RNA-binding protein SYNCRIP contributes to neuropathic pain through stabilising CCR2 expression in primary sensory neurones

Abstract

Background

Nerve injury-induced changes in gene expression in the dorsal root ganglion (DRG) contribute to the genesis of neuropathic pain. SYNCRIP, an RNA-binding protein, is critical for the stabilisation of gene expression. Whether SYNCRIP participates in nerve injury-induced alterations in DRG gene expression and nociceptive hypersensitivity is unknown.

Methods

The expression and distribution of SYNCRIP in mouse DRG after chronic constriction injury (CCI) of the unilateral sciatic nerve were assessed. Effect of microinjection of Syncrip small interfering RNA into the ipsilateral L3 and L4 DRGs on the CCI-induced upregulation of chemokine (C-C motif) receptor 2 (CCR2) and nociceptive hypersensitivity were examined. Additionally, effects of microinjection of adeno-associated virus 5 expressing full length Syncrip mRNA (AAV5-Syncrip) on basal DRG CCR2 expression and nociceptive thresholds were observed.

Results

SYNCRIP is expressed predominantly in DRG neurones, where it co-exists with CCR2. Levels of Syncrip mRNA and SYNCRIP protein in injured DRG increased time-dependently on days 3–14 after CCI. Blocking this increase through microinjection of Syncrip small interfering RNA into injured DRG attenuated CCI-induced upregulation of DRG CCR2 and development and maintenance of nociceptive hypersensitivities. Mimicking this increase through DRG microinjection of AAV5-Syncrip elevated CCR2 expression in microinjected DRGs, enhanced the responses to mechanical, heat, and cold stimuli, and induced ongoing pain in naive mice. Mechanistically, SYNCRIP bound to 3-UTR of Ccr2 mRNA and stabilised its expression in DRG neurones.

Conclusions

SYNCRIP contributes to the induction and maintenance of neuropathic pain likely through stabilising expression of CCR2 in injured DRG. SYNCRIP may be a potential target for treating this disorder.

Editor's key points.

• •Neuropathic pain is frequent and remains extremely difficult to treat.
• •Nerve injury-induced alterations of pain-associated gene expression in dorsal root ganglion (DRG) are believed to contribute to neuropathic pain pathogenesis.
• •Understanding the mechanisms underlying these alterations may enable development of new strategies for neuropathic pain.
• •In this preclinical study, the authors identified a new post-transcriptional mechanism triggered by SYNCRIP, an RNA-binding protein critical for the stabilisation of gene expression, by which peripheral nerve injury upregulates the expression of chemokines in injured DRG.
• •Their study strongly suggests that SYNCRIP may be a potential target for management of neuropathic pain.

Neuropathic pain (NP) is a chronic disease caused by damage, dysfunction, or both to the peripheral and central sensory nervous systems, and characterised clinically by spontaneous pain, allodynia, and hyperalgesia. It has become a global problem, impacting the quality of life of approximately ∼6.9–10% of the world population.^1,2 The total estimated costs on NP-related healthcare and productivity loss exceeded 100 billion dollars annually in USA.³ Direct and indirect costs of NP per patient were estimated to be €10,313 in France (69% of the total cost), €14,446 in Germany (78%), €9305 in Italy (69%), €10,597 in Spain (67%), and €9685 in the UK (57%).⁴ Although current pharmacological treatments for NP include opioids and non-opioid drugs (e.g. gabapentin, amitriptyline, and duloxetine),⁵ these medications are ineffective in >50% of NP patients, cause moderate to severe unwanted side-effects, or both, impacting the long-term adherence.5, 6, 7 Thus, identifying new targets and mechanisms underlying NP is clearly an urgent need.

Nerve injury-induced alterations of pain-associated gene expression in primary sensory neurones of dorsal root ganglion (DRG) are believed to the underlying molecular basis of NP pathogenesis.8, 9, 10 For example, the chemokine (C-C motif) receptor 2 (CCR2) and its binding ligand, C-C chemokine ligand 2 (CCL2), play an important role in the peripheral mechanisms of NP. CCL2 and CCR2 are upregulated in injured DRG neurones after peripheral nerve injury.11, 12, 13 CCR2 activation transactivates transient receptor potential channels to sensitise nociceptors.¹⁴ Genetic CCR2 knockout and intrathecal injection of the CCL2-neutralising antibody or CCR2 antagonists mitigated allodynia after peripheral nerve injury.14, 15, 16, 17, 18 Intrathecal administration of CCL2 led to mechanical allodynia and heat hyperalgesia.^16,18 CCR2 and its triggered intracellular signalling are likely required for NP genesis. However, how peripheral nerve injury upregulates CCR2 expression in injured DRG neurones is unknown. Understanding the mechanisms underlying this upregulation may enable us to develop a new strategy for NP management.

SYNCRIP (synaptotagmin binding, cytoplasmic RNA-interacting protein), also termed NSAP1¹⁹ or hnRNP Q1,²⁰ is a highly conserved RNA-binding protein that was originally discovered in the mouse brain.²¹ SYNCRIP is one of three alternative splicing variants (hnRNP Q1-3) and has high homology to hnRNP R.²¹ However, unlike hnRNP R and two other splicing variants (hnRNP Q2 and Q3), it is distributed predominantly in the cytoplasm instead of being in the nucleus.²¹ SYNCRIP may control the cytoplasmic fate of specific transcripts. Indeed, it interacts with RNA in vitro, preferentially with poly (A) or poly (U), in a phosphorylation-dependent manner.21, 22, 23 SYNCRIP was reported to stabilise proto-oncogene c-fos mRNA in mammalian cultured cells.²⁴ SYNCRIP plays a key role in neuronal upregulation of Prospero protein through binding to 3′-untranslated region of prospero mRNA to increase its stability.²⁵ It appears that SYNCRIP is implicated in post-transcriptional regulation of mRNAs, including mRNA stability. Nevertheless, whether SYNCRIP participates in nerve injury-induced changes in post-transcription of mRNAs remains unknown.

We first examined whether the expression of Syncrip mRNA and SYNCRIP protein was increased in the DRG using two well-established mouse models of NP induced by chronic constriction injury (CCI) of unilateral sciatic nerve or unilateral fourth lumbar spinal nerve ligation (SNL). We then determined if this increase was necessary for the development and maintenance of CCI-induced nociceptive hypersensitivities. Finally, we investigated the mechanism through which increased SYNCRIP contributed to NP.

Methods

Animal preparation

CD1 male and female mice (about 7–8 weeks, weight 20–25 g) were obtained from Charles River Laboratories (Wilmington, MA, USA). The animals were housed in the central facility at Rutgers New Jersey Medical School under a standard 12-h light/dark cycle, with ad libitum access to water and food. The experimental procedures were approved by the Animal Care and Use Committee at New Jersey Medical School and were consistent with the ethical guidelines of the US National Institutes of Health and the International Association for the Study of Pain. To minimise intra- and inter-individual variability in behavioural outcome measures, the animals underwent 2–3 days acclimation before behavioural testing. Every attempt was made to minimise animal suffering and limit the number of animals used. Additionally, the experimenters were blinded to the treatment condition during behavioural testing.

