit, 1:1000; ABclonal, Cat# AC015, RRID: AB_2773007).

Immunoprecipitation and immunoblotting

Cells were transfected and then lysed on ice in immunoprecipitation (IP) lysis buffer for 30 minutes. The cell lysates were then centrifuged at 12,000 × g at 4°C for 10 minutes to remove debris, and the supernatants containing whole cell extracts were incubated overnight with antibodies at 4°C on a rotator, followed by a 4-hour incubation with protein A agarose beads (Merck Millipore, Billerica, MA, USA). Next, the immunoprecipitated samples were rinsed with IP lysis buffer and separated by SDS-PAGE. The specific antibodies used in this experiments were as follows: anti-PRMT6 (rabbit, 1:200; Novus Biologicals, Cat# NB100-56642, RRID: AB_838734), anti-hnRNP-F (mouse, 1:200; Thermo Fisher Scientific, Cat# MA5-18024, RRID: AB_2539408), anti-Flag (rabbit, 2 µg; Proteintech, Wuhan, China, Cat# 80010-1-RR, RRID: AB_2882940), anti-His (rabbit, 1:1000; Proteintech, Cat# 10001-0-AP, RRID: AB_11232228), and anti-β-tubulin (rabbit, 1:1000; ABclonal, Cat# AC015, RRID: AB_2773007). The bands were detected and quantified using a Chemidoc XRS imager and laboratory imaging software (Bio-Rad).

Label-free proteomic analysis

Beijing Bio-Tech Pack Technology Company (Beijing, China) performed the label-free proteomics experiments. A high-intensity sonication machine was used to sonicate DRG tissue in lysis buffer on ice. Sonicated tissues were centrifuged at 12,000 × g at 4°C for 10 minutes. A BCA kit (Beyotime, Shanghai, China) was used to determine the protein concentrations according to the manufacturer’s recommended protocol. Protein samples were digested with trypsin, and the resulting peptides were analyzed using LC-MS/MS. MaxQuant (version 1.6.5.0; https://www.maxquant.org/) was used for data analysis. Differentially expressed proteins were defined as those that exhibited a fold change in expression of > 1.5 (P < 0.05).

Construction of plasmid and viral vectors

A plasmid vector (lentiviral shuttle plasmid pCD513B-1) containing mouse cDNA encoding PRMT6 or GFP with the cytomegalovirus promoter was constructed by Asia-Vector Biotechnology Corporation (Shanghai, China). The sequences of the primers used to detect wild-type PRMT6 [PRMT6(WT)], including LV-PRMT6 and LV-GFP, are shown in Table 1. Asia-Vector Biotechnology Corporation packaged the lentiviral particles.

PCR-based amplification was used to construct point-mutated, truncated, and deleted versions of PRMT6, using PRMT6 (WT) cDNA as the template. In addition, PRMT6(dead) carrying the oV86K, L87L, and D88A mutations, which is a catalytically inactive form of PRMT6(WT), was constructed. Versions of PRMT6 with different domains deleted, including PRMT6 (Δ1–88), PRMT6 (Δ89–188), PRMT6 (Δ189–318), and PRMT6 (Δ319–388), were cloned from the full-length PRMT6. All constructs were confirmed by sequencing. Table 1 lists all primers used for their construction.

Cell culture and transfection

Beyotime Biotechnology (Beijing, China) provided Neuro-2a (Cat# 4695S) and HEK293T (Cat# C6008) cells, which were cultured in a humidified incubator (37°C, 5% CO₂) in high-glucose Dulbecco’s modified Eagle’s medium (Gibco, Waltham, MA, USA) with 10% fetal bovine serum. Neuro-2a cells were used as a substitute for DRG primary neuronal culture in the cell biology experiments. HEK293T cells were transfected and subjected to co-immunoprecipitation and immunoblotting analysis. siRNAs to PRMT6 and hnRNP-F, as well as control siRNAs, were purchased from GenePharma (Shanghai, China) and diluted to 100 nM. The siRNA sequences are shown in Table 1. The Neuro-2a or HEK293T cells were transiently transfected with siRNAs or plasmid vectors using Lipofectamine-2000 (Invitrogen), following the manufacturer’s recommended protocol. Forty-eight hours following transfection, cells were collected for western blotting and quantitative reverse transcription-PCR analysis.

Dorsal root ganglion microinjection

Mice were anesthetized with sevoflurane, as described by Zhao et al. (2017). The details of this method are described in Additional file 1 ^{(111KB, pdf)} .

For knockdown experiments, siRNA was microinjected into the DRG of naive mice. Control-siRNA or Prmt6-siRNA was obtained from GenePharma. To suppress denaturation and increase siRNA delivery, In Vivo Transfection Reagent (Entranster^TM, Engreen Biosystem NZ Ltd., Auckland, New Zealand) was used, as previously reported. The Prmt6 target sequences were 5′-GAG CAC UCU AAU CUA AUA ATT-3′ (sense) and 5′-UUA UUA GAU UAG AGU GCU CTT-3′ (antisense). To test whether PRMT6 knockdown in DRGs induced pain hypersensitivity, we randomly assigned C57BL/6 male mice to the following groups using a random number table: PRMT6 siRNA-ipsi (n = 8), PRMT6 siRNA-cont (n = 8), PRMT6 NC-ipis (n = 8), and PRMT6 NC-cont (n = 8).

For overexpression experiments, 7 days before SNI/sham surgery, LV-PRMT6 and LV-GFP were microinjected into the DRG. To determine whether PRMT6 overexpression in the DRG attenuated pain sensitivity, we randomly assigned C57BL/6 male mice to the following groups using a random number table: Naive (n = 8), Sham + LV-GFP (n = 8), SNI + LV-GFP (n = 8), Sham + LV-PRMT6 (n = 8), and SNI + LV-PRMT6 (n = 8).

Statistical analysis

The data were evaluated for normality using the Kolmogorov-Smirnov test and are presented as the mean ± SEM. We performed unpaired or unpaired Student’s t-test or one-way/two-way analysis of variance with Tukey’s post hoc test for analysis of normally distributed data. The Mann–Whitney U test were applied for non-parametric data. All statistical data were analyzed using GraphPad Prism 6.0 (GraphPad, San Diego, CA, USA, www.graphpad.com). Statistical significance was defined as P < 0.05.

