Research Articles, Neurobiology of Disease

FUS Contributes to Nerve Injury-Induced Nociceptive Hypersensitivity by Activating NF-κB Pathway in Primary Sensory Neurons

Guang Han, Xiang Li, Chun-Hsien Wen, Shaogen Wu, Long He, Cynthia Tan, John Nivar, Alex Bekker, Steve Davidson and Yuan-Xiang Tao
Journal of Neuroscience 15 February 2023, 43 (7) 1267-1278; DOI: https://doi.org/10.1523/JNEUROSCI.2082-22.2022

Abstract

Dysregulation of pain-associated genes in the dorsal root ganglion (DRG) is considered to be a molecular basis of neuropathic pain genesis. Fused in sarcoma (FUS), a DNA/RNA-binding protein, is a critical regulator of gene expression. However, whether it contributes to neuropathic pain is unknown. This study showed that peripheral nerve injury caused by the fourth lumbar (L4) spinal nerve ligation (SNL) or chronic constriction injury (CCI) of the sciatic nerve produced a marked increase in the expression of FUS protein in injured DRG neurons. Blocking this increase through microinjection of the adeno-associated virus (AAV) 5-expressing Fus shRNA into the ipsilateral L4 DRG mitigated the SNL-induced nociceptive hypersensitivities in both male and female mice. This microinjection also alleviated the SNL-induced increases in the levels of phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) and glial fibrillary acidic protein (GFAP) in the ipsilateral L4 dorsal horn. Furthermore, mimicking this increase through microinjection of AAV5 expressing full-length Fus mRNA into unilateral L3/4 DRGs produced the elevations in the levels of p-ERK1/2 and GFAP in the dorsal horn, enhanced responses to mechanical, heat and cold stimuli, and induced the spontaneous pain on the ipsilateral side of both male and female mice in the absence of SNL. Mechanistically, the increased FUS activated the NF-κB signaling pathway by promoting the translocation of p65 into the nucleus and phosphorylation of p65 in the nucleus from injured DRG neurons. Our results indicate that DRG FUS contributes to neuropathic pain likely through the activation of NF-κB in primary sensory neurons.

SIGNIFICANCE STATEMENT In the present study, we reported that fused in sarcoma (FUS), a DNA/RNA-binding protein, is upregulated in injured dorsal root ganglion (DRG) following peripheral nerve injury. This upregulation is responsible for nerve injury-induced translocation of p65 into the nucleus and phosphorylation of p65 in the nucleus from injured DRG neurons. Because blocking this upregulation alleviates nerve injury-induced nociceptive hypersensitivity, DRG FUS participates in neuropathic pain likely through the activation of NF-κB in primary sensory neurons. FUS may be a potential target for neuropathic pain management.

Introduction

Neuropathic pain caused by a primary damage or injury in the somatosensory nervous system is a complex and refractory clinical problem. It affects the quality of life of 7% to 10% of the population in the United States with an estimated cost of over 600 billion dollars annually in neuropathic pain-related health care and productivity loss (Gilron et al., 2015). Current pharmacological management for this disorder includes antidepressants, nonsteroidal anti-inflammatory drugs, acetaminophen, and opioids, all of which only produce a modest effect (Cohen and Mao, 2014). The majority of neuropathic pain patients complain of inadequate pain relief and/or unwanted side effects (Vorobeychik et al., 2011). Neuropathic pain in the clinic is characterized by spontaneous pain, intermittent burning pain, allodynia (pain because of innocuous stimuli), and hyperalgesia (augmented pain from noxious stimuli). These persistent or long-lasting nociceptive hypersensitivities likely result from the nerve injury-caused dysregulation of pain-associated genes at the transcriptional and translational levels in primary sensory neurons of dorsal root ganglion (DRG; Campbell and Meyer, 2006; Lutz et al., 2014; Wang et al., 2014a, b; Liang et al., 2015; Wu et al., 2019; Ghosh and Pan, 2022). Therefore, understanding the mechanisms of how these DRG genes are dysregulated after nerve injury may provide a new potential avenue for therapeutic treatments of neuropathic pain.

Fused in sarcoma (FUS), a multifunctional DNA/RNA-binding protein, regulates diverse cellular processes including cell proliferation, DNA repair, transcription, pre-mRNA alternative splicing, mRNA transport, translation, and miRNA processing (Lagier-Tourenne et al., 2010; Konopka and Atkin, 2022). Under normal conditions, FUS is predominantly expressed in the cell nucleus (Andersson et al., 2008). FUS binds to RNA from >5500 genes through a GUGGU-binding motif and participates in neuronal plasticity and the maintenance of dendritic integrity in the central nervous system (Fujii et al., 2005; Lagier-Tourenne et al., 2012). Functional loss of FUS in the neuronal nucleus produces neuronal dysfunction and/or cell death; whereas dysfunction of FUS in the neuronal cytoplasm, particularly in the dendritic spines, causes mRNA destabilization (Ingolia et al., 2012). These findings indicate that FUS may be involved in multiple physiological and pathologic processes. Our recent work showed an increase of FUS protein in injured DRG on day 5 after peripheral nerve injury (Du et al., 2022). However, whether this increase participates in neuropathic pain is still unclear. Given that FUS is a critical regulator of gene transcription and translation (Lagier-Tourenne et al., 2010; Konopka and Atkin, 2022), we hypothesized that increased FUS might contribute to nerve injury-induced nociceptive hypersensitivity possibly by regulating pain-associated pathways and/or genes in injured DRG.

In the present study, we first assessed the expression and distribution of FUS in the DRG under normal conditions and its expressional change in the DRG after unilateral fourth lumbar (L4) spinal nerve ligation (SNL) or chronic constriction injury (CCI) of unilateral sciatic nerve. We then examined whether DRG FUS contributed to the development and maintenance of SNL-induced nociceptive hypersensitivity. Finally, we investigated the mechanism of how FUS participated in SNL-induced nociceptive hypersensitivity.

Materials and Methods

Animal preparations

CD1 male and female mice (seven to eight weeks; Charles River Laboratories) were used for this experiment. They were housed in the central housing facility under a standard 12/12 h light/dark cycle, with ad libitum food and water. The experimental procedures were approved by the Animal Care and Use Committee at Rutgers New Jersey Medical School and in accordance with the ethical guidelines of the National Institutes of Health and the International Association for the Study of Pain. To minimize intraindividual and interindividual variability of behavioral outcome measures, we trained mice for 1–2 d before behavioral testing. All efforts were made to minimize the suffering of animals and to reduce the number of animals used. The experimenters were blinded to treatment conditions.