Neuropathic pain models

The CCI- or SNL-induced NP models in mice were conducted following previously published methods.26, 27, 28, 29, 30 In the CCI model, the exposed sciatic nerve on one side was loosely ligated with 7-0 silk thread at three sites with an interval of about 1 mm proximal to trifurcation of the sciatic nerve. In the SNL model, after identifying and removing the unilateral fifth lumbar transverse process, the underlying lumbar (L) 4 spinal nerve was carefully isolated, tightly ligated with 7-0 silk suture, and transected distal to the ligature. The sham groups underwent identical procedures but without the ligature or transection of the respective nerve.

Behavioural tests

Evoked pain tests (including mechanical, heat, and cold tests), conditional place preference (CPP) test, and locomotor function test were conducted as previously described.26, 27, 28, 29, 30 Each evoked pain test was carried out at 30–60-min intervals. The CCP test was conducted 7 weeks after viral microinjection. The locomotor function test was performed before the tissue collection.

For the mechanical test, mice were individually placed in a Plexiglas chamber on an elevated mesh screen. Two calibrated von Frey filaments (0.07 and 0.4 g, Stoelting Co. Wood Dale, IL, USA) were applied to each hind paw 10 times at intervals of 5 min. A quick withdrawal of the paw was regarded as a positive response. Paw withdrawal frequency was calculated by dividing the number of paw withdrawal positive responses by 10 trials.

For the heat test, the animal was placed in an individual Plexiglas chamber on a glass plate. A beam of light through a hole in the light box of Model 336 Analgesic Meter (IITC Inc. Life Science Instruments. Woodland Hills, CA, USA) was directed onto the middle of the plantar surface of each hind paw. The light beam was automatically turned off when the animal withdrew its foot. Paw withdrawal latency (PWL) was recorded as the time between the start of the light beam and the foot withdrawal. Each test was repeated five times at 5-min intervals for each hind paw. To prevent tissue damage to the hind paw, a cut-off time of 20 s was applied.

For the cold test, the animal was placed in an individual Plexiglas chamber on a cold aluminium plate (−1 to 0°C), and the temperature of the plate was continuously monitored by a thermometer. PWL was recorded as the time between placing the hind paw on the plate and the paw's flinching, jumping or both. Each test was repeated three times at a 10-min interval for the paw on the ipsilateral side. To avoid tissue damage, a cut-off time of 20 s was applied.

For the CPP test, mice were first preconditioned with full access to two different Plexiglas chambers connected through an internal door (Med Associates Inc. Fairfax, VT, USA) for 30 min. At the end of the preconditioning phase, the basal duration of time spent in each chamber was recorded within 15 min. The conditioning protocol was then performed for the following 3 days with the internal door closed. The mice first received intrathecal injection of saline (5 μl) specifically paired with one conditioning chamber in the morning. Six hours later, lidocaine (0.8% in 5 μl of saline) was given intrathecally, paired with another conditioning chamber in the afternoon. The injection order of saline and lidocaine was switched each consecutive day. On the test day, the mice were placed in one chamber with free access to both chambers, and the duration of time that each mouse spent in each chamber was recorded for 15 min. Difference scores were calculated by subtracting the preconditioning time from the test time spent in the lidocaine chamber.

For the locomotor function test, placing, grasping, and righting reflexes were conducted. (1) Placing reflex was assessed by placing the dorsal surfaces of the hind paws into contact with the edge of a table and recording whether the hind paws reflexively placed on the table surface. (2) Grasping reflex was assessed by placing the animal on a wire grid and recording whether the hind paws grasped the wire upon contact. (3) Righting reflex was assessed by placing the animal on its back on a flat surface and recording whether it promptly returned to the normal upright position. Each trial was repeated five times at 5-min intervals and the scores for each reflex were recorded based on counts of each normal reflex.

Dorsal root ganglion microinjection

DRG microinjection was conducted as described.26, 27, 28, 29, 30 In brief, a dorsal midline incision was made in the lower lumbar region. Unilateral L3 and L4 articular processes were exposed and then removed. After exposing the DRGs, the viral solution (1 μl, 1.79×10¹³ viral genome ml⁻¹) or small interfering RNA (siRNA) solution (1 μl, 80 μM; dissolved in TurboFect; Thermo Fisher Scientific Inc., Waltham, MA, USA) was microinjected into the ipsilateral L3 and L4 DRGs using a glass micropipette connected to a Hamilton syringe. After each injection, a 10-min pipette retention was implemented before removing the glass pipette. After microinjection, the surgical field was irrigated with sterile saline, and the skin incision was closed with wound clips. Mice exhibiting abnormal locomotor activities were excluded from the experiment.

Cell culture and transfection

CAD cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco/Thermo Fisher Scientific) containing horse serum 5% and fetal bovine serum 5% v/v (FBS; Gibco/Thermo Fisher Scientific) at 37°C in a humidified incubator with CO₂ 5%. DRG neuronal cultures were prepared according to previously described methods.31, 32, 33, 34, 35 In brief, after 4-week-old male CD1 mice were euthanised with isoflurane, all DRGs were collected in cold Neurobasal Medium (Gibco/Thermo Fisher Scientific) with FBS 10% (JR Scientific, Woodland, CA, USA), penicillin 100 units ml⁻¹, and streptomycin 100 μg ml⁻¹ (Quality Biological, Gaithersburg, MD, USA). The DRGs were then treated with enzyme solution (dispase 5 mg ml⁻¹, collagenase type I 1 mg ml⁻¹ in Hanks' balanced salt solution (Gibco/Thermo Fisher Scientific). After trituration and centrifugation, the dissociated cells were resuspended in a mixed Neurobasal Medium and plated in a six-well plate coated with poly-D-lysine 50 μg ml⁻¹ (Sigma, St. Louis, MO, USA). The cells were incubated at O₂ 95%, CO₂ 5%, and 37°C. Virus (titre ≥1×10¹³ VC ml⁻¹; 5–10 μl) or siRNA (80 μM; 4–5 μl) was added to each 2-ml well after 24 h of incubation. Three days later, the cells were collected.