Results

Protein arginine methyltransferases localizes to dorsal root ganglion neurons, not astrocytes or microglia

To investigate the correlation between PRMT6 and neuropathic pain, we performed a double/triple-labeled immunofluorescence assay to evaluate PRMT6 distribution in the DRG. We observed co-localization of β-tubulin III (a neuron-specific marker) with PRMT6 within the DRG (Figure 1A); however, there was no significant overlap in staining between PRMT6 and GS (a marker of satellite glial cells; green) (Figure 1B). These results suggest that PRMT6 influences DRG neuronal activation. Furthermore, cross-sectional analysis of neuronal somata showed large (> 1200 µm²; 18.29%), medium (600–1200 µm²; 60.16%), and small (< 600 µm²; 21.95%) PRMT6-labeled neurons (Figure 1C). Subpopulation analysis demonstrated that neurons expressing PRMT6 were also positive for NF200 expression in myelinated Aβ fibers and medium and giant cells (32.70%), CGRP expression in small peptidergic neurons (48.90%), and IB4 expression in small non-peptidergic neurons (41.50%) (Figure 1D–F).

Figure 1.

PRMT6 is mainly expressed in mouse DRG nociceptive neurons.

(A) In DRG neurons, β-tubulin III (green) co-localized with PRMT6 (red). (B) Astrocyte GS (green) did not co-localize with PRMT6 (red). Nuclei were stained with DAPI (blue). (C) Distribution of PRMT6⁺ somata: large (18.29%), small (21.95%), and medium (60.16%). (D–F) PRMT6⁺ neurons were stained for NF200 (green), CGRP (green), or IB4 (green), scale bars: 50 µm. Five sections per mouse from three mice per group were evaluated. CGRP: Calcitonin gene-related peptide; DAPI: 4′,6-diamidino-2-phenylindole; DRG: dorsal root ganglion; GS: glutamine synthetase; IB4: isolectin B4; NF200: neurofilament-200; PRMT6: protein arginine methyltransferase-6.

Protein arginine methyltransferase-6 expression in dorsal root ganglions decreases with spared nerve injury–induced neuropathic pain

To examine the role of PRMT6 in neuropathic pain progression, we examined PRMT6 expression in the DRG of SNI and sham-operated mice. The experimental design is shown in Figure 2A. Consistent with results from a prior study (Zhang et al., 2023b), SNI, but not sham surgery, induced mechanical allodynia hyperalgesia on the ipsilateral side at 3, 7, and 14 days after surgery (Figure 2B and C). We also examined Prmt6 mRNA expression in the DRG at 3, 7, and 14 days post-SNI surgery and found that it was markedly decreased in the SNI group compared with the sham group (Figure 2D). Furthermore, western blotting showed decreased PRMT6 expression in the L3/4 DRG ipsilateral region of the SNI group (Figure 2E and F). Furthermore, PRMT6 expression in SNI mice was significantly reduced (0.81-fold that seen in the sham group) on day 3, most reduced (0.55-fold that seen in the sham group) on day 7, and still low (0.75-fold that seen in the sham group) on day 14 (Figure 2E and F). To investigate the role of PRMT6 in chronic pain, we also examined PRMT6 expression in the DRG of CCI and BCP animal models. PRMT6 was also markedly decreased in the CCI and BCP group compared to with the sham group (Figure 1 ^{(1.3MB, tif)} in Additional file 2 ^{(472.2KB, pdf)} ).

Figure 2.

PRMT6 expression is reduced in the injured DRG in a mouse model of neuropathic pain.

(A) Schematic diagram of the experimental procedure. (B, C) SNI increased the paw withdrawal frequency in response to stimulation with calibrated von Frey filaments (0.07 g and 0.4 g) at 3-, 7-, and 14-days post-surgery (n = 8 mice/group). (D) Prmt6 mRNA levels decreased in the injured DRG following SNI at each time point tested (n = 3 mice/group). (E) Western blot analysis of PRMT6 expression in the mouse ipsilateral L3/4 DRG at different time points following SNI. (F) Intensity analysis showed a marked decrease in PRMT6 expression following SNI (n = 4 mice/group). (G, H) Representative immunofluorescence images of neurons labeled for PRMT6 in the L3/4 DRG 7 days following sham or SNI surgery. Scale bar: 50 µm. (I) Immunofluorescence analysis showed a significant decrease in the number of PRMT6-positive neurons 7 days following SNI. The data shown are from three independent experiments. **P < 0.01, ***P < 0.001, vs. sham group (two-way analysis of variance followed by Tukey’s post hoc test for B–D, F; unpaired t-test for I). DRG: Dorsal root ganglion; H3: histone H3; PRMT6: protein arginine methyltransferase-6; qRT-PCR: quantitative reverse transcription-polymerase chain reaction; SNI: spared nerve injury.

Immunohistochemistry analysis revealed a 21.85% (P < 0.01) reduction in the number of PRMT6-labeled neurons in the SNI group 7 days postoperatively compared with the sham group (Figure 2G–I). We also assessed changes in the types of cells expressing PRMT6 in the SNI mice. The results showed that approximately 75% of β-tubulin III-positive neurons are labeled for PRMT6 in the sham mice. SNI surgery decreased the colocalization of β-tubulin III with PRMT6 to 51%. The distribution proportion of different neuron types did not change significantly. Approximately 48% of PRMT6-labeled neurons were positive for CGRP, 32% for IB4 and 23% for NF200 in Sham mice. While in SNI mice, approximately 52% of PRMT6-labeled neurons were positive for CGRP, 33% for IB4 and 20% for NF200 (Figure 2 ^{(1.1MB, tif)} A and B in Additional file 2 ^{(472.2KB, pdf)} ). These results indicate that PRMT6 protein and mRNA expression levels in the DRG correspond specifically to peripheral nerve injury, suggesting that PRMT6 could potentially be involved in neuropathic pain.

Prmt6^–/– mice exhibit pain hypersensitivity

Next, we examined whether Prmt6 loss causes pain hypersensitivity in mice. Treatment-naïve Prmt6^–/– mice were used in this experiment, and Prmt6 knockout was confirmed by PCR amplification of total DNA extracted from tail snips (Figure 6 in Additional file 2 ^{(472.2KB, pdf)} ). Testing the pain behaviors of male and female Prmt6^–/– mice showed a marked increase in sensitivity to mechanical, heat, and cold stimuli in both male and female Prmt6^–/– mice (Figure 3A–H). These data indicate that there was no sex difference in pain behavior changes affected by the absence of PRMT6 in the DRG. Next, we assessed PRMT6, MOR, and H3R2me2a expression levels and found decreased MOR expression (Figure 3I and J), but no change in H3R2me2a expression, in the DRG of Prmt6^–/– mice (Figure 3I and J).

