Endophilin A1 and Synaptojanin 1-Dependent Endocytosis of Synaptic Vesicles in Nociceptive Spinal Circuits Maintains Postoperative and Cancer Pain

Abstract

The continued release of neurotransmitters from central projections of nociceptors during chronic pain requires synaptic vesicle (SV) recycling. Mediators of SV endocytosis and recycling are thus pivotal for sustained pain transmission in nociceptive spinal circuits. We hypothesized that disruption of SV endocytosis in dorsal root ganglia (DRG) nociceptors would impede synaptic transmission and thereby provide sustained relief from multi-modalities of pain. Synaptojanin 1 (Synj1) and endophilin A1 (EndoA1), which mediate the neck formation of endocytic pit and subsequent endocytosis, were detected in primary sensory neurons of mouse DRG by immunofluorescence and RNAScope^® in situ hybridization. Intrathecal injection of Synj1 and EndoA1 siRNA or shRNA successfully knocked down Synj1 and Sh3gl2 (EndoA1) mRNA in DRG neurons and suppressed acute nociception induced by agonists of pronociceptive receptors and ion channels in male mice, without affecting normal motor functions. Synj1 and EndoA1 knockdown inhibited synaptic transmission between primary sensory neurons and neurons in lamina I/II of the spinal cord dorsal horn by suppressing SV release from presynaptic primary afferent neurons. Synj1 and EndoA1 silencing reversed mechanical allodynia and thermal hyperalgesia in preclinical models of postoperative and cancer pain. Knockdown of dynamin 1 (Dnm1) and adaptor-associated protein kinase 1 (AAK1), previously characterized mediators of SV endocytosis in nociceptive spinal circuits, also alleviated pain-like behavior in these models. Thus, Synj1, EndoA1, Dnm1 and AAK1 mediate SV recycling and are thus required for sustained synaptic transmission in nociceptive spinal circuits. Disruption of SV recycling effectively reduces nociceptive transmission, providing a novel strategy for pain relief.

Introduction

Chronic pain requires sustained synaptic transmission in nociceptive circuits in the dorsal horn of the spinal cord[27; 42]. Synaptic transmission depends on the balance between synaptic vesicle (SV) exocytosis, which releases neurotransmitters into the synaptic cleft, and SV endocytosis, which maintains a releasable pool of SVs in presynaptic nociceptor terminals[8; 36]. During chronic pain, this balance is disrupted, leading to enhanced transmission of pain signals[23; 35]. The molecular mechanisms of SV endocytosis and recycling in nociceptive circuits are not fully understood.

Clathrin-mediated endocytosis (CME), the most thorough mechanism of SV recycling in nerve terminals, is necessary for sustained synaptic transmission. The key components of this pathway are clathrin and adaptor protein-2 (AP-2) complex, which is required for SV budding, and dynamin (Dnm), which cleaves the budded vesicle from the plasma membrane[41]. Our previous studies revealed that Dnm1, Dnm3 and adaptor-associated protein kinase 1 (AAK1), which recruits clathrin and AP-2 to the plasma membrane enhancing SV assembly, mediate SV endocytosis in the central projections of nociceptors and thereby sustain pain transmission[37]. Inhibitors of this process deplete the releasable SV pool of neurotransmitters, disrupt synaptic transmission, and ameliorate inflammatory and neuropathic pain without affecting normal behavior[37]. Thus, knowledge of the molecular mechanisms of SV endocytosis and recycling provides a new understanding of the transmission of sustained pain and reveals possible therapeutic targets.

Several interacting proteins orchestrate endocytic membrane retrieval and SV reformation. Endophilin A (EndoA) is involved in endocytic membrane retrieval and uncoating in neurons[25]. The three endophilin proteins SH3GL2 (EndoA1), SH3GL1 (EndoA2), and SH3GL3 (EndoA3) have different patterns of expression but are all expressed in the brain, with EndoA1 being the most abundant isoform[20; 31; 32]. EndoA contains a BAR domain that senses and induces membrane curvature, and a SH3 domain that recruits the GTPase Dnm and the phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) phosphatase synaptojanin 1 (Synj1) to clathrin-coated pits[33; 38]. EndoA1, 2, 3 triple deletion impairs synaptic transmission and results in perinatal lethality. EndoA1, 2 double deletion causes severe neurological defects[25]. Along with EndoA, Synj1 is required for the neck formation of endocytic pits[11]. The SH3 domain of EndoA binds the proline-rich domain of Synj1 and recruits it to the membrane. EndoA and Synj1 work cooperatively through a positive feedback loop to promote endocytosis and subsequent uncoating of clathrin-coated vesicles[33; 38]. Besides decreasing the total SV number, Synj1 deletion results in accumulation of both clathrin-coated vesicles and pits[12; 18]. Although Synj1 and EndoA1 are known to control SV trafficking, their specific roles in synaptic transmission in nociceptive circuits and nociception are unknown. Whether Synj1 and EndoA1 are tractable targets for treatment of pain is unexplored.

This study sought to characterize previously unrecognized endocytic mediators necessary for nociception and to advance the understanding of the mechanisms and treatment of chronic pain. Anatomical, electrophysiological, and behavioral approaches show that Synj1 and EndoA1 are required for synaptic transmission of painful signals. Synj1 and EndoA1 silencing blocks synaptic transmission in nociceptive spinal circuits and blunts pain-like behavior in preclinical models of postoperative and cancer pain, without affecting normal behaviors.

Materials and Methods

Animals.

All experiments and procedures were approved by the New York University Institutional Animal Care and Use Committee and the Monash University Animal Ethics Committee. Experiments were carried out in accordance with the guidelines recommended by the National Institute of Health, the International Association for the Study of Pain, and the National Centre for the Replacement, Refinement, and Reduction of Animals in Research ARRIVE guidelines. Male C57BL/6 mice (8–10 weeks, Jackson Laboratory) were housed five per cage at 22 ± 0.5°C under a controlled 14/10 hours light/dark cycle with free access to food and water. Mice were randomly assigned to experimental groups, and the group size was based on our previous similar studies. Investigators were blind to treatments.

Collection of mouse tissue.

Mice were anesthetized (5% isoflurane) and perfused through the ascending aorta with PBS and then 4% paraformaldehyde in PBS. DRG (L4-L5) and spinal cord were removed, fixed in 4% paraformaldehyde in PBS at 4°C for 1 hour or overnight, respectively, cryoprotected in 30% sucrose (24 h, 4°C), and embedded in Optimal Cutting Temperature compound (Tissue Tek). Frozen sections (10–12 μm) were mounted onto Superfrost Plus slides (Fisher), dried (15 min) and stored (−20°C).

qRT-PCR.