Immunoblot analysis

Unilateral L3 and L4 DRGs from two mice were pooled to achieve enough proteins. The tissues were homogenised, and the cultured cells ultrasonicated in chilled lysis buffer (Tris 10 mM, phenylmethylsulfonyl fluoride 1 mM, MgCl₂ 5 mM, EGTA 5 mM, EDTA 1 mM, DTT 1 mM, leupeptin 40 μM, sucrose 250 mM). The homogenates were centrifuged at 4°C for 15 min at 1000×g, and the supernatant was collected. After measuring protein concentrations using the Bio-Rad protein assay (Bio-Rad Lab, Hercules, CA, USA), equal amounts of proteins were heated at 99°C for 5 min and loaded onto a 4–15% stacking/7.5% separating SDS-polyacrylamide gel (Bio-Rad Lab). The proteins were then electrophoretically transferred onto a polyvinylidene difluoride membrane (Bio-Rad Lab). The membrane was first blocked with non-fat milk 5% in Tris-buffered saline containing Tween-20 0.1% for 2 h at room temperature, and then incubated at 4°C overnight with the following primary antibodies: rabbit anti-SYNCRIP (1:2000; Abcam, Cambridge, UK), rabbit anti-CCR2 (1:1000; Abcam), rabbit anti-GAPDH (1:2000; Sigma), mouse anti-GFAP (1:2000; CST, Danvers, MA, USA), rabbit anti-phosphorylated ERK1 and 2 (p-ERK1 and 2; 1:1000; CST), and rabbit anti-total ERK1 and 2 (1:1000; CST). The proteins were detected by horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibody (1:3000; Jackson ImmunoResearch Lab; West Grove, PA, USA), observed using western peroxide reagent and luminol/enhancer reagent (Clarity Western ECL Substrate, Bio-Rad) and exposed using ChemiDoc XRS System with Image Lab software (Bio-Rad Lab). The intensity of blots was quantified with densitometry using Image Lab software (Bio-Rad). All cytosolic protein bands were normalised to the corresponding GAPDH.

Quantitative real-time PCR assay

Unilateral L3 and L4 DRGs from two mice were rapidly collected and pooled to achieve enough RNA. Total RNA was extracted using the miRNeasy kit (Qiagen, Valencia, CA, USA) following the manufacturer's instructions. Reverse transcription was performed using Thermo Script Reverse Transcriptase (Invitrogen, Waltham, MA, USA, Thermo Fisher Scientific) with oligo(dT) primers (Invitrogen, Thermo Fisher Scientific). The template (1 μl) was amplified in a Bio-Rad CFX96 real-time PCR system using specific primers listed in Supplementary Table 1. Each sample was run in triplicate in a 20 μl reaction volume containing 250 nM forward and reverse primers, 10 μl of Advanced Universal SYBR Green Supermix (Bio-Rad Laboratories), and 20 ng of cDNA. The PCR amplification consisted of 30 s at 95°C, 30 s at 60°C, and 30 s at 72°C for 40 cycles. Tuba1α mRNA was used as an internal control. Relative changes of mRNA levels were calculated using the ▵Ct method (2^−▵▵Ct).

Immunohistochemistry

Mice were anaesthetised with isoflurane and perfused with paraformaldehyde 4%, 100 ml in phosphate-buffered saline 0.1 M (PBS; pH7.4). After perfusion, the DRGs were dissected, postfixed at 4°C for 4 h and cryoprotected in sucrose 30% overnight. The sections were cut on a cryostat at a thickness of 20 μm and collected from each DRG by grouping every third section. After being blocked for 1 h at room temperature in PBS containing goat serum 10% and Triton X-100 0.3%, the sections from naive DRGs were incubated with mouse anti-SYNCRIP (1:50, Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-glutamine synthetase (GS, 1:500, EMD Millipore, Burlington, MA, USA), rabbit anti-NeuN (1:50, GeneTex, Irvine, CA, USA), rabbit anti-neurofilament 200 (NF200, 1:200, Sigma-Aldrich, St. Louis, MO, USA), biotinylated isolectin B4 (IB4, 1:200, Sigma-Aldrich), and rabbit anti-calcitonin gene-related peptide (CGRP, 1:200, Sigma-Aldrich) at 4°C for two nights. The sections from the ipsilateral L3 and L4 DRGs of CCI or sham mice were incubated with mouse anti-SYNCRIP (1:50, Santa Cruz Biotechology) and rabbit anti-CCR2 (1:100, Abcam) at 4°C for two nights. After being washed, the sections were incubated with species-appropriate fluro-488- or Cy3-conjugated secondary antibody (1:500, Jackson ImmunoResearch), or with FITC-labelled Avidin D (1:200, Sigma) for 2 h at room temperature. Control experiments included substitution of mouse serum or rabbit serum for the primary antiserum and omission of the primary antiserum. Finally, the sections were mounted using VectaMount permanent mounting medium or Vectashield plus 4′,6-diamidino-2-phenylindole (DAPI) mounting medium (Vector Laboratories, Burlingame, CA, USA). The images were captured using a Leica DMI4000 fluorescence microscope (Leica, Wetzlar, Germany) with DFC365 FX camera (Leica) and analysed using NIH Image J software after adjusting the contrast of the images in the same manner.

Plasmid construction and virus production

The SuperScript III One-Step RT–PCR System with the Platinum Taq High Fidelity Kit (Invitrogen) was used to reversely transcribe full-length Syncrip mRNA extracted from mouse DRG using the primers listed in Supplementary Table 1. After nested PCR using the primers (Supplementary Table 1) and Platinum™ Pfx DNA Polymerase (Invitrogen), the Syncrip coding sequences were amplified, digested by BspEI and NotI (New England Biolabs, Beverly, MA, USA), followed by gel purification and ligation into proviral plasmids. The full-length sequence was validated by sequencing. The control plasmid harbouring enhanced green fluorescent protein (GFP) was prepared in parallel. For packaging of AAV5 particles, HEK-293 cells were transfected with the pro-viral plasmid expressing SYNCRIP or GFP using a PEI transfection method with pHelper. Three days later, the transfected cells were collected, and AAV5 particles were purified using the AAV-pro Purification Kit (Takara, Mountain View, CA, USA).

RNA immunoprecipitation assay

The RNA immunoprecipitation (RIP) assay was conducted using the Magna RIP Kit (Upstate EMD Millipore, Darmstadt, Germany) as described previously.31, 32, 33, 34, 35 Homogenates from mouse CCI or sham DRGs were suspended in the RIP lysis buffer containing a protease inhibitor cocktail and RNase inhibitor. The RIP lysate was incubated on ice for 5 min and stored at −80°C. Magnetic Beads Protein A and G suspension for each immunoprecipitation was washed twice with the RIP wash buffer. Mouse anti-SYNCRIP antibody (4.0 μg; Abcam) or purified mouse IgG was incubated with Magnetic Beads Protein A and G resuspended in RIP wash buffer for 30 min at room temperature. After being washed three times with RIP wash buffer, the Beads Protein A and G-antibody complexes were resuspended in the RIP immunoprecipitation buffer. After the RIP lysate was thawed and centrifuged at 14 000 rpm at 4°C for 10 min, the supernatants were collected. About 10% (in volume) of the supernatants were used as Input. The remaining ones were incubated with the Beads Protein A/G-antibody complex in the RIP immunoprecipitation buffer overnight at 4°C by rotating. After the samples were washed five times with the RIP wash buffer, RNA was eluted from the beads by incubating in the proteinase K buffer at 55°C for 30 min with shaking, purified by phenol/chloroform extraction, and analysed by real-time quantitative PCR assay as described above.