Figure 6.

Effects of PRMT6 overexpression on neuropathic pain induced by SNI.

(A, B) Effects of LV-PRMT6 or LV-GFP microinjection into the L3/4DRG on ipsilateral and contralateral paw withdrawal frequency. (C, D) Paw withdrawal frequency following SNI at different time points (n = 8 mice/group). (E, F) PRMT6 and H3R2me2a expression levels in mice injected with LV-PRMT6 or LV-GFP 7 days after SNI. Ipsilateral L3/4 DRG tissue was pooled from two mice as one sample (n = 4 sample/group). (G) Images of PRMT6-positive neurons (red) in the lumbar DRG after microinjection with LV-PRMT6 or LV-GFP. Scale bar: 50 µm. (H) Immunofluorescence images showing a significant increase in the number of PRMT6-labeled neurons following injection with LV-PRMT6 (n = 3 mice/group). (I, J) P-ERK1/2 (and ERK1/2 expression in mice injected with LV-GFP or LV-PRMT6 on day 7 following SNI. Ipsilateral L3/4 spinal cord tissue was pooled from mice (n = 4 mice/group). The data shown are from three independent experiments. **P < 0.01, ***P < 0.001, vs. sham + LV-GFP group; #P < 0.05, ##P < 0.01, ###P < 0.001, vs. SNI + LV-GFP group in A and B, F, H, J. Two-way analysis of variance followed by Tukey’s post hoc test was used in A–D; one-way analysis of variance followed by Tukey’s post hoc test was used in F and J; Unpaired t-test was used in H. DRG: Dorsal root ganglion; ERK1/2: extracellular signal-regulated kinase1/2; GFP: green fluorescent protein; H3: histone H3; H3R2me2a: Asymmetric dimethylation of histone H3 arginine 2; LV: lentiviral; p-ERK1/2: phospho-extracellular signal-regulated kinase 1/2; PRMT6: protein arginine methyltransferase-6; SNI: spared nerve injury.

Figure 3.

Prmt6^–/–mice exhibit pain hypersensitivity.

(A, B) Male Prmt6^–/– mice showed hyperalgesia with increased frequency of paw withdrawal in response to stimulation with calibrated von Frey filaments (0.07 and 0.4 g). (C, D) Male Prmt6^–/– mice displayed shorter paw withdrawal latencies in response to thermal stimuli than WT mice. (E, F) Female Prmt6^–/– mice showed hyperalgesia with increased frequency of paw withdrawal in response to stimulation with von Frey filaments (0.07 and 0.4 g). (G, H) Female Prmt6^–/– mice exhibited thermal allodynia compared with WT mice. (I, J) PRMT6, MOR, and H3R2me2a protein expression levels of in the dorsal root ganglion of Prmt6^–/– mice. ***P < 0.001, vs. WT group (unpaired t-test). n = 6 mice/group. The data shown are from three independent experiments. H3R2me2a: Asymmetric dimethylation of histone H3 arginine 2; MOR: μ opioid receptor; PRMT6: protein arginine methyltransferase-6; Prmt6^–/–: PRMT6 knockout; WT: wild-type.

To explore whether the observed hyperalgesia occurred because of changes to the neural architecture in the transgenic mice, we immunostained L3–L4 spinal cord segments, DRG neurons, hindlimb skin, and sciatic nerve fibers to detect neuronal markers and examine innervation patterns and sensory nerve counts. Mice lacking Prmt6 showed no alterations in central or peripheral innervation density or total DRG neuron count (Figure 4A–D).

Figure 4.

Prmt6^–/– mice exhibit normal innervation patterns and sensory neuron numbers.

(A) L3–L4 spinal cord segments were harvested from WT or Prmt6^–/– mice and immunostained for IB4 (red) and CGRP (green) to label central nociceptive terminals. The pixel density of IB4 and CGRP in the dorsal horn of the spinal cord (expressed as arbitrary units (AUs)) was quantified in ImageJ and showed no change in central innervation density in Prmt6^–/–mice. (B) Analysis of the total number of neurons in the DRG in WT and Prmt6^–/– mice. L4–L5 DRGs were harvested from WT or Prmt6^–/– mice, and three sections from each mouse were immunostained with the pan-neural marker β-tubulin III (green) and counterstained with DAPI (blue). WT and Prmt6^–/– mice exhibited similar numbers of sensory neurons. (C) Immunostaining and quantification of WT and Prmt6^–/– mouse nerve fibers. β-tubulin III was used to label all nerve fibers, IB4 to label nonpeptidergic nociceptor fibers, and CGRP to label peptidergic nociceptor fibers. Following immunostaining, DAPI staining was performed to highlight the dermal–epidermal junction. Quantification of β-tubulin III⁺, IB4⁺, and CGRP⁺ nerve fibers showed that WT and Prmt6^–/– mice had similar levels of peripheral nerve density. (D) Sciatic nerves from WT and Prmt6^–/– mice were harvested and immunostained with the pan-neural marker β-tubulin III (red) and DAPI (blue). Scale bars: 200 µm in A, B, D and 100 µm in C. Quantification indicated that WT and Prmt6^–/– mice exhibited similar levels of peripheral nerve density. Unpaired t-test was used for all statistical comparisons. Five sections per mouse from four mice per group were evaluated. CGRP: Calcitonin gene-related peptide; DAPI: 4′,6-diamidino-2-phenylindole; DRG: dorsal root ganglion; IB4: isolectin B4; PRMT6: protein arginine methyltransferase-6; Prmt6^–/–: PRMT6 knockout; WT: wild-type.

Intrathecal administration of a protein arginine methyltransferase-6 inhibitor induces hyperalgesia

To further investigate the role of PRMT6 in pain generation and development, we treated mice with EPZ020411, a selective small-molecule PRMT6 inhibitor. EPZ020411 exacerbated the hypersensitivity response to mechanical stimuli induced by SNI. Furthermore, intrathecal administration of EPZ020411 to naive mice induced pain hypersensitivity (Figure 3 ^{(2.2MB, tif)} A–F in Additional file 2 ^{(472.2KB, pdf)} ). The detailed results are shown in Additional file 2 ^{(472.2KB, pdf)} . These results indicated that PRMT6 inhibition in the DRG may induce hyperalgesia.