Neuropathic pain models

The spinal nerve ligation (SNL)-induced neuropathic pain model in mice was generated as previous published protocol (S.H. Kim and Chung, 1992; Rigaud et al., 2008; He et al., 2020). Briefly, the experimental mice were anesthetized with 2–3% isoflurane and placed in a prone position. A dorsolateral skin incision was made on the lower back. The unilateral fourth lumbar (L4) transverse process was identified and removed after the surrounding tissues were dissected. The underlying L4 spinal nerve was isolated carefully, ligated with a 7–0 silk suture under a surgical microscope and then transected just distal to the ligature. The skin and muscles were closed in layers. Sham animals received an identical surgery but without transection and ligation of the L4 spinal nerve.

The CCI-induced neuropathic pain model in mice was created as described previously (Bennett and Xie, 1988; Yuan et al., 2019; L. Zhang et al., 2022). In brief, after mice were anesthetized as described above, the left sciatic nerve trunk was exposed above the hip and three ligatures were tied loosely with 7–0 silk thread around the nerve ∼1 mm apart proximal to the trifurcation. Ligatures were loosely tied until a subtle flick of the ipsilateral hindlimb was observed. The sham groups underwent identical procedures, but without the ligature of sciatic nerve.

Behavioral tests

The behavioral testing including mechanical, heat and cold tests was conducted as described previously (Bennett and Xie, 1988; Yuan et al., 2019; L. Zhang et al., 2022) in order with 1-h intervals. Conditioned place preference (CPP) test was performed as reported previously (Bennett and Xie, 1988; Yuan et al., 2019; L. Zhang et al., 2022) at the sixth week after PBS or viral microinjection. Locomotor functional testing was conducted as indicated (Bennett and Xie, 1988; Yuan et al., 2019; L. Zhang et al., 2022) after pain behavioral tests described below were completed.

For the mechanical test, the mouse was placed individually in a Plexiglas chamber on an elevated mesh screen and habituated for 30 min. Two calibrated von Frey filaments (calibrated 0.07 and 0.4 g, Stoelting Co) were used to stimulate the hind paw for 1–2 s and repeated 10 times on each hind paw with 5-min intervals. The occurrence of paw withdrawal in each of these 10 trials was expressed as a percent response frequency [(number of paw withdrawals/10 trials) × 100 = % response frequencies].

For the heat test, paw withdrawal latency to noxious heat was recorded with a Model 336 Analgesia Meter (IITC Inc., Life Science Instruments). The mouse was placed individually in a Plexiglas chamber on a glass plate. Radiant heat from a light box was applied to the middle of the plantar surface of each mouse's hind paw. When the animal lifted its foot, the light beam was automatically turned off. The length of time between the start of the light beam and withdrawal of the hind paw was defined as the paw withdrawal latency. Each trial was repeated three times at 5-min intervals for each side. An automatic cutoff time of 20 s was used to avoid tissue damage to the hind paw.

For the cold test, each mouse was placed in a Plexiglas chamber on a cold aluminum plate (0°C). The temperature was monitored continuously by digital thermometer. The length of time between the placement of the mouse and a positive nociceptive response (e.g., jumping or snapping at the relevant paw) was defined as the paw withdrawal latency. Each trial was repeated three times at 10-min intervals. A cutoff time of 20 s was used to avoid tissue damage.

For the CPP test, an apparatus (Med Associates Inc.) with two Plexiglas chambers connected with an internal door was used. Each chamber had unique floor texture and wall patterns. The movement of the mice and time spent in each chamber were monitored by photobeam detectors installed along the chamber walls and automatically recorded in MED-PC IV CPP software. Mice were first preconditioned to acclimatize to the test environment with free access to both chambers for 30 min. At the end of preconditioning, basal duration time spent in each chamber was recorded within 15 min to confirm whether mice had a preexisting chamber bias. Mice that spent >80% or <20% of the total time in any chamber were excluded from further testing. The following conditioning protocol was performed each day for 3 d when the internal door was closed. The mice were first given an intrathecal injection of saline (5 µl) specifically paired with one conditioning chamber in the morning for 15 min. Six hours later, lidocaine (0.8% in 5 µl of saline) was received intrathecally paired with another conditioning chamber in the afternoon for 15 min. The injection order of saline and lidocaine was alternated every day. On the test day, the mouse was randomly placed in one of the chambers with the interior door open. Movement and duration of time spent by each mouse in each chamber were recorded for 15 min for analysis of chamber preference. Difference in scores were analyzed through subtracting preconditioning time by test time spent in the lidocaine chamber.

Locomotor functions, including placing, grasping, and righting reflexes, were examined after the above-described behavioral tests. (1) Placing reflex: the hind limbs were placed slightly lower than the forelimbs, and the dorsal surfaces of the hind paws were brought into contact with the edge of a table. Whether the hind paws were placed on the table surface reflexively was recorded. (2) Grasping reflex: after the animal was placed on a wire grid, whether the hind paws grasped the wire on contact was recorded. (3) Righting reflex: when the animal was placed on its back on a flat surface, whether it immediately assumed the normal upright position was recorded. 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.

DRG microinjection

DRG microinjection was conducted as described in our previous studies (Pan et al., 2021; Du et al., 2022). In brief, a dorsal midline incision was made in the lower lumbar region. Unilateral L4 DRG or L3 and L4 DRGs were exposed after removing the corresponding articular processes. The adeno-associated virus type 5 (AAV5; 1 μl/DRG, 2 × 1013 GC/ml) was microinjected into the L4 DRG or L3 and L4 DRGs within 10 min through a micropipette connected to a Hamilton syringe under dissection microscopy. After microinjection, the micropipette was kept in place for 10 min before removal. The surgical field was irrigated with sterile saline and iodophor and closed with wound clips.

Cell culture and viral transduction

DRG neuronal cultures were prepared according to previously described methods (Pan et al., 2021; Du et al., 2022). Briefly, bilateral DRGs collected from four-week CD1 mice were collected in a cold Neurobasal Medium (Gibco/ThermoFisher Scientific) containing 10% fetal bovine serum (JR Scientific), 100 units/ml penicillin and 100 µg/ml streptomycin (Quality Biological). The DRGs were treated with enzyme solution (5 mg/ml dispase, 1 mg/ml collagenase Type I in HBSS without Ca2+ and Mg2+; Gibco/ThermoFisher Scientific) at 37°C for 20 min. After trituration and centrifugation, the dissociated cells were resuspended in cold Neurobasal Medium, plated onto 50 µg/ml poly-D-lysine (Sigma)-coated six-well plates and incubated at 37°C in 5% CO2 atmosphere. On the second day, 3–10 µl of AAV5 (titer ≥ 1 × 1013 GC/µl) were added to each well. Three days later, the cells were collected for Western blot analysis as described below.