Statistical analysis

All data are presented as mean (standard error of the mean). Data distribution was assumed to be normal, but this was not formally tested. The data were statistically analysed using two-tailed, unpaired Student's t-test or a one-way or two-way analysis of variance (anova). When anova showed significant difference, pairwise comparisons between means were tested by the post hoc Tukey method (Sigma-Plot v.12.5; San Jose, CA, USA). A value of P<0.05 was considered statistically significant in all analyses.

Results

Distribution of SYNCRIP in the primary sensory neurones of dorsal root ganglion

To examine the role of SYNCRIP in NP, we first observed its distribution pattern in the DRG. Double-labelling of SYNCRIP with NeuN (a specific neuronal marker) or glutamine synthetase (GS; a marker for satellite cells) was conducted. SYNCRIP-like immunoreactivity was detected predominantly in cytoplasm (Fig. 1a). SYNCRIP co-expressed with nuclear NeuN in individual cells (Fig. 1a) but was not expressed in the GS-positive cells (Fig. 1b), from the naive DRG, indicating that SYNCRIP is distributed predominantly in DRG sensory neurones. Cross-sectional area analysis of neuronal somata displayed that ∼34.16% of SYNCRIP-labelled neurones were small (<300 μm² in area), 25.42% were medium (300–600 μm² in area), and 40.42% were large (>600 μm² in area) (Fig. 1c). Furthermore, subpopulation analysis revealed that ∼57.5% of DRG neurones were positive for SYNCRIP (Fig. 1g), of which ∼24.6% were positive for CGRP (a marker for small DRG peptidergic neurones; Fig. 1d and 1g), 36.2% for IB4 (a marker for small non-peptidergic neurones; Fig. 1e and 1g) and 40.7% for NF200 (a marker for medium/large cells and myelinated Aβ-fibres; Fig. 1f and g).

Fig 1.

Distribution of SYNCRIP protein in the dorsal root ganglion (DRG) of naive male mice. (a–b) Double-labelled immunofluorescent staining showed co-localisation of cytoplasmic SYNCRIP (red) with nuclear NeuN (green; a), but not with cytoplasmic glutamine synthetase (GS; green; b), in individual DRG cells. Cellular nuclei were labelled by 4′,6-diamidino-2-phenylindole (DAPI; blue). (c) Size distribution of SYNCRIP-positive neuronal somata in naive DRG. Large, 40.4%; medium: 25.4%; small: 34.2%. (d–f) Double-labelled immunofluorescent staining (arrows) of SYNCRIP (red) with calcitonin gene-related peptide (CGRP; green; d), isolectin B4 (IB4; green; e), or neurofilament 200 (NF200; green; f) in DRG neurones. Scale bars: 50 μm. (g) Statistical summary of number of double-labelling neurones. n=8–12 sections marker⁻¹ from four mice. CGRP, calcitonin gene-related peptide; IB4, isolectin B4; NF200, neurofilament 200.

Upregulation of SYNCRIP expression in injured dorsal root ganglion after peripheral nerve injury

We next determined whether SYNCRIP expression was changed in the DRG and spinal cord after peripheral nerve injury. The levels of Syncrip mRNA and its protein SYNCRIP were time-dependently increased in the ipsilateral L3 and L4 (injured) DRG on days 3 (P<0.05), 7 (P<0.01), and 14 (P<0.05 for mRNA; P<0.01 for protein) after CCI, but not after sham surgery (Fig. 2a and b). In contrast, CCI did not alter the basal expression of SYNCRIP in the ipsilateral L3 and L4 spinal cords (Fig. 2c) and in the contralateral L3 and L4 DRGs (data not shown). Consistently, the number of SYNCRIP-positive neurones in the ipsilateral L3 and L4 DRG on day 7 post-CCI was 1.77-fold higher than that post-sham surgery (P<0.01; Fig. 2d). Interestingly, a cross-sectional area assessment of neuronal somata showed ∼29.74% of SYNCRIP-positive neurones were small, 42.53% were medium, and 27.73% were large on day 7 after CCI (Fig. 2e). Similarly, the amounts of Syncrip mRNA and SYNCRIP protein were increased by 1.8-fold (P<0.05) and 1.3-fold (P<0.05), respectively, in the ipsilateral L4 DRG on day 7 after SNL compared with the corresponding sham surgery group (Fig. 2f and g).

Fig 2.

Increase in expression of Snycrip mRNA and SYNCRIP protein in injured dorsal root ganglion (DRG) neurones after peripheral nerve injury. (a–b) Levels of Syncrip mRNA (a) and SYNCRIP protein (b) in the ipsilateral L3 and 4 DRGs on days 0, 3, 7, and 14 after CCI or sham surgery. Unilateral L3 and L4 DRGs from two mice were pooled. n=5 biological repeats (10 mice) group⁻¹ time point⁻¹ assay⁻¹. ∗P<0.05; ∗∗P<0.01 by two-way anova followed by post hoc Tukey test. (c) Level of SYNCRIP in the ipsilateral L3 and L4 spinal cord dorsal horn on days 0, 3, 7, and 14 after CCI. n=5 biological repeats (five mice) time point⁻¹. One-way anova followed by post hoc Tukey test. (d) Number of SYNCRIP-labelled neurones in the ipsilateral L3 and L4 DRGs on day 7 after CCI or sham surgery. Left: representative immunostaining images. Right: statistical summary. n=5 mice model⁻¹. ∗∗P<0.01 by two-tailed unpaired Student's t-test. Scale bar: 50 μm. (e) Size distribution of SYNCRIP-positive neuronal somata in the ipsilateral L3 and L4 DRGs on day 7 post-CCI. Large: 28%; medium: 42%; small: 30%. (f–g) Level of Syncrip mRNA (f) and SYNCRIP protein (g) in the ipsilateral L4 DRG on days 7 after spinal nerve ligation (SNL) or sham surgery. Unilateral L4 DRGs from four mice were pooled. n=5 biological repeats (20 mice) assay⁻¹ group⁻¹. ∗P<0.05 by two-tailed unpaired Student's t-test. anova, analysis of variance; CCI, chronic constriction injury; mRNA, messenger RNA.

Blocking increased SYNCRIP in injured dorsal root ganglion attenuated the development of chronic constriction injury-induced nociceptive hypersensitivity

Does the CCI-induced increase of SYNCRIP in injured DRG participate in the CCI-induced nociceptive hypersensitivity? To answer this question, we examined the effect of blocking this increase through microinjection of the Syncrip-specific siRNA (Syn-siRNA) into the ipsilateral L3 and L4 DRGs on the induction of CCI-induced nociceptive hypersensitivity in male mice. Control scrambled siRNA (Scr-siRNA) was used as a control. CCI or sham surgery was conducted 3 days after DRG microinjection of siRNA. As predicted, the levels of Syncrip mRNA and SYNCRIP protein in the ipsilateral L3 and L4 DRGs from the Scr-siRNA-microinjected male CCI mice were increased by 2.2-fold and 2.6-fold, respectively, compared with those in the Scr-siRNA-microinjected sham male mice on day 5 after surgery (P<0.05 for mRNA or 0.01 for protein; Fig. 3a and b). These significant increases were not observed in the Syn-siRNA-microinjected male CCI mice (Fig. 3a and b). Neither siRNA microinjection markedly altered basal expression of Syncrip mRNA and SYNCRIP protein in the ipsilateral L3 and L4 DRGs from male sham mice (Fig. 3a and b) and in the ipsilateral L3 and L4 spinal cords from male CCI or sham mice (Supplementary Fig. 1).

