CELF4 Modulates Neuropathic Pain Development by Regulating Pain-related Molecular Targets

CELF4 Modulates Neuropathic Pain Development by Regulating Pain-related Molecular Targets | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article CELF4 Modulates Neuropathic Pain Development by Regulating Pain-related Molecular Targets Guanxiang Kong, Rui Tan, Xianming Zeng, Hailong Zhang, Bin Zhang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8628072/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Despite significant advancements, the mechanisms underlying neuropathic pain remain poorly understood, making it challenging for effective pain management. This study investigated the role of CUGBP Elav-Like Family Member 4 (CELF4), an RNA-binding protein, in modulating pain pathways in neuropathic pain conditions. The results showed a reduction in both mRNA and protein levels of CELF4 in the spinal cord of a chronic constriction injury (CCI) mouse model, suggesting its potential role in response to nerve injury. To further explore the role of CELF4 in modulating pain pathways in vivo , an adeno-associated virus (AAV) was injected into mice to overexpress CELF4. The mice overexpressing CELF4 showed reduced hypersensitivity to mechanical and thermal stimuli as compared to controls, indicating effective mitigation of neuropathic pain symptoms. Mechanistically, the overexpression of CELF4 significantly downregulated the expressions of Transient Receptor Potential Vanilloid-1 (TRPV1), Voltage-Gated Sodium Channel Nav1.8 (Nav1.8), and Cyclooxygenase-2 (COX2), which are crucial mediators of pain signaling. In contrast, the knockdown of CELF4 rescued the TRPV1, Nav1.8, and COX2 expressions, indicating that CELF4 might act as a suppressive regulator in neuropathic pain pathways. These results suggested that CELF4 played a critical role in regulating neuropathic pain by modulating specific ion channels and inflammatory markers, thus offering potential targets for therapeutic intervention in pain management. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Neuropathic pain arises from a lesion or disease, affecting the somatosensory nervous system [ 1 , 2 ]​. Unlike nociceptive pain that results from actual or potential tissue damage, neuropathic pain is characterized by maladaptive neuronal activity, including ectopic discharges, central and peripheral sensitization, and neuroimmune interactions. Clinically, neuropathic pain is associated with several conditions, such as diabetic polyneuropathy, postherpetic neuralgia, trigeminal neuralgia, spinal cord injury, and chemotherapy-induced peripheral neuropathy​ [ 3 ]. Patients often present with spontaneous burning, shooting, or electric shock-like pain along with evoked pain, such as mechanical allodynia and hyperalgesia [ 2 ]​. The current treatment guidelines recommend first-line pharmacological interventions, such as serotonin-norepinephrine reuptake inhibitors (SNRIs), tricyclic antidepressants (TCAs), and calcium channel alpha-2-delta ligands like gabapentinoids​. The second- and third-line therapies include tramadol, opioids, and topical agents, such as capsaicin and lidocaine; however, their efficacy is often limited and varies among patients [ 4 – 6 ]​. Despite these treatment options, numerous patients with neuropathic pain experience inadequate pain relief, making it a current major clinical challenge [ 4 , 7 ]​. Understanding the underlying mechanisms of neuropathic pain is crucial for the development of targeted, mechanism-based therapies that can provide more effective pain relief and improve patient outcomes​. Gene regulation plays a critical role in the initiation and persistence of chronic pain. Gene regulation occurs at multiple levels, including the transcriptional and post-transcriptional controls of pain-related gene expression [ 8 – 10 ]. The post-transcriptional regulation involves RNA-binding proteins (RBPs) that modulate mRNA stability, splicing, transport, and translation, thus fine-tuning gene expression in response to nociceptive stimuli [ 11 – 13 ]. RBPs are a diverse protein group that can interact with RNA through specific domains, thereby regulating various aspects of RNA metabolism [ 13 ]. Emerging evidence suggests that RBPs are extensively involved in pain pathways. For instance, Methyltransferase Like 3 (METTL3) can regulate mRNA methylation and stability in the spinal cord [ 14 ], and Fragile X Mental Retardation Protein (FMRP) can modulate mRNA transport and translation in nociceptors [ 15 ]. Additionally, Human Antigen R (HuR) can prevent the association of repressive factors with Tropomyosin Receptor Kinase A (TrkA)-proximal mRNAs in sensory neurons, and its deletion increases the repression or destabilization of these transcripts [ 15 ]. The eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), an RBP, regulates protein synthesis by modulating cap-dependent translation. It also regulates the expression of neuroligin 1 and excitatory synaptic transmission in the spinal cord, thereby contributing to enhanced mechanical nociception [ 16 ]. Through these functions, RBPs play critical roles in regulating gene expression in response to physiological and pathological stimuli. In the nervous system, the dysregulation of specific RBPs has been linked to neurological disorders and chronic pain; however, the involvement of numerous RBPs in pain mechanisms remains poorly understood. The CUG-Binding Protein (CUGBP) Elav-like family (CELF) is a highly conserved group of RBPs that play critical roles in post-transcriptional regulations, such as mRNA splicing, stability, and translation [ 17 , 18 ]. Among its members, CELF4 is particularly notable for its expression in the nervous system, especially the excitatory neurons, such as the cerebral cortex and hippocampus neurons, suggesting its involvement in synaptic function and neural development [ 17 ]. It can regulate gene expression through multiple mechanisms, such as binding to the 3' untranslated regions (3' UTRs) of target mRNAs modulating their stability and translation [ 19 , 20 ]. In the nervous system, CELF4 is essential for maintaining neuronal excitability and synaptic transmission, and its dysfunction is linked to neurodevelopmental disorders [ 21 ]. It has also been implicated in modulating neuropathic pain [ 22 ]; however, its specific mechanism and impact on downstream molecular targets in the spinal cord remain unknown. Understanding the role of CELF4 in pain is crucial, as it may provide new insights into the molecular mechanisms of chronic pain and offer potential therapeutic targets for pain management. Methods Animal preparation C57/BL6 mice (n = 84), approximately 7-8 weeks old and weighing between 20-25 grams, were purchased from Shanghai Model Organisms (Shanghai, CHN). The mice were maintained at the central animal facility of Yangzhou University Medical College and housed under a regulated 12 h/12 h light/dark cycle with access to food and water ad libitum . The study procedure was approved by the Animal Care and Use Committee of Yangzhou University Medical College. The mice were acclimatized for 2–3 days before commencing behavioral assessments to reduce variability in behavioral responses as well as within and between individuals. Efforts were concentrated on reducing animal distress and minimizing the number of animals used in the study. Moreover, the researchers conducting the behavioral tests were blinded to the treatment assignments to ensure unbiased observations. Western blotting Total proteins were extracted from the spinal cord tissues using RIPA lysis buffer enriched with protease inhibitors. The protein levels were measured using the Bradford assay. Consistent quantities of protein (20-30 µg) were separated on a 10% SDS-PAGE and subsequently transferred to PVDF membranes. The membranes were then blocked using 5% non-fat milk in TBST for one hour at room temperature. The membranes were then incubated with primary antibodies against Celf4 (Invitrogen, 1:800, no. PA5-58196) and Gapdh (Invitrogen, 1:10000, no. MA5-35235) overnight at 4°C. After washing with 0.1% TBST, the membranes were incubated for 2 h at room temperature with HRP-goat anti-rabbit IgG (ABclonal, 1:10000, no. AS014). The protein bands were detected with enhanced chemiluminescence (ECL) and quantified using ImageJ software. Gapdh was employed as the internal standard for loading consistency. Immunofluorescence staining The mice's spinal cord tissues were cut and fixed in 4% paraformaldehyde for 24 hours, followed by cryoprotection in 30% sucrose at 4°C until the tissues were submerged. The samples were then embedded in an OCT compound and cut coronally into 20-µm-thick sections using a cryostat. These sections floated freely in PBS and were blocked with a blocking solution (5% normal goat serum and 0.3% Triton X-100 in PBS) for one hour at room temperature. The sections were then incubated at 4°C with primary antibodies against Celf4 (Invitrogen, 1:200, no. PA5-58196) and NeuN (Invitrogen, 1:500, no. MA5-33103) in the blocking solution. Following a rinse, the sections were incubated with fluorophore-tagged secondary antibodies, including Alexa Fluor 488 (Invitrogen, 1:10000, no. A-11008) and Alexa Fluor 647 (Invitrogen, 1:10000, no. A-31571) for one hour at room temperature. Finally, the sections were mounted on glass slides using an antifade mounting solution and visualized under a microscope. Quantitative PCR assay Total RNA was isolated from the mice's spinal cord tissues using the RNeasy Mini Kit (Qiagen, Hilden, Germany), following the manufacturer’s instructions. The RNA purity and concentration were determined using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Complementary DNA (cDNA) was synthesized with 1 µg of total RNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). Quantitative PCR (qPCR) was performed using a StepOnePlus Real-Time PCR System (Applied Biosystems) with SYBR Green PCR Master Mix (Applied Biosystems). The primers specific to CELF4 and GAPDH were used to identify the gene expression levels, which were normalized to GAPDH as the internal standard. Primer information is provided in Supplementary Table 1. Neuropathic pain models The neuropathic pain model in mice was established via the chronic constriction injury (CCI), following the established protocols [23]. Briefly, the mice were initially anesthetized using isoflurane The unilateral sciatic nerve was revealed and loosely ligated with a 7.0 silk thread at three sites with an interval of about 1 mm proximal to trifurcation of the sciatic nerve. The mice's body temperature was regulated via a heating pad throughout the surgical procedure. Post-operatively, the mice were frequently observed. To avoid any potential influence on the experimental outcomes, neither analgesics nor antibiotics were administered. Pain-related behavioral tests Assessment of mechanical sensitivity: To assess the mechanical sensitivity, each mouse was placed singly atop an elevated wire mesh grid enclosed by a Plexiglas compartment [24]. Following a 30-min acclimatization period, a series of calibrated von Frey filaments (0.07 g and 0.4 g) was administered to each hind paw (10 times), each lasting 1-2 seconds with a five-minute interval between the applications. A positive reaction was identified by a quick withdrawal of the paw. The rate of these responses was quantified as the proportion of positive reactions out of the ten trials. Evaluation of thermal hyperalgesia : Thermal sensitivity was determined using a Model 336 Analgesia Meter [25]. The mice were placed on a glass platform enclosed by a Plexiglas compartment. A concentrated beam of light was directed onto the plantar aspect of each hind paw. The withdrawal latency, which was defined as the duration before the mice swiftly retracted their paw, was recorded. This test was repeated five times for each mouse, with 5-min intervals between each session. A maximum exposure limit of 20 seconds was set to avoid tissue injury. Assessment of cold sensitivity: Cold sensitivity was evaluated by measuring the latency to withdrawal on a 0°C chilled aluminum plate [24]. The mice were placed on the chilled aluminum plate inside a Plexiglas compartment equipped with constant temperature monitoring. The time taken by the mice to react with a jump was noted. This procedure was repeated three times with 10-min intervals. A maximum duration of 20 seconds per trial was set to reduce the likelihood of tissue injury. Execution of the conditional place preference (CPP) test : The CPP test was performed using a dual-chamber setup, distinguished by unique textural and visual cues [26]. The mice underwent a 30-min acclimatization phase to familiarize them with the environment, followed by assessing their initial chamber preference for 15 minutes. Any mice showing pronounced bias were omitted from the study. The conditioning phase included alternating intrathecal administrations of saline and 0.8% lidocaine in saline, each associated with a designated chamber, and conducted over three consecutive days. The chamber preference post-conditioning was assessed after 20 hours of the final conditioning session. The duration spent in each chamber was measured over 15 minutes to calculate preference scores. Spinal cord microinjection Microinjections into the spinal cord were performed using standardized procedures. Mice were initially anesthetized with 2% isoflurane in oxygen for induction, followed by maintenance with 1.5% isoflurane. During the procedure, animals were placed on a temperature-controlled heating pad to maintain normothermia. An incision was made along the dorsal midline at the lumbar level to reveal the L5-L6 vertebrae. A partial laminectomy was then performed at these sites to access the spinal cord without disturbing the dura mater. A glass micropipette, connected to a Hamilton syringe, was used for the injections. This micropipette was precisely aligned perpendicular to the spinal cord surface at a 0.5-mm depth using a stereotaxic apparatus. Either a viral solution (1 µL, 1.79 × 10 13 viral genome per ml) or a shRNA solution was administered into the spinal cord at a rate of 0.1 µL per minute. After injection, the micropipette was maintained in position for 10 min before being carefully withdrawn to avoid pressure-related injury and solution reflux. Following the microinjection, the surgical area was cleansed with sterile saline, and the incision was secured with clips. Any mice that showed irregular locomotor behavior were removed from the study. Rotarod test Motor coordination and balance evaluations were performed utilizing the rotarod test, This test assesses the capacity of mice to sustain equilibrium on a revolving rod [27]. The mice underwent two pre-test sessions, each lasting five minutes at a steady speed of 4 rpm to familiarize them with the equipment. In the subsequent trials, the mice were placed on a rotating rod. The rotating speed incrementally increased from 4 to 40 rpm within five minutes. The duration each mouse managed to stay on the rod before falling off was noted. This was repeated three times with 15-min pause between each trial. The mean endurance time for each mouse was calculated and statistical analyses were performed to determine the effects of the experimental treatments on motor skills. Open field test Locomotion behavior in mice was assessed using open field tests [28]. Each mouse was introduced to an enclosed square area measuring 40 × 40 cm 2 , surrounded by high walls to inhibit escape. These sessions lasted for 10 minutes, with a video tracking system, recording the mice's movements. The recorded parameters included the total distance traversed, which served as an indicator of overall activity, and the duration spent in the center vs. the edges of the arena. The average distances and times spent in specific zones were derived using analytical software, which were then statistically analyzed. Statistical analyses The results are presented as mean ± standard error of the mean (SEM). While the assumption of data normality was made, this was not formally tested. The statistical analyses were conducted using either two-tailed, unpaired Student’s t -tests or one-way or two-way (ANOVA), based on the study design. When ANOVA revealed significant differences, subsequent post-hoc mean comparisons were made using the Tukey test (Sigma-Plot v.12.5; San Jose, CA, USA). A P -value of <0.05 was considered statistically significant for all tests. Results 1. Downregulation of CELF4 expression in the spinal cord after peripheral nerve injury CELF4, a known RNA-binding protein, has been extensively studied for its regulatory roles in various neurological disorders [21]. This study aimed to explore its role in the CCI model, particularly focusing on its expression levels in the spinal cord. Immunofluorescence analysis in the control mice, which did not undergo CCI surgery, showed that CELF4 was primarily localized in the spinal cord neurons (Fig. 1A). The merged images of the neuronal marker NeuN (red) and CELF4 (green) showed significant overlap, indicating widespread expression of CELF4 in normal spinal cord neurons (Fig. 1A). In order to assess the effects of peripheral nerve injury on CELF4 expression, its mRNA and protein levels were quantitatively analyzed at various post-operative days (3, 7, and 14 days) following CCI surgery. The qPCR analysis revealed a significant reduction in the mRNA expression levels of CELF4 in the spinal cord of mice with peripheral nerve injury as early as 3 days post-surgery, with further significant reductions observed by day 7. By day 14, CELF4 protein levels appeared to stabilize, showing no further decrease as compared to day 7 (Fig. 1B). Concurrently, Western blot analysis also showed that CELF4 protein levels decreased significantly on day 3 after surgery and were further downregulated on days 7 and 14 (Fig. 1C and 1D). These findings suggested that CELF4 might have a suppressive role in the pathophysiological processes triggered by neural injury, potentially affecting recovery or exacerbation of neuropathic pain states. 