Western blot analysis

Protein extraction and Western blotting were conducted according to our previously published protocol (Pan et al., 2021; Du et al., 2022). Briefly, to achieve enough protein, unilateral L4 DRGs from four mice or unilateral L3 and L4 DRGs from two mice were pooled together. The tissues or the cultured cells were homogenized and ultrasonicated in chilled lysis buffer (10 mm Tris, 1 mm phenylmethylsulfonyl fluoride, 5 mm MgCl2, 5 mm EGTA, 1 mm EDTA, 1 mm DTT, 40 μm leupeptin, 250 mm sucrose). Approximately 10% of the homogenate (in volume) was used for total protein. The remaining was centrifuged at 4°C for 15 min at 1000 × g. The supernatant was collected for cytosolic protein and the pellet for nuclear protein. After the protein concentration was measured, the samples were heated at 99°C for 5 min and loaded onto a 4–15% stacking/7.5% separating SDS-polyacrylamide gel (Bio-Rad Laboratories). The proteins were electrophoretically transferred onto a polyvinylidene difluoride membrane (Bio-Rad). The membrane was blocked with 3% nonfat milk in Tris-buffered saline containing 0.1% Tween 20 for 1 h at room temperature and then incubated at 4°C overnight with the following primary antibodies: mouse anti-FUS (1:1000; Abcam), mouse anti-phosphorylated p65 (p-p65; 1:1000; CST), rabbit anti-p65 (1:1000; CST), rabbit anti-GAPDH (1:1000; Santa Cruz), rabbit anti-H3 (1:1000; Santa Cruz), mouse anti-glial fibrillary acidic protein (GFAP; 1:1000; Abcam), rabbit anti-total ERK1/2 (1:1000; CST), or rabbit anti-phosphorylated ERK1/2 (p-ERK1/2; 1:1000; CST). The proteins were detected by horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibody (1:3000; Jackson ImmunoResearch). The proteins were visualized by Western peroxide reagent and luminol/enhancer reagent (Clarity Western ECL Substrate, Bio-Rad) and exposed using ChemiDoc XRS and System with Image Lab software (Bio-Rad). The intensity of Western blottings was quantified with densitometry using Image Lab software (Bio-Rad). The nuclear protein bands were normalized to total histone H3, whereas the cytosol/total protein bands were normalized to GAPDH.

Quantitative real-time RT-PCR

Total RNA extraction and quantitative real-time RT-PCR assays were conducted as described previously (Pan et al., 2021; Du et al., 2022). Briefly, the ipsilateral L4 DRGs from four adult mice were pooled together to obtain enough RNA. Total RNA was extracted using the miRNeasy kit with on-column digestion of genomic DNA (QIAGEN) according to the manufacturer's instructions and then reverse-transcribed using ThermoScript reverse transcriptase (Invitrogen/Thermo Fisher Scientific) using oligo (dT) primers. Template (1 µl) was amplified with Bio-Rad CFX96 real-time PCR system by using the following primers for Fus mRNA (forward: 5′-GGCTACTCCCAACAGAGCAG-3′, reverse: 5′-ATATCCCTGGGGAGCTGACT-3′) or for Tuba1a mRNA (forward: 5′-GTGCATCTCCATCCATGTTG-3′, reverse: 5′-GTGGGTTCCAGGTCTACGAA-3′). Each sample was run in triplicate in a 20 μl reactive volume containing 250 nm forward and reverse primers, 10 μl of SsoAdvanced Universal SYBR Green Supermix (Bio-Rad Laboratories) and 20 ng of cDNA. The PCR amplification was made up from 30 s at 95°C, 30 s at 60°C, 30 s at 72°C, and 5 min at 72°C for 39 cycles. The ratios of mRNA levels at other time points to those at 0 d were calculated by using the △Ct method (2−△△Ct) after normalization to the corresponding Tuba-1a, as the expression of Tuba1a, an internal control, was demonstrated to be stable after peripheral nerve injury (Pan et al., 2021; Du et al., 2022).

Immunohistochemistry

Immunohistochemistry was conducted as previously described (Pan et al., 2021; Du et al., 2022). Briefly, after the mice were deeply anesthetized with isoflurane, they were transcardially perfused with 20–30 ml of 0.01 m PBS and then with 50–100 ml of 4% paraformaldehyde in 0.1 m phosphate buffer (pH 7.4). The DRG was harvested, postfixed for 2–4 h in same fixative solution and then cryoprotected in 30% sucrose in 0.1 m phosphate buffer at 4°C overnight. DRGs were sectioned at a thickness of 20 μm on a cryostat. The sections were blocked for 1 h at room temperature with 5% goat serum and 0.3% Triton X-100 in PBS and then incubated with rabbit anti-FUS (1:200, Abcam), mouse anti-NeuN (1:100, Temecula), biotinylated isolectin B4 (IB4; 1:200, Sigma), mouse anti-glutamine synthetase (1:1000, Sigma), mouse anti-NF200 (1:200, Sigma), mouse anti-calcitonin gene-related peptide (CGRP; 1:200, Abcam) and rabbit anti-p65 (1:1000; CST), respectively, at 4°C overnight. On the second day, the sections were incubated with species-appropriate Cy3-conjugated secondary antibody (1:500, Jackson ImmunoResearch) or with FITC-labeled Avidin D (1:200, Sigma) at room temperature for 2 h. The images were captured using a DMI 4000 fluorescence microscope (Leica) with a DFC365 FX camera (Leica) and analyzed using NIH ImageJ software.

Statistical analysis

The mice were grouped randomly. The results were analyzed by one-way or two-way ANOVA or two-tailed independent Student's t tests. When ANOVA showed a significant difference, pairwise comparisons between means was performed using the post hoc Tukey's method (GraphPad Prism 8). All data were presented as means ± SEM. The significant difference was set at p < 0.05.

Results

FUS expression is increased in injured DRG after peripheral nerve injury

To investigate the potential function of FUS in neuropathic pain, we first examined whether FUS expression was changed in the DRG and spinal cord, two major pain-associated regions, after unilateral SNL. SNL, but not sham surgery, significantly increased the expression of FUS protein in the ipsilateral L4 DRG (Fig. 1A). The amounts of FUS protein were increased by 1.8-fold on day 3, 2.1-fold on day 7, and 1.4-fold on day 14 after SNL, compared with those at the corresponding time points after sham surgery (Fig. 1A). Interestingly, the level of Fus mRNA was not significantly altered in the ipsilateral L4 DRG during the observation period (Fig. 1B). Neither SNL nor sham surgery changed basal amounts of FUS protein in the contralateral L4 DRG, ipsilateral L3 DRG, or ipsilateral L4 spinal cord during the experimental period (Fig. 1C). Similar results were observed after CCI to unilateral sciatic nerve. The level of FUS protein was elevated by 2.1-fold in the ipsilateral L3/4 DRGs on day 7 post-CCI, as compared with the corresponding sham mice (Fig. 1D).

Figure 1.
Figure 1.