Fig 3.

Effect of DRG pre-microinjection of Syncrip siRNA on CCI-induced nociceptive hypersensitivities during the development period in male mice. (a–b) Levels of Syncrip mRNA (a) and SYNCRIP protein (b) in the ipsilateral L3 and L4 DRGs on day 5 after CCI or sham surgery in mice pre-microinjected with Syncrip siRNA (Syn-siRNA) or control scrambled siRNA (Scr-siRNA) for 3 days. n=5 biological repeats (10 mice) assay⁻¹ group⁻¹. ∗P<0.05 by two-way anova followed by post hoc Tukey test. (c–i) Effect of pre-micoinjection of Syncrip siRNA (Syn-siRNA) or control scrambled siRNA (Scr-siRNA) into the ipsilateral L3 and L4 DRGs on paw withdrawal frequency (PWF) to 0.07 g (c and g) and 0.4 g (d and h) von Frey filament stimuli and on paw withdrawal latency (PWL) to heat (f and i) and cold (f) stimuli on the ipsilateral (c–f) and contralateral (g–i) sides at the different days after CCI or sham surgery. n=12 mice group⁻¹. Three-way anova with repeated measures followed by post hoc Tukey test, ∗∗P<0.01 vs the Scr-siRNA plus sham group at the corresponding time points. ^##P<0.01 vs the Scr-siRNA plus CCI group at the corresponding time points. (j) Effect of pre-microinjection of Syncrip siRNA (Syn-siRNA) or control scrambled siRNA (Scr-siRNA) into the ipsilateral L3 and L4 DRGs on the expression of p-ERK1 and 2, total ERK1 and 2 and GFAP in the ipsilateral L3 and L4 dorsal horn on day 5 after CCI or sham surgery. Left: representative Western blots. Right: statistical summary of densitometric analysis. n=5 biological repeats (five mice) group⁻¹. ∗P<0.05, ∗∗P<0.01 by two-way anova followed by post hoc Tukey test. anova, analysis of variance; CCI, chronic constriction injury; DRG, dorsal root ganglion; ERK, extracellular signal-regulated kinase; GFAP, glial fibrillary acidic protein; mRNA, messenger RNA; siRNA, small interfering RNA.

Similar to our previous studies,^26,27,30 behavioural observations showed that CCI, but not sham surgery, produced mechanical allodynia as evidenced by significant increases in paw withdrawal frequencies in response to 0.07 g and 0.4 g von Frey filaments, and heat and cold hyperalgesia as demonstrated by marked decreases in paw withdrawal latencies to heat and cold stimuli, respectively, on the ipsilateral side 3 and 5 days post-surgery in the Scr-siRNA-microinjected male mice (Fig. 3c–f). These nociceptive hypersensitivities were significantly attenuated in the Syn-siRNA- microinjected male CCI mice (Fig. 3c–f). DRG microinjection of neither siRNA affected basal paw responses to mechanical, heat or cold stimuli on the contralateral side of male CCI mice and on both ipsilateral and contralateral sides of male sham mice during the observation period (Fig. 3c–i).

Peripheral nerve injury-induced neuronal hyperexcitability in injured DRG triggers the hyperactivation of neurones and astrocytes in spinal cord dorsal horn through augmenting the release of neurotransmitters, neuromodulators, or both in primary afferents under NP conditions.³⁶ We also determined whether DRG pre-microinjection of Syn-siRNA affected the CCI-induced neuronal and astrocyte hyperactivations in the dorsal horn of male mice to further confirm our behavioural observations above. In line with our previous reports,^26,27,30 the amounts of p-ERK1 and 2 (a marker for neuronal hyperactivation) and GFAP (a marker for astrocyte hyperactivation), but not total ERK1 and 2, were markedly increased in the ipsilateral L3 and L/4 dorsal horns on day 5 post-CCI in the Scr-siRNA-microinjected male mice (Fig. 3j). These increases were substantially blocked in the Syn-siRNA-microinjected male CCI mice (Fig. 3J). Neither siRNA altered basal levels of total ERK1 and 2, p-ERK1 and 2, and GFAP in the ipsilateral L3 and L4 dorsal horns of male sham mice (Fig. 3j).

The results were similar after DRG microinjection of Syn-siRNA or Scr-siRNA in female CCI or sham mice (Supplementary Fig. S2a–i). All microinjected male and female mice displayed normal locomotor functions (Supplementary Table S2).

Taken together, our findings indicate that DRG increased SYNCRIP may participate in the induction of CCI-induced nociceptive hypersensitivity in both male and female mice.

Blocking increased SYNCRIP in injured dorsal root ganglion attenuated the maintenance of chronic constriction injury-induced nociceptive hypersensitivity

We also examined whether the increased SYNCRIP in DRG was required for the maintenance of CCI-induced pain hypersensitivities. Scr-siRNA or Syn-siRNA was microinjected 7 days after CCI in male mice, a time point when CCI-induced nociceptive hypersensitivities were fully developed.^26,27,30 In the Scr-siRNA-microinjected male CCI mice, mechanical allodynia and heat and cold hyperalgesia were observed on the ipsilateral side 7, 10, 12, and 14 days post-CCI (Fig. 4a–d). In contrast, these nociceptive hypersensitivities were significantly impaired on days 12 and 14, but not on days 7 and 10, post-CCI in the Syn-siRNA-microinjected male CCI mice (Fig. 4a–d). As expected, post-microinjection of neither siRNA changed basal paw withdrawal responses on the contralateral side (Fig. 4a–c). Compared with naive mice, CCI increased the level of SYNCRIP protein in the ipsilateral L3 and L4 DRGs on day 14 post-CCI in the Scr-siRNA-microinjected male CCI mice (Fig. 4e), but this increase was robustly attenuated in the Syn-siRNA-microinjected male CCI mice (Fig. 4e). Moreover, CCI-induced increases in the levels of the p-ERK1 and 2 (but not total ERK1 and 2) and GFAP in the ipsilateral L3 and L4 dorsal horn on day 14 post-CCI from the Scr-siRNA-microinjected male CCI mice were not observed in the Syn-siRNA-microinjected mice (Fig. 4f). All microinjected mice displayed normal locomotor activities (Supplementary Table S2). The evidence revealed an important role of DRG increased SYNCRIP in the maintenance of CCI-induced pain hypersensitivities.

Fig 4.