2. Restoration of spinal CELF4 levels attenuates hyperalgesia development after chronic constriction injury The role of CCI-induced decrease in CELF4 expression in spinal cords in the CCI-induced nociceptive hypersensitivity was further investigated. CELF4 was overexpressed by microinjecting Celf4 -expressing AAV (AAV- Celf4 ) into the spinal cord of naive mice. As a control, AAV encoding GFP (AAV-GFP) was used. After 8 weeks of the AAV injections, either CCI or sham surgery was performed (Fig. 2A). After 5 days of surgery, the mRNA and protein expression levels of CELF4 in the spinal cords of CCI mice injected with AAV-GFP decreased as compared to the sham group mice injected with AAV-GFP (Fig. 2B and 2C). Notably, this decrease was absent in the CCI mice injected with AAV- Celf4 (Fig. 2B and 2C). The AAV- Celf4 microinjection did not markedly alter the mRNA and protein expression of CELF4 in the spinal cords of sham group mice (Fig. 2B and 2C). In order to evaluate the effects of spinal cord microinjections on motor function, the post-procedure motor functions were assessed using both open field and rotarod tests. The open field test data showed no substantial alterations in the total distance traversed, suggesting that the microinjections did not negatively impact the overall locomotor activities of the mice (Fig. 2D). Similarly, rotarod performance also showed no significant decrease in the ability to maintain balance and coordination in post-injection assessments (Fig. 2E). Behavioral assessments revealed that, unlike the sham surgery, CCI induced mechanical allodynia in mice, which was depicted by a significant increase in the frequency of paw withdrawals in response to 0.07 g and 0.4 g von Frey filaments. Moreover, the AAV-GFP-injected CCI mice exhibited heat and cold hyperalgesia, as evidenced by substantial reductions in paw withdrawal latencies to thermal and cold stimuli on days 3, 5, and 7 post-operations (Fig. 3A-3D). These heightened pain sensitivities were markedly reduced in the CCI mice injected with AAV5- Celf4 (Fig. 3A-3D). Collectively, these results suggested that the reduced CELF4 expression in the spinal cord might contribute to the development of nociceptive hypersensitivity following CCI in mice. 3. Mimicking peripheral nerve injury-induced decrease in CELF4 expression in the spinal cord resulted in neuropathic pain-like symptoms Next, this study investigated whether the CCI-induced reduction in CELF4 expression in the spinal cord was adequate to cause peripheral nerve injury-induced nociceptive hypersensitivity. The effects of downregulating Celf4 expression by microinjecting Celf4 -specific shRNA ( Celf4 -shRNA) into the spinal cords on the development of hypersensitivity in male mice were investigated. A scrambled shRNA (Scr-shRNA) was used as a control (Fig. 4A). Consistent with the hypothesis, after 7 days post microinjection, the mRNA and protein expression levels of Celf4 in the spinal cords of Celf4 -shRNA-injected mice significantly increased as compared to those injected with Scr-shRNA (Fig. 4B and 4C). Furthermore, open field and rotarod tests were performed after the procedure to evaluate the influence of these microinjections on motor abilities. Open field analysis showed no significant alterations in total distance traveled, suggesting that the microinjections neither impaired general locomotor activity nor provoked anxiety-like behaviors in the mice (Fig. 4D). Similarly, rotarod evaluations indicated no significant decrease in balance and coordination following the injections (Fig. 4E) Unlike the Scr-shRNA-injected mice, those injected with Celf4 -shRNA in their spinal cords showed a significant increase in the frequencies of paw withdrawals in response to 0.07 g and 0.4 g von Frey filament stimuli (Fig. 5A and 5B) as well as pronounced reductions in the latencies of paw withdrawals to both heat and cold stimuli (Fig. 5C and 5D). These enhanced responses were observed starting 3 days after the microinjection and continued for at least 14 days (Fig. 5A-5D). Notably, 7 days following the Celf4 -shRNA microinjection, the mice exhibited evoked, stimulation-independent spontaneous pain, which was evidenced by a marked preference for the lidocaine-paired chamber (Fig. 5E). In contrast, injections with Scr-shRNA did not lead to a significant preference for either the saline-paired or the lidocaine-paired chamber (Fig. 5E), suggesting an absence of spontaneous pain. Collectively, these findings demonstrated that increased CELF4 levels in the spinal cord led to both spontaneous and evoked nociceptive hypersensitivities, symptoms that are commonly seen in neuropathic pain clinics. 4. Effects of CELF4 modulation on expression levels of pain-related markers in spinal cord Previous research has shown the protective role of CELF4 in neuropathic pain. Therefore, the current study delved into the underlying mechanisms by evaluating the effects of CELF4 modulation on the expression of key pain-associated molecular markers. Using AAV- Celf4 , CELF4 was overexpressed in CCI model, and the expression levels of several nociceptive markers, including TRPV1, P2X3, Nav1.8, Nav1.9, CGRP, and COX2, were subsequently assessed using qPCR (Fig. 6A). The results demonstrated that as compared to the control CCI group, the AAV- Celf4 group showed a notable downregulation in the expression of TRPV1, Nav1.8, and COX2. This suggested a targeted modulation of these channels and enzymes that are directly implicated in pain signaling pathways. Interestingly, the expression levels of P2X3, Nav1.9, and CGRP remained unchanged, highlighting a selective effect of CELF4 overexpression on specific nociceptive markers. In order to further elucidate the CELF4's role, shRNA was used to knock down CELF4 expression in the normal WT mice. The qPCR analysis showed a significant upregulation of TRPV1, Nav1.8, and COX2 expressions in the shRNA-treated group as compared to the WT control (Fig. 6B). This indicated a reversal of CELF4's suppressive effects on these pain-related molecules. Although P2X3 and Nav1.9 showed an increasing tendency following CELF4 knockdown, the changes were not statistically significant, suggesting a more complex interaction of CELF4 with these particular molecular targets. These results suggested the significant regulatory role of CELF4 in managing neuropathic pain by modulating specific molecular pathways. Discussion Neuropathic pain is a significant clinical challenge due to its devastating effects and difficulty in achieving effective long-term treatment [ 29 ]. Current studies often struggle to fully elucidate the underlying molecular mechanisms, thus limiting the development of targeted therapies. The current study demonstrated that CELF4 expression was significantly reduced in the spinal cord during CCI, and its overexpression via AAV could mitigate neuropathic pain symptoms by downregulating key nociceptive markers, such as TRPV1, Nav1.8, and COX2. These findings highlighted the potential role of CELF4 as a therapeutic target, offering new avenues for the treatment of neuropathic pain through molecular modulation (Fig. 7 ). Previous studies have shown that CELF4 is a critical RBP involved in synaptic function and neuronal development, emphasizing its regulatory capacity within neuronal populations [ 19 , 21 ]. Recent investigations found that CELF4 is expressed in dorsal root ganglia (DRG) and may have a potential role in the pain pathway. This study revealed a notable decrease in CELF4 expression in the spinal cord within a neuropathic pain model, suggesting alterations in central processing. Furthermore, the microinjection of AAV- Celf4 in the spinal cord significantly attenuated pain hypersensitivity, highlighting its pivotal role in modulating neuropathic pain. These findings not only corroborated CELF4's role in pain mechanisms but also highlighted its therapeutic potential for addressing neuropathic pain. CELF4 can regulate gene expression through multiple mechanisms, thus participating in a broad range of physiological processes [ 30 – 33 ]. In this study, following the AAV-mediated overexpression and knockdown of CELF4, changes were observed in the expression levels of several pain-related genes, such as TRPV1 , Nav1.8 , and COX2 , suggesting that CELF4 could regulate neuropathic pain pathways. There are certain limitations to this study. First, the precise mechanisms by which CELF4 regulated the mRNA and protein levels of these genes remain unclear. Second, the motifs that are involved in the interactions between CELF4 and its target mRNAs have not been explored in this study, leaving a significant gap in understanding how CELF4 exerted its effects at the molecular level. Our future studies will focus on addressing these unresolved questions about the precise regulatory mechanisms of CELF4, as they are crucial for fully elucidating the role of CELF4 in neuropathic pain and potentially developing targeted therapeutic strategies. In conclusion, this study revealed a crucial role of CELF4 in modulating neuropathic pain pathways in the spinal cord; its downregulation correlated with increased pain sensitivity. By enhancing CELF4 expression through AAV-mediated delivery, the expression levels of specific pain-related genes decreased, thus diminishing the development of neuropathic pain hypersensitivity. These findings enhanced the understanding of CELF4's role in neuropathic pain regulation and highlighted its potential as a target for therapeutic intervention to alleviate chronic pain conditions. Declarations Acknowledgements Not applicable. Funding The present study was supported by Jiangsu Provincial Health Commission (grant no. Z2023083). Availability of data and materials The data generated in the present study may be requested from the corresponding author. Author contributions Conceptualization: Rushan Xie, Guanxiang Kong. Methodology: Rushan Xie, Rui Tan. Formal analysis: Guanxiang Kong, Xianming Zeng. Investigation: Hailong Zhang, Guanxiang Kong. Writing-original draft preparation: Xianming Zeng, Hailong Zhang. Writing-review and editing: Guanxiang Kong, Rui Tan, Rushan Xie. Ethics approval and consent to participate The present study was approved by the Research Ethics Committee of Yangzhou University School of Medicine (approval number: YXYLL-2023-026) and the Research Ethics Committee of Shuyang Hospital of Traditional Chinese Medicine (approval number: 2024-16). Patient consent for publication Not applicable. Competing interests The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. 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Supplementary Files 1SupplementaryTable1.