Peripheral nerve injury led to an increase in FUS protein in injured DRG. A, Expression of FUS protein in the ipsilateral L4 DRG from mice on days as indicated after SNL or sham surgery. n = 12 mice/time point/group. **p < 0.01, by two-way ANOVA followed by post hoc Tukey's test. B, Expression of Fus mRNA in the ipsilateral L4 DRG from mice on days as shown after SNL or sham surgery. n = 12 mic/time point/group. Two-way ANOVA followed by post hoc Tukey's test. C, Expression of FUS protein in the ipsilateral (Ipsi) L3 DRG, contralateral (Con) L4 DRG and ipsilateral L4 spinal cord (SC) from mice on days as indicated after SNL or sham surgery. n = 12 mic/time point/group. One-way ANOVA followed by post hoc Tukey's test. D, Expression of FUS protein in the ipsilateral L3/4 DRGs from mice on day 7 after CCI or sham surgery. n = 6 mic/time point/group. **p < 0.01, by two-tailed, independent Student's t test.

We also examined the distribution pattern of FUS and the alteration in number of FUS-positive cells in the DRG following SNL. FUS was expressed in cellular nuclei and co-localized with NeuN (a specific neuronal marker) in individual cells of DRG (Fig. 2A). It was undetected in the cells labeled by glutamate synthetase (GS; a marker for satellite glial cells; Fig. 2B). Approximately 48% of DRG neurons were labeled by FUS. A cross-sectional area analysis of DRG neuronal somata showed that ∼51.4% of FUS-labeled neurons were small (<600 μm2 in area), 30.5% were medium (600–1200 μm2 in area) and 18.1% were large (>1200 μm2 in area; Fig. 2C). Further analysis revealed that ∼39.8% of FUS-labeled neurons were positive for isolectin B4 (IB4; a marker for small nonpeptidergic neurons; Fig. 2D), 31.2% were positive for calcitonin gene-related peptide (CGRP; a marker for small peptidergic neurons; Fig. 2E), and 26.7% were positive for neurofilament 200 (NF200, a marker for medium/large cells and myelinated Aβ fibers; Fig. 2F). In line with Western blot analysis above, on day 7 after SNL, the number of FUS-labeled neurons in the ipsilateral L4 DRG was 1.5-fold higher than that after sham surgery (Fig. 2G).

Figure 2.
Figure 2.

Distribution pattern of FUS protein in DRG of naive mice and changes in number of FUS-positive neurons in injured DRG after SNL. A, B, Representative photographs showing that FUS (red color) is co-expressed exclusively with NeuN (green color) in cellular nuclei (A; arrows) and undetected in glutamine synthetase (GS; green color)-labeled cells (B). Cellular nuclei were labeled by 4′,6-diamidino-2-phenylindole (DAPI; blue color). n = 3 mice. Scale bars: 25 µm (A) and 20 µm (B). C, Histogram showing the distribution of FUS-positive neuronal somata in DRG. Small: 51%. Medium: 31%. Large: 18%. D–F, FUS-positive neurons were labeled by isolectin B4 (IB4; D), calcitonin gene-related peptide (CGRP; E), or neurofilament-200 (NF200; F) in naive DRG. Arrows: double labeling. n = 3 mice. Scale bars: 25 µm. G, Changes in number of FUS-positive neurons in the ipsilateral L4 DRG on day 7 after SNL or sham surgery. n = 3 mice/group. **p < 0.01, by two-tailed, independent Student's t test. Scale bar: 20 μm.

Our findings suggest that nerve injury-induced FUS upregulation in injured DRG may have a functional role in neuropathic pain.

Blocking the increased DRG FUS attenuates the development of SNL-induced nociceptive hypersensitivity

We next examined whether nerve injury-induced increase of DRG FUS participated in the development of neuropathic pain in male mice. To this end, we blocked this increase through microinjection of AAV5 expressing Fus shRNA (AAV5-Fus shRNA) into the ipsilateral L4 DRG 35 d before SNL or sham surgery, because AAV5 takes three to four weeks to be expressed (Pan et al., 2021; Du et al., 2022). AAV5 harboring scrambled shRNA (AAV5-scrambled shRNA) was used as a control. Consistent with the observation above, the level of FUS protein was increased by 2.8-fold in the ipsilateral L4 DRG of the AAV5-scrambled shRNA-microinjected SNL male mice on day 14 post-SNL as compared with that in the AAV5-scramble shRNA-microinjected sham male mice (Fig. 3A). This increase was significantly reduced in the AAV5-Fus shRNA-microinjected SNL male mice (Fig. 3A). No significant reduction in the basal amount of FUS protein was observed in the ipsilateral L4 DRG of the AAV5-Fus shRNA-microinjected sham male mice on day 14 postsham surgery (Fig. 3A).

Figure 3.
Figure 3.

Blocking the SNL-induced increase in DRG FUS protein attenuated the SNL-induced development of nociceptive hypersensitivity and dorsal horn central sensitization in male mice. A, Level of FUS protein in the ipsilateral L4 DRG from the AAV5-Fus shRNA (shRNA)-microinjected or AAV5-scrambled shRNA (scramble)-microinjected male mice on day 14 post-SNL or sham surgery. n = 12 mice/group. **p < 0.01, by two-way ANOVA followed by post hoc Tukey's test. B–H, Paw withdrawal frequency to low (0.07 g; B, F) and median (0.4 g; C, G) force von Frey filament stimuli and paw withdrawal latency to heat (D, H) and cold (E) stimuli on the ipsilateral side (B–E) and contralateral side (F–H) of male mice with microinjection of AAV5-Fus shRNA (shRNA) or AAV5-scrambled shRNA (Scramble) into the ipsilateral L4 DRG at different days post-SNL or sham surgery. n = 12 mice/group. **p< 0.01 versus the AAV5-scrambled shRNA-microinjected sham male mice at the corresponding time point. ##p < 0.01 versus the AAV5-scrambled shRNA-microinjected SNL male mice at the corresponding time point. Two-way ANOVA with repeated measures followed by post hoc Tukey's test. I, Levels of phosphorylated ERK1/2 (p-ERK1/2), total ERK1/2 and GFAP in the ipsilateral L4 dorsal horn from male mice on day 14 post-SNL or sham surgery. n = 3 mice/group. **p < 0.01, by two-way ANOVA followed by Tukey's post hoc test.