Effect of DRG post-microinjection of Syncrip siRNA on CCI-induced nociceptive hypersensitivities during the maintenance period in male mice. (a–g) Effect of post-microinjection of Syncrip siRNA (Syn-siRNA) or control scrambled siRNA (Scr-siRNA) into the ipsilateral L3 and L4 DRGs on paw withdrawal frequency (PWF) to 0.07 g (a) and 0.4 g (b) von Frey filament stimuli and on paw withdrawal latency (PWL) to heat (c) and cold (d) stimuli on the ipsilateral (Ipsi) and contralateral (Con) sides at the different days after CCI. n=8 mice group⁻¹. ∗∗P<0.01 vs the CCI plus Scr-siRNA group on the ipsilateral side at the corresponding time points. Two-way anova with repeated measures followed by post hoc Tukey test. (e) Level of SYNCRIP protein in naive L3 and L4 DRGs and ipsilateral L3 and L4 DRGs on day 14 after CCI in mice post-microinjected with Syncrip siRNA (Syn-siRNA) or control scrambled siRNA (Scr-siRNA). n=5 biological repeats (10 mice) group⁻¹. ∗P<0.05 by one-way anova followed by post hoc Tukey test. (f) Effect of post-microinjection of Syncrip siRNA (Syn-siRNA) or control scrambled siRNA (Scr-siRNA) into the ipsilateral L3 and L4 DRGs on the expression of p-ERK1 and 2, total ERK1 and 2, and GFAP in naive L3 and L4 dorsal horn and ipsilateral L3 and L4 dorsal horn on day 14 after CCI. Left: representative Western blots. Right: statistical summary of densitometric analysis. n=5 biological repeats (five mice) group⁻¹. ∗P<0.05, ∗∗P<0.01 by one-way anova followed by post hoc Tukey test. anova, analysis of variance; CCI, chronic constriction injury; DRG, dorsal root ganglion; ERK, extracellular signal-regulated kinase; GFAP, glial fibrillary acidic protein; siRNA, small interfering RNA.

Mimicking peripheral nerve injury-induced increase of dorsal root ganglion SYNCRIP produces neuropathic pain-like symptoms

To determine whether CCI-induced increase of SYNCRIP in injured DRG was sufficient for peripheral nerve injury-induced nociceptive hypersensitivity, we overexpressed SYNCRIP in the DRGs through the microinjection of AAV5 that expresses full-length Syncrip mRNA (AAV5-Syn) into the unilateral L3 and L4 DRGs of naive male mice. AAV5 expressing GFP (AAV5-GFP) was used as a control. As expected, the amounts of Syncrip mRNA and SYNCRIP protein were significantly increased by 1.8-fold and 1.9-fold, respectively, in the ipsilateral L3 and L4 DRGs 8 weeks after AAV5-Syn microinjection, compared with those after AAV5-GFP microinjection (Fig. 5a and b). DRG microinjection of AAV5-Syn, but not AAV5-GFP, produced significant increases in paw withdrawal frequencies in response to 0.07 g and 0.4 g von Frey filament stimuli (Fig. 5c and d) and marked decreases in paw withdrawal latencies in response to heat and cold stimuli (Fig. 5e and f) on the ipsilateral side. These augmented responses occurred 4 weeks post-microinjection and persisted for at least 8 weeks (Fig. 5c–f). This phenomenon was consistent with the fact that AAV5 required 3–4 weeks to become expressed and lasted for at least 3 months.31, 32, 33, 34, 35 On the contralateral side, basal paw withdrawal responses were not altered in either AAV5-microinjected male mouse (Fig. 5c–f). More importantly, DRG microinjection of AAV5-Syn produced evoked stimulation-independent spontaneous pain, as demonstrated by significant preference for the lidocaine-paired chamber on week 7 post-AAV5 microinjection (Fig. 5g and h). As predicted, DRG microinjection of AAV5-GFP did not produce marked preference to either saline-paired or lidocaine-paired chamber (Fig. 5g and h), indicating no spontaneous pain. In addition, DRG microinjection of AAV5-Syn, but not AAV5-GFP, elevated the amounts of p-ERK1 and 2 (but not total ERK1 and 2) and GFAP in the ipsilateral L3 and L4 spinal cord dorsal horns 8 weeks after AAV5 microinjection (Fig. 5i). Similar results were also observed after DRG microinjection of AAV5-Syn or AAV5-GFP in naive female mice (Supplementary Fig. S3). All viral microinjected male and female mice exhibited normal locomotor activity (Supplementary Table S2).

Fig 5.

Effect of DRG SYNCRIP overexpression on nociceptive thresholds in naive male mice. (a–b) Levels of Syncrip mRNA (a) and SYNCRIP protein (b) in the ipsilateral L3 and L4 DRGs 8 weeks after microinjection of AAV5-Syncrip (AAV5-Syn) or control AAV5-GFP. n=5 biological repeats (10 mice) assay⁻¹ group⁻¹. ∗P<0.05 by two-tailed independent Student's t-test. (c–f) Effect of microinjection of AAV5-Syncrip (AAV5-Syn) or AAV5-GFP into the unilateral L3 and L4 DRGs on paw withdrawal frequencies (PWF) to 0.07 g (c) and 0.4 g (d) von Frey filament stimuli and on paw withdrawal latencies (PWL) to heat (e) and cold (f) stimuli on the ipsilateral (Ipsi) and contralateral (Con) sides at the different weeks post-viral microinjection. n=16 mice group⁻¹. ∗∗P<0.01 vs the AAV5-Gfp group at the corresponding time points on the ipsilateral side. Two-way anova with repeated measures followed by post hoc Tukey test. (g–h) Effect of microinjection of AAV5-Syncrip (AAV5-Syn) or AAV5-GFP into the unilateral L3 and L4 DRGs on spontaneous ongoing pain as assessed by the CPP paradigm. Pre: preconditioning. Post: post-conditioning. n=12 mice group⁻¹. ∗∗P<0.01 by three-way anova with repeated measures followed by Tukey post hoc test (g) or two-tailed, independent Student's t-test (h). (i) Effect of microinjection of AAV5-Syncrip (AAV5-Syn) or AAV5-GFP into the unilateral L3 and L4 DRGs on the levels of p-ERK1 and 2, total ERK1 and 2 and GFAP in the ipsilateral L3 and L4 dorsal horn 8 weeks after viral microinjection. Representative Western blots (left panels) and a summary of the densitometric analysis (right graphs) are shown. n=5 biological repeats (five mice) group⁻¹. ∗P<0.05, ∗∗P<0.01 by two-tailed, independent Student's t-test. AAV5, adeno-associated virus 5; anova, analysis of variance; CCI, chronic constriction injury; DRG, dorsal root ganglion; ERK, extracellular signal-regulated kinase; GFP, green fluorescent protein; GFAP, glial fibrillary acidic protein; mRNA, messenger RNA; siRNA, small interfering RNA.

Collectively, these findings demonstrated that increased SYNCRIP in the DRG leads to both spontaneous and evoked nociceptive hypersensitivities, symptoms commonly seen in NP clinics.