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 09 Feb, 2026 Reviews received at journal 09 Feb, 2026 Reviews received at journal 01 Feb, 2026 Reviewers agreed at journal 21 Jan, 2026 Reviewers agreed at journal 20 Jan, 2026 Reviewers invited by journal 20 Jan, 2026 Editor assigned by journal 20 Jan, 2026 Submission checks completed at journal 20 Jan, 2026 First submitted to journal 17 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8628072","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":578093114,"identity":"d609cf05-a69f-475a-ab48-c01f64d2761b","order_by":0,"name":"Guanxiang Kong","email":"","orcid":"","institution":"Shuyang Hospital of Traditional Chinese Medicine, Affiliated to Yangzhou University","correspondingAuthor":false,"prefix":"","firstName":"Guanxiang","middleName":"","lastName":"Kong","suffix":""},{"id":578093115,"identity":"5dc6b8dd-627a-4fc2-8b7a-64d4f4cf1999","order_by":1,"name":"Rui Tan","email":"","orcid":"","institution":"Shuyang Hospital of Traditional Chinese Medicine, Affiliated to Yangzhou University","correspondingAuthor":false,"prefix":"","firstName":"Rui","middleName":"","lastName":"Tan","suffix":""},{"id":578093116,"identity":"d0cd5b4c-1579-4e93-8e12-a6672032fc30","order_by":2,"name":"Xianming Zeng","email":"","orcid":"","institution":"Shuyang Hospital of Traditional Chinese Medicine, Affiliated to Yangzhou University","correspondingAuthor":false,"prefix":"","firstName":"Xianming","middleName":"","lastName":"Zeng","suffix":""},{"id":578093117,"identity":"ec352db2-56e5-49ec-b319-325c3f3392ca","order_by":3,"name":"Hailong Zhang","email":"","orcid":"","institution":"Shuyang Hospital of Traditional Chinese Medicine, Affiliated to Yangzhou University","correspondingAuthor":false,"prefix":"","firstName":"Hailong","middleName":"","lastName":"Zhang","suffix":""},{"id":578093118,"identity":"86a401f2-e22b-4f68-9c36-05b8c968a800","order_by":4,"name":"Bin Zhang","email":"","orcid":"","institution":"Shuyang Hospital of Traditional Chinese Medicine, Affiliated to Yangzhou University","correspondingAuthor":false,"prefix":"","firstName":"Bin","middleName":"","lastName":"Zhang","suffix":""},{"id":578093119,"identity":"11f58b7b-caa7-4707-b007-3b690cb66bc2","order_by":5,"name":"Jiasheng Yu","email":"","orcid":"","institution":"Shuyang Hospital of Traditional Chinese Medicine, Affiliated to Yangzhou University","correspondingAuthor":false,"prefix":"","firstName":"Jiasheng","middleName":"","lastName":"Yu","suffix":""},{"id":578093120,"identity":"1d91e770-7440-4f9c-b8cf-5fef63af3b64","order_by":6,"name":"Rushan Xie","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA10lEQVRIie3PsQrCMBCA4ZNAXA67niDxFQoF8XESBCeFjh1ECooOIl31LRwdWwJxqbtjfQPddBGrjoqpm0P+4aZ8XA7A5frDuDdJ9fVGyFk9K2Q0spMGGaWBd0Wjjj2/yI2dCBgEJYkC4UGneZyyCh8DI3WIpGYM+pGKOXjzhfxOWHnLih6kZg5q2wLK9xvrlhT915aDyjn4NLSRgZ+ifJJO+JiViMaUAl4SqEbIyGwdk+AMeyRzg9Zb2slEn87xGNvJLjtfopHw5svv5C387bnL5XK5PnYHWlJGjVlPKFkAAAAASUVORK5CYII=","orcid":"","institution":"Shuyang Renci Hospital","correspondingAuthor":true,"prefix":"","firstName":"Rushan","middleName":"","lastName":"Xie","suffix":""}],"badges":[],"createdAt":"2026-01-17 20:08:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8628072/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8628072/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":100855896,"identity":"46a1155a-2a0b-4609-9f5b-458c46d234f3","added_by":"auto","created_at":"2026-01-22 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06:57:22","extension":"html","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":99771,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8628072/v1/18a5b69552edc9c26d0c3575.html"},{"id":100855938,"identity":"87437e99-82b5-4222-9db9-c979d9d8a219","added_by":"auto","created_at":"2026-01-22 06:57:24","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":267288,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDownregulation of CELF4 expression in spinal cord after peripheral nerve injury. \u003c/strong\u003e(A) Immunofluorescence analysis revealed CELF4 and NeuN localization in the spinal cord. NeuN and CELF4 are colored red and green, respectively, while their colocalization is shown in yellow, illustrating CELF4's extensive presence in spinal cord neurons. Scale bars: 50 mm. (B-D) Measurements of \u003cem\u003eCELF4\u003c/em\u003e mRNA (B) and protein (C) levels in the spinal cord on days 0, 3, 7, and 14. (D) Analysis of CELF4 protein concentrations using Western blot analysis. *\u003cem\u003eP\u003c/em\u003e \u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt;0.001, ***\u003cem\u003eP\u003c/em\u003e \u0026lt;0.0001, determined with two-way ANOVA with subsequent Tukey post hoc tests.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8628072/v1/be561656fd592af60c9c4a29.png"},{"id":100855954,"identity":"9f0f468c-4bd5-4f49-956f-5f894d0c449a","added_by":"auto","created_at":"2026-01-22 06:57:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":183344,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRescuing the reduced CELF4 expression in spinal cords attenuated the development of CCI-induced hyperalgesia. \u003c/strong\u003e(A) Diagram of the experimental setup. (B-D) Concentrations of \u003cem\u003eCelf4\u003c/em\u003e mRNA (B) and CELF4 protein (C) in spinal cord tissues of sham and CCI mice after 5 days of surgery; both groups were pre-injected with either AAV-\u003cem\u003eCelf4\u003c/em\u003e or control AAV-GFP four weeks before surgery. (D) Assessment of CELF4 protein levels via Western blot analysis. ***\u003cem\u003eP \u003c/em\u003e\u0026lt;0.001, ****\u003cem\u003eP\u003c/em\u003e \u0026lt;0.0001, analyzed using two-way ANOVA and subsequent Tukey post hoc test. (E) Outcomes of the open field test following spinal cord microinjection to measure the locomotor behavior of mice. The graphs display the total distance covered (meters). Results are presented as the mean ± SEM. Statistical analysis was conducted using a repeated measures \u003cem\u003et\u003c/em\u003e-test, revealing no significant differences (\u003cem\u003eP\u003c/em\u003e \u0026gt;0.05). (F) Rotarod test results following spinal cord microinjection. The endurance time (seconds) is shown on the accelerating rotarod at predetermined intervals after microinjection. The graph shows the mean duration for which each mouse could remain on the rod; error bars represent SEM. Statistical analysis was performed using repeated measures \u003cem\u003et\u003c/em\u003e-test, demonstrating no significant decline in motor coordination and balance post-injection (\u003cem\u003eP\u003c/em\u003e \u0026gt;0.05).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8628072/v1/cab425fde2e6cd1414c09441.png"},{"id":100855886,"identity":"4f820df6-e577-4732-880c-60e2bd1cf0c8","added_by":"auto","created_at":"2026-01-22 06:57:20","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":134573,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRescuing the reduced CELF4 in spinal cords attenuated the development of CCI-induced hyperalgesia. \u003c/strong\u003e(A-D) Measurements of paw withdrawal frequency (PWF) to 0.07 g (A) and 0.4 g (B) von Frey filaments, along with paw withdrawal latency (PWL) to heat (C) and cold (D) stimuli were recorded at various intervals post-CCI or sham surgery. The data analysis involved a three-way ANOVA with repeated measures, followed by a post hoc Tukey test, **\u003cem\u003eP \u003c/em\u003e\u0026lt;0.01 compared to the AAV5-GFP plus sham group at equivalent time points.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8628072/v1/2ee417de950b06b131232a70.png"},{"id":100856006,"identity":"27cf4f69-31e3-4aaf-85bb-d03ce357ca5e","added_by":"auto","created_at":"2026-01-22 06:57:30","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":141152,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMimicking peripheral nerve injury-induced decrease in CELF4 expression in the spinal cord resulted in neuropathic pain-like symptoms. \u003c/strong\u003e(A) Experimental flow chart. (B-D) Levels of \u003cem\u003eCelf4\u003c/em\u003e mRNA (B) and CELF4 protein (C) in the spinal cord tissues 8 days after the microinjection of \u003cem\u003eCelf4\u003c/em\u003e-shRNA or control Scr-shRNA. (D) Determination of CELF4 protein levels using Western blot analysis. ****\u003cem\u003eP \u003c/em\u003e\u0026lt;0.0001, evaluated using two-way ANOVA, followed by Tukey post hoc test. (E) Locomotor activity in mice was evaluated in an open field after microinjection. The graph shows the total distance traveled, expressed as mean ± SEM. Statistical significance was assessed using repeated measures \u003cem\u003et\u003c/em\u003e-test, showing no significant differences (\u003cem\u003eP\u003c/em\u003e \u0026gt;0.05). (F) Rotarod test results following spinal cord microinjection. Endurance times on an accelerating rotarod were recorded at intervals post-microinjection after microinjection. Data, shown as mean duration ± SEM, indicate no significant decrease in motor coordination or balance (repeated measures \u003cem\u003et\u003c/em\u003e-test, \u003cem\u003eP\u003c/em\u003e \u0026gt;0.05).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8628072/v1/a881515613bd0d62c2434793.png"},{"id":100855895,"identity":"02e996f1-0f3e-4aa9-a65d-c072eb595814","added_by":"auto","created_at":"2026-01-22 06:57:20","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":156969,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMimicking peripheral nerve injury-induced decrease in CELF4 expression in the spinal cord showed neuropathic pain-like symptoms. \u003c/strong\u003e(A-D) Impact of spinal cord microinjection with \u003cem\u003eCelf4\u003c/em\u003e-shRNA or control Scr-shRNA on the frequencies of paw withdrawal (PWF) to 0.07 g (A) and 0.4 g (B) von Frey filaments and paw withdrawal latency (PWL) to thermal (C) and cold (D) stimuli at various intervals after the microinjection. **\u003cem\u003eP\u003c/em\u003e \u0026lt;0.01 compared to the Scr-shRNA group at equivalent time points. Analysis was performed using a two-way ANOVA with repeated measures, followed by a Tukey post hoc test. (E) Influence of spinal cord microinjection with \u003cem\u003eCelf4\u003c/em\u003e-shRNA or control Scr-shRNA on spontaneous pain, evaluated using the Conditioned Place Preference (CPP) paradigm. Pre: before conditioning. Post: after conditioning. **\u003cem\u003eP\u003c/em\u003e \u0026lt;0.01, analyzed using a three-way ANOVA with repeated measures and a subsequent Tukey post hoc test.