Knocking down protein arginine methyltransferase-6 expression in the dorsal root ganglion causes pain hypersensitivity

Intrathecal injection of the PRMT6 inhibitor EPZ020411 may lack anatomical specificity. Therefore, to determine whether PRMT6 in the DRG alone alters nociceptive thresholds, we microinjected Prmt6-specific siRNA unilaterally into the L3/4 DRG of naive mice. The experimental design is shown in Figure 5A in Additional file 2 ^{(472.2KB, pdf)} . Behaviorally, microinjection of Prmt6-siRNA, but not a negative siRNA (NC-siRNA), resulted in mechanical allodynia, as evidenced by an ipsilateral increase in the frequency of paw withdrawal in response to mechanical stimulation (Figure 5A and B). As expected, PRMT6 expression levels in the ipsilateral L3/4 DRG of Prmt6-siRNA–microinjected mice were reduced compared with NC-siRNA–microinjected mice 2 days post-siRNA microinjection (Figure 5C–F).

Figure 5.

Effect of Prmt6-siRNA microinjection into the DRG on nociceptive thresholds in naïve mice.

(A, B) Effect of Prmt6-siRNA or NC-siRNA microinjection into the L3/4 DRG on paw withdrawal frequency in response to mechanical stimulation (n = 8 mice/group). (C, D) PRMT6 and H3R2me2a expression 2 days following Prmt6-siRNA or NC-siRNA microinjection into the L3/4 DRG. Unilateral L3/4 DRG tissue was harvested from two mice as one sample for analysis (n = 4 sample/group). (E) Representative images of PRMT6-labeled neurons in the lumbar DRG after NC-siRNA or Prmt6-siRNA injection. Scale bar: 50 µm. (F) Immunofluorescence analysis showed a significant decrease in the number of PRMT6-positive neurons following Prmt6-siRNA injection. (G) Representative traces of the movements of mice with spontaneous continuous pain that received either saline or lidocaine during the CPP conditioning period. (H, I) Effects of unilateral microinjection of Prmt6-siRNA or NC-siRNA into the L3/4 DRG on spontaneous continuous pain, as assessed by the CPP test. (J, K) Phospho-ERK1/2 (p-ERK1/2) and ERK1/2 expression levels in mice injected with Prmt6-siRNA or NC-siRNA. Unilateral L3/4 spinal cord tissue was pooled from mice (n = 3 mice/group). The data shown are from three independent experiments. **P < 0.01, ***P < 0.001, vs. NC-ipsi group in A and B; ***P < 0.001, vs. NC group in D; **P < 0.01, vs. sham plus LV-GFP group in F; ***P < 0.01, vs. saline-paired in H; ***P < 0.01, vs. NC in H and K. Two-way analysis of variance followed by Tukey’s post hoc test was used in A, B, and H; one-way analysis of variance followed by Tukey’s post hoc test was used in D and F; Unpaired t-test was used in H and K. con: Contralateral side; CPP: conditioned place preference; ERK1/2: extracellular signal-regulated kinase1/2; H3R2me2a: Asymmetric dimethylation of histone H3 arginine 2; ipsi: ipsilateral side; LV-GFP: lentivirus encoding green fluorescent protein; NC: negative control; p-ERK1/2: phospho-extracellular signal-regulated kinase 1/2; Prmt6^–/–: PRMT6 knockout; PRMT6: protein arginine methyltransferase-6.

Next, we performed a conditioned place preference test to assess whether knocking down Prmt6 expression in the DRG induced spontaneous continuous pain. Prmt6-siRNA–microinjected mice spent more time in the lidocaine-paired chamber (Figure 5G–I), suggesting that they were experiencing stimulus-independent pain. As expected, NC-siRNA-microinjected mice demonstrated no significant preference for either the saline- or the lidocaine-paired chamber (Figure 5G–I). Furthermore, compared with the NC-siRNA group, p-ERK1/2 levels were significantly increased in the ipsilateral L3/4 dorsal horn in the Prmt6-siRNA group (Figure 5J and K). These findings suggest that decreasing PRMT6 expression in the DRG evoked spontaneous pain hypersensitivity, a typical clinical manifestation of neuropathic pain, despite the absence of nerve damage.

Protein arginine methyltransferase-6 overexpression in dorsal root ganglions alleviates spared nerve injury–induced pain hypersensitivity

We then examined whether PRMT6 overexpression affects SNI-induced neuropathic pain. The experimental design is shown in Figure 5B in Additional file 2 ^{(472.2KB, pdf)} . Seven days before SNI or sham surgery, lentiviruses expressing full-length Prmt6 mRNA (LV-PRMT6) or a control (LV-GFP) were microinjected unilaterally into the L3/4 DRGs. Ipsilateral L3/4 DRGs were harvested to assess PRMT6 expression 14 days following sham or SNI surgery. Consistent with our previous observations, SNI significantly increased the frequency of ipsilateral paw withdrawal in response to mechanical stimuli from day 3 to day 14 following surgery compared with baseline (Figure 6A and B). In contrast, compared with LV-GFP–treated SNI mice, LV-PRMT6–treated SNI mice showed a lower ipsilateral paw withdrawal frequency from day 3 to day 14 post-SNI (Figure 6A and B). LV-PRMT6 microinjection did not alter the basal paw withdrawal response of sham mice to mechanical stimulation (Figure 6A and B). Neither LV-PRMT6 nor LV-GFP affected either contralateral basal responses (Figure 6C and D) or motor function (Table 2).

Table 2.

Mean changes in locomotor function in mice

Group	Locomotor function test
	Placing	Grasping	Righting
Sham + EPZ020411	5(0)	5(0)	5(0)
Sham + vehicle	5(0)	5(0)	5(0)
SNI + EPZ020411	5(0)	5(0)	5(0)
SNI + vehicle	5(0)	5(0)	5(0)
SNI + LV-GFP	5(0)	5(0)	5(0)
SNI + LV-PRMT6	5(0)	5(0)	5(0)
Sham + LV-GFP	5(0)	5(0)	5(0)
Sham + LV-PRMT6	5(0)	5(0)	5(0)
PRMT6 siRNA	5(0)	5(0)	5(0)
PRMT6 NC	5(0)	5(0)	5(0)

SEM given in parentheses. n = 8 mice per group; five trials. EPZ020411: Selective small-molecule PRMT6 inhibitor; GFP: green fluorescent protein; LV: lentiviral; NC: negative control; PRMT6: protein arginine methyltransferase-6; SNI: spared nerve injury.