Consistent with the previous reports (Pan et al., 2021; Du et al., 2022), SNL led to mechanical allodynia as evidenced by increases in paw withdrawal frequencies in response to mechanical stimuli (0.07 and 0.4 g von Frey filaments) and heat and cold hyperalgesia as documented by reductions in paw withdrawal latencies in response to heat and cold stimuli, respectively, from days 3 to 14 post-SNL surgery on the ipsilateral (but not contralateral) side of the AAV5-scrambled shRNA-microinjected SNL male mice (Fig. 3B–H). However, these nociceptive hypersensitivities were markedly reduced in the AAV5-Fus shRNA-microinjected SNL male mice (Fig. 3B–E). Neither AAV5-Fus shRNA nor AAV5-scrambled shRNA altered basal paw withdrawal responses to mechanical, heat, and cold stimuli on the contralateral side of SNL male mice and on both ipsilateral and contralateral sides of sham male mice (Fig. 3B–H). All microinjected male mice displayed normal locomotor activity (Table 1).

View this table:
Table 1.

Locomotor function

Given that DRG neuronal hyperexcitability triggers the hyperactivation of spinal cord dorsal horn neurons and astrocytes through enhancing the release of neurotransmitters/neuromodulators in primary afferents under neuropathic pain conditions (Campbell and Meyer, 2006), we also examined whether DRG microinjection of AAV5-Fus shRNA blocked the SNL-induced hyperactivities of neurons and astrocytes in the dorsal horn to further confirm our behavioral observations above. The levels of phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2, a marker for neuronal hyperactivation) and glial fibrillary acidic protein (GFAP; a marker for astrocyte hyperactivation) were significantly increased in the ipsilateral L4 dorsal horn of the AAV5-scrambled shRNA-microinjected SNL male mice, as compared with those in the AAV5-scrambled shRNA-microinjected sham male mice (Fig. 3I). These increases were absent in the AAV5-Fus shRNA-microinjected SNL male mice (Fig. 3I). DRG microinjection of AAV5-Fus shRNA did not change basal levels of p-ERK1/2 and GFAP in the ipsilateral L4 dorsal horn of sham male mice (Fig. 3I).

Similar results were observed in SNL/sham female mice with DRG microinjection of AAV5-Fus shRNA or AAV5-scrambled shRNA (Fig. 4A–I; Table 1). Our findings strongly suggest that the increased FUS in injured DRG of both male and female mice is required for neuropathic pain development.

Figure 4.
Figure 4.

Blocking the SNL-induced increase in DRG FUS protein attenuated the SNL-induced development of nociceptive hypersensitivity and dorsal horn central sensitization in female mice. A, Level of FUS protein in the ipsilateral L4 DRG from the AAV5-Fus shRNA (shRNA)-microinjected or AAV5-scrambled shRNA (scramble)-microinjected female mice on day 14 post-SNL or sham surgery. n = 12 mice/group. **p < 0.01, by two-way ANOVA followed by post hoc Tukey's test. B–H, Paw withdrawal frequency to low (0.07 g; B, F) and median (0.4 g; C, G) force von Frey filament stimuli and paw withdrawal latency to heat (D, H) and cold (E) stimuli on the ipsilateral side (B–E) and contralateral side (F–H) of female mice with microinjection of AAV5-Fus shRNA (shRNA) or AAV5-scrambled shRNA (scramble) into the ipsilateral L4 DRG at different days post-SNL or sham surgery. n = 12 mice/group. **p < 0.01 versus the AAV5-scrambled shRNA-microinjected sham female mice at the corresponding time point. ##p < 0.01 versus the AAV5-scrambled shRNA-microinjected SNL female mice at the corresponding time point. Two-way ANOVA with repeated measures followed by post hoc Tukey's test. I, Levels of phosphorylated ERK1/2 (p-ERK1/2), total ERK1/2 and GFAP in the ipsilateral L4 dorsal horn from female mice on day 14 post-SNL or sham surgery. n = 3 mice/group. **p < 0.01, by two-way ANOVA followed by Tukey's post hoc test.

Blocking the increased DRG FUS attenuates the maintenance of SNL-induced nociceptive hypersensitivity

The role of increased DRG FUS in the maintenance of SNL-induced nociceptive hypersensitivity was also examined. AAV5-Fus shRNA or AAV5-scrambled shRNA was microinjected into the ipsilateral L4 DRG two weeks before SNL surgery in male mice. Mechanical, heat and cold nociceptive hypersensitivities were seen on days 3, 5, and 7 post-SNL on the ipsilateral (not contralateral) side in both AAV5-Fus shRNA-microinjected and AAV5-scrambled shRNA-microinjected male mice (Fig. 5A–G). However, these nociceptive hypersensitivities were significantly reduced on days 14 and 21 post-SNL in the AAV5-Fus shRNA-microinjected SNL male mice, as compared with those in the AAV5-scrambled shRNA-microinjected SNL male mice (Fig. 5A–D). As expected, SNL increased the expression of FUS protein by 1.6-fold in the ipsilateral L4 DRG on day 21 post-SNL in the AAV5-scrambled shRNA-microinjected SNL male mice (Fig. 5H), but this increase was entirely absent in the AAV5-Fus shRNA-microinjected SNL male mice (Fig. 5H). Additionally, SNL-induced increases in p-ERK1/2 (but not total ERK1/2) and GFAP in the ipsilateral L4 dorsal horn on day 21 after SNL from the AAV5-scrambled shRNA-microinjected SNL male mice were not observed in the AAV5-Fus shRNA-microinjected SNL male mice (Fig. 5I). These results indicate the critical role of increased DRG FUS in neuropathic pain maintenance.

Figure 5.
Figure 5.

Blocking the SNL-induced increase in DRG FUS mitigated the maintenance of SNL-induced nociceptive hypersensitivity and dorsal horn central sensitization in male mice. Male mice were subjected to SNL 14 d after DRG microinjection of AAV5-Fus shRNA (shRNA) or AAV5-scrambled shRNA (Scramble). A–G, Paw withdrawal frequency to low (0.07 g; A, E) and median (0.4 g; B, F) force von Frey filament stimuli and paw withdrawal latency to heat (C, G) and cold (D) stimuli on both ipsilateral (A–D) and contralateral (E–G) sides of male mice with microinjection of AAV5-Fus shRNA or AAV5-scrambled shRNA into the ipsilateral L4 DRG at different days after SNL. n = 12 mice/group. **p < 0.01 versus the AAV5-scrambled shRNA-microinjected SNL mice at the corresponding time point on the ipsilateral side. Two-way ANOVA with repeated measures followed by post hoc Tukey's test. H, Level of FUS protein in the ipsilateral L4 DRG from the AAV5-Fus shRNA-microinjected or AAV5-scrambled shRNA-microinjected mice on day 21 post-SNL or sham surgery. n = 12 mice/group. **p < 0.01, by one-way ANOVA followed by Tukey's post hoc test. I, Levels of total ERK1/2, phosphorylated ERK1/2 (p-ERK1/2) and GFAP in the ipsilateral L4 dorsal horn on day 21 post-SNL or sham surgery in male mice with DRG microinjection of AAV5-Fus shRNA or AAV5-scrambled shRNA. n = 3 mice/group. **p < 0.01, by one-way ANOVA followed by Tukey's post hoc test.