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8628072/v1/b820eb905533a24db11f0aae.png"},{"id":100856007,"identity":"46c0b536-90c3-4d22-b864-cde12bdce4c3","added_by":"auto","created_at":"2026-01-22 06:57:31","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":79405,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of CELF4 modulation on expression levels of pain-related markers in the spinal cord.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) qPCR assessment of the expression levels of \u003cem\u003eTRPV1\u003c/em\u003e, \u003cem\u003eP2X3\u003c/em\u003e, \u003cem\u003eNav1.8\u003c/em\u003e, \u003cem\u003eNav1.9\u003c/em\u003e, \u003cem\u003eCGRP\u003c/em\u003e, and \u003cem\u003eCOX2\u003c/em\u003e following the overexpression of CELF4 via AAV. (B) qPCR evaluation of \u003cem\u003eTRPV1, P2X3, Nav1.8, Nav1.9, CGRP\u003c/em\u003e, and \u003cem\u003eCOX2\u003c/em\u003eexpression levels after CELF4 knockdown using shRNA in wild-type mice. The results are presented as mean ± SEM for each analyzed marker, with significant differences indicated where appropriate, **\u003cem\u003eP \u003c/em\u003e\u0026lt;0.01, determined by a two-tailed independent Student’s \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8628072/v1/a6338b72b99912989499b858.png"},{"id":100856010,"identity":"64cfa2fe-5334-4129-ab47-6de21584981b","added_by":"auto","created_at":"2026-01-22 06:57:31","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":105689,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic diagram illustrating the role of CELF4 in modulating neuropathic pain.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn a mouse model of chronic constriction injury (CCI), spinal injection of adeno-associated virus encoding CELF4 (AAV-CELF4) leads to overexpression of CELF4 in the spinal cord. This upregulation of CELF4 significantly attenuates neuropathic pain symptoms. Mechanistically, CELF4 overexpression suppresses the expression of key pain-related molecules including TRPV1, Nav1.8, and COX2 in sensory neurons, thereby reducing neuronal excitability and inflammatory responses. These findings support CELF4 as a potential therapeutic target for neuropathic pain intervention.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8628072/v1/234b5779a6de7aad22f38fbd.png"},{"id":100952829,"identity":"49419882-bcbd-459e-8cca-ccf3abef3f92","added_by":"auto","created_at":"2026-01-23 07:18:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1841352,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8628072/v1/7229469e-2cdd-4746-83f5-6610cbc45278.pdf"},{"id":100855821,"identity":"61b86e89-567a-4d77-9564-b05c983e081b","added_by":"auto","created_at":"2026-01-22 06:57:09","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":16168,"visible":true,"origin":"","legend":"","description":"","filename":"1SupplementaryTable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8628072/v1/f457a28a161be6c27855dfca.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"CELF4 Modulates Neuropathic Pain Development by Regulating Pain-related Molecular Targets","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNeuropathic pain arises from a lesion or disease, affecting the somatosensory nervous system [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]​. Unlike nociceptive pain that results from actual or potential tissue damage, neuropathic pain is characterized by maladaptive neuronal activity, including ectopic discharges, central and peripheral sensitization, and neuroimmune interactions. Clinically, neuropathic pain is associated with several conditions, such as diabetic polyneuropathy, postherpetic neuralgia, trigeminal neuralgia, spinal cord injury, and chemotherapy-induced peripheral neuropathy​ [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Patients often present with spontaneous burning, shooting, or electric shock-like pain along with evoked pain, such as mechanical allodynia and hyperalgesia [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]​. The current treatment guidelines recommend first-line pharmacological interventions, such as serotonin-norepinephrine reuptake inhibitors (SNRIs), tricyclic antidepressants (TCAs), and calcium channel alpha-2-delta ligands like gabapentinoids​. The second- and third-line therapies include tramadol, opioids, and topical agents, such as capsaicin and lidocaine; however, their efficacy is often limited and varies among patients [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]​. Despite these treatment options, numerous patients with neuropathic pain experience inadequate pain relief, making it a current major clinical challenge [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]​. Understanding the underlying mechanisms of neuropathic pain is crucial for the development of targeted, mechanism-based therapies that can provide more effective pain relief and improve patient outcomes​.\u003c/p\u003e \u003cp\u003eGene regulation plays a critical role in the initiation and persistence of chronic pain. Gene regulation occurs at multiple levels, including the transcriptional and post-transcriptional controls of pain-related gene expression [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The post-transcriptional regulation involves RNA-binding proteins (RBPs) that modulate mRNA stability, splicing, transport, and translation, thus fine-tuning gene expression in response to nociceptive stimuli [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRBPs are a diverse protein group that can interact with RNA through specific domains, thereby regulating various aspects of RNA metabolism [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Emerging evidence suggests that RBPs are extensively involved in pain pathways. For instance, Methyltransferase Like 3 (METTL3) can regulate mRNA methylation and stability in the spinal cord [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], and Fragile X Mental Retardation Protein (FMRP) can modulate mRNA transport and translation in nociceptors [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Additionally, Human Antigen R (HuR) can prevent the association of repressive factors with Tropomyosin Receptor Kinase A (TrkA)-proximal mRNAs in sensory neurons, and its deletion increases the repression or destabilization of these transcripts [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), an RBP, regulates protein synthesis by modulating cap-dependent translation. It also regulates the expression of neuroligin 1 and excitatory synaptic transmission in the spinal cord, thereby contributing to enhanced mechanical nociception [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Through these functions, RBPs play critical roles in regulating gene expression in response to physiological and pathological stimuli. In the nervous system, the dysregulation of specific RBPs has been linked to neurological disorders and chronic pain; however, the involvement of numerous RBPs in pain mechanisms remains poorly understood.\u003c/p\u003e \u003cp\u003eThe CUG-Binding Protein (CUGBP) Elav-like family (CELF) is a highly conserved group of RBPs that play critical roles in post-transcriptional regulations, such as mRNA splicing, stability, and translation [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Among its members, CELF4 is particularly notable for its expression in the nervous system, especially the excitatory neurons, such as the cerebral cortex and hippocampus neurons, suggesting its involvement in synaptic function and neural development [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. It can regulate gene expression through multiple mechanisms, such as binding to the 3' untranslated regions (3' UTRs) of target mRNAs modulating their stability and translation [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In the nervous system, CELF4 is essential for maintaining neuronal excitability and synaptic transmission, and its dysfunction is linked to neurodevelopmental disorders [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. It has also been implicated in modulating neuropathic pain [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]; however, its specific mechanism and impact on downstream molecular targets in the spinal cord remain unknown. Understanding the role of CELF4 in pain is crucial, as it may provide new insights into the molecular mechanisms of chronic pain and offer potential therapeutic targets for pain management.