As expected, in mice microinjected with LV-GFP, PRMT6 expression was significantly reduced in the ipsilateral L3/4 DRG on day 14 post-SNI compared with mice microinjected with LV-GFP and subjected to sham surgery (Figure 6E and F). This pattern of decreased expression was not observed in mice microinjected with LV-PRMT6 and subjected to SNI (Figure 6E and F). Furthermore, the expression basal PRMT6 expression level in in the ipsilateral L3/4 DRG was significantly increased in mice microinjected with LV-PRMT6 and subjected to sham surgery (Figure 6E and F).

Considering that PRMT6 catalyzes H3R2 methylation and represses gene transcription, we next assessed H3R2me2a levels following microinjection of LV-GFP or LV-PRMT6 into the DRG. No marked alteration in H3R2me2a expression was detected in either group (Figure 6E and F), indicating that the effect of PRMT6 on peripheral neuropathic pain is independent of H3R2 methylation. Furthermore, compared with the Sham + GFP group, the number of PRMT6-positive cells was significantly increased following microinjection of LV-PRMT6 (1.35-fold; Figure 6G and H) into the L3/4 DRG, as detected by immunofluorescence staining.

The role of DRG PRMT6 in neuropathic pain was further supported by increased expression of biomarkers of cell activity in the spinal cord dorsal horn. Levels of p-ERK1/2, a marker of neuronal hyperactivation, were significantly reduced in the ipsilateral L3/4 dorsal horn of SNI mice microinjected with LV-PRMT6 compared with those microinjected with LV-GFP at 7 days post-microinjection (Figure 6I and J). These data indicated that PRMT6 overexpression in the DRG alleviates the pain hypersensitivity induced by SNI.

Heterogeneous nuclear ribonucleoprotein F is required for protein arginine methyltransferase-6-mediated modulation of neuropathic pain

To identify the downstream regulatory targets of PRMT6 in DRGs, label-free proteomics analysis was performed using DRG samples from three groups: the PRMT6-knockout group, the PRMT6-overexpression group, and the sham group. Of the 4186 proteins detected, 405 exhibited differential expression among the three groups and were grouped into four clusters based on protein expression ratio and used to generate a heat map. There were 354 differentially expressed proteins between the PRMT6-knockout group and the sham group, and 60 differentially expressed proteins between the PRMT6-overexpression group and the sham group. Nine differentially expressed proteins were common to both groups (HIST1H1E, hnRNP-F, MIH1, MYLFP, POSTN, RPS16, RPS16.1, TNNC2, and VTN; Figure 7A and B).

Figure 7.

HnRNP-F is required for PRMT6 mediation of neuropathic pain.

(A) LC-MS/MS analysis identified nine differentially expressed proteins that were common to both comparisons. (B) Heatmap showing the expression of significantly differentially expressed proteins in all samples, as determined by label-free proteomics. (C) Western blot showing that hnRNP-F expression was significantly increased at all time points following SNI (n = 4 mice/group). (D) hnRNP-F and MOR expression in mice injected with LV-PRMT6 or LV-GFP, 7 days following SNI (n = 4 mice/group). (E) Prmt6-siRNA or NC-siRNA was microinjected into the L3/L4 DRG, and hnRNP-F and MOR expression were assessed 2 days later (n = 4 mice/group). (F) Prmt6 and hnRNP-F mRNA expression following injection with LV-PRMT6 or LV-GFP, 7 days following SNI (n = 4 mice/group). (G) Prmt6 and hnRNP-F mRNA expression in naïve mice following injection with Prmt6-siRNA or NC-siRNA (n = 4 mice/group). (H, I) Relative protein expression levels of PRMT6, hnRNP-F, and MOR in Neuro-2a cells treated with Prmt6-siRNA, hnRNP-F-siRNA, or Prmt6-siRNA and hnRNP-F-siRNA (n = 4 repeats/group). The data shown are from three independent experiments. **P < 0.01, ***P < 0.001, vs. sham group in C and ***P < 0.001, vs. NC group in E and G; *P < 0.05, **P < 0.01, ***P < 0.001, vs. sham + LV-GFP group and #P < 0.05, ###P < 0.001, vs. SNI + LV-GFP group in D and F; ***P < 0. 001, vs. NC + LV-GFP group and ###P < 0.001, vs. Prmt6-siRNA + NC group in I. Two-way analysis of variance followed by Tukey’s post hoc test was used in C; one-way analysis of variance followed by Tukey’s post hoc test was used in D, F, I; unpaired t-test was used in E, G. GFP: Green fluorescent protein; H3: histone H3; hnRNP-F: heterogeneous nuclear ribonucleoprotein F; LV: lentiviral; MOR: μ opioid receptor; NC: negative control; PRMT6: protein arginine methyltransferase-6; si-hn: hnRNP-F siRNA; si-PR: PRMT6 siRNA; SNI: spared nerve injury.

These results suggested that hnRNP-F is a downstream target protein of PRMT6. A previous study showed that hnRNP-F functions as a translational repressor of MOR in an M4 (ATG upstream region, spanning -71 to -75 bp) sequence–dependent manner (Song et al., 2012). In addition, MOR downregulation in DRG neurons caused by peripheral nerve injury promotes the release of pain-causing neurotransmitters from primary sensory afferent terminals, an important mechanism leading to neuropathic pain (Wu et al., 2019). These findings imply that PRMT6 mediates the occurrence and development of neuropathic pain via hnRNP-F. To test this hypothesis, we assessed hnRNP-F protein levels in the DRG after SNI surgery by western blot and detected a marked increase in hnRNP-F expression on days 3, 7, and 14 after SNI compared with the sham group (Figure 7C).

We further investigated whether PRMT6 mediates neuropathic pain via hnRNP-F by performing in vivo and in vitro experiments. In vivo, hnRNP-F protein expression in the DRG was inhibited by injection with LV-PRMT6 or Prmt6-siRNA (Figure 7D and E). SNI increased hnRNP-F expression, but injection with LV-PRMT6 abrogated this increase. MOR downregulation was observed in the LV-GFP plus SNI group, but not in the LV-PRMT6 plus SNI group (Figure 7D). Furthermore, microinjection with Prmt6-siRNA significantly increased hnRNP-F expression and decreased MOR expression in the DRG (Figure 7E). The results from the in vivo experiments were consistent with the results from the behavioral experiments. However, hnRNP-F mRNA levels appeared to be unaffected by PRMT6 (Figure 7F and G).