Mimicking the SNL-induced increase in DRG FUS leads to nociceptive hypersensitivity

We also asked whether the increased DRG FUS was sufficient for SNL-induced nociceptive hypersensitivity. To mimic nerve injury-induced increase of DRG FUS, we microinjected AAV5 expressing full-length Fus mRNA (AAV5-Fus) into unilateral L3/4 DRGs of naive male mice. AAV5 expressing green fluorescent protein (AAV5-Gfp) was used as a control. DRG microinjection of AAV5-Fus, but not AAV5-Gfp, led to marked increases in paw withdrawal frequencies in response to 0.07 and 0.4 g von Frey filament stimuli and decreases in paw withdrawal latencies in response to heat and cold stimuli from week 5 to at least week 7 postmicroinjection on the ipsilateral side (Fig. 6A–D). Neither AAV5 changed basal responses on the contralateral side (Fig. 6E–G) and locomotor function (Table 1). In addition to evoked nociceptive hypersensitivities, DRG microinjection of AAV5-Fus led to evoked stimulation-independent nociceptive hypersensitivity demonstrated by obvious preference (that is, spent more time) for the lidocaine-paired chamber on week 7 after viral microinjection in male mice (Fig. 6H,I). As predicted, DRG microinjection of AAV5-Gfp did not exhibit significant preference toward either the saline-paired or lidocaine-paired chamber (Fig. 6H,I), indicating no spontaneous pain. The level of FUS protein was increased by 1.6-fold in the ipsilateral L3/4 DRGs of AAV5-Fus-microinjected male mice as compared with the AAV5-Gfp-microinjected male mice seven weeks after microinjection (Fig. 6J). DRG microinjection of AAV5-Fus, but not AAV5-Gfp, also elevated the levels of p-ERK1/2 (but not total ERK1/2) and GFAP in the ipsilateral L3/4 dorsal horn of male mice seven weeks after microinjection (Fig. 6K). Similar findings were observed in naive female mice after DRG microinjection of AAV5-Fus or AAV5-Gfp (Fig. 7A–K; Table 1). Collectively, these data indicate that mimicking the nerve injury-induced FUS increase in the DRG of male and female naive mice leads to both spontaneous and evoked nociceptive hypersensitivities.

Figure 6.
Figure 6.

DRG FUS overexpression produced the enhanced nociceptive response and dorsal horn central sensitization in naive male mice. A–G, Effect of microinjection of AAV5-Fus or AAV5-Gfp into the unilateral L3/4 DRGs on paw withdrawal frequencies to low (0.07 g; A, E) and median (0.4 g; B, F) force von Frey filament stimuli and paw withdrawal latencies to heat (C, G) and cold stimuli (D) on both ipsilateral (A–D) and contralateral (E–G) sides at the different weeks after AAV5 microinjection. n = 10 mice/group. **p < 0.01 versus control AAV5-Gfp group at the corresponding time points on the ipsilateral side. Two-way ANOVA with repeated measures followed by Tukey's post hoc test. H, I, Effect of microinjection of AAV5-Fus or AAV5-Gfp into the unilateral L3/4 DRGs on spontaneous ongoing pain as assessed by the CPP paradigm. n = 10 mice/group. **p < 0.01, by two-way ANOVA with repeated measures followed by Tukey's post hoc test (H) or by two-tailed, independent Student's t test (I). J, Level of FUS in the ipsilateral L3/4 DRGs seven weeks after microinjection of AAV5-Fus or control AAV5-Gfp. n = 6 mice/group. **p < 0.01, by two-tailed, independent Student's t test. K, Effect of microinjection of AAV5-Fus or AAV5-Gfp into the unilateral L3/4 DRGs on dorsal horn neuronal and astrocyte hyperactivities evidenced by the increases in the phosphorylated ERK1/2 (p-ERK1/2) and GFAP abundance, respectively, in the ipsilateral L3/4 dorsal horn seven weeks after viral microinjection. n = 6 mice/group. **p < 0.01, by two-tailed, independent Student's t test.

Figure 7.
Figure 7.

DRG FUS overexpression produced the enhanced nociceptive response and dorsal horn central sensitization in naive female mice. A–G, Effect of microinjection of AAV5-Fus or AAV5-Gfp into the unilateral L3/4 DRGs on paw withdrawal frequencies to low (0.07 g; A, E) and median (0.4 g; B, F) force von Frey filament stimuli and paw withdrawal latencies to heat (C, G) and cold stimuli (D) on both ipsilateral (A–D) and contralateral (E–G) sides at the different weeks after AAV5 microinjection. n = 10 mice/group. **p < 0.01 versus the control AAV5-GFP group at the corresponding time points on the ipsilateral side. Two-way ANOVA with repeated measures followed by Tukey's post hoc test. H, I, Effect of microinjection of AAV5-Fus or AAV5-Gfp into the unilateral L3/4 DRGs on spontaneous ongoing pain as assessed by the CPP paradigm. n = 10 mice/group. **p < 0.01, by two-way ANOVA with repeated measures followed by Tukey's post hoc test (H) or by two-tailed, independent Student's t test (I). J, Level of FUS in the ipsilateral L3/4 DRGs seven weeks after microinjection of AAV5-Fus or control AAV5-Gfp. n = 6 mice/group. **p < 0.01, by two-tailed, independent Student's t test. K, Effect of microinjection of AAV5-Fus or AAV5-Gfp into the unilateral L3/4 DRGs on dorsal horn neuronal and astrocyte hyperactivities evidenced by the increases in the phosphorylated ERK1/2 (p-ERK1/2) and GFAP abundance, respectively, in the ipsilateral L3/4 dorsal horn seven weeks after viral microinjection. n = 6 mice/group. **p < 0.01, by two-tailed, independent Student's t test.