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eAnimal preparation\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC57/BL6 mice (n = 84), approximately 7-8 weeks old and weighing between 20-25 grams, were purchased from Shanghai Model Organisms (Shanghai, CHN). The mice were maintained at the central animal facility of Yangzhou University Medical College and housed under a regulated 12 h/12 h light/dark cycle with access to food and water \u003cem\u003ead libitum\u003c/em\u003e. The study procedure was approved by the Animal Care and Use Committee of Yangzhou University Medical College. The mice were acclimatized for 2\u0026ndash;3 days before commencing behavioral assessments to reduce variability in behavioral responses as well as within and between individuals. Efforts were concentrated on reducing animal distress and minimizing the number of animals used in the study. Moreover, the researchers conducting the behavioral tests were blinded to the treatment assignments to ensure unbiased observations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blotting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal proteins were extracted from the spinal cord tissues using RIPA lysis buffer enriched with protease inhibitors. The protein levels were measured using the Bradford assay. Consistent quantities of protein (20-30 \u0026micro;g) were separated on a 10% SDS-PAGE and subsequently transferred to PVDF membranes. The membranes were then blocked using 5% non-fat milk in TBST for one hour at room temperature. The membranes were then incubated with primary antibodies against Celf4 (Invitrogen, 1:800, no. PA5-58196) and Gapdh (Invitrogen, 1:10000, no. MA5-35235) overnight at 4\u0026deg;C. After washing with 0.1% TBST, the membranes were incubated for 2 h at room temperature with HRP-goat anti-rabbit IgG (ABclonal, 1:10000, no. AS014). The protein bands were detected with enhanced chemiluminescence (ECL) and quantified using ImageJ software. Gapdh was employed as the internal standard for loading consistency.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence staining\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mice\u0026apos;s spinal cord tissues were cut and fixed in 4% paraformaldehyde for 24 hours, followed by cryoprotection in 30% sucrose at 4\u0026deg;C until the tissues were submerged. The samples were then embedded in an OCT compound and cut coronally into 20-\u0026micro;m-thick sections using a cryostat. These sections floated freely in PBS and were blocked with a blocking solution (5% normal goat serum and 0.3% Triton X-100 in PBS) for one hour at room temperature. The sections were then incubated at 4\u0026deg;C with primary antibodies against Celf4 (Invitrogen, 1:200, no. PA5-58196) and NeuN (Invitrogen, 1:500, no. MA5-33103) in the blocking solution. Following a rinse, the sections were incubated with fluorophore-tagged secondary antibodies, including Alexa Fluor 488 (Invitrogen, 1:10000, no. A-11008) and Alexa Fluor 647 (Invitrogen, 1:10000, no. A-31571) for one hour at room temperature. Finally, the sections were mounted on glass slides using an antifade mounting solution and visualized under a microscope.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative PCR assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was isolated from the mice\u0026apos;s spinal cord tissues using the RNeasy Mini Kit (Qiagen, Hilden, Germany), following the manufacturer\u0026rsquo;s instructions. The RNA purity and concentration were determined using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Complementary DNA (cDNA) was synthesized with 1 \u0026micro;g of total RNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). Quantitative PCR (qPCR) was performed using a StepOnePlus Real-Time PCR System (Applied Biosystems) with SYBR Green PCR Master Mix (Applied Biosystems). The primers specific to \u003cem\u003eCELF4\u003c/em\u003e and \u003cem\u003eGAPDH\u003c/em\u003e were used to identify the gene expression levels, which were normalized to \u003cem\u003eGAPDH\u003c/em\u003e as the internal standard. Primer information is provided in Supplementary Table 1.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNeuropathic pain models\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe neuropathic pain model in mice was established via the chronic constriction injury (CCI), following the established protocols [23]. Briefly, the mice were initially anesthetized using isoflurane The unilateral sciatic nerve was revealed and loosely ligated with a 7.0 silk thread at three sites with an interval of about 1 mm proximal to trifurcation of the sciatic nerve. The mice\u0026apos;s body temperature was regulated via a heating pad throughout the surgical procedure. Post-operatively, the mice were frequently observed. To avoid any potential influence on the experimental outcomes, neither analgesics nor antibiotics were administered.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePain-related behavioral tests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAssessment of mechanical sensitivity:\u003c/em\u003e To assess the mechanical sensitivity, each mouse was placed singly atop an elevated wire mesh grid enclosed by a Plexiglas compartment [24]. Following a 30-min acclimatization period, a series of calibrated von Frey filaments (0.07 g and 0.4 g) was administered to each hind paw (10 times), each lasting 1-2 seconds with a five-minute interval between the applications. A positive reaction was identified by a quick withdrawal of the paw. The rate of these responses was quantified as the proportion of positive reactions out of the ten trials.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEvaluation of thermal hyperalgesia\u003c/em\u003e: Thermal sensitivity was determined using a Model 336 Analgesia Meter [25]. The mice were placed on a glass platform enclosed by a Plexiglas compartment. A concentrated beam of light was directed onto the plantar aspect of each hind paw. The withdrawal latency, which was defined as the duration before the mice swiftly retracted their paw, was recorded. This test was repeated five times for each mouse, with 5-min intervals between each session. A maximum exposure limit of 20 seconds was set to avoid tissue injury.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAssessment of cold sensitivity:\u003c/em\u003e Cold sensitivity was evaluated by measuring the latency to withdrawal on a 0\u0026deg;C chilled aluminum plate [24]. The mice were placed on the chilled aluminum plate inside a Plexiglas compartment equipped with constant temperature monitoring. The time taken by the mice to react with a jump was noted. This procedure was repeated three times with 10-min intervals. A maximum duration of 20 seconds per trial was set to reduce the likelihood of tissue injury.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eExecution of the conditional place preference (CPP) test\u003c/em\u003e: The CPP test was performed using a dual-chamber setup, distinguished by unique textural and visual cues [26]. The mice underwent a 30-min acclimatization phase to familiarize them with the environment, followed by assessing their initial chamber preference for 15 minutes. Any mice showing pronounced bias were omitted from the study. The conditioning phase included alternating intrathecal administrations of saline and 0.8% lidocaine in saline, each associated with a designated chamber, and conducted over three consecutive days. The chamber preference post-conditioning was assessed after 20 hours of the final conditioning session. The duration spent in each chamber was measured over 15 minutes to calculate preference scores.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSpinal cord microinjection\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMicroinjections into the spinal cord were performed using standardized procedures. Mice were initially anesthetized with 2% isoflurane in oxygen for induction, followed by maintenance with 1.5% isoflurane. During the procedure, animals were placed on a temperature-controlled heating pad to maintain normothermia. An incision was made along the dorsal midline at the lumbar level to reveal the L5-L6 vertebrae. A partial laminectomy was then performed at these sites to access the spinal cord without disturbing the dura mater. A glass micropipette, connected to a Hamilton syringe, was used for the injections. This micropipette was precisely aligned perpendicular to the spinal cord surface at a 0.5-mm depth using a stereotaxic apparatus. Either a viral solution (1 \u0026micro;L, 1.79 \u0026times; 10\u003csup\u003e13\u003c/sup\u003e viral genome per ml) or a shRNA solution was administered into the spinal cord at a rate of 0.1 \u0026micro;L per minute. After injection, the micropipette was maintained in position for 10 min before being carefully withdrawn to avoid pressure-related injury and solution reflux. Following the microinjection, the surgical area was cleansed with sterile saline, and the incision was secured with clips. Any mice that showed irregular locomotor behavior were removed from the study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRotarod test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMotor coordination and balance evaluations were performed utilizing the rotarod test, This test assesses the capacity of mice to sustain equilibrium on a revolving rod [27]. The mice underwent two pre-test sessions, each lasting five minutes at a steady speed of 4 rpm to familiarize them with the equipment. In the subsequent trials, the mice were placed on a rotating rod. The rotating speed incrementally increased from 4 to 40 rpm within five minutes. The duration each mouse managed to stay on the rod before falling off was noted. This was repeated three times with 15-min pause between each trial. The mean endurance time for each mouse was calculated and statistical analyses were performed to determine the effects of the experimental treatments on motor skills.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOpen field test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLocomotion behavior in mice was assessed using open field tests [28]. Each mouse was introduced to an enclosed square area measuring 40 \u0026times; 40 cm\u003csup\u003e2\u003c/sup\u003e, surrounded by high walls to inhibit escape. These sessions lasted for 10 minutes, with a video tracking system, recording the mice\u0026apos;s movements. The recorded parameters included the total distance traversed, which served as an indicator of overall activity, and the duration spent in the center \u003cem\u003evs.