To further validate the role of hnRNP-F in neuropathic pain, we performed in vitro transfection experiments. Prmt6-siRNA treatment significantly decreased PRMT6 expression, increased hnRNP-F expression, and decreased MOR expression. hnRNP-F-siRNA rescued the effect of Prmt6-siRNA on hnRNP-F and MOR protein expression levels (Figure 7H and I). These data indicate that hnRNP-F was required for PRMT6-mediated inhibition of MOR expression.

To explore the relationship between hnRNP-F and PRMT6 in the genesis and development of neuropathic pain, hnRNP-F and PRMT6 co-localization in DRG neurons was assessed by immunofluorescence staining (Figure 8A). Approximately 40% of β-tubulin III-positive neurons were also positive for hnRNP-F. Prmt6 knockout in the DRG increased the proportion of cells expressing both β-tubulin III and hnRNP-F to 69% (Figure 8B). The proportions of different neuron types did not change significantly. Consistently, approximately 29% of hnRNP-F-positive neurons were positive for CGRP, 40% were positive for IB4, and 27% were positive for NF200 in wild-type mice (Figure 8B), while in Prmt6^–/– mice approximately 30% of hnRNP-F-labeled neurons were positive for CGRP, 40% were positive for IB4, and 30% were positive for NF200 (Figure 8B).

Figure 8.

hnRNP-F and PRMT6 co-localize in DRG neurons.

(A) Immunofluorescence images showing co-localization of hnRNP-F (red) and PRMT6 (green) in DRG neuronal nuclei. (B) Distribution of hnRNP-F (red) within DRG neurons in Prmt6 knockout mice. Approximately 40% of β-tubulin III–positive neurons (green) were also positive for hnRNP-F immunofluorescence in WT mice. Prmt6 knockout in the DRG increased the proportion of cells exhibiting β-tubulin III and hnRNP-F colocalization to 69%. There was no significant change in the relative proportions of different neuron types. Approximately 29% of hnRNP-F-positive neurons were positive for CGRP (green), 40% for IB4 (green), and 27% for NF200 (green) in WT mice, while in Prmt6 knockout mice, approximately 30% of hnRNP-F-positive neurons were positive for CGRP, 40% for IB4, and 30% for NF200. Scale bars: 50 µm in A and B. Unpaired t-test was used. ***P < 0.001, vs. WT. Five sections per mouse from three mice per group were evaluated.

These findings further verify that hnRNP-F is upregulated following Prmt6 knockout in the DRG. These results suggested that PRMT6 plays a critical role in the occurrence and development of neuropathic pain via hnRNP-F.

Residues 189-318 of protein arginine methyltransferase-6 are critical for regulating heterogeneous nuclear ribonucleoprotein F expression

To explore how PRMT6 modulates hnRNP-F, we first performed exogenous and endogenous immunoprecipitation assays and found that hnRNP-F interacts directly with PRMT6 (Figure 9A and B). The data shown in Figures 1–8 demonstrate that the regulatory effect of PRMT6 on hnRNP-F expression is independent of H3R2 methylation.

Figure 9.

PRMT6 regulation of hnRNP-F expression does not require methyltransferase activity but does require amino acids 319–388.

(A, B) Assessment of the interaction of PRMT6 with hnRNP-F by exogenous (A) and endogenous (B) immunoprecipitation assays. (C) Western blot showing relative protein expression levels of hnRNP-F and MOR in Neuro-2a cells overexpressing PRMT6(WT) or PRMT6(dead), a catalytically inactive form of PRMT6. (D) Intensity analysis showed that PRMT6(WT) and PRMT6(dead) had similar effects on the ratio of MOR to hnRNP-F (n = 3/group). One-way analysis of variance followed by Tukey’s post hoc test was used. ***P < 0.001, vs. hnRNP-F OE + GFP group; ###P < 0.001, vs. GFP group. (E) Structure of WT and mutant PRMT6 constructs. (F) Co-immunoprecipitation and immunoblotting analysis of HEK293T cells transfected with PRMT6-Flag and hnRNP-F-His mutants. The PRMT6-Flag mutants included PRMT6(Δ1–88), PRMT6(Δ89–188), PRMT6(Δ189–318), PRMT6(Δ319–388), and PRMT6 (dead). The data shown are from three independent experiments. GFP: Neuro-2a cells transfected with a plasmid encoding green fluorescent protein; hnRNP-F OE: Neuro-2a cells transfected with an hnRNP-F-His plasmid; hnRNP-F: heterogeneous nuclear ribonucleoprotein F; IP: immunoprecipitation; MOR: μ opioid receptor; OE: overexpression; PRMT6: protein arginine methyltransferase-6; WT: wild-type.

Next, to clarify the role of PRMT6 in neuropathic pain regulation, we overexpressed or PRMT6(WT) or PRMT6(dead), a catalytically inactive form of PRMT6, in Neuro-2a cells and assessed relative hnRNP-F and MOR expression levels. The results showed that PRMT6(WT) and PRMT6(dead) had similar effects on the ratio of MOR to hnRNP-F (Figure 9C and D), suggesting that PRMT6 governs hnRNP-F expression independently of methyltransferase activity.

To identify the specific domains of PRMT6 required to regulate hnRNP-F expression, we generated the deletion mutants PRMT6(Δ1–88), PRMT6(Δ89–188), PRMT6(Δ189–318), and PRMT6(Δ319–388), which lack the N-terminus, methyltransferase domain, AA189–318 domain, and C-terminus, respectively. Co-immunoprecipitation and immunoblotting showed that deletion of amino acids 319–388 significantly reduced PRMT6 binding to hnRNP-F, while PRMT6(Δ1–88), PRMT6(Δ89–188), and PRMT6(Δ189–318) were still able to interact with hnRNP-F (Figure 9E and F). These findings demonstrate that PRMT6-mediated regulation of hnRNP-F expression is dependent on PRMT6 residues 319–388.

Discussion

The present study showed that SNI reduces PRMT6 expression in the ipsilateral DRG, which contributes to neuropathic pain via modulation of hnRNP-F in DRG neurons. These findings suggest that PRMT6 is a possible target for peripheral neuropathic pain therapy.