Increased FUS activates the NF-κB pathway in injured DRG after SNL

Finally, we explored the mechanisms by which the increase in FUS protein in injured DRG contributed to the SNL-induced nociceptive hypersensitivity. It is well evidenced that nerve injury-induced activation of the NF-κB pathway in injured DRG is critical for neuropathic pain genesis (H.H. Zhang et al., 2015; Chen et al., 2016; Xu et al., 2017; Yu et al., 2017). The activation of NF-κB pathway was documented by the translocation of p65, a key member of the NF-κB family, from the cellular cytoplasm to the nucleus (Tomita et al., 2016; Y.C. Zhang et al., 2017; Bao et al., 2018). Consistent with our previous studies (Huang et al., 2019; He et al., 2020), SNL produced significant increases in the levels of p65 and phosphorylated p65 (p-p65) in the nuclear fraction from the ipsilateral L4 DRG on day 14 post-SNL in the AAV5-scrambled shRNA-microinjected SNL male or female mice (Fig. 8A; Extended Data Fig. 8-1A), although the amount of p65 in total (including cytoplasm, membrane, and nuclear) fraction was not altered in the ipsilateral L4 DRG from these microinjected mice (Fig. 8B; Extended Data Fig. 8-1B), when compared with naive male mice. Interestingly, these increases were not seen in the AAV5-Fus shRNA-microinjected SNL male or female mice on day 14 after SNL (Fig. 8A; Extended Data Fig. 8-1A). DRG microinjection of neither AAV5-Fus shRNA nor AAV5-scrambled shRNA altered basal levels of p-65 and p-p65 in nuclear fraction or basal level of p65 in total fraction from the ipsilateral L4 DRG of sham male or female mice (Fig. 8A,B; Extended Data Fig. 8-1A,B). Moreover, microinjection of AAV5-Fus into unilateral L3/4 DRGs elevated the levels of p65 and p-p65 in the nuclear fraction from these microinjected DRGs seven weeks after microinjection, as compared with those in the AAV5-Gfp-microinjected group, in male or female mice (Fig. 8C; Extended Data Fig. 8-1C). As expected, no marked change in basal expression of p65 in total fraction was observed in the ipsilateral L3/4 DRGs of male or female mice between AAV5-Fus-microinjected and AAV5-Gfp-microinjected groups (Fig. 8D; Extended Data Fig. 8-1D).

Figure 8.
Figure 8.

Increased FUS activated the NF-κB pathway in injured DRG of male mice after SNL. A, B, Levels of phosphorylated p65 (p-p65) and p-65 in the nuclear fraction (A) and the amount of p65 in total (cytosolic, membrane, and nuclear) fraction (B) from the ipsilateral L4 DRG of male mice with microinjection of AAV5-Fus shRNA (shRNA) or AAV5-scrambled shRNA (Scramble) 14 d after SNL or sham surgery. n = 12 mice/group. **p < 0.01 by two-way ANOVA followed by Tukey's post hoc test. C, D, Levels of p65 and p-p65 in the nuclear fraction (C) and the amount of p-65 in total fraction (D) from the microinjected L3/4 DRGs of male mice seven weeks after microinjection of AAV5-Fus or control AAV5-Gfp. n = 6 mice/group. **p < 0.01, by two-tailed, independent Student's t test. E, F, Levels of FUS, p65 and p-p65 in the nuclear fraction (E) and the amount of p-65 (F) in total fraction from mouse cultured DRG neurons transduced as indicated. GFP: AAV5-Gfp. FUS: AAV5-Fus. Scra: AAV5-scrambled shRNA. shRNA: AAV5-Fus shRNA. n = 3 biological repeats/treatment. **p < 0.01 by one-way ANOVA followed by Tukey's post hoc test. G, Double labeling staining (arrows) showed co-localization of FUS with p65 in mouse L4 DRG neurons. 4′,6-diamidino-2-phenylindole (DAPI) was used to label cellular nuclei. Scale bar: 25 µm. Increased FUS-activated NF-κB pathway in injured DRG of female mice after SNL is shown in Extended Data Figure 8-1.

Extended Data Figure 8-1

Increased FUS activated the NF-κB pathway in injured DRG of female mice after SNL. Related to Figure 8. A, B, Levels of phosphorylated p65 (p-p65) and p-65 in the nuclear fraction (A) and the amount of p65 in total (cytosolic, membrane, and nuclear) fraction (B) from the ipsilateral L4 DRG of female mice with microinjection of AAV5-Fus shRNA (shRNA) or AAV5-scrambled shRNA (Scramble) 14 days after SNL or sham surgery. n = 12 mice/group. **p < 0.01 by two-way ANOVA followed by Tukey's post hoc test. C, D, Levels of p65 and p-p65 in the nuclear fraction (C) and the amount of p-65 in total fraction (D) from the microinjected L3/4 DRGs of female mice seven weeks after microinjection of AAV5-Fus or control AAV5-Gfp. n = 6 mice/group. **p < 0.01, by two-tailed, independent Student's t test. Download Figure 8-1, TIF file.

In addition, cultured DRG neurons co-transduced with AAV5-Fus plus AAV5 scrambled shRNA, but not AAV5-Gfp plus AAV5 scrambled shRNA, displayed significant increases in the amounts of FUS, p65 and p-p65 in the nuclear fraction, when compared with naive group (Fig. 8E). These increases were absent in the cultured DRG neurons co-transduced with AAV5-Fus plus AAV5-Fus shRNA (Fig. 8E). Co-transduction of AAV5-Fus shRNA plus AAV5-Gfp also reduced basal levels of FUS, p65 and p-p65 in the nuclear fraction of cultured DRG neurons (Fig. 8E). As predicted, basal level of p65 in total fraction was not changed in any AAV5-transduced cultured DRG neurons (Fig. 8F). Given that p65 co-expressed with FUS in DRG neurons (Fig. 8G), our in vitro results suggest that the translocation of p65 into the nucleus is specific in response to an increase of FUS in DRG neurons.

Taken together, our in vivo and in vitro findings indicate that nerve injury-induced increase of FUS expression may contribute to activation of the NF-κB pathway in injured DRG under neuropathic pain conditions.

Discussion

Peripheral nerve injury caused by SNL or CCI leads to persistent/chronic nociceptive hypersensitivity including spontaneous ongoing pain, mechanical allodynia, heat hyperalgesia, and cold hyperalgesia in the preclinical mouse models, similar to the symptoms in peripheral nerve trauma-induced neuropathic pain in the clinic. Studying how peripheral nerve injury produces nociceptive hypersensitivity may provide potential avenues for neuropathic pain treatment. In this study, we demonstrated that peripheral nerve injury led to an increase in FUS protein expression in injured DRG. This increase contributed to the development and maintenance of SNL-induced nociceptive hypersensitivity likely through the activation of NF-κB pathway in injured DRG neurons. Our findings suggest that FUS may be a target for neuropathic pain management.

Similar to other RNA-binding proteins such as RALY and HuR (Borgonetti and Galeotti, 2021; Pan et al., 2021), FUS expression can be regulated in the DRG following peripheral nerve injury. The present study revealed that FUS was expressed exclusively in the nuclei of DRG neurons. SNL time-dependently increased the expression of FUS protein, but not its mRNA, in injured DRG. This increase may occur in most small-diameter and medium-diameter neurons of injured DRG, as FUS is distributed predominantly in these neurons under normal conditions. Since this increase was correlated to the development and maintenance of SNL-induced nociceptive hypersensitivity and that small-diameter and medium-diameter DRG neurons participate in nerve injury-induced nociceptive hypersensitivity (Sakai et al., 2013; Gong et al., 2014; D. Kim et al., 2020; Shin et al., 2022), our results indicate that increased FUS in injured DRG may be implicated in neuropathic pain. The present findings suggest that Fus gene in injured DRG is post-transcriptionally activated after peripheral nerve injury. Although detailed mechanisms by which peripheral nerve injury increases FUS protein expression in injured DRG are still unclear, this increase may be related to the changes in RNA modifications (e.g., m6A methylation; Li et al., 2020) and splicing (Liang et al., 2020) under neuropathic pain conditions. These possibilities will be addressed in our future study.