\u003c/em\u003e the edges of the arena. The average distances and times spent in specific zones were derived using analytical software, which were then statistically analyzed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analyses\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe results are presented as mean \u0026plusmn; standard error of the mean (SEM). While the assumption of data normality was made, this was not formally tested. The statistical analyses were conducted using either two-tailed, unpaired Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-tests or one-way or two-way (ANOVA), based on the study design. When ANOVA revealed significant differences, subsequent post-hoc mean comparisons were made using the Tukey test (Sigma-Plot v.12.5; San Jose, CA, USA). A \u003cem\u003eP\u003c/em\u003e-value of \u0026lt;0.05 was considered statistically significant for all tests.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e1.\u0026nbsp; \u0026nbsp; \u0026nbsp;Downregulation of CELF4 expression in the spinal cord after peripheral nerve injury\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCELF4, a known RNA-binding protein, has been extensively studied for its regulatory roles in various neurological disorders [21]. This study aimed to explore its role in the CCI model, particularly focusing on its expression levels in the spinal cord. Immunofluorescence analysis in the control mice, which did not undergo CCI surgery, showed that CELF4 was primarily localized in the spinal cord neurons (Fig. 1A). The merged images of the neuronal marker NeuN (red) and CELF4 (green) showed significant overlap, indicating widespread expression of CELF4 in normal spinal cord neurons (Fig. 1A). In order to assess the effects of peripheral nerve injury on CELF4 expression, its mRNA and protein levels were quantitatively analyzed at various post-operative days (3, 7, and 14 days) following CCI surgery. The qPCR analysis revealed a significant reduction in the mRNA expression levels of \u003cem\u003eCELF4\u003c/em\u003e in the spinal cord of mice with peripheral nerve injury as early as 3 days post-surgery, with further significant reductions observed by day 7. By day 14, CELF4 protein levels appeared to stabilize, showing no further decrease as compared to day 7 (Fig. 1B). Concurrently, Western blot analysis also showed that CELF4 protein levels decreased significantly on day 3 after surgery and were further downregulated on days 7 and 14 (Fig. 1C and 1D). These findings suggested that CELF4 might have a suppressive role in the pathophysiological processes triggered by neural injury, potentially affecting recovery or exacerbation of neuropathic pain states.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.\u0026nbsp; \u0026nbsp; \u0026nbsp;Restoration of spinal CELF4 levels attenuates hyperalgesia development after chronic constriction injury\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe role of CCI-induced decrease in CELF4 expression in spinal cords in the CCI-induced nociceptive hypersensitivity was further investigated. CELF4 was overexpressed by microinjecting\u003cem\u003e\u0026nbsp;Celf4\u003c/em\u003e-expressing AAV (AAV-\u003cem\u003eCelf4\u003c/em\u003e) into the spinal cord of naive mice. As a control, AAV encoding GFP (AAV-GFP) was used. After 8 weeks of the AAV injections, either CCI or sham surgery was performed (Fig. 2A). After 5 days of surgery, the mRNA and protein expression levels of CELF4 in the spinal cords of CCI mice injected with AAV-GFP decreased as compared to the sham group mice injected with AAV-GFP (Fig. 2B and 2C). Notably, this decrease was absent in the CCI mice injected with AAV-\u003cem\u003eCelf4\u003c/em\u003e (Fig. 2B and 2C). The AAV-\u003cem\u003eCelf4\u003c/em\u003e microinjection did not markedly alter the mRNA and protein expression of CELF4 in the spinal cords of sham group mice (Fig. 2B and 2C). In order to evaluate the effects of spinal cord microinjections on motor function, the post-procedure motor functions were assessed using both open field and rotarod tests. The open field test data showed no substantial alterations in the total distance traversed, suggesting that the microinjections did not negatively impact the overall locomotor activities of the mice (Fig. 2D). Similarly, rotarod performance also showed no significant decrease in the ability to maintain balance and coordination in post-injection assessments (Fig. 2E).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBehavioral assessments revealed that, unlike the sham surgery, CCI induced mechanical allodynia in mice, which was depicted by a significant increase in the frequency of paw withdrawals in response to 0.07 g and 0.4 g von Frey filaments. Moreover, the AAV-GFP-injected CCI mice exhibited heat and cold hyperalgesia, as evidenced by substantial reductions in paw withdrawal latencies to thermal and cold stimuli on days 3, 5, and 7 post-operations (Fig. 3A-3D). These heightened pain sensitivities were markedly reduced in the CCI mice injected with AAV5-\u003cem\u003eCelf4\u003c/em\u003e (Fig. 3A-3D). Collectively, these results suggested that the reduced CELF4 expression in the spinal cord might contribute to the development of nociceptive hypersensitivity following CCI in mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.\u0026nbsp; \u0026nbsp; \u0026nbsp;Mimicking peripheral nerve injury-induced decrease in CELF4 expression in the spinal cord resulted in neuropathic pain-like symptoms\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNext, this study investigated whether the CCI-induced reduction in CELF4 expression in the spinal cord was adequate to cause peripheral nerve injury-induced nociceptive hypersensitivity. The effects of downregulating \u003cem\u003eCelf4\u003c/em\u003e expression by microinjecting \u003cem\u003eCelf4\u003c/em\u003e-specific shRNA (\u003cem\u003eCelf4\u003c/em\u003e-shRNA) into the spinal cords on the development of hypersensitivity in male mice were investigated. A scrambled shRNA (Scr-shRNA) was used as a control (Fig. 4A). Consistent with the hypothesis, after 7 days post microinjection, the mRNA and protein expression levels of Celf4 in the spinal cords of \u003cem\u003eCelf4\u003c/em\u003e-shRNA-injected mice significantly increased as compared to those injected with Scr-shRNA (Fig. 4B and 4C). Furthermore, open field and rotarod tests were performed after the procedure to evaluate the influence of these microinjections on motor abilities. Open field analysis showed no significant alterations in total distance traveled, suggesting that the microinjections neither impaired general locomotor activity nor provoked anxiety-like behaviors in the mice (Fig. 4D). Similarly, rotarod evaluations indicated no significant decrease in balance and coordination following the injections (Fig. 4E)\u003c/p\u003e\n\u003cp\u003eUnlike the Scr-shRNA-injected mice, those injected with \u003cem\u003eCelf4\u003c/em\u003e-shRNA in their spinal cords showed a significant increase in the frequencies of paw withdrawals in response to 0.07 g and 0.4 g von Frey filament stimuli (Fig. 5A and 5B) as well as pronounced reductions in the latencies of paw withdrawals to both heat and cold stimuli (Fig. 5C and 5D). These enhanced responses were observed starting 3 days after the microinjection and continued for at least 14 days (Fig. 5A-5D). Notably, 7 days following the \u003cem\u003eCelf4\u003c/em\u003e-shRNA microinjection, the mice exhibited evoked, stimulation-independent spontaneous pain, which was evidenced by a marked preference for the lidocaine-paired chamber (Fig. 5E). In contrast, injections with Scr-shRNA did not lead to a significant preference for either the saline-paired or the lidocaine-paired chamber (Fig. 5E), suggesting an absence of spontaneous pain. Collectively, these findings demonstrated that increased CELF4 levels in the spinal cord led to both spontaneous and evoked nociceptive hypersensitivities, symptoms that are commonly seen in neuropathic pain clinics.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.\u0026nbsp; \u0026nbsp; \u0026nbsp;Effects of CELF4 modulation on expression levels of pain-related markers in spinal cord\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrevious research has shown the protective role of CELF4 in neuropathic pain. Therefore, the current study delved into the underlying mechanisms by evaluating the effects of CELF4 modulation on the expression of key pain-associated molecular markers. Using AAV-\u003cem\u003eCelf4\u003c/em\u003e, CELF4 was overexpressed in CCI model, and the expression levels of several nociceptive markers, including TRPV1, P2X3, Nav1.8, Nav1.9, CGRP, and COX2, were subsequently assessed using qPCR (Fig. 6A). The results demonstrated that as compared to the control CCI group, the AAV-\u003cem\u003eCelf4\u003c/em\u003e group showed a notable downregulation in the expression of TRPV1, Nav1.8, and COX2. This suggested a targeted modulation of these channels and enzymes that are directly implicated in pain signaling pathways. Interestingly, the expression levels of P2X3, Nav1.9, and CGRP remained unchanged, highlighting a selective effect of CELF4 overexpression on specific nociceptive markers.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn order to further elucidate the CELF4's role, shRNA was used to knock down CELF4 expression in the normal WT mice. The qPCR analysis showed a significant upregulation of TRPV1, Nav1.8, and COX2 expressions in the shRNA-treated group as compared to the WT control (Fig. 6B). This indicated a reversal of CELF4's suppressive effects on these pain-related molecules. Although P2X3 and Nav1.9 showed an increasing tendency following CELF4 knockdown, the changes were not statistically significant, suggesting a more complex interaction of CELF4 with these particular molecular targets. These results suggested the significant regulatory role of CELF4 in managing neuropathic pain by modulating specific molecular pathways.