PRMT6 is associated with various cellular functions and diseases, but its role in neuropathic pain remains unclear (Guccione and Richard, 2019). Our findings showed that PRMT6 expression decrease markedly in the ipsilateral L3/4 DRG in association with SNI-induced neuropathic pain. PRMT6 mainly localized to the nuclei of medium and small neurons in DRG and co-localizes with CGRP, IB4, and NF200. Large neurons mainly produce thick myelinated Aα and Aβ fibers, which play an essential role in transmitting non-nociceptive signals and inhibiting nociception. In contrast, small and medium neurons mainly produce thin Aδ and unmyelinated C fibers, which primarily transmit nociception signals and may be closely involved in pain signal transmission (Xu et al., 2015). Our observation of PRMT6 expression in small and medium neurons in the DRG suggests that PRMT6 is involved in regulating nociception.

To investigate the role of PRMT6 in chronic pain, we also examined PRMT6 expression in the DRG of CCI and BCP models. The results showed that, consistent with the SNI model, PRMT6 expression was significantly downregulated in the DRG of the two different chronic pain models. We used the SNI model to further explore the molecular mechanism of PRMT6 regulation of chronic pain.

Transcriptional regulation may modulate PRMT6 activation in DRGs through peripheral nerve injury-specific responses. As expected, the SNI-induced reduction in DRG PRMT6 expression was specifically and selectively blocked by injecting LV-PRMT6 into the mouse DRG, suggesting that PRMT6 overexpression alleviated SNI-induced mechanical allodynia and that reducing PRMT6 expression in the injured DRG is required for SNI-induced pain hypersensitivity. Furthermore, we showed that microinjection of Prmt6-siRNA into the DRG of naïve mice evoked pain hypersensitivity and spontaneous hyperalgesia, indicating that PRMT6 downregulation in the DRG is involved in pain hypersensitivity.

Various epigenetic mechanisms cooperatively modulate pain hypersensitivity (Hsieh et al., 2023). The function of PRMT6 in transcriptional regulation was evaluated in an earlier study (Gupta et al., 2021). Methyltransferase activity is generally critical for the physiological roles of PRMT-family proteins. Recently, some non-histone and histone proteins have been discovered to be PRMT6 substrates (Bouchard et al., 2018; Chan et al., 2018). PRMT6 typically functions as a transcriptional repressor, and this negative regulatory effect depends on its catalytic function (Kim et al., 2020b). PRMT6 is the primary enzyme that mediates methylation of H3R2 and is involved in regulating overall H3R2me2a levels (Bouchard et al., 2018). The H3R2me2a modification is a repressive post-translational modification, and its presence in promoter regions is closely correlated with the transcription level of the associated target gene (Damez-Werno et al., 2016). Thus, PRMT6 generally modifies the expression of downstream target genes by inducing H3R2 arginine methylation. For example, DNMT1 and PRMT6 reduced the expression of the cancer suppressor genes Cdknla (p21) and Cdknlb (p27) through histone H3R2 dimethylation (Luo et al., 2019). However, we found that H3R2me2a levels were not affected by PRMT6, suggesting that H3R2 arginine methylation levels are not significantly altered in the context of neuropathic pain. Therefore, we speculated that PRMT6 regulated the onset of neuropathic pain in SNI mice via a mechanism that does not involve histone arginine methylation.

Upregulating the expression of non-histone proteins also involves methylation modification, suggesting that methylation modification is likely a universal form of post-translational regulation of protein function (Gao et al., 2022). Arginine methyltransferases also catalyze the methylation of many non-histone substrates (according to PhosphoSite Plus statistics, over 2000 methylation sites are present in approximately 1300 human proteins) (Massignani et al., 2022). Many known arginine methylases have a G-R-rich motif (defined as the GAR motif), which is the primary catalytic site of arginase (Yuan et al., 2018). RNA-binding proteins have GAR domains and are significant substrates for PRMTs (Doron-Mandel et al., 2021). RNA-binding protein (RBP) methylation may be a signal for hnRNP maturation. hnRNPA1, hnRNP-U, SMD1, snRBP, and other RBPs are enriched in this motif and can be methylated by PRMTs (Gupta et al., 2021), which can modify RNA processing by affecting RNA-protein interactions. Arginine methyltransferases are also involved in initiating and prolonging gene transcription by covalently modifying the activity of non-histone proteins. CREB-binding protein (CBP), a histone acetyltransferase, is an essential transcriptional coactivator. CARM1 can methylate the CBP-KIX domain in vitro, inhibiting its transcriptional activation (Wei et al., 2003). In addition, CARM1 can be activated in vitro and in vivo by internal catalytic methylation of other CBP domains (Ceschin et al., 2011).

Importantly, RBPs can affect RNA cleavage, maturation, transport, localization, stability, and translation and function as transcriptional regulators of various signaling molecules (Sachse et al., 2023). Defects in RBP expression can lead to the development of various diseases and are linked to chronic pain. Chronic pain in many animal models is known to be affected by RBPs, which modulate mRNA decay, repression, simulation, and stabilization, as well as mRNA cap recognition. For example, staufen, a double-stranded RBP, is expression in peripheral sensory neurons, where it is involved in axonal mRNA transport (Gardiol and St Johnston, 2014). hnRNPs is also found in axons (hnRNP-R), where it serves as a post-transcriptional regulators of opioid receptor expression (Song et al., 2012).

To explore the mechanism by which PRMT6 mediates neuropathic pain without modulating histone methylation, we performed label-free -proteomics analysis to screen for differentially expressed proteins in PRMT6-overexpressing versus PRMT6-knockout DRG tissues. hnRNP-F was differentially expressed in both the PRMT6-overexpression and the PRMT6-knockout groups compared with the sham group, suggesting that hnRNP-F may be an important downstream target of PRMT6. hnRNPs belong to a family of nuclear RBPs that interact with nascent mRNAs transcribed by RNA polymerase II and form a stable complex with other RNA-proteins (Li et al., 2019). hnRNPs are complex, diverse, and multifunctional, and mediate nuclear RNA maturation to mRNA, as well as transcription (Li et al., 2019). A previous study showed that hnRNP-F functions as a translational repressor of MOR, depending on the M4 sequence (Song et al., 2012). Here, we provide evidence that PRMT6 downregulation contributes to MOR downregulation in DRGs damaged by nerve injury. We found that PRMT6 mediates changes in MOR levels by regulating hnRNP-F expression at the onset and during progression of neuropathic pain. Thus, our findings demonstrate a potentially critical link between neuropathic pain and PRMT6, indicating that PRMT6 is a potential therapeutic target for chronic pain.

Our findings also revealed a role for PRMT6 in regulating the hnRNP-F expression in vitro and in vivo, as hnRNP-F and PRMT6 co-localized in DRG neurons. Furthermore, exogenous and endogenous immunoprecipitation assays showed that PRMT6 interacts directly with hnRNP-F. To clarify the mechanism by which PRMT6 regulates hnRNP-F, we first investigated whether the methyltransferase activity of PRMT6 was involved and found that overexpression of a catalytically inactive version of PRMT6 in HEK393T cells could not reverse hnRNP-F-mediated inhibition of MOR expression, suggesting that PRMT6 regulates neuropathic pain by PRMT6 may a mechanism that is independent of its methyltransferase activity. These results confirm the distinct functional features of PRMT6 in the regulation of neuropathic pain.

Additionally, deletion of PRMT6 residues 319–388 significantly reduced PRMT6 binding to hnRNP-F, suggesting that the PRMT6 C-terminal domain mediates this protein-protein interaction.

Treating neuropathic pain at the level of the peripheral nervous system is a viable alternative to systemic administration of therapeutic agents. The DRG carries all nociceptive signals from the peripheral nervous system to the spinal cord. Treatments targeting the DRG are widely used for pain relief (Ji et al., 2023). A previous study demonstrated that applying combinatorial adeno-associated virus (AAV)-mediated gene therapy to the DRG has a long-term analgesic effect on neuropathic pain (Kim et al., 2020a) that involves modifying the expression of specific genes. Hence, a gene therapy approach involving recombinant AAV encoding PRMT6 applied to the DRG could be a new treatment option for neuropathic pain; however, further research is needed to demonstrate its safety and effectiveness.

This study had several limitations. First, we only evaluated the PRMT6 expression in the DRG, and future studies should investigate whether PRMT6 expression in the spinal cord. Second, we did not investigate the exact mechanism by which PRMT6 modifies hnRNP-F. Third, we did not explore the impact of MOR modification by hnRNP-F. Finally, while PRMT6 downregulation may affect the expression of multiple downstream proteins, we only examined hnRNP-F; thus, further studies should investigate whether and how PRMT6 is involved in mediating the expression of other proteins.

In conclusion, our findings demonstrate that PRMT6 contributes to SNI-induced neuropathic pain by regulating hnRNP-F expression and suggest new therapeutic targets for alleviating neuropathic pain.

Additional files:

Additional file 1: ^{(111KB, pdf)} Supplementary methods.

Additional file 2: ^{(472.2KB, pdf)} Supplementary results.

Additional Figure 1

Levels of PRMT6 protein in injured DRGs are reduced due to chronic pain.

(A) PRMT6 protein expression on day seven following CCI or sham surgery in ipsilateral L3/4 DRG (n = 4 mice/group). (B) Western blotting for expression of PRMT6 protein by 21 days following BCP surgery in ipsilateral L3/4 DRGs (n = 4 mice/group). (C) Nerve injury-mediated neuropathic pain decreases Prmt6 mRNA levels in injured DRGs following SNI surgery at each time point (n = 3 mice/group). ^**P < 0.01, ^***P < 0.001, vs. sham group (unpaired t-test for A and B; two-way analysis of variance followed by Tukey’s post hoc test for C).

Additional Figure 2

The distribution proportion of different neuron types in the DRG after SNI surgery.

(A) Representative images of different neuron types in L3/4 DRGs by 7 days following sham or SNI surgery. Scale bars: 50 μm. (B) The cell distribution of PRMT6 in DRG neurons in SNI mice. Approximately 75% of β-tubulin III-positive neurons are labeled for PRMT6 in the sham mice. SNI surgery decreased the co-existed of β-tubulin III with PRMT6 to 51%. The distribution proportion of different neuron types did not change significantly. Approximately 48% of PRMT6-labeled neurons were positive for CGRP, 32% for IB4, and 23% for NF200 in Sham mice. While in SNI mice, approximately 52% of PRMT6-labeled neurons were positive for CGRP, 33% for IB4, and 20% for NF200. n = 4 mice/group. CGRP: Calcitonin gene-related peptide; DRG: dorsal root ganglion; PRMT6: protein arginine methyltransferase-6; SNI: spared nerve injury.

Additional Figure 3

Effect of intrathecal PRMT6 inhibitor EPZ020411 on neuropathic pain induced by SNI.

(A, B) Effect of intrathecal EPZ020411 (50 μmol/mice) or vehicle on paw withdrawal frequencies after application of mechanical stimuli (calibrated von Frey hair 0.07 and 0.4 g) after SNI or sham surgery. n=8 mice per group. (C–F) Dose-dependent effects of EPZ020411 or vehicle on the frequency of paw withdrawal following application of mechanical stimuli (0.07 g and 0.4 g of calibrated von Frey hairs) and latency of paw withdrawal to heat or cold stimuli. n = 8 mice per group. ^***P < 0.001, vs. sham + EPZ020411 group; ^##P < 0.01, ^###P < 0.001, vs. sham + vehicle group (two-way analysis of variance followed by Tukey’s post hoc test). PRMT6: Protein arginine methyltransferase-6; SNI: spared nerve injury.

Additional Figure 4

PRMT6, MOR, and H3R2me2a expression in the DRG of SNI-induced neuropathic pain model.

(A) Western blotting results show the expression of PRMT6, MOR, and H3R2me2a protein expression at different time points in mouse ipsilateral L3/4 DRGs following SNI surgery. (B, C) Based on the intensity analysis, it showed markedly decreased abundance in PRMT6 and MOR expression following SNI surgery. (D) Based on the intensity analysis, it showed no marked alteration in the H3R2me2a expression following SNI surgery. n = 4 mice per group. ^**P < 0.01, ^***P < 0.001, vs. sham + EPZ020411 group (two-way analysis of variance followed by Tukey’s post hoc test).

Funding Statement

Funding: This work was supported by the National Natural Science Foundation of China, Nos. 82001178 (to LW), 81901129 (to LH), 82001175 (to FX); Shanghai Sailing Program, No. 20YF1439200 (to LW); the Natural Science Foundation of Shanghai, China, No. 23ZR1450800 (to LH); and the Fundamental Research Funds for the Central Universities, No. YG2023LC15 (to ZX).

Data availability statement:

Materials will be made available upon request (contact Lina Huang, honilla@163.com). All data needed to evaluate the conclusions in the paper are present in the paper or its Additional files.