The present study conducted the AAV5-Fus shRNA strategy to investigate the role of DRG FUS in peripheral nerve injury-induced nociceptive hypersensitivity. Microinjection of AAV5-Fus shRNA into the ipsilateral L4 DRG attenuated the SNL-induced increase of DRG FUS and nociceptive hypersensitivity during the development and maintenance periods. Given that actual or basal nociceptive responses and locomotor activity were not affected in the AAV5-Fus shRNA-microinjected mice, this indicates the specificity and selectivity of AAV5-Fus shRNA effect. Unexpectedly, in vivo DRG microinjection of AAV5-Fus shRNA did not alter basal expression of FUS in the ipsilateral L4 DRG of sham mice, although transduction of AAV5-Fus shRNA into the in vitro cultured DRG neurons significantly knocked down basal expression of FUS. The reason of why AAV5-Fus shRNA did not affect basal FUS expression in in vivo DRG remains elusive, but it is possible that the lower expression of DRG FUS under normal conditions cannot be further knocked down markedly by AAV5-Fus shRNA because of the limited microinjection volume of AAV5 (1 µl/DRG). Another possibility is that the remaining Fus mRNA after its knockdown may have a high translational efficacy. The latter likely maintains normal level of basal FUS protein in sham DRG.

The increased FUS participates in the SNL-induced nociceptive hypersensitivity by activating the NF-κB pathway in injured DRG. NF-κB, a ubiquitous rapid response transcription factor, contributes to the production of several inflammatory cytokines, chemokines and nociceptive mediators under neuropathic pain conditions (Zelenka et al., 2005; Chen et al., 2016; Xu et al., 2017; Huang et al., 2019; He et al., 2020). Peripheral nerve injury activated the NF-κB in injured DRG neurons (Zelenka et al., 2005; H.H. Zhang et al., 2015; Yu et al., 2017; Huang et al., 2019; He et al., 2020). Intrathecal injection of pyrrolidine dithiocarbamate, a specific NF-κB inhibitor, alleviated the nerve injury-induced nociceptive hypersensitivity and blocked the nerve injury-induced generations of pro-inflammatory cytokines (e.g., TNF-α, IL-1β, and IL-6), brain-derived neurotrophic factor and nerve growth factor in the DRG (H.H. Zhang et al., 2015; Xu et al., 2017; Yu et al., 2017). The present work revealed that blocking the increased DRG FUS expression prevented the SNL-induced nuclear translocation of NF-κB subunit p65 and elevation of nuclear phosphorylated p-65 in injured DRG neurons and attenuated the SNL-induced nociceptive hypersensitivity. This blocking also attenuated the CCI-induced increase of DRG C-C chemokine ligand (CCL2; Du et al., 2022). DRG overexpression of FUS activated the NF-κB signaling pathway by triggering nuclear translocation of p65 and increasing the level of CCL2 (Du et al., 2022) in DRG neurons and produced enhanced responses to nociceptive stimuli in naive mice. The double-labeling assay showed the co-expression of FUS and p65 in the nuclei of individual DRG neurons. These data strongly indicate that FUS is critical for the activation of the NF-κB signaling pathway and the subsequent generation of pro-inflammatory cytokines and chemokines (e.g., CCL2) in the DRG following peripheral nerve injury. Our recent study revealed that FUS interacted with the promoter of Ccl2 mRNA and transcriptionally triggered Ccl2 mRNA expression through a nerve injury-specific long noncoding RNA-dependent mechanism in DRG neurons under neuropathic pain conditions (Du et al., 2022). NF-κB may involve in Ccl2 gene transcription activation, as FUS functioning as an RNA-binding protein may potentially bind to NF-κB, recruit it to the promoter of Ccl2 mRNA and increase CCL2 expression. This conclusion is supported by the fact that NF-κB was required for CCL2 upregulation in the DRG under neuropathic pain conditions (Kanngiesser et al., 2012; Y.P. Zhang et al., 2013). Given that CCL2 is documented as an endogenous initiator of neuropathic pain by directly sensitizing nociceptors (Jung et al., 2008), exciting DRG neurons (Sun et al., 2006; Jung et al., 2008; Van et al., 2011) and enhancing glutamate release of primary afferents (Gao et al., 2009), we conclude that the antinociception caused by blocking the increased DRG FUS following peripheral nerve injury may result from inactivation of the NF-κB and subsequent Ccl2 gene, silence of CCL2 protein expression and decrease of neuronal excitability in injured DRG. The latter may lead to a reduction in release of primary afferent transmitters/neuromodulators and consequent impairment of spinal cord central sensitization formation. In support of these conclusions, the present study showed that blocking the increased DRG FUS reduced the SNL-induced hyperactivity of dorsal horn neurons and astrocytes. It should be noted that, besides CCL2, the role of other NF-κB-controlled chemokines, cytokines, and nociceptive mediators in the antinociceptive effect of blocking the increased DRG FUS after peripheral nerve injury cannot be ruled out. In addition, the contribution of increased DRG FUS to neuropathic pain through NF-κB-independent mechanisms in injured DRG should also be considered.

In summary, the present study demonstrated that blocking the nerve injury-induced increase in DRG FUS abundance attenuated the development and maintenance of the nerve injury-induced nociceptive hypersensitivity, without altering basal or acute nociceptive responses and locomotor functions. FUS may be a potential target for therapeutic treatment of neuropathic pain. However, potential side effects should be paid an attention, because FUS is a widely expressed protein in the body.

Footnotes

  • This work was supported by National Institutes of Health Grants R01NS111553 (to Y.-X.T.) and RFNS113881 (to S.D. and Y.-X.T.).

  • The authors declare no competing financial interests.

  • Correspondence should be addressed to Yuan-Xiang Tao at yuanxiang.tao{at}njms.rutgers.edu

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FUS Contributes to Nerve Injury-Induced Nociceptive Hypersensitivity by Activating NF-κB Pathway in Primary Sensory Neurons
Guang Han, Xiang Li, Chun-Hsien Wen, Shaogen Wu, Long He, Cynthia Tan, John Nivar, Alex Bekker, Steve Davidson, Yuan-Xiang Tao
Journal of Neuroscience 15 February 2023, 43 (7) 1267-1278; DOI: 10.1523/JNEUROSCI.2082-22.2022
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