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eNeuropathic pain is a significant clinical challenge due to its devastating effects and difficulty in achieving effective long-term treatment [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Current studies often struggle to fully elucidate the underlying molecular mechanisms, thus limiting the development of targeted therapies. The current study demonstrated that CELF4 expression was significantly reduced in the spinal cord during CCI, and its overexpression via AAV could mitigate neuropathic pain symptoms by downregulating key nociceptive markers, such as TRPV1, Nav1.8, and COX2. These findings highlighted the potential role of CELF4 as a therapeutic target, offering new avenues for the treatment of neuropathic pain through molecular modulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePrevious studies have shown that CELF4 is a critical RBP involved in synaptic function and neuronal development, emphasizing its regulatory capacity within neuronal populations [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Recent investigations found that CELF4 is expressed in dorsal root ganglia (DRG) and may have a potential role in the pain pathway. This study revealed a notable decrease in CELF4 expression in the spinal cord within a neuropathic pain model, suggesting alterations in central processing. Furthermore, the microinjection of AAV-\u003cem\u003eCelf4\u003c/em\u003e in the spinal cord significantly attenuated pain hypersensitivity, highlighting its pivotal role in modulating neuropathic pain. These findings not only corroborated CELF4's role in pain mechanisms but also highlighted its therapeutic potential for addressing neuropathic pain.\u003c/p\u003e \u003cp\u003eCELF4 can regulate gene expression through multiple mechanisms, thus participating in a broad range of physiological processes [\u003cspan additionalcitationids=\"CR31 CR32\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In this study, following the AAV-mediated overexpression and knockdown of CELF4, changes were observed in the expression levels of several pain-related genes, such as \u003cem\u003eTRPV1\u003c/em\u003e, \u003cem\u003eNav1.8\u003c/em\u003e, and \u003cem\u003eCOX2\u003c/em\u003e, suggesting that CELF4 could regulate neuropathic pain pathways.\u003c/p\u003e \u003cp\u003eThere are certain limitations to this study. First, the precise mechanisms by which CELF4 regulated the mRNA and protein levels of these genes remain unclear. Second, the motifs that are involved in the interactions between CELF4 and its target mRNAs have not been explored in this study, leaving a significant gap in understanding how CELF4 exerted its effects at the molecular level. Our future studies will focus on addressing these unresolved questions about the precise regulatory mechanisms of CELF4, as they are crucial for fully elucidating the role of CELF4 in neuropathic pain and potentially developing targeted therapeutic strategies.\u003c/p\u003e \u003cp\u003eIn conclusion, this study revealed a crucial role of CELF4 in modulating neuropathic pain pathways in the spinal cord; its downregulation correlated with increased pain sensitivity. By enhancing CELF4 expression through AAV-mediated delivery, the expression levels of specific pain-related genes decreased, thus diminishing the development of neuropathic pain hypersensitivity. These findings enhanced the understanding of CELF4's role in neuropathic pain regulation and highlighted its potential as a target for therapeutic intervention to alleviate chronic pain conditions.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThe present study was supported by Jiangsu Provincial Health Commission (grant no. Z2023083).\u003c/p\u003e\n\u003cp\u003eAvailability of data and materials\u003c/p\u003e\n\u003cp\u003eThe data generated in the present study may be requested from the corresponding author.\u003c/p\u003e\n\u003cp\u003eAuthor contributions\u003c/p\u003e\n\u003cp\u003eConceptualization: Rushan Xie, Guanxiang Kong. Methodology: Rushan Xie, Rui Tan. Formal analysis: Guanxiang Kong, Xianming Zeng. Investigation: Hailong Zhang, Guanxiang Kong. Writing-original draft preparation: Xianming Zeng, Hailong Zhang. Writing-review and editing: Guanxiang Kong, Rui Tan, Rushan Xie.\u003c/p\u003e\n\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eThe present study was approved by the Research Ethics Committee of Yangzhou University School of Medicine (approval number: YXYLL-2023-026) and the Research Ethics Committee of Shuyang Hospital of Traditional Chinese Medicine (approval number: 2024-16).\u003c/p\u003e\n\u003cp\u003ePatient consent for publication\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFinnerup NB, Kuner R and Jensen TS: Neuropathic Pain: From Mechanisms to Treatment. 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Biochem Biophys Res Commun 605: 39-44, 2022.\u003c/li\u003e\n\u003cli\u003eAlors-P\u0026eacute;rez E, Pedraza-Arevalo S, Bl\u0026aacute;zquez-Encinas R, et al.: Altered CELF4 splicing factor enhances pancreatic neuroendocrine tumors aggressiveness influencing mTOR and everolimus response. Mol Ther Nucleic Acids 35: 102090, 2024.\u003c/li\u003e\n\u003cli\u003eRagab SM, El-Hawy MA, El-Hefnawy SM, El-Deeb HMA, Elfalah AS and Mahmoud AA: CELF4 (rs1786814) gene polymorphism and speckle-tracking Echocardiography for cardiovascular complications in childhood cancer survivors. Pediatr Res2024.\u003c/li\u003e\n\u003cli\u003eSingh G, Charlet BN, Han J and Cooper TA: ETR-3 and CELF4 protein domains required for RNA binding and splicing activity in vivo. Nucleic Acids Res 32: 1232-1241, 2004.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-molecular-neuroscience","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jomn","sideBox":"Learn more about [Journal of Molecular Neuroscience](https://www.springer.com/journal/12031)","snPcode":"12031","submissionUrl":"https://submission.nature.com/new-submission/12031/3","title":"Journal of Molecular Neuroscience","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8628072/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8628072/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDespite significant advancements, the mechanisms underlying neuropathic pain remain poorly understood, making it challenging for effective pain management. This study investigated the role of CUGBP Elav-Like Family Member 4 (CELF4), an RNA-binding protein, in modulating pain pathways in neuropathic pain conditions. The results showed a reduction in both mRNA and protein levels of CELF4 in the spinal cord of a chronic constriction injury (CCI) mouse model, suggesting its potential role in response to nerve injury. To further explore the role of CELF4 in modulating pain pathways \u003cem\u003ein vivo\u003c/em\u003e, an adeno-associated virus (AAV) was injected into mice to overexpress CELF4. The mice overexpressing CELF4 showed reduced hypersensitivity to mechanical and thermal stimuli as compared to controls, indicating effective mitigation of neuropathic pain symptoms. Mechanistically, the overexpression of CELF4 significantly downregulated the expressions of Transient Receptor Potential Vanilloid-1 (TRPV1), Voltage-Gated Sodium Channel Nav1.8 (Nav1.8), and Cyclooxygenase-2 (COX2), which are crucial mediators of pain signaling. In contrast, the knockdown of CELF4 rescued the TRPV1, Nav1.8, and COX2 expressions, indicating that CELF4 might act as a suppressive regulator in neuropathic pain pathways. These results suggested that CELF4 played a critical role in regulating neuropathic pain by modulating specific ion channels and inflammatory markers, thus offering potential targets for therapeutic intervention in pain management.\u003c/p\u003e","manuscriptTitle":"CELF4 Modulates Neuropathic Pain Development by Regulating Pain-related Molecular Targets","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-22 06:55:22","doi":"10.21203/rs.3.rs-8628072/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-10T03:22:49+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-09T20:02:30+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-01T14:42:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"296758241482294873154642615599098529313","date":"2026-01-21T11:38:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"103112974646088287764633300361380891907","date":"2026-01-20T16:50:27+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-20T11:21:11+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-20T11:10:31+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-20T11:09:35+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Molecular Neuroscience","date":"2026-01-17T19:50:10+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-molecular-neuroscience","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jomn","sideBox":"Learn more about [Journal of Molecular Neuroscience](https://www.springer.com/journal/12031)","snPcode":"12031","submissionUrl":"https://submission.nature.com/new-submission/12031/3","title":"Journal of Molecular Neuroscience","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"6a1a01d1-dcd5-4cc6-ace9-57f74959cd77","owner":[],"postedDate":"January 22nd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-05T12:43:26+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-22 06:55:22","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8628072","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8628072","identity":"rs-8628072","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}