Intranasal CRMP2-Ubc9 Inhibitor Regulates Na_V1.7 to Alleviate Trigeminal Neuropathic Pain

1. Introduction

The trigeminal ganglion (TG) innervates craniofacial areas via the ophthalmic, maxillary, and mandibular nerves [83] and plays a crucial role in craniofacial nociception [3]. Post-traumatic trigeminal pain (PTNP) is a severe unilateral pain with limited long-term improvement [65; 72] caused by nerve damage, dental procedures or craniofacial/oral traumas [31; 34; 40; 50; 52; 62; 66; 67; 73; 77]. Unfortunately, PTNP management drugs [29; 43; 84] have central side effects due to their broad molecular targets. Understanding the molecular components contributing to PTNP pathophysiology is crucial for developing more effective, targeted drugs.

Craniofacial noxious stimulation activates intracellular signaling cascades, altering membrane ionic channel activity [38; 75; 76], leading to increased peripheral nerve excitability. For instance, dysregulation of N-type voltage-gated calcium (Ca_V2.2) channels has been linked to cephalic pain [23; 70]. Preclinical evidence suggests that inhibiting their interaction with collapsin response-mediated protein 2 (CRMP2) can reduce excitatory neurotransmitter release [64] and decrease periorbital allodynia [10; 55]. The T-type voltage-gated calcium (Ca_V3.2) channels are also important mediators of trigeminal neuropathic pain [30; 61]. Patients with trigeminal neuralgia exhibit point mutations in the Ca_V3.2 channel gene [19] leading to altered funtion of the channel [30; 61].Voltage-gated sodium channels (VGSCs) also play a critical role in trigeminal pain transduction [85; 86]. In rats with temporomandibular joint inflammation, Na_V1.7 mRNA and protein levels increase in the TG [85], correlating with enhanced sodium currents in TG neurons [86]. However, no link between CRMP2 and Na_V1.7 in the trigeminal system has been established.

We previously demonstrated CRMP2 regulation of Na_V1.7 in dorsal root ganglion (DRG) neurons [14; 15; 20; 22; 26], with pain increasing CRMP2 expression [53; 54]. SUMOylation of CRMP2 enhances functional expression of Na_V1.7 [20; 22], promoting DRG excitability [53; 54]. We have developed a small molecule called 194 that targets the CRMP2-Na_V1.7 interaction [21; 27], and inhibits CRMP2 SUMOylation. By reducing trafficking and hyperactivity of Na_V1.7 in DRG neurons across multiple species, including humans, 194 provides a non-opioid alternative for neuropathic pain treatment [9; 13; 44]. Our preclinical studies have shown that 194 decreases spinal nociceptive transmission, normalizing the response to mechanical stimulation in acute and neuropathic pain models in rodents [9; 13; 44].

Evidence points to the potential of investigating the CRMP2-Na_V1.7 interaction in TG neurons to address trigeminal pain treatment. Based on shared characteristics between DRG and TG, our study explored if regulation of Na_V1.7 by CRMP2 also occurs in TG neurons and if 194 can disrupt this regulation to alleviate PTNP. Our work revealed the presence and interaction of both CRMP2 and Na_V1.7 in TGs and showed that exposing TG neurons to 194 decreased (i) difussion and surface expression of Na_V1.7 channels, (ii) density of Na_V1.7 currents, and (ii) TG neuron exctibaility. Lastly, we observed that intranasal administration of compound 194 reduced chronic constriction injury of the infraorbital nerve (ION-CCI)-induced nociceptive behaviors, a model of chronic trigeminal neuropathic pain. These findings highlight the importance of the Na_V1.7/CRMP2 interaction in TG sensory neurons and demonstrate the efficacy of 194 in inhibiting established neuropathic trigeminal pain.

2. Materials and methods

2.1. Animals

Pathogen-free adult male and female Sprague-Dawley rats (150–200 g; Charles River) were housed in temperature-controlled (23 ± 3 ⁰C) and light-controlled (12-h light/12-h dark cycle; lights on 7:00–19:00) rooms with standard rodent chow and water available ad libitum. The Institutional Animal Care and Use Committee of New York University approved all experiments. All procedures were conducted in accordance with the Guide for Care and Use of Laboratory Animals published by the National Institutes of Health.

For the surgical procedures and behavioral experiments: adult (56 days old, 362.55 ± 16.06 g, Charles River Laboratories) male and female Sprague Dawley rats were used. Rats were pair-housed in animal care system IVC M.I.C.E. cages with access to food and hyper-chlorinated reverse osmosis water ad libitum. Facility was maintained on a 12:12 light: dark cycle at 23 ± 3 ⁰C (humidity between 30–50%). Two wooden blocks and an enviro-pack was provided per cage for environmental enrichment. Experimental procedures were approved by the Rutgers University Institutional Animal Care and Use Committee protocol NO. 999901099. Animal experiments were performed in accordance with the International Association for the Study of Pain guidelines for research involving animals.

2.2. Chemicals

All chemicals, unless noted, were purchased from Sigma (St Louis, MO). 194 (benzoylated 2-(4-piperidinyl)-1,3-benzimidazole analog; molecular weight 567.6) was obtained from TCG Lifesciences (Kolkata, India) and >99% purity was confirmed using HPLC and resuspended in DMSO for in vitro use. The synthesis of 194 has been previously reported by us [13].

2.3. Isolation, culture, and transfection of rat trigeminal ganglion (TG) neurons

Female Sprague-Dawley rats were euthanized through an isoflurane overdose and decapitated. Trigeminal ganglia were identified, collected, enzymatically digested in DMEM F12 media (cat. no. 11765, GIBCO) with dispase type II (2 mg mL −1, MB, cat. no. 165859) and collagenase type II (3 mg mL −1, cat. no. 4176, Worthington) for ~40 min at 37 ⁰C under gentle agitation and centrifuged at 800 × g for 7 minutes. The supernatant was discarded, and the pellet was resuspended in complete TG media (DMEM F12 media containing 1% penicillin/streptomycin sulfate (10,000 μL⁻¹, stock) and 10% fetal bovine serum (Hyclone)).

For the corresponding set of experiments, trigeminal ganglion neurons (TGs) were transfected with EGFP and Invitrogen Stealth RNAi^™ siRNA Negative Control (scramble siRNA) (cat. no. 12935300, Thermo Fisher Scientific) or CRMP2 siRNA (5′ GTAAACTCCTTCCTCGTGT-3′; obtained from Thermo Fisher Scientific) using the 4D-Nucleofector (P3 Primary Cell Solution, program DR 114; Lonza Biosciences). We have previously validated efficiency of knockdown of CRMP2 protein by CRMP2 siRNA in a variety of neurons [12; 16; 57; 81]. For the total internal reflection (TIR) microscopy, TG trigeminal ganglion neurons were transfected with 3 μg of Na_V1.7-PEPCy3 plasmid and 0.75 μg green fluorescent protein (GFP) using the rat neuron 4D-Nucleofector solution (P3 Primary Cell Solution, program DR 114; Lonza Biosciences). Neurons were plated on round bottom glass dishes (cat. no. P35G-1.5–10-C, Mattek) and maintained at 37 °C and 5% CO₂ in complete DMEM F12 media. Successfully transfected cells (transfection rate was ~10–15% in these conditions) were identified by GFP fluorescence.

2.4. In situ hybridization

TG tissue was extracted fresh and flash frozen on dry ice before being embedded in optimal cutting temperature gel (Cat. No. 23–730-571, Fisher Scientific). Tissue was sectioned at 20 μm on a cryostat at −20°C and mounted directly onto SuperFrost Plus microscope slides (cat. no22–037-246, Fisher Scientific). Slides were air dried at room temperature for 24 hours before undergoing RNAScope in situ hybridization. Slides were immersed in distilled water for 5 minutes to remove OCT before immersion in chilled 10% neutral buffered formalin (STL286001, Fisher Scientific) for 15 minutes.

Slides then underwent dehydration in a series ethanol baths at 50% (5 min), 70% (5 min), and finally 100% (10 min) ethanol [32]. Slides were air dried, and a hydrophobic barrier was drawn around the tissue sections. RNAScope was carried out according to ACD instructions. Briefly, tissue was digested using Protease IV for 3 minutes at room temperature. Slides were washed in RNAScope wash buffer for 4 minutes before being incubated in RNAScope probes for 2 hours at 40C in HybEZ oven. Probes for Rbfox3 (ACD Bio. 436351), Scn9a (ACD Bio. 317851), Calca (ACD Bio. 317511), Nefh (ACD Bio. 474241), Dpysl2 (ACD Bio. 1235621), and Ube2i (ACD Bio. 1253901) were purchased from ACD Bio. Slides then underwent a series of amplification via incubation in AMP1 (30 min), AMP2 (30 min), and AMP3 (15 min) at 40°C, with 4-minute wash buffer baths at room temperature between each step. Each channel was developed for 15 minutes at 40°C (HRP C1, C2 or C3) and underwent a 4-minute bath in wash buffer at room temperature before incubation with a tyrosine signal amplification (TSA) based fluorophore for 30 minutes at 40°C. After a 4-minute wash buffer bath, HRP blocker was applied for 15 minutes at 40°C before developing the next channel. Slides were incubated with DAPI for 30 seconds before coverslipping with EverBrite (Biotium, 23003) hardset mounting medium and drying overnight at room temperature [32].

Stitched images encompassing the TG were collected on a Leica DMI8 microscope (Wetzlar, Germany) using a 40x objective and analyzed using QuPath software v0.4.3.

Only neurons were counted, defined by a DAPI-labeled nucleus surrounded by Rbfox3 signal. Sub analysis quantified neurons expressing Scn9a, Calca, Nefh, Dpysl2, or Ube2i. Neurons were considered positive for a marker if they expressed greater than 10 fluorescent puncta. Each individual data point represents the mean of 4–6 sections from one animal.

2.5. Immunofluorescence, confocal microscopy, and quantification of Na_V1.7

Female rat TG neurons in culture were incubated overnight with either DMSO (as control) or 5 μM of 194. Immunofluorescence was performed as described previously [20; 28; 53; 59]. Briefly, cells were fixed for 5 min using ice-cold methanol and allowed to dry at room temperature. Cells were rehydrated in phosphate buffered saline (PBS) and incubated overnight with anti-Na_V1.7 antibody (1/200; Cat #: MABN41, Sigma-Aldrich) in PBS with 3% bovine serum albumin at 4°C. Cells were then washed 3 times in PBS and incubated with PBS containing 3% bovine serum albumin and secondary antibody Alexa 594 goat anti-mouse (Thermo Fisher Scientific, Waltham, MA) for 1 hour at room temperature. After washing with PBS, cells were stained with 49,6-diamidino-2-phenylindole (DAPI, 50 mg/mL) and mounted in ProLong Diamond Antifade Mountant (Cat #: P36961, Life Technologies Corporation). Immunofluorescent micrographs were acquired using a Plan-Apochromat 63x/1.4 oil CS2 objective on a Leica SP8 confocal microscope operated by the LAS X microscope software (Leica). Camera gain and other relevant settings were kept constant. Membrane immunoreactivity was calculated by measuring the signal intensity in the area contiguous to the boundary of the cell. The membrane to cytosol ratio was determined by defining regions of interest in the cytosol and on the membrane of each cell using Image J. Total fluorescence was normalized to the area analyzed and before calculating the ratios. The experimenter was blinded to the experimental conditions. Two individual cultures from 4 rats were used for this experiment, and at least three coverslips per cultures were analyzed.

2.6. Total internal reflection (TIR) microscopy of TG neurons

2.6.1. Fusion of Na_V1.7 to a Cy3 binding polypeptide PEPCy

Genetically encoded photostable dyes are a key requirement to observe single Na_V1.7 dynamics for extended time scales. In this regard, we isolated single chain variable fragments (scFv) that bind the Cyanine dye Cy3 with <100 nM affinity. Briefly, a library of yeast cells optimized for surface expression of proteins was created by mutagenizing previously published dye [35] and fluorogen binding scFvs [68; 74]. Cy3 binding clones were selected via flow cytometry, enriched, and further mutagenized to obtain the 29 kDa scFv refered to as Photostability Enhancing Peptide (PEPCy) in this study. To create the Na_V1.7-PEPCy3 plasmid, the complete open reading frame of mouse Na_V1.7 was cloned into the pcDNA3.1(+)-C-DYK vector (GenScript) with several modifications. The PEPCy3 tag was fused in-frame at the N-terminus of the Na_V1.7 sequence using a small 8 amino acid G linker. Additionally, to promote membrane localization and orient the PEPCy3 toward the extracellular membrane, the N-terminus portion of β subunit was fused to the N-terminus of the full sequence.

2.6.2. Total internal reflection (TIR) microscopy of TG neurons

TG neurons transfected with the Na_V1.7-PEPCy scFv plasmid were plated on round bottom glass dishes (cat. no. P35G-1.5–10-C, Mattek) were incubated with 0.1% DMSO or compound 194 (5 μM) overnight at 37°C. Prior to imaging, the cells were washed 1x with 1mL Colorless Tyrode’s buffer followed by incubating the samples with 50 nM Sulfo-Cy3 carboxylic acid (Lumiprobe) for 20 mins at 37°C. The cells were gently washed 2x with colorless Tyrode solution followed by imaging in the same medium with or without compound 194 as appropriate. The mattek dish containing TG neurons was then placed in a temperature and humidity-controlled chamber (OkoLabs) on a custom-built microscope stand (Nikon). Microscopy was performed at 37°C. An excitation laser line of 561 nm was used with approximate intensity of 500 W/cm² at the sample. For total internal reflection (TIR) microscopy, a super apochromat objective (PlanApo, 100x, 1.45 N.A. oil immersion, Nikon) was used for collecting fluorescence to maximize the photon collection efficiency. TIR was achieved by changing the angle of the incident laser on the back focal plane. The fluorescence emission from the sample passed through a dichroic (C-TIRF ultra high signal to noise 405/488/561/638 nm, Semrock) and band pass emission filters for Cy3 (Cat. No. ET607/36, Brightline, Semrock). Finally, fluorescence images were recorded on a scientific cMOS camera (Prime95B, Photometrix). For each sample, transfected neurons were located using low laser power (~50 W/cm²) to minimize sample bleaching. 1000 frames were acquired for a 512 by 512-pixel region with an exposure time of 20 ms and without any delay between the frames. Analyses of TIR data were performed using Thunderstorm [63] and Trackmate [25] plugin in Fiji followed by plotting of diffusion coefficients using bespoke Python codes.

2.7. Proximity ligation assay

The proximity ligation assay (PLA) was performed as described previously [53; 56; 58] to visualize protein–protein interactions by microscopy. This assay is based on paired complementary oligonucleotide‐labelled secondary antibodies that can hybridize and amplify a red fluorescent signal only when bound to two corresponding primary antibodies whose targets are in close proximity (within 40 nm). Briefly, trigeminal ganglion neurons were incubated overnight with 0.1% DMSO (as control) or 194 (5 μM). The next day, neurons were fixed using ice-cold methanol for 5 minutes and allowed to dry at room temperature.

The proximity ligation assay was performed according to the manufacturer’s protocol using the Duolink Detection Kit with PLA PLUS and MINUS probes for mouse and rabbit antibodies (Duolink in situ detection reagents red, cat. no. DUO92008; Duolink in situ PLA probe anti-rabbit MINUS, cat. no. DUO92005; Duolink in situ PLA probe anti-mouse PLUS, cat. no. DUO92001, Sigma-Aldrich). Primary antibodies (1/1000 dilution) were incubated for 1 hour at RT; Na_V1.7 (cat. no. MABN41; Millipore, Research Resource Identifiers (RRID):AB_10808664), CRMP2 (cat. no. C2993; Sigma-Aldrich, RRID:AB_1078573), SUMO1 (cat. no. S8070; Sigma-Aldrich, RRID:AB_477543) and CRMP2 (cat. no. 11096; Tecan, immunobiological lab, RRID:AB_494511). Cells were then incubated overnight with anti- NeuN antibody (1/500; Cat #: D3S3I, Cell Signaling, RRID:AB_2630395) in PBS with 3% bovine serum albumin at 4°C. Cells were then washed 3 times in PBS and incubated with PBS containing 3% bovine serum albumin and secondary antibody, Alexa 488 goat anti-rabbit (Thermo Fisher Scientific, Waltham, MA) for 1 hour at room temperature. After washing with PBS, cells were stained with 49,6-diamidino-2-phenylindole (DAPI, 50 mg/mL) to detect cell nuclei and mounted in ProLong Diamond Antifade Mountant (cat. no. P36961, Life Technologies Corporation). Immunofluorescent micrographs were acquired using a Plan-Apochromat 63x/1.4 oil CS2 objective on a Leica SP8 confocal microscope operated by the LAS X microscope software (Leica). Camera gain and other relevant settings were kept constant throughout imaging sessions. Image J was used to count the number of PLA puncta per cell by an experimenter blinded to the treatment condition. Three individual cultures from 6 rats were used for this experiment, and at least three coverslips per cultures were analyzed.

2.8. Patch-clamp electrophysiology

Whole-cell voltage-clamp and current-clamp recordings were performed between 18 h after culture or 36 h after transfection at room temperature by using an EPC 10 HEKA amplifier. In experiments where CRMP2 SUMOylation was prevented by the addition of 5 μM 194, the compound was incubated in the tissue culture wells overnight, ~14 h before the experiment. In experiments in which Na_V1.7 channels were blocked with 5 nM ProTxII, the compound was added to the recording external solution.

2.8.1. Voltage-clamp recordings

Trigeminal ganglion neurons were subjected to current-voltage (I-V) and activation/inactivation voltage protocols as follows: a) for the I-V protocol, cells were held at a potential of −60 mV and depolarized by 150-ms voltage steps from −70 mV to +60 mV in 5-mV increments, the resulting currents were normalized to the cell size, expressed as cell capacitance in pF, to get the corresponding current density and also infer the peak current density.

The activation voltage dependence of sodium channels was analyzed as a function of current versus voltage; b) inactivation protocol: from a holding potential of −60 mV, cells were subjected to 1-s hyperpolarizing/repolarizing pulses between−120 and 10 mV (+10 mV steps) followed by a 200-ms test pulse to +10 mV. This incremental increase in membrane potential conditioned various proportions of sodium channels into a state of fast inactivation. The external solution for voltage-clamp sodium recordings contained 140 mM NaCl, 30 mM tetraethylammonium chloride, 10 mM D-glucose, 3 mM KCl, 1 mM CaCl₂, 0.5 mM CdCl₂, 1 mM MgCl₂, and 10 mM HEPES (pH 7.3 and 310–315 mOsm); and the internal solution consisted of: 140 mM CsF, 10 mM NaCl, 1.1 mM Cs-EGTA, and 15 mM HEPES (pH 7.3 and 290–310 mOsm). Analysis was performed by using Fitmaster software (HEKA).

For I−V curves, functions were fitted to data using a non-linear least-squares analysis. I−V curves were fitted using a double Boltzmann function:

$$\text{f}=\text{a}+\text{g}1/(1+\text{exp}\left(\left(\text{x}-{\text{V}}_{1/2}1/\text{k}1\right)\right)+\text{g}2/(1+\text{exp}\left(-\left(\text{x}-{\text{V}}_{1/2}2/\text{k}2\right)\right)$$

where x is the prepulse potential, V_1/2 is the midpoint potential, and k is the corresponding slope factor for single Boltzmann functions. Double Boltzmann fits were used to describe the shape of the curves, not to imply the existence of separate channel populations. Numbers 1 and 2 simply indicate the first and second midpoints; a along with g are fitting parameters.

Activation curves were obtained from the I−V curves by dividing the peak current at each depolarizing step by the driving force according to the equation: G= I/ (V_mem − E_rev), where I is the peak current, V_mem is the membrane potential, and Er_ev is the reversal potential.

The conductance (G) was normalized against the maximum conductance (G_max). Inactivation curves were obtained by dividing the peak current recorded at the test pulse by the maximum current (I_max). Activation and inactivation curves were fitted with the Boltzmann equation.

2.8.2. Current-clamp recordings

Trigeminal ganglion neurons were held at their resting membrane potential and then subjected to a 100-millisecond depolarizing current step. The intensity of this current step was adjusted until the neurons fired an action potential to determine the rheobase of the cells. Additionally, depolarizing current injections of 0–120 pA were applied to trigeminal ganglion cells to collect data on relative excitability, analyzed by counting the number of evoked action potentials during a 300-millisecond current step.

For whole-cell current-clamp experiments, the external solution contained 154 mM NaCl, 5.6 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM D-glucose and 8 mM HEPES (pH 7.4 and 310–315 mOsm); and the internal solution consisted of: 137 mM KCl, 10 mM NaCl, 1 mM MgCl₂, 1 mM EGTA, and 10 mM HEPES (pH 7.3 and 290–310 mOsm). The counting of the number of action potentials was performed by using Easy Electrophysiology software. A waveform analysis was carried out to calculate rise time, amplitude, and threshold by utilizing Easy Electrophysiology software.

Electrodes were pulled from filamented borosilicate glass capillaries (Warner Instruments) with a P-97 electrode puller (Sutter Instruments) to final resistances of 2.5–3.5 MΩ when filled with internal solutions. Whole-cell capacitance and series resistance were compensated.

Linear leak currents were digitally subtracted by the P/4 method for voltage clamp experiments, and bridge balance was compensated in current clamp experiments.

Signals were filtered at 10 kHz and digitized at 10–20 kHz. Cells in which series resistance or bridge balance was more than 15 MΩ or fluctuated by more than 30% over the course of an experiment were omitted from datasets.

2.9. Surgical Procedures

2.9.1. Anesthesia

Rats of both sexes were anesthetized with intraperitoneal injections of ketamine (50mg/kg)/xylazine (7.5mg/kg) solution prior to any surgical procedure. A single investigator (OAK) performed the surgeries to minimize variability.

2.9.2. ION-CCI surgery

Unilateral chronic constriction injury to the infraorbital nerve was performed to induce trigeminal neuropathic pain in rats as previously described [9; 13; 20; 22; 31; 36; 38; 40; 44; 50; 52–56; 58; 62; 65–67; 72; 73; 75; 76; 85; 86]. Briefly, following anesthesia, an approximately 1cm-long incision was made along the left gingivobuccal sulcus beginning just proximal to the first molar. The infraorbital nerve (ION) was exposed (~0.5 cm) and freed from the surrounding tissue. Two chromic gut (4–0) ligatures were loosely tied around the exposed nerve. The incision was closed with absorbable sutures.

2.10. Intranasal compound administration

Twenty-five days post-ION-CCI, one group of rats received compound 194 intranasally (200 μg in 20 μL isotonic saline) whereas another group received 20 μL of isotonic saline (vehicle-control). Intranasal delivery was performed with a pipette and a disposable plastic tip. Immediately after administration, the head of the animal was held in a tilted back position for ~15 seconds to prevent loss of solution from the nares. Behavioral assessments were done at 30 minutes, 1-, 2-, and 3-hours post-administration.

2.11. Assessment of somatosensory pain-like behavior

Von Frey detection threshold and pinprick response score assays were carried out to measure the hypersensitivity to mechanical stimulation applied over the ION dermatome. Somatosensory assessments were performed by a single investigator blinded to the experimental conditions. Animals were habituated to testing cages for three days prior to behavioral testing. Habituation included bringing animals to the laboratory, allowing them to settle in their home cages for at least 30 minutes. Animals were then placed in testing cages for 15 minutes after which they were returned to their home cages.

Mechanical allodynia was measured by applying von Frey monofilaments. The force applied by the von Frey monofilaments is measured in grams, and to transform this linear scale into a logarithmic one, the log score was calculated as the logarithm (usually base 10) of the force applied. The logarithmic transformation allows for a more accurate representation of the perceived differences in touch sensitivity across a wide range of forces. Using the von Frey log scores allows for more precise assessment of the sensory thresholds. A log score of 1.65 corresponds to 0.008g.

Here, the von Frey monofilaments delivered a calibrated amount of force ranging from 0.0008g to 1g or converted to log units 1.65 to 4.08 (EXACTA Precision & Performance monofilaments, Stoelting) within the ION dermatome in ascending order of intensity [45]. The lowest filament that evoked at least one withdrawal response was designated as the withdrawal threshold. A decrease in the withdrawal threshold is indicative of development of hypersensitivity.

Pinprick test was measured by scoring the response to stimulation with a blunted acupuncture needle applied within the vibrissal pad of rats. The scores were assigned as follows: 0 = no response, 1 = non-aversive response, 2 = mild aversive response, 3 = strong aversive response, 4 = prolonged aversive behavior [6; 79]. An increase in the response score is indicative of development of hypersensitivity.

2.12. Data analysis

All data sets corresponding to the electrophysiological, surface expression, PLA and in situ experiments were graphed and analyzed with GraphPad Prism (Version 9) [4]. They were checked for normality using the D’Agostino−Pearson test. Details of statistical tests, significance, and sample sizes are reported in the appropriate figure legends. All data plotted represent mean ± standard error of the mean (SEM). The statistical significance of differences between means was calculated by performing nonparametric Mann Whitney test or parametric analysis of variance (ANOVA) with a post hoc comparisons test (Tukey).

The statistical analyses for the behavioral data were performed using JMP Pro 16.0.0 software (JMP^®, Version 16.0.0 SAS Institute Inc., 1989–2007). Goodness-of-fit test was used to evaluate normality of data. Analysis of variance was used to measure the between groups variability in normally distributed data and Wilcoxon/Kruskal Wallis Rank Sum’s nonparametric test was used to measure between groups differences of not normally distributed data. Results are reported as mean ± SEM unless otherwise specified. Wilcoxon Signed Rank test, a non-parametric alternative to paired t-test, was used to measure development of hypersensitivity following ION-CCI.

3. Results

3.1. Molecular identification and characterization of Na_V1.7 neurons in the rat trigeminal ganglion

The expression and function of Na_V1.7 channels as well as CRMP2 in rat TG neurons have been previously demonstrated [55; 85; 86]. Nevertheless, a comprenhensive search on their co-localization and the identity of the trigeminal nociceptor populations where they can be found has not been performed. We utilized in situ hybridization to characterize Scn9a-expressing TG neurons (defined by a DAPI-labeled nucleus surrounded by Rbfox3 signal), using Nefh as a marker for A-∂ nociceptors and Calca to identify peptidergic C nociceptors (Figure 1A–B). Approximately 73% of all TG neurons were Scn9a positive, suggesting that 3/4 of TG neurons express the Na_V1.7 channel (Figure 1C). Of Scn9a labeled neurons, approximately 68% were co-labeled for Calca and approximately 77% were co-labeled for Nefh (Figure 1D–E), indicating that Scn9a is highly expressed in both peptidergic C and A-∂ neurons.

Figure 1. Characterization of Scn9a cells in rat trigeminal ganglion.

Representative expression of Scn9a with Nefh (A) and Calca (B) in trigeminal ganglion neurons. Yellow arrowheads represent Scn9a negative Nefh or Calca neurons, purple arrowheads represent Nefh or Calca negative Scn9a neurons, blue arrowheads represent Scn9a neurons co-expressing either Nefh or Calca. C. Percentage of all neurons in the trigeminal ganglion expressing Calca, Nefh, Scn9a, or a combination of Scn9a and Calca or Nefh. D. Percent of Scn9a, Calca, or Nefh neurons co-expressing other markers. E. Visual representation of Scn9a co-expression with Calca or Nefh. F. Representative expression of Dpysl2, Ube2i, and Scn9a in the trigeminal ganglion. Yellow arrowheads represent Scn9a negative cells co-expressing Dpysl2 and Ube2i, blue arrowheads represent cells co-expressing Dpysl2, Ube2i, and Scn9a. G. Quantified percentages of Ube2i, Dpysl2, and Scn9a cells co-expressing other markers in the trigeminal ganglion. H. Visual representation of co-expression of Dpysl2, Ube2i, and Scn9a. All scale bars represent 50 μm. Each individual data point represents the mean of 4–6 sections from one animal. N= 3 to 6 rats per condition.

Here, we used Nefh as a marker to help characterize expression of Scn9a. However, Nefh expression in the soma does not necessarily define these as A fiber neurons. Because RNAscope is restricted to mRNA expression in the soma, we are unable to comment on axonal expression. Therefore, while many of these Scn9a TG neurons are likely A fibers, we are unable to categorically define all the Nefh-expressing neurons as A fibers. Previous TG studies have shown that transcriptomic identity, and similarly in situ expression identity, does not solely identify a neuron’s role (for example, see [78]). Further characterization with a wider variety of probes is needed to provide a more complete neurochemical identity of these Scn9a TG neurons.

In order to explore the incidence of Na_V1.7 with CRMP2 and the SUMO-conjugating enzyme Ubc9, we performed in situ hybridization in rat TG tissue for Scn9a, Dpysl2, and UBE2I (Figure 1F). Scn9a positive cells expressed high levels of both Dpysl2 abd UBE2I (approximately 70%), suggesting that the majority of Na_V1.7 TG neurons can be potentially regulated by CRMP2 SUMOylation (Figure 1G–H). Further, only 36% of UBE2I labeled cells expressed both Scn9a and Dpysl2 and 50% and Dpysl2 labeled cells expressed both Scn9a and UBE2I (Figure 1G–H), indicating that both UBE2I and Dpysl2 are highly expressed in Scn9a negative neurons as well and also function in other TG populations.

3.2. CRMP2 and Na_V1.7 physically interact in rat TG neurons and the small molecule 194 can uncouple this interaction

The SUMOylation of CRMP2 is a postranslational modification that modifies the surface expression, function, and promotes internalization of Na_V1.7 channels [20; 22; 33; 53; 54]. Inhibition of this mechanism, both pharmacologically and genetically, has been demonstrated to promote the endocytosis of Na_V1.7 in DRG neurons, reduce the excitability of dorsal horn neurons, and exhibit antinociceptive effects in animal pain models [20; 22; 33; 53; 54]. In the TG, however, no molecular correlation has been established between these two proteins. To address this, we employed proximity ligation assay (PLA) to examine the interactions between Na_V1.7 and CRMP2, determine the SUMOylation status of CRMP2, and assess the disruptive effect of these two biological functions by compound 194 in TG neurons. PLA allows detection and quantification of protein complexes when proteins are within 40 nm of each other. The generation of a PLA signal can be interpreted as a protein-protein interaction. In cells treated with 0.1% DMSO, a positive PLA signal was found in TG neurons for SUMO1/CRMP2 and Na_V1.7/CRMP2 (Figure 2A–D).

Figure 2. Compound 194 reduces CRMP2 SUMOylation decreasing binding of SUMO1 to CRMP2 and of Na_V1.7 to CRMP2 in rat trigeminal ganglion (TG) neurons.

Representative images of rat TG neuron cultures treated overnight with 0.1% DMSO (as a control) or 5 μM 194 following proximity ligation assay (PLA) between CRMP2 and SUMO1 (A), or between Na_V1.7 and CRMP2 (C). NeuN signal was used to identify neurons. The PLA immunofluorescence labeled sites of interaction between CRMP2 and SUMO1 or Na_V1.7 and CRMP2 (red puncta). In addition, nuclei are labeled with the nuclear labeling dye 4’,6’-diamidino-2-phenylindole (DAPI). Scale bar: 10 μm. Quantification of PLA puncta per neuron show that in TG neurons treated with 194, SUMO1-CRMP2 (B) and Na_V1.7-CRMP2 (D) interactions are significantly reduced compared with the control condition. Each individual data point represents PLA puncta per neuron. n= 190–245 neurons p values as indicated; Mann Whitney test; Error bars indicate mean ± SEM.

Next, we tested if 194 could block the SUMOylation of CRMP2 and if this treatment could lead to Na_V1.7/CRMP2 uncoupling. Overnight incubation of TGs with 194 significantly decreased both SUMO1/CRMP2 and Na_V1.7/CRMP2 PLA signals per neuron compared to the control condition (0.1% DMSO; Figure 2A–D). These findings indicate that the interaction between Na_V1.7 and CRMP2 is preserved in TG neurons and that 194 is able to disrupt this interaction by blocking the addition of SUMO1 to CRMP2 by SUMO conjugating enzyme Ubc9.

3.3. CRMP2 SUMOylation inhibitor compound 194 decreases the membrane expression, but increases clustering, of Na_V1.7 channels in rat trigeminal ganglion (TG) neurons

Our group has reported that the CRMP2 SUMOylation inhibitor, compound 194, can reduce the cell surface expression of Na_V1.7 in DRG neurons, facilitating the internalization of the channel [13]. We used immunofluorescent labeling followed by confocal imaging to evaluate if the membrane expression of Na_V1.7, expressed as membrane to cytosol immunoreactivity ratio per neuron, is affected by 194 treatment of cultured rat TG neurons. Results showed that the surface expression of this channel was decreased in cells treated with 194, compared to the control condition (Figure 3A, B). These data suggest that 194 is promoting the internalization of Na_V1.7 in rat TG neurons.

Figure 3. Compound 194 reduces CRMP2 SUMOylation causing a decrease in Na_V1.7 surface expression in rat trigeminal ganglion (TG) neurons.

A Three representative confocal images of rat TG neuron cultures treated overnight with 0.1% DMSO (as a control) or 5 μM of 194 and labeled with an antibody against Na_V1.7. Scale bar: 10 μm. B Quantification of normalized membrane to cytosol ratio of Na_V1.7 per neuron shows that TG neurons treated with 194 have decreased surface expression of Na_V1.7 compared with control condition. Each individual data point represents one neuron, quantified neurons from the same coverslip are represented in different colors. n = 50–51 neurons cells; p values as indicated; Unpaired t-test; error bars indicate mean ± SEM.

To independently corroborate this effect on surface expression of Na_V1.7, we used super-resolution microscopy to resolve Na_V1.7 channels at single molecule resolution. We fused a 29 kilodalton Photostability Enhancing Peptide (PEP) for the membrane impermeable dye cyanine (PEPCy) to the N-terminus of Na_V1.7 (Figure 4A). To test the functionality of this channel, we trasfected HEK293 cells with Na_V1.7-PEPCy and measured macroscopic sodium currents. Depolarizing pulses ranging from −70 to +60 mV produced a rapidly activating and inactivating family of sodium currents (Figure 4B) with a peak between −20 and 0 mV (Figure 4C) and a peak current density of −46.48 ± 9.74 pA/pF (n = 14; Figure 4D). Normalized conductance values were fit with the Boltzmann equation to determine the voltage dependence of activation. Half-activation value was −19.64 ± 1.34 (n=12) and (slope) k = 7.81 ± 1.33 (n=12)(Figure 4E). Steady-state fast-inactivation curves were generated by applying 500 ms inactivating potentials from −120 to −10 mV, followed by a 40 ms step to −10 mV. Normalized conductance values were fit with the Boltzmann equation to determine the voltage dependence of steady-state fast-inactivation. Half-inactivation value was: −75.18 ± 1.45 (n=12) and k = 9.85 ± 1.36 (n=12)(Figure 4E). The combined information affirms that the introduction of PEPCy3 into the channel did not affect its capability to generate functional Na_V1.7 channels.

Figure 4. TIR microscopy reveals effects on reduced diffusivity of single Na_V1.7 molecules upon treatment with compound 194.

A. Schematic of the NaV1.7-PEPCy construct and site of binding of cell impermeant Cy3 dye. The schematic was generated with BioRender. B. Representative traces of sodium currents (I_Na⁺) recorded from HEK 293 cells transiently expressing Na_V1.7-PEPCy and green fluorescent protein (GFP). Cells were identified with GFP fluorescence. C. Summary of I_Na⁺ density versus voltage relationship. D. Bar graphs of total peak I_Na⁺ density from HEK 293 cells transiently expressing Na_V1.7-PEPCy. E. Boltzmann fits for normalized conductance voltage relationship for voltage dependent activation (G/Gmax) and inactivation (I/Imax) from HEK293 cells transiently expressing Na_V1.7-PEPCy. Error bars indicate mean ± SEM; n= 12 cells. F. Schematic of Total Internal Reflection (TIR) Microscopy used for imaging TG neurons transfected with Na_V1.7-PEPCy. G. Super-resolved images showing single molecule localizations in a representative field of TG neurons incubated without (left) or with (right) 5 μM of compound 194. Without drug treatment single molecules of Na_V1.7 assemble in large clusters and as mobile single molecules compared to more clustered Na_V1.7 molecules in the presence of 194. Scale bar is 1 μm. H. Distribution of cluster areas shown as a violin plot for control and 194 treated TG neuron samples. The white dot within the bars in the violin plot denotes the median of the distribution. Between 36000–50000 clusters were analyzed from fifteen regions for each distribution. I. Super-resolved images showing single molecules in first frame overlaid with their trajectories in a representative field of TG neurons incubated without (left) or with (right) 5 μM of compound 194. Each trajectory is color-coded by track displacement in μm. Without drug treatment, single molecules of Na_V1.7 are more diffusive compared to Na_V1.7 molecules in the presence of 194. Scale bar is 2 μm. J. Distribution of two-dimensional diffusion coefficients shown as a violin plot for control and 194 treated TG neuron samples. The white dot within the bars in the violin plot denotes the median of the distribution. Between 4200–4300 single molecules were analyzed from twelve regions for each distribution.

Total internal reflection (TIR) microscopy on TG neurons transfected with Na_V1.7-PEPCy3 labeled with the membrane impermeable dye Cy3 revealed single molecules and clusters of Na_V1.7 on the cell surface (Figure 4F). Single molecule localization analyses from super-resolution microscopy revealed the presence of clusters of various sizes (Figure 4G). Interestingly, the observed Na_V1.7-PEPCy3 cluster sizes were dependent on the presence or absence of compound 194. In the presence of 194 we observed a median cluster size of 0.0008 μm² versus 0.003 μm² in the untreated case (Figure 4H). A smaller cluster could arise from slow diffusion of molecules. To test this hypothesis, we performed a trajectory analysis of single NaV1.7-PEPCy3 proteins. Visual inspection of trajectories revealed that TG neurons transfected with Na_V1.7-PEPCy3 and treated with 194 exhibited lower mobility compared to untreated cells (Figure 4I, Supplementary Videos 1 and 2). We analyzed the motion of single molecules from both samples using mean squared displacement (MSD) analysis [51]. Using the first three points of the MSD versus lag time plot, we computed the distribution of two-dimensional diffusion coefficients (D) for the trajectories. For the 194-treated samples, the distribution of D had a median value of 0.0009 μm²/s, which was approximately two-fold lower than the median D for the untreated samples (D = 0.001 μm²/s) (Figure 4J). Additionally, the untreated samples exhibited many highly diffusive molecules (D values greater than 0.05 μm²/s)(Supplementary Video 1). Together, these data demonstrate that 194 treatment inhibits Na_V1.7 diffusion on the membrane leading to the formation of tightly localized membrane clusters.

3.4. Preventing CRMP2 SUMOylation with compound 194 decreases the activity of Na_V1.7 channels in rat trigeminal ganglion neurons

To assess the functional implications of CRMP2 SUMOylation on Na_V1.7 channels in TG neurons, we initially examined the impact of 194 on total sodium currents. Sodium currents from TG cells held at −60 mV and depolarized by 150-ms voltage steps from −70 mV to +60 mV in 5-mV increments were normalized to the cell’s capacitance to obtain the current-voltage relationships shown in Figure 5. Large transient inward currents were seen in response to depolarizing steps (Figure 5A). Mean peak current density (at −5 mV) of DMSO treated TG neurons was −525.3 ± 40.3 pA/pF (DMSO, n = 29, Figure 5B–C). Blocking CRMP2 deSUMOylation by overnight incubation with 194 (5 μM) decreased total sodium currents and current density, causing a significant diminution in peak sodium currents of ~ 58% (−220.8 ± 29.8 pA/pF, n = 30, Figure 5A–C) with respect to control (i.e., DMSO-treated TG neurons). Analysis of voltage-dependence of activation revealed that treating TG neurons with 194 caused a significant ~6 mV shift in the activation curve towards more positive potentials. Moreover, 194 induced a significant ~5 mV leftward shift in the voltage-dependence of inactivation (Figure 5D and Table 1). The observed reduction in total sodium currents in DRGs after exposure to compound 194 can be attributed to the remaining slow-inactivating TTX-R sodium currents that are unaffected by 194, as previously reported by us [13].

Figure 5. Blocking CRMP2 SUMOylation with compound 194 reduces sodium currents of rat trigeminal ganglion sensory neurons.

A. Representative traces of sodium currents (I_Na⁺) from rat trigeminal ganglion neurons (TGs) incubated with 0.1% DMSO (control; black circles), 5 μM 194 (red squares), 5 nM ProTxII (blue triangles) and 5 μM 194 + 5 nM ProTxII (green diamonds). B. Summary of total I_Na⁺ density versus voltage relationship. C. Bar graphs of total peak I_Na⁺ density from TGs pre-treated as indicated. Significant differences were observed in the I_Na⁺ density of each group, when compared to the control (one-way ANOVA followed by a Tukey’s post hoc test, n= 14 to 30 cells per condition from four different animals). D. Boltzmann fits for normalized conductance voltage relationship for voltage dependent activation (G/Gmax) and inactivation (I/Imax) of rat TG neurons pre-treated as indicated. Significant differences were observed in the V_1/2act and V_1/2inact of each group, when compared to the control (one-way ANOVA followed by a Tukey’s post hoc test, n= 14 to 30 cells per condition). Error bars indicate mean ± SEM.

Table 1.

Gating properties of voltage-gated sodium channels in rat trigeminal ganglion neurons.

Condition	Activation		Inactivation
	V 0.5	k	V 0.5	k
Figure 5D
DMSO	−19.626 ± 0.598 (29)	5.201 ± 0.535 (29)	−47.260 ± 1.348 (29)	−13.943 ± 1.337 (29)
194 (5 μM)	−13.742 ± 0.907 (30) p < 0.0001 vs. DMSO	6.343 ± 0.841 (30)	−52.294 ± 1.463 (30) p = 0.0440 vs. DMSO	−12.521 ± 1.403 (30)
DMSO+ ProTx-II (5 nM)	−15.439 ± 1.021 (13) p = 0.0040 vs. DMSO	6.446 ± 0.951 (13)	−56.828 ± 1.625 (13) p = 0.0009 vs. DMSO	−11.461 ± 1.513 (13)
194 (5 μM) + ProTx-II (5 nM)	−14.307 ± 0.722 (23) p < 0.0001 vs. DMSO	6.854 ± 0.683 (23)	−54.979 ± 1.424 (23) p = 0.0014 vs. DMSO	−12.416 ± 1.357 (23)
Figure 6D
Scramble siRNA	−21.225 ± 0.565 (24)	3.908 ± 0.494 (24)	−41.770 ± 1.722 (24)	−16.145 ± 1.758 (24)
CRMP2 siRNA	−21.519 ± 1.132 (35)	7.718 ± 1.071 (35) p = 0.0192 vs. Scramble siRNA	−49.580 ± 1.648 (35) p = 0.0072 vs. Scramble siRNA	−17.560 ± 1.852 (35) p=0.9369
Scramble siRNA + 194 (5 μM)	−17.052 ± 0.730 (18) p = 0.0480 vs. scramble siRNA	5.941 ± 0.668 (18)	−54.172 ± 1.171 (18) p = 0.0001 vs. Scramble siRNA	−12.332 ± 1.115 (18)
CRMP2 siRNA + 194 (5 μM)	−14.844 ± 1.261 (18) p = 0.0007 vs. Scramble siRNA	8.155 ± 1.259 (18) p = 0.0295 vs. Scramble siRNA	−46.227 ± 2.414 (18)	−17.975 ± 2.662 (18)

Values are means ± S.E.M. calculated from fits of the data from the indicated number (in parentheses) of cells to the Boltzmann equation. V_0.5, midpoint potential (mV) for voltage-dependent activation or inactivation; k, slope factor. Data were analyzed with a one-way ANOVA followed by a Tukey’s post hoc test.

We next utilized Protoxin-II (ProTx-II), a selective Na_V1.7 channel blocker [69], to assess the contribution of Na_V1.7 channels to the reduction in total sodium currents caused by 194. Acute application of ProTx-II revealed a similar decrease in sodium currents and current density (~46%, −284 ± 39.5 pA/pF, n = 13, Figure 5A–C) to that observed in the presence of compound 194. When analyzing the kinetics of activation and inactivation, we found that ProTxII promoted a significant ~4 mV shift in the activation curve towards more positive potentials as well as a ~9 mV leftward shift in the voltage-dependence of inactivation (Figure 5D and Table 1).

Next, to test if compound 194 and ProTx-II converge on modulating Na_V1.7 channels, we performed oclussion experiments where we first incubated TG neurons with 194 and then applied ProTx-II. We found that co-application of both compounds did not cause a further reduction in sodium currents (~ 66% respect to control, −175.6 ± 20.8 pA/pF, n = 23, Figure 5A–C) when compared to cells treated with 194 and ProTx-II alone. This suggests that Na_V1.7 channels had already been maximally blocked/regulated. Importantly, no significant changes were observed in tetrodotoxin-resistant sodium channels —Na_V1.8 and Na_V1.9— in TG neurons in the presence of 194 (data not shown).

Overall, these data support the conclusion that modulation of Na_V1.7 channels by CRMP2 SUMOylation occurs in TG neurons, and that sodium current reductions imposed by 194 are mainly due to reduced current influx through Na_V1.7 channels.

3.5. CRMP2 knockdown decreases sodium currents and recapitulates the effect of 194 on rat trigeminal ganglion neurons

Our data thus far demonstrates that Na_V1.7 channels represent an important component in rat TG neurons and that the SUMOylation of a cytosolic regulator CRMP2, can modify the function of these channels, ultimately regulating neuronal excitability. These observations position CRMP2 as a central driver of Na_V1.7 activity and underscore the inhibitory effect exerted by compound 194 [20]. Considering that CRMP2’s loss of SUMOylation inhibits Na_V1.7 activity, a similar reduction in Na_V1.7 currents would be expected when CRMP2 expression is reduced. To test if CRMP2 is the target protein of 194, we proceeded to evaluated if silencing CRMP2 in TG neurons would modify the effect of compound 194 on sodium current density. Firstly, we knocked-down the expression of CRMP2 in TG neurons by using a previously validated siRNA against CRMP2 [11; 20] and measured sodium currents by performing whole-cell voltage clamp electrophysiology. Silencing CRMP2 led to a significant reduction (~35%) in peak sodium currents and current densities (−209.4 ± 21.5 pA/pF, n = 35, Figure 6A–C) when compared to the control condition (−323.2 ± 30.6 pA/pF, n = 24, Figure 6A–C). Analysis of voltage-dependence showed a significant ~8-mV shift in the V_0.5 of inactivation towards more negative potentials (Figure 6D). Overnight incubation of 194 alone (5 μM) caused a significant reduction of sodium currents (~ 53%, −150.5 ± 24.4 pA/pF, n = 18,) which did not decrease further when the compound was applied to CRMP2-silenced cells (~ 57%, −136.9 ± 21.6 pA/pF, n = 18) (Figure 6A–C). Compound 194 promoted a rightward shift (~4 mV) in the V_0.5 of activation of control cells and a ~13-mV shift in the V_0.5 of inactivation towards more negative voltages (Figure 6D, Table 1). A ~7-mV rightward shift in the V_0.5 of activation was observed when 194 was added to the CRMP2-silenced cells (Figure 6D, Table 1). These data functionally validate the role of CRMP2 in modulating Na_V1.7 function in TG neurons. Furthermore, our findings suggest that the target effector protein of compound 194 is indeed CRMP2, which subsequently allosterically modulates Na_V1.7 channels.

Figure 6. Knockdown of CRMP2 reduces sodium currents in rat trigeminal ganglion (TG) sensory neurons to a similar extent as compound 194.

A. Representative traces of sodium currents (I_Na⁺) from rat TG neurons transfected with scramble siRNA (black circles), CRMP2 siRNA (red squares), scramble siRNA + 194 (blue triangles), or CRMP2 siRNA + 194 (green diamonds). B. Summary of total I_Na⁺ density versus voltage relationship. C. Bar graphs of total peak I_Na⁺ density from TGs pre-treated as indicated. Significant differences were observed in the V_1/2act of each group, when compared to the control (one-way ANOVA followed by a Tukey’s post hoc test, n= 18 to 35 cells per condition). D. Boltzmann fits for normalized conductance voltage relationship for voltage dependent activation (G/Gmax) and inactivation (I/Imax) of rat TG neurons pre-treated as indicated. Significant differences were observed in the V_1/2act and V_1/2inact of each group (Table 1), when compared to the control (one-way ANOVA followed by a Tukey’s post hoc test, n= 18 to 35 cells per condition from four different animals). Error bars indicate mean ± SEM.

3.6. The CRMP2 SUMOylation inhibitor 194 decreases excitability of rat trigeminal ganglion neurons

As Na_V1.7 channels play a crucial role in determining the threshold for action potential firing in sensory neurons [49; 80], the observed regulation of their activity in our voltage-clamp experiments is likely to have a direct impact on neuronal excitability. To test if preventing the SUMOylation of CRMP2 and resulting Na_V1.7 inhibition have an impact on the excitability properties of TG neurons, we performed current-clamp experiments in the absence and presence of 194. We observed that overnight incubation of TG neurons with 5 μM of 194 significantly decreased the number of action potentials from 80 to120 pA-current injection steps (Figure 7A, B). When analyzing the maximum number of action potentials, quantified at 120 pA, we observed a ~50% decrease in the presence of 194 (3.23 ± 2.029) compared to the control group (6.4 ± 3.544; Figure 7A, B). No changes were observed in the rheobase, which is the minimum current required to evoke a single action potential, or in the basal resting membrane potential (Figure 7C, D). A waveform analysis of the evoked action potentials revealed that 194 significantly increases the rise time to peak and the threshold at every current step while, in turn, the amplitude was found to be decreased (Figure 7E–G). Collectively, these findings strongly indicate that 194 exerts a significant influence on the excitability of TG neurons, primarily by inhibiting Na_V1.7 activity through the reduction of CRMP2 SUMOylation.

Figure 7. Preventing CRMP2 SUMOylation with compound 194 decreases excitability of rat trigeminal ganglion sensory neurons.

A Representative traces of evoked action potentials at 120 pA from rat trigeminal ganglion neurons (TGs) incubated overnight with 0.1% DMSO (control; black circles) or 5 μM 194 (red squares). B Summary of the current-evoked action potentials in response to current injections between 0–120 pA in the presence of overnight 0.1% DMSO or 5 μM 194. C Quantification of the rheobase in the presence of overnight 0.1% DMSO or 5 μM 194 D Quantification of the resting membrane potential in the presence of overnight 0.1% DMSO or 5 μM 194. Waveform parameters, (E) rise time (in ms), (F) amplitude (in mV) and (G) threshold (in mV) of such evoked action potentials. Unpaired single and multiple t tests, n= 15 to 21 cells per condition from four different animals. Error bars indicate mean ± SEM.

3.7. Intranasal administration of CRMP2 SUMOylation inhibitor 194 decreases unilateral ION-CCI-mediated mechanical allodynia in rats

Our previous study demonstrated that the small molecule 194 effectively reverses pain in six different models across two rodent species, administered via four routes [13]. However, treating craniofacial pain, like PTNP, poses challenges as common drug delivery methods may not effectively reach cephalic areas to alleviate pain [8; 17]. To manage trigeminal pain, intranasal administration offers a non-invasive drug delivery route, where drugs are absorbed by the nasal cavity’s mucus membrane, reaching systemic blood circulation and trigeminal nerves [8; 17]. Given 194’s proven in vitro effects on rat trigeminal Na_V1.7 channels and excitability, we investigated its potential for ameliorating trigeminal neuropathic pain through intranasal delivery in rats.

3.7.1. Establishment of the unilateral ION-CCI-mediated mechanical allodynia model in rats

We first measured the development of mechanical allodynia at 17-, 21- and 24-days post ION-CCI. Male and female animals developed significant hypersensitivity as indicated by a significant drop in von Frey detection thresholds (vF) at 17 (1.65 ± 0.0 for males and females), 21 (1.65 ± 0.0, for male and females), and 24 (1.65 ± 0.0, for male and females) days following ION-CCI compared to baseline (2.68 ± 0.28 for males and 2.43 ± 0.01 for females) (Figures 8A, B). Furthermore, there was a significant increase in pin prick (PP) score at 17 (2.90 ± 0.45 for males and 3.00 ± 0.00 for females), 21 (2.85 ± 0.49 for males and 3.00 ± 0.00 for females), and 24 (2.8 ± 0.41 for males and 3.00 ± 0.00 for females) days following ION-CCI compared to baseline (1.25 ± 0.44 for males and 1.60 ± 0.18 for females) (Figures 8C, D). These behavioral results show that both male and female animals exhibited significant hypersensitivity following ION-CCI.

Figure 8. Intranasally-administered CRMP2 SUMOylation inhibitor 194 decreases mechanical hypersensitivity in rats with trigeminal neuropathic pain.

A. Timeline of the experimental paradigm indicating that rats developed significant hypersensitivity to von Frey monofilaments at days 17, 21 and 24 post chronic constriction of the infraorbital nerve (ION-CCI) compared to baseline (BL). Intranasal compound 194 resulted in a significant increase in the von Frey detection threshold in the ipsilateral side of ION-CCI rats compared to ION-CCI rats who received vehicle (saline). Von Frey detection threshold was evaluated at 30 min, 1-, 2- and 3-hours post compound 194 administration. p value as indicated; Steel-Dwass test. N = 9 −10 rats per treatment; error bars indicate mean ± SEM. B. Timeline of the experimental paradigm indicating that rats developed significant hypersensitivity as indicated by the increase in pinprick response scores at days 17, 21 and 24 post ION-CCI compared to baseline (BL). Pinprick response scores were observed at 30 min, 1-, 2- and 3-hours post compound 194 administration. p value as indicated; Steel-Dwass test. N= 9 −10 rats per treatment. Error bars indicate mean ± SEM.

3.7.2. Effects of intranasal administration of compound 194 on von Frey detection thresholds in rats with ION-CCI

Intranasally delivery of compound 194 (200 μg / 20 μL) effectively increased mechanical detection threshold in the side of the face ipsilateral to ION-CCI compared to vehicle (i.e., saline) treated animals. A significant increase in detection threshold was observed beginning at 30 min post administration (194: 2.01 ± 0.38, saline 1.65 ± 0.00), and the effect remained significant at 1 hour (194: 2.16 ± 0.36, saline 1.65 ± 0.00), 2 hours (194: 2.13 ± 0.44, saline 1.65 ± 0.00) and 3 hours (194: 1.98 ± 0.45, saline 1.65 ± 0.00) post administration for males (Figure 8A) and 30 minutes (194: 2.01 ± 0.37, saline 1.65 ± 0.00), 1 hour (194: 2.15 ± 0.34, saline 1.65 ± 0.00) and 2 hours (194: 2.15 ± 0.34, saline 1.65 ± 0.00) post administration in female rats (Figure 8B). This reduction demonstrates that intranasal delivery of 194 reduces mechanical allodynia induced by ION-CCI.

3.7.3. Effects of intranasal administration of compound 194 on pinprick response in rats with ION-CCI

Treatment with intranasal 194 resulted in a significant decrease in the pinprick response score in the ipsilateral side of the face in rats exposed to ION-CCI compared to those treated with saline. The decrease in pinprick score was observed as early as 30 minutes post administration (194: 1.60 ± 0.84, saline 3.00 ± 0.47), and the effect remained significant at 1 hour (194: 1.7 ± 0.82, saline 2.90 ± 0.57), 2 hours (194: 1.80 ± 0. 79, saline 2.60 ± 0.52) and 3 hours (194: 1.80 ± 0. 91, saline 2.90 ± 0.32) post administration for males (Figure 8C) and 30 minutes (194: 1.70 ± 0.26, saline 2.90 ± 0.11) and 1 hour post administration for females (194: 2.00 ± 0.30, saline 3.00 ± 0.00) (Figure 8D). These findings indicate that compound 194 effectively reduces acute nociception in male and female rats with ION-CCI.

The above results highlight that intranasal administration is an effective route for delivering compound 194 to target cranial neuropathic pain.

4. Discussion

Despite the incidence of PTNP [5; 7], much of pain research has focused on the study of spinal nerves, and as a result, the management of craniofacial pain remains poor. In this study, we use molecular, electrophysiological, and behavioral approaches to demonstrate that regulation of Na_V1.7 via blocking SUMOylation of CRMP2 by the small molecule 194 can be used as a therapeutic target for the alleviation of trigeminal neuropathic pain.

Although increased functionality of Na_V1.7 is a known contributor to chronic pain, efforts to directly target Na_V1.7 as a therapeutic have failed [24; 48; 71]. To circumvent these failures, we have advanced a differentiated strategy to allosterically regulate Na_V1.7 [14; 15; 20; 22; 26], resulting in the discovery of compound 194 [13]. Compound 194 is a benzoylpiperidylbenzimidazole that blocks CRMP2 SUMOylation in DRG neurons and promotes internalization of Na_V1.7, thereby decreasing sodium currents and was shown to reduce pain-like behaviors across multiple pain models, including formalin, postsurgical, spared nerve injury, spared nerve ligation, oxaliplatin- and paclitaxel-induced peripheral neuropathy, and HIV-induced sensory neuropathy in two species (mice and rats) via four routes of administration (intrathecal, intraperitoneal, subcutaneous, and oral) [13]. The efficay of 194 was not accompanied by any tolerance, addiction, rewarding properties, or neurotoxicity [13]. Here, we evaluated whether 194’s mechanism of regulating Na_V1.7 extended to the trigeminal ganglion. Although the presence and activity of both CRMP2 and Na_V1.7 channels have been reported in TG neurons [55; 85; 86], this work is the first to demonstrate direct functional and molecular interactions between these two proteins in TG.

The mechanism of analgesia of 194 reportedly involves a central component and is predicted to have a high likelihood of passively diffusing through the blood-brain barrier (BBB) [13]. Studies have demonstrated that intranasal delivery can rapidly and efficiently transport drugs from the nasal cavity to the brain. This noninvasive method follows the olfactory and trigeminal nerve pathways, effectively circumventing the blood-brain barrier. Experiments conducted on rats revealed that when lidocaine is administered intranasally, structures innervated by the trigeminal nerve accumulate significantly higher concentrations of lidocaine, up to 20 times greater than those found in the brain and bloodstream. This phenomenon allows targeted therapeutic treatment for orofacial disorders, minimizing unwanted side effects in the brain and the rest of the body [37]. Furthermore, intranasal lidocaine has demonstrated analgesic effects in patients suffering from second-division trigeminal neuralgia, without causing serious adverse reactions [39]. As 194 reduced trigeminal neuropathic pain-like mechanical allodynia and acute nociception demonstrates that intranasal administration is an effective and safe route of administration for 194 to target cranial neuropathic pain.

We found that the genes for Na_V1.7 (Scn9a), CRMP2 (Dpysl2), and Ubc9 (Ube2i) co-localize moslty in trigeminal A-∂ and peptidergic C fibers, which are responsible for the transmission of noxious stimuli, and our PLA experiments suggest a physical interaction between these two proteins. Approximately 75% of Scn9a neurons express both Ube2i and Dpysl2, which is supported by our PLA results demonstrating a physical interaction between Na_V1.7 and CRMP2 as a result of CRMP2 SUMOylation. A more detailed characterization of this Scn9a/Dpysl2/Ube2i subpopulation could further direct the development of more specifically targeted drugs. Intranasal 194 was also able to effectively reduce behavioral signs of PTNP in both male and female rats in the CCI-ION model. Interestingly, we found approximately 25% of Scn9a cells that do not express Ube2i, suggesting that some Scn9a/Dpysl2 TG cells do not undergo Ubc9-mediated SUMOylation. Therefore, the ability of 194 to reduce the behavioral signs of ION-CCI suggests that targeting a subpopulation of Na_V1.7 is sufficient for reduction of PTNP.

We also demonstrate that over half of Ube2i is expressed in cells that are Scn9a and/or Dpysl2 negative. This illustrates that Ubc9 SUMOylates proteins other than CRMP2 in the TG, which may include transcription factors, ion channels, and signaling proteins [2]. While many peripheral sensory neurons in the DRG undergo SUMOylation and as a result have altered functionality, the role of SUMOylation in the TG is largely understudied. Although TG and DRG are homologs of each other, differences in their transcriptional profiles have been reported at baseline [41] as well as following corresponding peripheral nerve injuries [42] suggesting that TG and DRG may respond differently to nerve injury. Therefore, aside from CRMP2, Ubc9 may contribute to SUMOylation of other molecular targets in the TG that ultimately contribute to the manifestation of craniofacial pain.

Mechanistically, we have previously shown that inhibition of CRMP2 SUMOylation triggers the assembly of an endocytic protein complex around Na_V1.7, facilitating its internalization through a clathrin-mediated pathway ultimately leading to reduced surface expression and current density of Na_V1.7 [33; 53]. Similarly, 194 engages this mechanism to reduce Na_V1.7 currents by promoting clathrin-mediated internalization of Na_V1.7 through blocking SUMOylation of CRMP2 in DRGs [13] as well as in TGs as shown in this study. Bridging the gap from molecular to organismal length scales, here, using total internal reflection fluorescence (TIRF) combined with super resolution microscopy (SIM), we report that the reduction in surface expression of Na_V1.7 caused by 194 is due to decreased diffusivity of channels, observed at single molecule resolution for the first time, as well as an increased clustering of the immobile particles. Based on the observations of single Na_V1.7 channels, we can infer that 194 inhibits rapid diffusion of the receptor on the membrane, which could in turn be related to channel activity as suggested by work from Akin and colleagues [1]. This remains an avenue for further investigation.

We used electrophysiology to evaluate the functional implications of Na_V1.7/CRMP2 uncoupling, which revealed that Na_V1.7 accounts for aproximately 50% of the total sodium currents in TG, similar to what has been previously reported in DRGs (57%) [13]. Based on the prominent involvement of Na_V1.7 in TGs and the selectivity of 194 for Na_V1.7 over other Na_V channels [13], we assessed the effect of 194 on TG neurons. Overnight treatment with 194 significantly decreased the total sodium currents of TG neurons and did not show further inhibition when coapplied along with Pro-TxII, demonstrating that 194 can modulate the entirety of Na_V1.7 current in TG neurons. This is similar to the effect of 194 in DRG neurons but opposite to what has been found in nodose ganglia neurons, where CRMP2 does not regulate Nav1.7 and 194 has no effect [13; 47]. These conflicting findings point to a cell-specific mechanism of regulation of Na_V1.7 channels by CRMP2 SUMOylation.

The literature offers inconsistent findings on the levels of Na_V1.7expression in TG neurons following nerve damage. One study reported a notable rise in TG axonal mRNA Na_V1.7 levels after an infraorbital nerve entrapment model for trigeminal pain. This increase was observed along with an increase in A- and C-fiber axonal signal transmission, which was subsequently reduced following TTX treatment [60]. In contrast, another study reported a minor, though statistically insignificant, increase of Na_V1.7 protein levels in the TG, 14 days post-infrastructure orbital nerve chronic constriction injury (ION-CCI), when compared to the sham group [46]. Yet another study noted that Na_V1.7 mRNA and protein levels were diminished 12 days after ION-CCI surgery [82]. The conflicting results could potentially be attributed to varying factors, such as the timing of assessment, different pain models, or the methodologies employed to gauge Na_V1.7 expression.

The initiation of the action potentials is due to the amplification of subthreshold depolarizations provided by Na_V1.7 channels in sensory neurons [80]. AP firing could be different between the heterogeneous populations of DRG neurons and needs to be invesgtigated in reported animals usign specific types of neurons. Notwithstanding this limitation, the role of Na_V1.7 in contributing to neuronal excitability via increased action potential firing is manifested in the human gain-of-function mutations of these channels [18]. We found that inhibition of trigeminal Na_V1.7 via 194 decreased the number of evoked action potentials in TG neurons, suggesting that the inhibition of Na_V1.7 via suppression of CRMP2 SUMOylation dampens the excitability of trigeminal neurons. Comparable results were observed in lumbar dorsal horn neurons, where 194 significantly decreased the number of evoked action potentials [13]. Together, the present results support the conclusion that 194 prevents the Ubc9-CRMP2 interaction and represents an indirect path to regulate Na_V1.7 channels in TG sensory neurons.

Overall, our work demonstrates that 194 is an effective inhibitor of Na_V1.7/CRMP2 interaction, a mechanism that is conserved among different nociceptive cellular types, with efficacy in the treatment of neuropathic pain not only spinal but also of trigeminal origin. Intranasal formulation of 194 may be a palusible route to improve treatment of trigeminal neuralgia.

Data availability statement:

All data is presented in the manuscript or available in the two Supplementary Videos.