RNA was isolated from snap-frozen mouse tissues using Direct-zol RNA MiniPrep kit (cat#R2051 Zymo Research). cDNA was prepared using MultiScribe Reverse Transcriptase (cat#4311235 Thermo Fisher). cDNA (50 ng) was amplified for 40 cycles by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) using Synj1 (Mn01210539_m1), Sh3gl2 (EndoA1, Mn01273606_m1), Sh3gl1 (EndoA2, Mn00490393_m1), Sh3gl3 (EndoA3, Mn00658910_m1) and GAPDH (Mn99999915_g1) primers, QuantStudio 3 Real-Time PCR System, and TaqMan Fast Advanced PCR Mastermix (cat#4444556 Thermo Fisher). All samples were analyzed at least in duplicate and normalized by GAPDH expression. The relative expression ratio per condition was calculated as described[28].

RNAScope^® in situ hybridization and immunofluorescence.

The RNAScope^® system (Advanced Cell Diagnostics) was used per manufacturer’s directions for fresh-frozen tissue except for omission of the initial on-slide fixation step. Probe hybridization and detection using the Multiplex Fluorescent Kit v2 followed the manufacturer’s directions. Probes to Mm-Sh3gl2 (#492641-C3) and Mm-Synj1 (#829951-C1) were used. Sections were incubated with TSA VividTM Fluorophore 520 (1:1500, cat#323271, Advanced Cell Diagnostics) for detection. To detect total neurons, hybridized slides were incubated with NeuroTrace^™ 500/525 Green Fluorescent Nissl Stain (1:500, cat#N21480, Invitrogen) (10 min, RT). To detect peptidergic neurons, hybridized slides were incubated with rabbit anti-calcitonin gene-related peptide (CGRP) (1:1000, cat#C8198, Sigma) overnight (4°C). Slides were washed and incubated with goat anti-rabbit Alexa Fluor^® 488 (1:1000, ThermoFisher) (1 hour, RT). To detect non-peptidergic neurons, hybridized slides were incubated with isolectin GS-IB4, Alexa Fluor^™ 488 Conjugate (1:1000, cat#I21411, Invitrogen) (10 min, RT). Slides were washed and incubated with DAPI, 4′,6-diamidino-2-phenylindole (1 μg/ml, 5 min) and mounted in ProLong^® Gold Antifade (Thermo Fisher). Sections were observed using a Leica SP8 confocal microscope with HCX PL APO 20x objective and 40x oil objective. The percentage of hybridized positive peptidergic or non-peptidergic neurons were quantified and normalized by the total number of CGRP+ or IB4+ neurons, respectively.

RNAScope^® quantification.

Sh3gl2 and Synj1 were localized by RNAScope^®. Confocal images were analyzed using Fiji ImageJ (NIH) according to ACD Bio-Techne Technical Note. Regions of interest were defined by applying a threshold with the moments setting (Min & Max) and analyzing particles with sizes ranging from 0 to infinity. Regions of interest were overlaid on the original micrograph and the number of dots per area was quantified. Results are expressed as dots/mm² tissue. A total of 3 images (20X magnification) were analyzed for each mouse (N=4 or 5 mice for control and treatment groups; 12 images were analyzed per experimental group).

Immunofluorescence.

DRG tissue sections were blocked with 2% bovine serum albumin, BSA, and 0.2% Triton X-100 in PBS for 1 hour, at RT. Sections were incubated (4°C, overnight) with polyclonal rabbit anti-Sh3gl2 (1:200; cat# NBP2–20345, Novus Biologicals), then washed and incubated with donkey anti-rabbit Alexa Fluor^® 488 (1:1000, ThermoFisher). Slides were incubated with DAPI (1 μg/ml, 5 min) and mounted (ProLong^® Gold Antifade). Sections were imaged using a Leica SP8 confocal microscope with HCX PL APO 40x oil objective.

Intrathecal administration of siRNA or shRNA to mice.

Mouse Synj1 siRNA (L-053808-00-0005), Sh3gl2 siRNA (EndoA1, L-060213-01-0005), or nontargeting control (CTR) siRNA (D-001810-10-05) were from Dharmacon (Table S1). Mouse Dnm1 (cat# TL500548), AAK1 (cat# TL508098), Synj1 (cat# HC108991), Sh3gl2 (cat# TL512981) shRNA plasmids and noneffective 29-mer scrambled shRNA cassette in pGFP-C-shLenti Vector were from OriGene (Table S1). The Synj1, EndoA1 or CTR siRNA or Dnm1, AAK1, Synj1, EndoA1 or CTR shRNA (1.25 μg) was mixed with polyethyleneimine-based transfection reagent (in vivo-jetPEI, 201–50 G; Polyplus) in an 8:1 N:P ratio (polyethyleneimine nitrogen to DNA phosphate ratio)[37]. The siRNA or shRNA in vivo jetPEI mixture was administered to unanesthetized mice by intrathecal (i.t.) injection (L4-L5, 5 μL). Synj1 and Sh3gl2 mRNA expression in DRG and spinal cord (L4-L5) were analyzed by RNAScope^® in situ hybridization 12 hours after siRNA injection or 72 hours after shRNA. Sh3gl2 protein expression in DRG was analyzed by immunofluorescence 48 hours after siRNA injection. Dnm1 and AAK1 mRNA expressions in DRG were downregulated 72 hours after shRNA injection[37].

Acute pain.

Mice were pre-treated with Synj1, EndoA1 or CTR siRNA intrathecally. After 48 hours, capsaicin (CPS, 0.1 nmol/10 μl), nerve growth factor (NGF, 50 ng/10 μl), trypsin (80 nM/10 μl) or vehicle (control) was administered by intraplantar (i.pl.) injection into the right hindpaw of mice. Mechanical allodynia and thermal hyperalgesia were assessed 1 up to 24 hours after injection.

Non-evoked pain-like behavior.

Non-evoked nociception was assessed using a behavioral spectrometer, which eliminates operator bias (Behavior Sequencer, Behavioral Instruments)[6; 37]. The spectrometer comprised a 40 cm² arena with a CCD camera mounted in the center of the ceiling and a door aperture in the front area of the arena. Mouse movement was assessed by a floor mounted vibration sensor and 32 wall mounted infrared transmitter and receiver pairs. Mice were individually placed in the center of the behavioral spectrometer and their behavior was recorded, tracked, evaluated and analyzed using a computerized video tracking system (Viewer3, BiObserve) for 20 min. Total distance traveled (track length) in the open field, average velocity of locomotion, ambulation, and locomotory activity were recorded and analyzed. Non-evoked pain-like behavior was assessed 48 hours after Synj1, EndoA1 or CTR siRNA injection into normal mice.

Incisional pain.

Postoperative pain was induced by plantar incision, as previously described[29]. Mice were anesthetized with 2% isoflurane, and 100% O₂ 1 L/min via a nose cone. After antiseptic preparation of the right hind paw with 10% povidone–iodine solution (Betadine Solution), a 5-mm longitudinal incision was made with a no. 11 blade through the skin and fascia of the plantar foot. The incision started 2 mm from the proximal edge of the heel and extended toward the toes. The skin was closed with a single mattress suture. Control mice underwent a sham procedure involving anesthesia and antiseptic preparation without an incision. Synj1, EndoA1, Dnm1, AAK1 or CTR siRNA or shRNA was injected intrathecally 24 hours before the plantar incision. Mechanical allodynia and thermal hyperalgesia were assessed on 2, 24, 48,72 and 144 hours after the incision. Paw guarding behavior was assessed 2 hours after the incision.

Cancer pain.

B16-F10 (CRL-6475; ATCC) murine melanoma cells were cultured in DMEM containing 10% FBS and 1% penicillin-streptomycin (10,000 U/100 μg/mL) at 37 °C with 5% CO₂ in a humidified atmosphere and were used without further authentication. For inoculation, B16-F10 melanoma cells (2 × 10⁵ cells) were suspended in PBS and injected (20 μL) into the plantar region of the mouse right hindpaw[14]. Control groups were injected with 20 μL of PBS. The cell line was syngeneic with the C57BL/6J mouse strain. Synj1, EndoA1, Dnm1, AAK1 or CTR siRNA or shRNA was injected intrathecally 14 days after melanoma inoculation. Mechanical allodynia and thermal hyperalgesia were assessed on 24, 48, 72, 96 and 168 hours after treatment.

Mechanical allodynia.

Mechanical allodynia was assessed by measuring hindpaw withdrawal response to von Frey filament stimulation using the up-and-down method [15]. Mice were acclimatized to the testing apparatus, which comprised individual clear Plexiglass boxes on an elevated wire mesh platform to facilitate access to the plantar surface of the hindpaws, for 1 h/d for 2 days. A series of von Frey filaments (0.02, 0.07, 0.16, 0.4, 1.0, and 2 g; Stoelting) were applied perpendicular to the plantar surface of hindpaw. The test began with an application of 0.4 g filament. A positive response was defined as a clear paw withdrawal or shaking. Whenever a positive response occurred, the next lower filament was applied, and whenever a negative response occurred, the next higher filament was applied. The testing consisted of 6 stimuli, and the pattern of response was converted to a 50% von Frey threshold[9].

Thermal hyperalgesia.

The Hargreaves apparatus was used to evaluate hypersensitivity to heat (Ugo Basile)[17]. Mice were acclimatized to the testing apparatus, which comprised individual clear Plexiglass chambers and a radiant heat source, for 1 h/d for 2 days. The infrared intensity was set at 50% and cut off time to a maximum of 30 seconds. The time between stimulus onset and paw withdrawal was measured automatically, giving an index of the thermal nociceptive threshold. Significant decreases in paw withdrawal latency were interpreted as evidence of thermal hyperalgesia. The latency, expressed in seconds, was evaluated before (basal) and at different time points after the treatment.

Guarding behavior.

Paw guarding behavior was evaluated 2 hours after plantar incision. Mice were placed in the testing apparatus, which comprised individual clear Plexiglass boxes on an elevated wire mesh platform to facilitate visibility to the plantar surface of the hindpaws. Incised hind paws were closely observed during a 1 min period. Guarding behavior was scored based on the hind paw position during the majority of the 1 min scoring period: 0 when the incised area was touching the mesh and the area was blanched or distorted by the mesh; 1 when the incised area touched the mesh without blanching or distortion; and 2 when the incised area was completely off of the mesh.

Spinal cord slice preparation.

Adult C57BL/6J mice were anesthetized (5% isoflurane), decapitated and the lumbar region of the spinal cord with the dorsal root exposed by laminectomy was removed. Parasagittal spinal cord slices with the dorsal root attached (300 μm) were sectioned on a vibratome (Leica VT 1200s) in ice cold (0–4°C) oxygenated sucrose-based ACSF that contained (mM): 100 sucrose, 63 NaCl, 2.5 KCl, 1.2 NaH₂PO₄, 1.2 MgCl₂, 25 glucose, 25 NaHCO₃ and 5 Na ascorbate. Slices were then incubated for 15 min at 34°C in NMDG-based recovery ACSF composed of (mM): 93 NMDG, 2.5 KCl, 1.2 NaH₂PO₄, 30 NaHCO₃, 20 HEPES, 25 glucose, 5 Na ascorbate, 2 thiourea, 3 Na pyruvate, 10 MgSO₄ and 0.5 CaCl₂ and adjusted to pH 7.4 with HCl. After the recovery incubation, slices were transferred to oxygenated ACSF with the following composition (mM): 125 NaCl, 2.5 KCl, 1.25 NaH₂PO₄, 1.2 MgCl₂, 2.5 CaCl₂, 25 glucose and 25 NaHCO₃ for 45 min at 36°C and then maintained at RT prior to transfer to the recording chamber. All ACSF solutions were equilibrated with 95% O₂ and 5% CO₂. Spinal cord slices were collected 48 hours after i.t. Synj1, EndoA1 or CTR siRNA.

Spinal cord electrophysiology.

Slices were transferred to the recording chamber and continuously superfused with ACSF equilibrated with 95% O₂/5% CO₂ at a rate of 2ml/min at RT. Dodt-contrast optics were used to identify dorsal horn neurons in the translucent substantia gelatinosa layer of the superficial dorsal horn. Evoked excitatory post-synaptic currents (eEPSCs) were recorded in whole-cell voltage clamp using a CsCl-based internal solution composed of (mM): 140 CsCl, 10 EGTA, 5 HEPES, 2 CaCl₂, 2 MgATP, 0.3 NaGTP, 5 QX-314.Cl and 0.1% biocytin (osmolarity 285–295 mosmol/l). Patch clamp electrodes had resistances between 3–5 MΩ and neurons were held at −65 mV (not corrected for the liquid junction potential of 4 mV). A bipolar stimulating electrode was placed in the dorsal root entry zone for electrical stimulation of evoked post-synaptic currents. For paired-pulse experiments, evoked currents were elicited by two consecutive stimuli of identical strength separated by 40 ms. Paired pulse ratio was calculated by dividing the second pulse by the first (PSC2/PSC1). The decay time constant was determined using a single exponential fit, a simplex algorithm and Chi-squared optimization in Axograph X. All eEPSCs were recorded in gabazine (10 μM) and strychnine (0.5 μM).

SV imaging.

Lumbar spinal cord slices were incubated in Mg²⁺-free ACSF that contained (mM) 125 NaCl, 2.5 KCl, 1.25 NaH₂PO₄, 25 NaHCO₃, 2.5 CaCl₂, 10 Glucose and 0.1 4-aminopyridine (4-AP) for 10 min to increase neuronal activity. Slices were then transferred to Mg²⁺-free ACSF with 4-AP containing 8 μM FM1–43 (Abcam, Australia) for 3 min, washed with ACSF for 2 min and then incubated in 1 mM ADVASEP-7 (Sigma) for 2 min, washed with ACSF for 2 min and the ADVASEP-7 incubation repeated. Slices were placed in the recording chamber and washed for a further 15 min in ACSF prior to imaging. A bipolar stimulating electrode was placed in the dorsal root entry zone of the spinal cord and stimulated for 10 seconds at 1Hz, 4–6V to facilitate the release of vesicles from the presynaptic terminals. Optical recordings were completed on an upright fluorescence microscope (BX51W1, Olympus) under 40x magnification using a Cy3/TRITC filter set CCD camera (C11440 Orca Flash 4.0, Hamamatsu). Image sequences were analyzed using Fiji NIH image software.

Experimental Design and Statistical Analysis.

The group size for each experiment was based on our previous studies[37]. We estimated a group size of 6 mice for the acute nociceptive tests and 8 mice for the postoperative and cancer pain models tests. Mice were allocated to treatment using a randomization procedure (http://www.randomizer.org/). To specifically downregulate gene expression in DRGs, i.t. injection was used for delivery of siRNA or shRNA as described previously[37]. Double-blind tests were adopted in all behavioral experiments. Additionally, to exclude the effect of locomotor impairment on the pain behavior data, locomotor function was included in this study. Data are presented as mean ± standard error of the mean (SEM). Differences were assessed using Student’s two-tailed t-test for two comparisons and 1- or 2-way ANOVA and Sídák, Tukey, Newman-Keuls or Dunnett’s post-hoc test for multiple comparisons. P<0.05 was considered significant at the 95% confidence level. Sample sizes and statistical tests are specified in figure legends.

Results

Synj1 and EndoA1 are expressed in neurons of DRG and spinal cord dorsal horn

Expression of Synj1 and EndoA isoforms in DRG and spinal cord of mice was assessed by qRT-PCR. Levels of Synj1 and Sh3gl2 (EndoA1) mRNA were higher in DRG and spinal cord than Sh3gl1 and Sh3gl3 (EndoA2 and EndoA3) mRNA (Figure 1a, b). RNAScope^® in situ hybridization was used to localize Synj1 and Sh3gl2 (EndoA1) mRNAs in DRG and spinal cord of mice. Neurons were identified by Nissl staining. Synj1 and Sh3gl2 mRNAs were detected in neurons of DRG (Figure 1c) and in neurons of superficial laminae (LI, LII and LIII) and deeper laminae of the dorsal horn of the spinal cord (Figure 1d). Synj1 was detected in 97% of small-diameter peptidergic nociceptors of mouse DRG that expressed immunoreactive CGRP and in 97% of small-diameter non-peptidergic fibers of neurons that expressed immunoreactive IB4. Sh3gl2 was detected in 86% of mouse DRG that expressed immunoreactive CGRP and 58% of neurons expressing IB4 (Figure 1e, f, g). Thus, Synj1 and EndoA1 are expressed in small peptidergic and non-peptidergic neurons, two subpopulations of primary afferent neurons that are involved in nociceptive sensory transduction[4].

Figure 1. Localization of Synj1, EndoA1 mRNA in DRG and spinal cord.

Quantification of expression of Sh3gl2 (EndoA1), Sh3gl1 (EndoA2), Sh3gl3 (EndoA3) and Synj1 mRNA in the DRG (a) and spinal cord (SC) (b) of mice determined by qRT-PCR, n=5 mice per group. RNAScope^® localization of Synj1 and Sh3gl2 (EndoA1) mRNA in DRG (c) and dorsal horn of the spinal cord (d) of mice. Arrows indicate mRNA expression within DRG and spinal cord neurons. Scale bar, 50 μm and in the detail box 20 μm. (e, f) Immunofluorescence detection of CGRP or IB4 and RNAScope^® detection of Synj1 or Sh3gl2 (EndoA1) mRNA in mouse DRG. Arrows indicate mRNA expression within CGRP or IB4 neurons. Scale bar, 20 μm. Representative images, n=4–6 mice per group. (g) Percentage of mouse DRG neurons expressing Synj1 or Sh3gl2 in CGRP+ve or IB4+ve cells. Hybridized positive neurons (%) from n=5–6 mice.

Synj1 and EndoA1 siRNA suppress CPS-, NGF- and trypsin-induced nociception

The contribution of Synj1 and EndoA1 to nociception was studied by administration (i.t. injection) of Synj1, EndoA1 or CTR siRNAs to mice. RNAScope^® revealed that Synj1 or EndoA1 siRNA reduced expression of Synj1 and Sh3gl2 mRNA in DRG neurons by 26 ± 6% and 31 ± 7%, respectively, after 12 hours compared with CTR siRNA (Figure 2a). Expression of Synj1 and Sh3gl2 mRNA in the spinal cord was unaffected (Suppl. Figure 1). The protein levels of EndoA1 were reduced by 44 ± 10%, 48 hours post treatment when evaluated by immunofluorescence (Figure 2b). Due to limitations in antibody specificity, we were unable to measure Synj1 protein levels.

Figure 2. Synj1 and EndoA1 knockdown prevent CPS, NGF and trypsin nociceptive effect.

RNAScope localization and quantification (number of dots per area) of Synj1 and Sh3gl2 (EndoA1) mRNA expression (a) and immunofluorescence detection of Sh3gl2 (b) in mouse DRG at 12 and 48 hours, respectively, after administration of Synj1, EndoA1 or control (CTR) siRNA, n= 4 mice per group. Scale bar, 50 μm. Experimental timeline (c). Effects of Synj1, EndoA1 or CTR siRNA injected intrathecally 48 hours before CPS (0.1 nmol/10 μl, i.pl., d, e), NGF (50 ng/10 μl, i.pl., f, g) or Trypsin (80 nM /10 μl, i.pl., h, i) on mechanical allodynia and thermal hyperalgesia of the ipsilateral paw in mice. Area under the curve (AUC) of time courses (j, k). n=6 mice per group. Mean±SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs. CTR siRNA. Parametric unpaired 2-tailed t-test, 1-way ANOVA, Dunnett multiple comparison test or 2-way ANOVA, Sídák multiple comparisons test.

Acute nociception was assessed after administration of agonists of transient receptor potential vanilloid 1 (TRPV1, CPS), tropomyosin receptor kinase A (TrkA, NGF) or protease-activated receptor 2 (PAR₂, trypsin) (Figure 2c). These channels and receptors mediate multiple modalities of acute and chronic pain[4; 7; 39]. Intraplanar injection of CPS (0.1 nmol/10 μl, Figure 2d, e), NGF (50 ng/10 μl, Figure 2f, g) or trypsin (80 nM /10 μl, Figure 2h, i) in mice reduced ipsilateral paw withdrawal thresholds to von Frey filaments and reduced ipsilateral paw withdrawal latency to radiant heat for at least 24 hours, consistent with mechanical allodynia and thermal hyperalgesia, respectively. Intrathecal injection of Synj1 or EndoA1 siRNA 48 hours before CPS, NGF or trypsin prevented mechanical allodynia for at least 24 hours, and partially inhibited thermal hyperalgesia, when compared to CTR siRNA. Measurement of the integrated withdrawal responses (area under the curve (AUC) of time courses) confirmed the inhibitory actions of Synj1 or EndoA1 siRNA on nociception (Figure 2j, k). None of the treatments (i.pl. CPS, NGF or trypsin, i.t. siRNAs) affected withdrawal responses of the contralateral (non-injected) paw to mechanical stimuli (Suppl. Figure 2a, b, c).

To determine whether Synj1 or EndoA1 siRNA could impair motor function and thus artefactually affect paw withdrawal responses to painful stimuli, locomotor and exploratory behaviors were measured using a behavioral spectrometer. Behavior was monitored for 20 minutes at 48 hours after administration (i.t. injection) of Synj1, EndoA1 or CTR siRNA to naïve mice (i.e., not treated with a painful stimulus). Synj1 and EndoA1 siRNAs did not affect average velocity, track length, ambulation or locomotory activity when compared with CTR siRNA (Figure 3a, b, c). Thus, Synj1 and EndoA1 downregulation in DRG neurons suppress acute nociception without discernible effects on normal motor functions or behavior.

Figure 3. Synj1 and EndoA1 knockdown do not impair locomotor activity.

Non-evoked pain-like behavior was recorded for 20 min at 48 hours after intrathecal injection of Synj1 (a), EndoA1 (b) or CTR siRNA. n=5 mice per group. (c) Representative images of the track records.

Synj1 and EndoA1 siRNA increase the threshold for dorsal root-evoked synaptic currents in spinal neurons

To investigate the mechanism by which Synj1 and EndoA1 contribute to synaptic transmission in nociceptive circuits, we recorded dorsal root-evoked synaptic currents in spinal neurons from parasagittal spinal cord slices collected from mice that had been injected (i.t.) with Synj1, EndoA1 or CTR siRNA. eEPSCs were recorded from superficial dorsal horn (lamina I-II) neurons in whole-cell voltage clamp configuration and the stimulus increased from 1 to 10V, to determine the intensity required to elicit a synaptic response. The threshold to elicit a response was lower in the CTR siRNA treated group compared to the Synj1 and EndoA1 siRNA treated groups, which had lower current amplitudes across all stimulus intensities (Figure 4a, b). In the control group, the 50% maximal eEPSC amplitude was reached at 2.99 V, which was lower than both Synj1 and Endo1 siRNA treated at 6.86 V and 3.50 V, respectively. When grouped, the data showed significant differences between the CTR siRNA group and the Synj1 and Endo1 siRNA treated animals (P=0.0173 and P=0.0176 respectively, one-way ANOVA with Tukey’s multiple comparisons test). The decay time constant was significantly faster in the Synj1 and Endo1 siRNA groups (P = <0.0001 for each compared to control, one-way ANOVA with Tukey’s multiple comparisons test), reflecting the decrease in vesicle release (Figure 4c, d). These results show that downregulating Synj1 and EndoA1 increases the threshold required to induce synaptic currents evoked by dorsal root stimulation in spinal neurons.

Figure 4. Effect of Synj1 and Endo1 knockdown on synaptic transmission in the spinal dorsal horn.

Input-output responses of electrically evoked excitatory post-synaptic currents (eEPSCs) from spinal cord slices of mice 48 hours after intrathecal injection with Synj1 (n=12), EndoA1 (n=9) and CTR siRNA (n=14). Representative traces show eEPSCs recorded in whole-cell voltage clamp in dorsal horn neurons, in response to increasing intensity of a dorsal-root electrical stimulus (1–10 V) (a, b). Traces showing overlayed eEPSCs in response to increasing intensity and mean eEPSC decay time (c, d) for the data shown in (b). Traces showing average paired-pulse responses from a representative neuron of Synj1, EndoA1 and CTR siRNA groups (e). Mean paired-pulse responses of Synj1 (n=12), EndoA1 (n=9) and CTR siRNA (n=14) groups (f). Traces showing eEPSC responses to 1 Hz presynaptic stimulation in Synj1, EndoA1 and CTR siRNA groups (g). Normalized amplitude of eEPSCs over 80 seconds (h) and the initial 10 seconds (i). Normalized FM1–43 fluorescence over 10 seconds of 1 Hz electrical stimulation of the dorsal roots of spinal cord slices from Synj1 (n=24), EndoA1 (n=25) and CTR (n=21) siRNA treated groups (j). Images of FM1–43 fluorescence showing activity at individual synapses before and following 1 Hz stimulation (k). Scale bar = 4 um. Mean±SEM. *<P=0.05, **<P=0.01, ***<P=0.001 vs CTR siRNA. Parametric unpaired 2-tailed t-test or 1-way ANOVA, Dunnett or Tukey’s multiple comparison test or 2-way ANOVA, Sídák multiple comparisons test.

Synj1 and EndoA1 siRNA suppresses SV release probability and sustained release in presynaptic afferent neurons

To determine the effect of Synj1 and EndoA1 knockdown on the probability of SV release, we measured paired-pulse ratios of eEPSCs in dorsal horn neurons by applying electrically evoked dorsal root stimulation at 40 ms intervals. Compared to controls, the Synj1 and EndoA1 siRNA significantly reduced paired-pulse ratio, suggesting that release probability from primary afferent neurons was impaired through a presynaptic mechanism (Figure 4e, f, P = 0.0026 and P = 0.0088 for Synj1 and EndoA1 siRNA compared to control, one-way ANOVA with Dunnett’s multiple comparisons test). Since a reduction in release probability is likely to affect sustained responses from repetitive stimuli, we tested changes in eEPSC amplitude while applying a 1 Hz stimulation of the dorsal roots, as we previously described[37]. Compared to neurons from mice treated with CTR siRNA, both the Synj1 and EndoA1 siRNA treated groups showed a significant reduction in current amplitude throughout the 80 seconds recording (Figure 4g, h), which developed rapidly over the first 10 seconds (Figure 4i).

To further investigate the effect of Synj1 and EndoA1 knockdown on SV recycling, we used the styryl dye FM1–34 to label newly formed SVs. When these labeled vesicles undergo exocytosis in dye-free medium, the dye molecules dissociate from the plasma membrane and lose fluorescence. Spinal cord slices were incubated in Mg²⁺-free ACSF containing 4-AP to enhance calcium-dependent neurotransmitter release. Tissues were then incubated with FM1–43 for 3 min, washed, and incubated with ADVASEP-7 to scavenge unincorporated FM1–43. Fluorescence at synaptic sites was imaged while stimulating the dorsal roots at 1 Hz, 4–6V to facilitate SV release from presynaptic terminals. A significant reduction in fluorescence at individual synapses was detected in the CTR siRNA group, compared to both Synj1 and EndoA1 siRNA groups (Figure 4j, k).

Taken together, these results show that Synj1 and EndoA1 siRNA knockdown reduces the probability of neurotransmitter release by disrupting SV recycling in presynaptic primary afferent neurons. This mechanism likely accounts for the inhibitory effects of Synj1 and EndoA1 siRNA on acute nociceptive responses mediated by TRPV1, TrkA and PAR₂. We next determined whether inhibitors of SV recycling would disrupt persistent nociception in preclinical models of postoperative and cancer pain.

Synj1 and EndoA1 siRNA prevent postoperative pain

We investigated the effects of Synj1 and EndoA1 siRNA in a preclinical mouse model of postoperative pain induced by plantar incision of the left hindpaw (Figure 5a). Synj1, EndoA1 or CTR siRNA was administered by i.t. injection 24 hours before the incision. Spontaneous nociception was assessed 2 hours after incision by scoring paw guarding behavior. Withdrawal responses of the incision (ipsilateral) and non-incision (contralateral) hindpaws to stimulation with von Frey filaments and radiant heat were assessed from 2 – 72 hours after surgery to evaluate mechanical allodynia and thermal hyperalgesia, respectively. Plantar incision caused paw guarding behavior after 2 hours (Figure 5b). Synj1 and EndoA1 siRNA reduced this effect. Plantar incision reduced both the withdrawal threshold to von Frey filaments and the withdrawal latency to heat in the ipsilateral paw for at least 3 days, consistent with mechanical allodynia and thermal hyperalgesia (Figure 5c, d). Synj1 siRNA prevented mechanical allodynia for 2, 24 and 48 hours after incision when compared to CTR siRNA (Figure 5c). Mechanical allodynia was 78 ± 13% of baseline after 24 hours. Likewise, EndoA1 siRNA reversed incision-induced mechanical allodynia 2 and 24 hours when compared to CTR siRNA (Figure 5c). Mechanical allodynia was 69 ± 11% of baseline after 2 hours. EndoA1 siRNA prevented thermal hyperalgesia after 2, 24 and 48 hours of incision, with an 81 ± 23% of baseline at 48 hours, while Synj1 siRNA inhibited thermal hyperalgesia by 48 ± 20% after 2 hours (Figure 5d). None of the treatments (i.t. siRNAs) affected withdrawal responses of the contralateral (non-incision) paw to mechanical and thermal stimuli (Suppl. Figure 3a, b). Thus, downregulation of Synj1 and EndoA1 ameliorates nociception in a preclinical model of postoperative pain.

Figure 5. Synj1 and EndoA1 knockdown inhibited postoperative and cancer pain.

Postoperative pain experimental timeline (a). Paw guarding behavior (b), mechanical allodynia (c) and thermal hyperalgesia (d) in plantar incision mice measured 2 – 72 hours after plantar incision. Synj1, EndoA1 or CTR siRNA was injected intrathecally 24 hours before incision, n=7–8 mice per group. Cancer pain experimental timeline (e). Mechanical allodynia (f) and thermal hyperalgesia (g) of B16-F10 melanoma cell inoculated in the paw, measured 14 days (time 0) after cell inoculation and 24 – 72 hours after intrathecal injection of Synj1, EndoA1 or CTR siRNA, n=8 mice per group. Mean±SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs. CTR siRNA. 1-way ANOVA, Dunnett multiple comparison test or 2-way ANOVA, Sídák multiple comparisons test.

Synj1 and EndoA1 siRNA reverse cancer pain

Melanoma cell inoculation is widely used to study cancer pain[3; 14; 34]. To investigate the effect of Synj1 and EndoA1 siRNA in a cancer pain model, B16-F10 melanoma cells were inoculated into the hindpaw of mice (Figure 5e). Melanoma cells inoculation induced mechanical allodynia and thermal hyperalgesia for at least 17 days in the ipsilateral paw (Figure 5f, g). Synj1, EndoA1 or CTR siRNA was administrated (i.t. injection) 14 days after inoculation, and mechanical allodynia and thermal hyperalgesia of the injected (ipsilateral) and non-injected (contralateral) hindpaws were evaluated daily. Synj1 and EndoA1 siRNA partially reversed cancer-evoked mechanical allodynia at 48 hours and -evoked thermal hyperalgesia for 24 and 48 hours after i.t. injection when compared with CTR siRNA (Figure 5f, g). Synj1 and EndoA1 siRNA respectively reversed mechanical allodynia to 40 ± 10% and 32 ± 4% of baseline and thermal hyperalgesia to 64 ± 8% and 58 ± 9% of baseline after 48 hours. None of the treatments (i.t. siRNAs) affected withdrawal responses of the contralateral (non-injected) paw to mechanical and thermal stimuli (Suppl. Figure 3c, d). Thus, the downregulation of Synj1 and EndoA1 ameliorates nociception in a pre-clinical model of cancer pain.

Synj1 and EndoA1 shRNA induce sustained inhibition of postoperative and cancer pain

To determine whether prolonged knockdown of Synj1 or EndoA1 would produce a greater and longer lasting antinociceptive effect in preclinical models of postoperative and cancer pain, we administered shRNA to Synj1, EndoA1 or CTR by i.t. injection in mice (Figure 6). Synj1 and EndoA1 shRNA depleted Synj1 mRNA by 22 ± 2% and EndoA1 mRNA by 37 ± 9%, respectively, in DRG neurons after 72 hours, determined by RNAScope (Suppl. Figure 4). Synj1, EndoA1 or CTR shRNA was administered by i.t. injection 24 hours before the plantar incision (Figure 6a). Synj1 and EndoA1 shRNA reduced paw guarding behavior caused by the plantar incision 2 hours after the incision (Figure 6b). Synj1 and EndoA1 shRNA caused a large and long-lasting inhibition of incision-evoked mechanical allodynia and thermal hyperalgesia for 7 days when compared with CTR shRNA (Figure 6c, d). Synj1 and EndoA1 shRNA respectively reversed mechanical allodynia to 78 ± 12% and 75 ± 12% of baseline and thermal hyperalgesia to 100 % and 96 ± 14% of baseline after 48 hours. Moreover, Synj1, EndoA1 or CTR shRNA was administered by i.t. injection 14 days after melanoma cells inoculation (Figure 6e). Synj1 and EndoA1 shRNA caused a long-lasting inhibition of cancer-evoked mechanical and thermal hyperalgesia (Figure 6f, g). Synj1 and EndoA1 shRNA respectively reversed mechanical allodynia to 65 ± 6% and 76 ± 9% of baseline and thermal hyperalgesia to 84 ± 10% and 100 % of baseline at 48 hours. Synj1 or EndoA1 shRNA did not affect withdrawal responses of the contralateral paw (Suppl. Figure 5a, b). Thus, Synj1 and EndoA1 shRNA cause sustained inhibition of nociception in preclinical models of postoperative and cancer pain.

Figure 6. Synj1 and EndoA1 shRNA inhibited postoperative and cancer pain.

Postoperative pain experimental timeline (a). Paw guarding behavior (b), mechanical allodynia (c) and thermal hyperalgesia (d) in plantar incision mice measured 2 – 144 hours after plantar incision. Synj1, EndoA1 or CTR shRNA was injected intrathecally 24 hours before incision, n=8 mice per group. Cancer pain experimental timeline (e). Mechanical allodynia (f) and thermal hyperalgesia (g) of B16-F10 melanoma cell inoculated in the paw, measured 14 days (time 0) after cell inoculation and 24 – 168 hours after intrathecal injection of Synj1, EndoA1 or CTR shRNA, n= 8 mice per group. Mean±SEM. *P<0.05, **p<0.01, ***P<0.001, ****P<0.0001 vs. CTR shRNA. 1-way ANOVA, Dunnett multiple comparison test or 2-way ANOVA, Sídák multiple comparisons test.

Dnm1 and AAK1 shRNA induce sustained inhibition of postoperative and cancer pain

Our previous studies demonstrated that sustained shRNA knockdown of Dnm or AAK1 has a larger and longer lasting antinociceptive effect in inflammatory and neuropathic pain[37]. To determine whether shRNA knockdown of Dnm or AAK1 may prolong the antinociceptive effect in postoperative and cancer pain, Dnm1, AAK1 or CTR shRNA was administered to mice (Figure 7). When injected 24 hours before the plantar incision, Dnm1 and AAK1 shRNA reduced paw guarding behavior 2 hours after the plantar incision (Figure 7a, b). Dnm1 and AAK1 shRNA caused a long-lasting (7 days) inhibition of incision-evoked mechanical allodynia and thermal hyperalgesia (Figure 7c, d). Dnm1 and AAK1 shRNA respectively reversed mechanical allodynia to 87 ± 8% and 83 ± 6% of baseline and thermal hyperalgesia to 99 ± 17% and 94 ± 20% of baseline after 48 hours. Moreover, Dnm1 and AAK1 shRNA caused a long-lasting (7 days) inhibition of cancer-evoked mechanical allodynia and thermal hyperalgesia when injected 14 days after melanoma cells inoculation (Figure 7e, f, g). Dnm1 and AAK1 shRNA respectively reversed mechanical allodynia to 76 ± 9% and 46 ± 10% of baseline at 24 hours and reversed thermal hyperalgesia to 98 ± 19% and 100 % of baseline at 72 hours. Dnm1 or AAK1 shRNA did not affect withdrawal responses of the contralateral paw (Suppl. Figure 5c, d). Thus, Dnm1 and AAK1 shRNA cause sustained inhibition of nociception in preclinical models of postoperative and cancer pain.

Figure 7. AAK1 and Dnm1 shRNA inhibited postoperative and cancer pain.

Postoperative pain experimental timeline (a). Paw guarding behavior (b), mechanical allodynia (c) and thermal hyperalgesia (d) in plantar incision mice measured 2 – 144 hours after plantar incision. AAK1, Dnm1 or CTR shRNA was injected intrathecally 24 hours before incision, n=8 mice per group. Cancer pain experimental timeline (e). Mechanical allodynia (f) and thermal hyperalgesia (g) of B16-F10 melanoma cell inoculated in the paw, measured 14 days (time 0) after cell inoculation and 24 – 168 hours after intrathecal injection of AAK1, Dnm1 or CTR shRNA, n= 8 mice per group. Mean±SEM. *P<0.05, **p<0.01, ***P<0.001, ****P<0.0001 vs. CTR shRNA. 1-way ANOVA, Dunnett multiple comparison test or 2-way ANOVA, Sídák multiple comparisons test.

Discussion

The identification of mediators of SV endocytosis in the central projections of nociceptors and the elucidation of their role in nociception provide insights into the mechanisms of pain transmission and reveal possible therapeutic targets. Previously, we found that Dnm1, Dnm3 and AAK1 mediate SV recycling in nociceptive spinal circuits and demonstrated that this process is necessary for sustained synaptic transmission and persistent inflammatory and neuropathic pain-like behavior[37]. Herein, we explore the role of Synj1 and EndoA1 in synaptic transmission in nociceptive circuits. Synj1 and EndoA1 were localized to mouse DRG and spinal cord neurons. Downregulation of Synj1 and EndoA1 in DRG neurons suppressed acute nociception induced by agonists of pronociceptive receptors and ion channels and ameliorated nociception in pre-clinical models of postoperative and cancer pain. Synj1 and EndoA1 disruption inhibited synaptic transmission between primary sensory neurons and neurons in lamina I/II of the spinal cord dorsal horn by suppressing the release of SVs from presynaptic primary afferent neurons. Downregulation of Dnm1 and AAK1 with shRNA also inhibited nociception in preclinical models of postoperative and cancer pain. However, baseline synaptic transmission and normal behavior were unaffected by these treatments.

Molecular mechanism of SV recycling in nociceptors

Synj1 and EndoA1 work cooperatively during SV recycling, promoting endocytic membrane retrieval and subsequent uncoating after vesicle fission[38; 40]. Anatomical, behavioral and electrophysiological studies of the current investigation support the conclusion that Synj1 and EndoA1 mediate synaptic transmission in nociceptive spinal circuits. Synj1 and EndoA1 were prominently expressed in primary sensory neurons of DRGs, including peptidergic and non-peptidergic nociceptors, and in spinal cord neurons. In support of these findings, recent transcriptomic analyses of the DRG, which integrate data from various prior omics studies, have revealed that Synj1 and Sh3gl2 are expressed in subsets of CGRP- and IB4-positive nociceptors in both mouse and human DRG[5].

Nociceptor sensitization is triggered by a diverse array of chemical signals that act through G protein-coupled receptors and receptor tyrosine kinase. These receptors affect the expression or sensitivity of cation channels, particularly TRPV1 channel, to evoke pain[4; 16]. Intrathecal injection of Synj1 and EndoA1 siRNA downregulated Synj1 and Sh3gl2 mRNA in DRG neurons and prevented acute nociception induced by local administration of trypsin, NGF and CPS, agonists of pronociceptive PAR₂, TrkA and TRPV1, respectively. Thus, Synj1 and EndoA1 mediate different modalities of nociception triggered by the activation of diverse receptors and ion channels. Distinct mechanisms of nociceptive transmission may explain the differences in the strength of the antinociceptive actions of Synj1 or EndoA1 siRNA on mechanical allodynia and thermal hyperalgesia; further studies are needed to explore this possibility. Synj1 or EndoA1 neuronal downregulation also inhibited mechanical and thermal nociception in mice with postoperative and cancer pain. Prolonged knockdown of Synj1 or EndoA1 through shRNA administration resulted in sustained antinociceptive effects for at least seven days in both pain models.

Electrophysiological studies revealed that Synj1 and EndoA1 knockdown suppressed eEPSCs in superficial dorsal horn neurons and depressed the paired-pulse ratio of eEPSCs, which reduced the probability of SV release from presynaptic primary afferent neurons. Imaging SV recycling with a styryl dye demonstrated that Synj1 or EndoA1 siRNA disrupts SV recycling, likely as a result of impaired SV endocytosis. Together with our anatomical and behavioral studies, these results support the conclusion that Synj1 and EndoA1 sustain synaptic transmission in presynaptic nerve terminals in nociceptive circuits.

Our recent study showed that i.t. injection of Dnm and AAK1 siRNA or shRNA in mice effectively knockdown Dnm1 and AAK1 mRNA in DRG neurons[37]. This disruption reversed mechanical and thermal hyperalgesia and normalized non-evoked behavior in preclinical models of inflammatory and neuropathic pain[37]. Here Dnm1 and AAK1 shRNA had large antinociceptive effects that were sustained for at least 7 days in models of postoperative and cancer pain. These findings suggest that the disruption of synaptic transmission in nociceptors could ameliorate different types of pain.

Therapeutic targeting SV recycling for pain treatment

Targeting endocytosis for the treatment of pain is challenging because of the widespread and multiple functions of CME beyond pain. By injecting siRNA or shRNA intrathecally, we preferentially knockdown mediators of SV recycling in DRG neurons, thereby reducing the possibility of disruption of vital cellular processes reliant on endocytosis. Remarkably, the treatments did not affect the locomotor activity or spontaneous behavior of mice, as observed with a behavioral spectrometer.

Completely disrupting synaptic transmission would be detrimental, as evidenced by the fact that Dnm1, 3 and EndoA1, 2, 3 knockout mice do not survive[25; 30]. A key advantage of the partial downregulation of endocytic proteins observed in our study is that it spares basal synaptic transmission, affecting it only under strong stimulation. Indeed, spontaneous neurotransmission remained largely unaffected by Dnm depletion, while SV retrieval was compromised at higher levels of activity[2]. Thus, partial disruption of the activity of SV endocytosis mediators may preserve basal synaptic transmission while inhibiting the excessive synaptic activity that is a feature of chronic pain.

Our study has certain limitations. siRNA or shRNA delivered intrathecally may target non-nociceptive neurons in addition to nociceptors. Although this method is widely used in preclinical studies to silence specific gene expression, future investigations should consider more targeted delivery approaches, such as the use of cell-type-specific promoters or viral vectors, to increase precision in targeting nociceptors. The finding that Synj1 and EndoA1 siRNA or shRNA effectively knockdown Synj1 and Sh3gl2 mRNA in DRG, rather than the spinal cord, does not eliminate the potential significance of endocytosis in spinal neurons, which deserves further investigation. The translational value of the cancer model used in this study is limited because pain is infrequent in patients with early-stage melanoma[26]. Nonetheless, in metastatic melanoma over 50% of patients present pain and require treatment[13; 21; 22]. The robust tumor growth and pronounced inflammatory response observed in this model may better simulate the pain conditions associated with metastatic disease[14]. In the current study, we investigated the contribution of SV endocytosis in nociceptors to pain transmission in male mice, providing new insights into the mechanisms of pain transmission. We tested whether the knockdown of endocytic mediator would prevent the development of postoperative pain and reverse pre-established cancer pain. Further studies will be required to evaluate the effectiveness of inhibiting SV endocytosis in reversing nociception in preclinical disease models in both male and female mice.

Inhibition of SV endocytosis presents a promising target mechanism to treat chronic pain. Clinical trials targeting endocytic proteins have increased in the last decade. Notably, the AAK1 inhibitor LX9211 showed promising efficacy and safety in phase II clinical trials for post-herpetic neuralgia and painful diabetic neuropathy[19; 24]. Beyond pain management, prochlorperazine, an antitumor and antipsychotic drug, enhances cancer immunotherapy by inhibiting Dnm[1; 10], further highlighting the therapeutic potential of endocytic protein inhibitors.

Endocytic proteins are key molecular components of SV endocytosis and recycling. Notably, the disruption of Dnm, AAK1, Synj1, and EndoA1 in nociceptors inhibits synaptic transmission, resulting in reduced transduction of the painful stimulus, regardless of the pain’s origin. This study characterized previously unrecognized endocytic mediators necessary for nociception, advancing our understanding of the mechanisms and treatment of chronic pain.

Data availability:

Contact the corresponding author (R. Tonello at rt2368@nyu.edu) to obtain original data.