Increased SYNCRIP participates in the chronic constriction injury-induced upregulation of chemokine receptor CCR2 through stabilising Ccr2 mRNA in injured dorsal root ganglion

Finally, we elucidated the mechanisms by which the increased SYNCRIP in injured DRG contributed to the CCI-induced nociceptive hypersensitivities. Using the online software STRING (https://string-db.org), we identified several mRNA candidates, including chemokine C-C motif receptor 2 (Ccr2) mRNA, as potential binding partners of SYNCRIP. Given that peripheral nerve injury-induced increases of Ccr2 mRNA and CCR2 protein in injured DRG were required for NP,11, 12, 13, 14, 15, 16, 17, 18 we hypothesised that increased SYNCRIP participated in the CCI-induced increase of Ccr2 mRNA in the injured DRG. To test our hypothesis, we first conducted the RIP assay and found the binding of SYNCRIP to the 3′-UTR of Ccr2 mRNA in the ipsilateral L3 and L4 DRGs of male sham mice (Fig. 6a). This binding was significantly elevated on day 7 post-CCI (Fig. 6a). This elevation is attributed to the increases in the levels of not only SYNCRIP but also Ccr2 mRNA in the ipsilateral L3 and L4 DRGs post-CCI (Fig 2, Fig 6b). Consistent with the previous reports,11, 12, 13 CCI increased the expression of Ccr2 mRNA and CCR2 protein in the ipsilateral L3 and L4 DRGs on day 5 after CCI in the Scr-siRNA-microinjected male mice (Fig. 6c and d). These increases were markedly attenuated in the Syn-siRNA-microinjected male CCI mice (Fig. 6c and d). DRG microinjection of Syn-siRNA did not obviously change basal levels of Ccr2 mRNA and CCR2 in the ipsilateral L3 and L4 DRGs on day 5 post-sham surgery (Fig. 6c and d). Consistently, the amounts of Ccr2 mRNA and CCL2 protein in the ipsilateral L3 and L4 DRGs from the AAV5-Syn-microinjected male mice were significantly increased compared with those from the AAV5-GFP-microinjected male mice (Fig. 6e and f). In in vitro cultured DRG neurones, transduction with AAV5-Syn, but not AAV5-GFP, increased the levels of SYNCRIP and CCR2 in the Scr-siRNA-transfected neurones (Fig. 6g). These increases were blocked in the AAV5-Syn-transduced plus Syn-siRNA-transfected neurones (Fig. 6g), suggesting that the induction of CCR2 in DRG neurones is specific in responses to SYNCRIP. Syn-siRNA transfection plus AAV5-GFP transduction reduced basal levels of SYNCRIP and CCR2 in the cultured DRG neurones (Fig. 6g). Furthermore, when the transcription was halted with actinomycin D, the decay rate of Ccr2 mRNA was much slower, and the amount of Ccr2 mRNA was significantly elevated in the AAV5-Syn-transducted plus Scr-siRNA-transfected cultured CAD cells compared with those in naive CAD cells (Fig. 6h and 6i). These effects were attenuated in the AAV5-Syn-transducted plus Syn-siRNA-transfected cultured CAD cells (Fig. 6h and i). Given that SYNCRIP co-expressed with CCR2 in the same DRG neurones (Fig. 6j), our findings suggest that DRG increased SYNCRIP contributes to CCI-induced nociceptive hypersensitivity likely by stabilising Ccr2 mRNA expression in injured DRG neurones.

Fig 6.

Participation of increased DRG SYNCRIP in the CCI-induced upregulation of chemokine receptor 2 (CCR2) through stabilising Ccr2 mRNA in injured DRG. (a) 3′-UTR fragment of Ccr2 mRNA immunoprecipitated by mouse anti-SYNCRIP, but not normal mouse serum (IgG), in the ipsilateral L3 and L4 DRGs on day 5 post-CCI or sham surgery. Input: total purified fragment. M: ladder marker. NC: negative control (no template). n=3 biological repeats (nine mice repeat⁻¹) group⁻¹. ∗∗P<0.01 by two-tailed unpaired Student's t-test. (b) Level of Ccr2 mRNA in the ipsilateral L3 and L4 DRGs on days 0, 3, 7, and 14 after CCI or sham surgery. Unilateral L3 and L4 DRGs from two mice were pooled. n=5 biological repeats (10 mice) group⁻¹ time point⁻¹. ∗P<0.05, ∗∗P<0.01 by two-way anova followed by post hoc Tukey test. (c–d) Levels of Ccr2 mRNA (c) and CCR2 protein (d) in the ipsilateral L3 and L4 DRGs on day 5 after CCI or sham surgery in mice pre-microinjected with Syncrip siRNA (Syn-siRNA) or control scrambled siRNA (Scr-siRNA). n=5 biological repeats (10 mice) group⁻¹ assay⁻¹. ∗P<0.05, ∗∗P<0.01 by two-way anova followed by post hoc Tukey test. (e–f) Levels of Ccr2 mRNA (e) and CCR2 protein (f) in the ipsilateral L3 and L4 DRGs 8 weeks after microinjection of AAV5-Syncrip (AAV5-Syn) or control AAV5-GFP. n=5 mice/assay/group. ∗P<0.05 by two-tailed independent Student's t-test. (g) Levels of SYNCRIP and CCR2 proteins in mouse cultured DRG neurones transduced/transfected as indicated. GFP: AAV5-GFP. Syn: AAV5-Syncrip. Scr-siRNA: control scrambled siRNA. Syn-siRNA: Syncrip siRNA. Representative Western blots (left) and a summary of the densitometric analysis (right) are shown. n=5 biological repeats/group. ∗P<0.05 vs the corresponding GFP plus Scr-siRNA group. ^#P<0.05 vs the corresponding Syn plus Scr-siRNA group. One-way anova followed by Tukey post hoc test. (h–i) CAD cells transfected/transduced as shown were treated with actinomycin D (Act D; 5 μg ml⁻¹) for the indicated times. (h) Levels of Syncrip and Ccr2 mRNAs 24 h after treatment. (i) Level of Ccr2 mRNA at different times after treatment. AAV5-Syn: AAV5-syncrip. Scr-siRNA: Syncrip siRNA. Scr-siRNA: control scrambled siRNA. n=5 biological repeats. ∗P<0.05, ∗∗P<0.01 vs naive group at the corresponding time points. ^#P<0.05, ^##P<0.01 vs AAV5-Syn plus Scr-siRNA group at the corresponding time points. One (for h) or two (for i)-way anova followed by Tukey post hoc test. (j) Double-labelled immunofluorescent staining (yellow) of SYNCRIP (red) with CCR2 (green) in DRG neurones. n=3 mice. Scale bar: 50 μm. AAV5, adeno-associated virus 5; anova, analysis of variance; CCI, chronic constriction injury; CCR2, chemokine (C-C motif) receptor 2; DRG, dorsal root ganglion; GFP, green fluorescent protein; mRNA, messenger RNA; siRNA, small interfering RNA.

Discussion

Peripheral nerve injury-induced persistent nociceptive hypersensitivities, including spontaneous pain, mechanical allodynia, and heat and cold hyperalgesia, in preclinical mouse models of NP mimic the conditions observed in trauma- or surgery-induced NP in clinical settings.³⁷ Exploring the mechanism underlying nociceptive hypersensitivity after peripheral nerve injury holds promise for developing innovative approaches to prevent and treat this challenging disorder. In our study, we observed a time-dependent upregulation of SYNCRIP at both mRNA and protein levels within ipsilateral DRG sensory neurones after peripheral nerve injury. Importantly, this upregulation actively contributed to the development and maintenance of nerve injury-induced nociceptive hypersensitivity by enhancing the stability of Ccr mRNA in the injured DRG. SYNCRIP may participate in the peripheral mechanism of NP and be a potential target for the therapy of this disorder.

Similar to other RNA-binding proteins such as RALY,²⁶ FUS,³¹ and HuR,³⁸ SYNCRIP expression undergoes regulation in injured DRG after peripheral nerve injury. SYNCRIP was expressed in the neurones, but not satellite cells, within the DRG. CCI and SNL produced significant increases of SYNCRIP at both mRNA and protein levels in injured DRG, but not in the spinal cord. These increases occurred likely in injured medium-sized DRG neurones, as more SYNCRIP-like immunoreactivity was detected in these DRG neurones on the ipsilateral side 5 days post-CCI. Given that these increases are temporally correlated with the induction and maintenance of NP and that medium-sized DRG neurones participate in the development and maintenance of mechanical, heat and cold hypersensitivities after peripheral nerve injury,^39,40 our results indicate that the increased SYNCRIP in injured DRG may be associated with NP. Although detailed mechanisms by which peripheral nerve injury triggers transcriptional activation of the Syncrip gene in injured DRG neurones are still unclear, this activation is likely related to the changes in expression of some transcription factors, epigenetic modifications, RNA stability, or all three. Further investigations into these possibilities are warranted in future studies.

In the present study, we used the Syn-siRNA strategy to investigate the role of DRG SYNCRIP in CCI-induced nociceptive hypersensitivities. To prevent the siRNA degradation and promote its efficiency, we used TurboFect in vivo transfect reagent to dissolve siRNA as reported previously.41, 42, 43, 44, 45 The results indicate that microinjection of Syn-siRNA into injured DRG attenuated the CCI-induced increase of DRG SYNCRIP and nociceptive hypersensitivity. Given that Syn-siRNA did not alter basal (acute) responses to noxious stimuli and locomotor activity, this strongly suggests the specificity and selectivity of Syn-siRNA's effect. Notably, DRG microinjection of Syn-siRNA did not significantly reduce basal expression of SYNCRIP in sham DRG, whereas transfection of Syn-siRNA into cultured DRG neurones produced a significant reduction in basal level of SYNCRIP in the AAV5-GFP-transducted DRG neurones. This discrepancy might be attributed to the limited volume (1 μl) of DRG Syn-siRNA microinjection. While this small volume may effectively block the CCI-induced increase of DRG SYNCRIP, it might not be sufficient to knock down the basal level of DRG SYNCRIP under normal or sham conditions. In addition, this phenomenon may be associated with the difference in the second structure between already existing and newly transcribed Syncrip mRNAs. It is possible that Syn-siRNA access of already existing Syncrip mRNA is blocked by other RNA-binding proteins or the secondary structure of the mRNA, thereby preventing access of Syn-siRNA, whereas the newly transcribed Syncrip mRNA is accessible for Syn-siRNA to knock down the Syncrip mRNA. This speculation remains to be confirmed in our future study.

Increased SYNCRIP stabilises Ccr2 mRNA expression in injured DRG under NP conditions. SYNCRIP, identified as a component of cytoplasmic RNA granules in dendrites of mammalian neurones,⁴⁶ remains poorly elucidated with respect to its functional characteristics. It was shown that SYNCRIP was partially colocalised with synaptotagmin 7, a Ca²⁺ sensor, in somata of Purkinje neurones in the cerebellum cortex,⁴⁷ suggesting possible functional connections between two proteins in Purkinje neurones. Recent research has demonstrated that that SYNCRIP bound to the 3′-UTR of a long prospero mRNA, stabilising its expression.²⁵ In the present study, we observed that SYNCRIP interacted with 3′-UTR of Ccr2 mRNA in sham DRG. Notably, this interacting activity was substantially increased in CCI DRG 5 days post-surgery. Our in vitro study further demonstrated that SYNCRIP significantly delayed the degradation of Ccr2 mRNA and increased its expression. These findings suggest that the upregulation of SYNCRIP may contribute to the CCI-induced increases of CCR2 at both mRNA and protein levels by stabilising Ccr2 mRNA expression and subsequently facilitating translation of more Ccr2 mRNA in injured DRG. Indeed, CCI-induced increases in Ccr2 mRNA and CCR2 protein were markedly blocked in injured DRGs microinjected with Syn-siRNA. DRG overexpression of SYNCRIP through DRG microinjection of AAV5-Syn resulted in an augmentation of Ccr2 mRNA and CCR2 protein expression in microinjected DRG. These in vivo findings were further validated in in vitro cultured DRG neurones, which were transduced with AAV5-Syn, transfected with Syn-siRNA, or both. Considering the co-expression of SYNCRIP with CCR2 in the cytoplasm of individual DRG neuronal somata and the pivotal role of CCR2 in NP,11, 12, 13, 14, 15, 16, 17, 18 it is plausible that SYNCRIP contributes to NP, likely through stabilising Ccr2 mRNA expression in DRG sensory neurones. Given that CCR2 expression was also increased in macrophages in injured DRG,⁴⁸ the SYNCRIP-stabilised expression of Ccr2 mRNA in DRG macrophages, immune cells, or both cannot be ruled out.

In summary, we have identified a SYNCRIP-triggered post-transcriptional mechanism by which peripheral nerve injury upregulates CCR2 expression in injured DRG. Blocking increased DRG SYNCRIP has been shown to mitigate nerve injury-induced nociceptive hypersensitivity without affecting acute/basal pain and locomotor function, suggesting that SYNCRIP may be a potential target for NP management. Although the siRNA strategy used in the present study has not been applied in a clinical setting, chemically modified long-acting antisense oligonucleotides have been approved by the US Food and Drug Administration (FDA) for clinical therapy of neurological diseases.^49,50 We will design the modified antisense oligonucleotide specifically targeting SYNCRIP and examine its effect on NP in our future study. However, we should pay more attention to unwanted potential side-effects considering that SYNCRIP is widely expressed in various body tissues.

Authors’ contributions

Study conception and design: YXT, YZ, BW, XF, HW

Data acquisition and analysis: YZ, BW, XF, HW, XL, XH, YXT

Drafting figures: YZ, BW, XF, HW

Drafting manuscript: YZ, BY, JG, AB, HH, YXT

Finalising manuscript: YXT

Declaration of interest

The authors declare that they have no conflicts of interest.

Funding

Grants R01NS117484, R01NS111553, and RFNS113881 from the US National Institutes of Health (Bethesda, MD, USA).

Handling Editor: Nadine Attal

Appendix ASupplementary data

The following is the Supplementary data to this article: