Similar excitability through different sodium channels and implications for the analgesic efficacy of selective drugs.

Nociceptive sensory neurons convey pain-related signals to the CNS using action potentials. Loss-of-function mutations in the voltage-gated sodium channel NaV1.7 cause insensitivity to pain (presumably by reducing nociceptor excitability) but clinical trials seeking to treat pain by inhibiting NaV1.7 pharmacologically have struggled. This may reflect the variable contribution of NaV1.7 to nociceptor excitability. Contrary to claims that NaV1.7 is necessary for nociceptors to initiate action potentials, we show that nociceptors can achieve similar excitability using different combinations of NaV1.3, NaV1.7, and NaV1.8. Selectively blocking one of those NaV subtypes reduces nociceptor excitability only if the other subtypes are weakly expressed. For example, excitability relies on NaV1.8 in acutely dissociated nociceptors but responsibility shifts to NaV1.7 and NaV1.3 by the fourth day in culture. A similar shift in NaV dependence occurs in vivo after inflammation, impacting ability of the NaV1.7-selective inhibitor PF-05089771 to reduce pain in behavioral tests. Flexible use of different NaV subtypes exemplifies degeneracy - achieving similar function using different components - and compromises reliable modulation of nociceptor excitability by subtype-selective inhibitors. Identifying the dominant NaV subtype to predict drug efficacy is not trivial. Degeneracy at the cellular level must be considered when choosing drug targets at the molecular level.

A novel isoquinoline alkaloid HJ-69 isolated from Zanthoxylum bungeanum attenuates inflammatory pain by inhibiting voltage-gated sodium and potassium channels.

ETHNOPHARMACOLOGY RELEVANCE: Zanthoxylum bungeanum Maxim. (Z. bungeanum), a member of the Rutaceae family, has a rich history of traditional use in Asia for treating arthritis and toothache conditions. As characteristic chemical components, numerous kinds of alkaloids have been extracted from plants and their diverse biological activities have been reported. However, research on the isoquinoline alkaloid, a specific type of alkaloids, in Z. bungeanum was scarce. AIM OF THE STUDY: The study aimed to isolate a novel isoquinoline alkaloid from Z. bungeanum and explore its pharmacological activity in vitro and analgesic activity in vivo. MATERIALS AND METHODS: Isoquinoline alkaloid isolation and identification from Z. bungeanum were conducted using chromatographic and spectroscopic methods. The whole-cell patch-clamp technique was applied to assess its impact on neuronal excitability, and endogenous voltage-gated potassium (Kv) and sodium (Nav) currents in acutely isolated mouse small-diameter dorsal root ganglion (DRG) neurons. Its inhibitory impacts on channels were further validated with HEK293 cells stably expressing Nav1.7 and Nav1.8, and Chinese hamster ovary (CHO) cells transiently expressing Kv2.1. The formalin inflammatory pain model was utilized to evaluate the potential analgesic activity in vivo. RESULTS: A novel isoquinoline alkaloid named HJ-69 (N-13-(3-methoxyprop-1-yl)rutaecarpine) was isolated and identified from Z. bungeanum for the first time. HJ-69 significantly suppressed the firing frequency and amplitudes of action potentials in DRG neurons. Consistently, it state-dependently inhibited endogenous Nav currents of DRG neurons, with half maximal inhibitory concentration (IC50) values of 13.06 ± 2.06 µM and 30.19 ± 2.07 µM for the inactivated and resting states, respectively. HJ-69 significantly suppressed potassium currents in DRG neurons, which notably inhibited the delayed rectifier potassium (IK) currents (IC50 = 6.95 ± 1.29 µM) and slightly affected the transient outward potassium (IA) currents (IC50 = 523.50 ± 39.16 µM). Furtherly, HJ-69 exhibited similar potencies on heterologously expressed Nav1.7, Nav1.8, and Kv2.1 channels, which correspondingly represent the main components in neurons. Notably, intraperitoneal administration of 30 mg/kg and 100 mg/kg HJ-69 significantly alleviated pain behaviors in the mouse inflammatory pain model induced by formalin. CONCLUSION: The study concluded that HJ-69 is a novel and active isoquinoline alkaloid, and the inhibition of Nav and Kv channels contributes to its analgesic activity. HJ-69 may be a promising prototype for future analgesic drug discovery based on the isoquinoline alkaloid.

Nav1.7 as a chondrocyte regulator and therapeutic target for osteoarthritis.

Osteoarthritis (OA) is the most common joint disease. Currently there are no effective methods that simultaneously prevent joint degeneration and reduce pain1. Although limited evidence suggests the existence of voltage-gated sodium channels (VGSCs) in chondrocytes2, their expression and function in chondrocytes and in OA remain essentially unknown. Here we identify Nav1.7 as an OA-associated VGSC and demonstrate that human OA chondrocytes express functional Nav1.7 channels, with a density of 0.1 to 0.15 channels per µm2 and 350 to 525 channels per cell. Serial genetic ablation of Nav1.7 in multiple mouse models demonstrates that Nav1.7 expressed in dorsal root ganglia neurons is involved in pain, whereas Nav1.7 in chondrocytes regulates OA progression. Pharmacological blockade of Nav1.7 with selective or clinically used pan-Nav channel blockers significantly ameliorates the progression of structural joint damage, and reduces OA pain behaviour. Mechanistically, Nav1.7 blockers regulate intracellular Ca2+ signalling and the chondrocyte secretome, which in turn affects chondrocyte biology and OA progression. Identification of Nav1.7 as a novel chondrocyte-expressed, OA-associated channel uncovers a dual target for the development of disease-modifying and non-opioid pain relief treatment for OA.

Machine learning study of the extended drug-target interaction network informed by pain related voltage-gated sodium channels.

ABSTRACT: Pain is a significant global health issue, and the current treatment options for pain management have limitations in terms of effectiveness, side effects, and potential for addiction. There is a pressing need for improved pain treatments and the development of new drugs. Voltage-gated sodium channels, particularly Nav1.3, Nav1.7, Nav1.8, and Nav1.9, play a crucial role in neuronal excitability and are predominantly expressed in the peripheral nervous system. Targeting these channels may provide a means to treat pain while minimizing central and cardiac adverse effects. In this study, we construct protein-protein interaction (PPI) networks based on pain-related sodium channels and develop a corresponding drug-target interaction network to identify potential lead compounds for pain management. To ensure reliable machine learning predictions, we carefully select 111 inhibitor data sets from a pool of more than 1000 targets in the PPI network. We employ 3 distinct machine learning algorithms combined with advanced natural language processing (NLP)-based embeddings, specifically pretrained transformer and autoencoder representations. Through a systematic screening process, we evaluate the side effects and repurposing potential of more than 150,000 drug candidates targeting Nav1.7 and Nav1.8 sodium channels. In addition, we assess the ADMET (absorption, distribution, metabolism, excretion, and toxicity) properties of these candidates to identify leads with near-optimal characteristics. Our strategy provides an innovative platform for the pharmacological development of pain treatments, offering the potential for improved efficacy and reduced side effects.

Elucidating molecular mechanisms of protoxin-II state-specific binding to the human NaV1.7 channel.

Human voltage-gated sodium (hNaV) channels are responsible for initiating and propagating action potentials in excitable cells, and mutations have been associated with numerous cardiac and neurological disorders. hNaV1.7 channels are expressed in peripheral neurons and are promising targets for pain therapy. The tarantula venom peptide protoxin-II (PTx2) has high selectivity for hNaV1.7 and is a valuable scaffold for designing novel therapeutics to treat pain. Here, we used computational modeling to study the molecular mechanisms of the state-dependent binding of PTx2 to hNaV1.7 voltage-sensing domains (VSDs). Using Rosetta structural modeling methods, we constructed atomistic models of the hNaV1.7 VSD II and IV in the activated and deactivated states with docked PTx2. We then performed microsecond-long all-atom molecular dynamics (MD) simulations of the systems in hydrated lipid bilayers. Our simulations revealed that PTx2 binds most favorably to the deactivated VSD II and activated VSD IV. These state-specific interactions are mediated primarily by PTx2's residues R22, K26, K27, K28, and W30 with VSD and the surrounding membrane lipids. Our work revealed important protein-protein and protein-lipid contacts that contribute to high-affinity state-dependent toxin interaction with the channel. The workflow presented will prove useful for designing novel peptides with improved selectivity and potency for more effective and safe treatment of pain.

Structural basis for severe pain caused by mutations in the voltage sensors of sodium channel NaV1.7.

Voltage-gated sodium channels in peripheral nerves conduct nociceptive signals from nerve endings to the spinal cord. Mutations in voltage-gated sodium channel NaV1.7 are responsible for a number of severe inherited pain syndromes, including inherited erythromelalgia (IEM). Here, we describe the negative shifts in the voltage dependence of activation in the bacterial sodium channel NaVAb as a result of the incorporation of four different IEM mutations in the voltage sensor, which recapitulate the gain-of-function effects observed with these mutations in human NaV1.7. Crystal structures of NaVAb with these IEM mutations revealed that a mutation in the S1 segment of the voltage sensor facilitated the outward movement of S4 gating charges by widening the pathway for gating charge translocation. In contrast, mutations in the S4 segments modified hydrophobic interactions with surrounding amino acid side chains or membrane phospholipids that would enhance the outward movement of the gating charges. These results provide key structural insights into the mechanisms by which these IEM mutations in the voltage sensors can facilitate outward movements of the gating charges in the S4 segment and cause hyperexcitability and severe pain in IEM. Our work gives new insights into IEM pathogenesis at the near-atomic level and provides a molecular model for mutation-specific therapy of this debilitating disease.

Evaluated periodontal tissues and oxidative stress in rats with neuropathic pain-like behavior.

BACKGROUND: Oxidative stress has a critical effect on both persistent pain states and periodontal disease. Voltage-gated sodium NaV1.7 (SCN9A), and transient receptor potential ankyrin 1 (TRPA1) are pain genes. The goal of this study was to investigate oxidative stress markers, periodontal status, SCN9A, and TRPA1 channel expression in periodontal tissues of rats with paclitaxel-induced neuropathic pain-like behavior (NPLB). METHODS AND RESULTS: Totally 16 male Sprague Dawley rats were used: control (n = 8) and paclitaxel-induced pain (PTX) (n = 8). The alveolar bone loss and 8-hydroxy-2-deoxyguanosine (8-OHdG) levels were analyzed histometrically and immunohistochemically. Gingival superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activities (spectrophotometric assay) were measured. The relative TRPA1 and SCN9A genes expression levels were evaluated using quantitative real-time PCR (qPCR) in the tissues of gingiva and brain. The PTX group had significantly higher alveolar bone loss and 8-OHdG compared to the control. The PTX group had significantly lower gingival SOD, GPx and CAT activity than the control groups. The PTX group had significantly higher relative gene expression of SCN9A (p = 0.0002) and TRPA1 (p = 0.0002) than the control in gingival tissues. Increased nociceptive susceptibility may affect the increase in oxidative stress and periodontal destruction. CONCLUSIONS: Chronic pain conditions may increase TRPA1 and SCN9A gene expression in the periodontium. The data of the current study may help develop novel approaches both to maintain periodontal health and alleviate pain in patients suffering from orofacial pain.

Meta-Analysis of Mechano-Sensitive Ion Channels in Human Hearts: Chamber- and Disease-Preferential mRNA Expression.

The cardiac cell mechanical environment changes on a beat-by-beat basis as well as in the course of various cardiac diseases. Cells sense and respond to mechanical cues via specialized mechano-sensors initiating adaptive signaling cascades. With the aim of revealing new candidates underlying mechano-transduction relevant to cardiac diseases, we investigated mechano-sensitive ion channels (MSC) in human hearts for their chamber- and disease-preferential mRNA expression. Based on a meta-analysis of RNA sequencing studies, we compared the mRNA expression levels of MSC in human atrial and ventricular tissue samples from transplant donor hearts (no cardiac disease), and from patients in sinus rhythm (underlying diseases: heart failure, coronary artery disease, heart valve disease) or with atrial fibrillation. Our results suggest that a number of MSC genes are expressed chamber preferentially, e.g., CHRNE in the atria (compared to the ventricles), TRPV4 in the right atrium (compared to the left atrium), CACNA1B and KCNMB1 in the left atrium (compared to the right atrium), as well as KCNK2 and KCNJ2 in ventricles (compared to the atria). Furthermore, 15 MSC genes are differentially expressed in cardiac disease, out of which SCN9A (lower expressed in heart failure compared to donor tissue) and KCNQ5 (lower expressed in atrial fibrillation compared to sinus rhythm) show a more than twofold difference, indicative of possible functional relevance. Thus, we provide an overview of cardiac MSC mRNA expression in the four cardiac chambers from patients with different cardiac diseases. We suggest that the observed differences in MSC mRNA expression may identify candidates involved in altered mechano-transduction in the respective diseases.

Identification and targeting of a unique NaV1.7 domain driving chronic pain.

Small molecules directly targeting the voltage-gated sodium channel (VGSC) NaV1.7 have not been clinically successful. We reported that preventing the addition of a small ubiquitin-like modifier onto the NaV1.7-interacting cytosolic collapsin response mediator protein 2 (CRMP2) blocked NaV1.7 function and was antinociceptive in rodent models of neuropathic pain. Here, we discovered a CRMP2 regulatory sequence (CRS) unique to NaV1.7 that is essential for this regulatory coupling. CRMP2 preferentially bound to the NaV1.7 CRS over other NaV isoforms. Substitution of the NaV1.7 CRS with the homologous domains from the other eight VGSC isoforms decreased NaV1.7 currents. A cell-penetrant decoy peptide corresponding to the NaV1.7-CRS reduced NaV1.7 currents and trafficking, decreased presynaptic NaV1.7 expression, reduced spinal CGRP release, and reversed nerve injury-induced mechanical allodynia. Importantly, the NaV1.7-CRS peptide did not produce motor impairment, nor did it alter physiological pain sensation, which is essential for survival. As a proof-of-concept for a NaV1.7 -targeted gene therapy, we packaged a plasmid encoding the NaV1.7-CRS in an AAV virus. Treatment with this virus reduced NaV1.7 function in both rodent and rhesus macaque sensory neurons. This gene therapy reversed and prevented mechanical allodynia in a model of nerve injury and reversed mechanical and cold allodynia in a model of chemotherapy-induced peripheral neuropathy. These findings support the conclusion that the CRS domain is a targetable region for the treatment of chronic neuropathic pain.

Pain-causing stinging nettle toxins target TMEM233 to modulate NaV1.7 function.

Voltage-gated sodium (NaV) channels are critical regulators of neuronal excitability and are targeted by many toxins that directly interact with the pore-forming α subunit, typically via extracellular loops of the voltage-sensing domains, or residues forming part of the pore domain. Excelsatoxin A (ExTxA), a pain-causing knottin peptide from the Australian stinging tree Dendrocnide excelsa, is the first reported plant-derived NaV channel modulating peptide toxin. Here we show that TMEM233, a member of the dispanin family of transmembrane proteins expressed in sensory neurons, is essential for pharmacological activity of ExTxA at NaV channels, and that co-expression of TMEM233 modulates the gating properties of NaV1.7. These findings identify TMEM233 as a previously unknown NaV1.7-interacting protein, position TMEM233 and the dispanins as accessory proteins that are indispensable for toxin-mediated effects on NaV channel gating, and provide important insights into the function of NaV channels in sensory neurons.

Structural basis for severe pain caused by mutations in the S4-S5 linkers of voltage-gated sodium channel NaV1.7.

Gain-of-function mutations in voltage-gated sodium channel NaV1.7 cause severe inherited pain syndromes, including inherited erythromelalgia (IEM). The structural basis of these disease mutations, however, remains elusive. Here, we focused on three mutations that all substitute threonine residues in the alpha-helical S4-S5 intracellular linker that connects the voltage sensor to the pore: NaV1.7/I234T, NaV1.7/I848T, and NaV1.7/S241T in order of their positions in the amino acid sequence within the S4-S5 linkers. Introduction of these IEM mutations into the ancestral bacterial sodium channel NaVAb recapitulated the pathogenic gain-of-function of these mutants by inducing a negative shift in the voltage dependence of activation and slowing the kinetics of inactivation. Remarkably, our structural analysis reveals a common mechanism of action among the three mutations, in which the mutant threonine residues create new hydrogen bonds between the S4-S5 linker and the pore-lining S5 or S6 segment in the pore module. Because the S4-S5 linkers couple voltage sensor movements to pore opening, these newly formed hydrogen bonds would stabilize the activated state substantially and thereby promote the 8 to 18 mV negative shift in the voltage dependence of activation that is characteristic of the NaV1.7 IEM mutants. Our results provide key structural insights into how IEM mutations in the S4-S5 linkers may cause hyperexcitability of NaV1.7 and lead to severe pain in this debilitating disease.

Integrative miRNA-mRNA profiling of human epidermis: unique signature of SCN9A painful neuropathy.

Personalized management of neuropathic pain is an unmet clinical need due to heterogeneity of the underlying aetiologies, incompletely understood pathophysiological mechanisms and limited efficacy of existing treatments. Recent studies on microRNA in pain preclinical models have begun to yield insights into pain-related mechanisms, identifying nociception-related species differences and pinpointing potential drug candidates. With the aim of bridging the translational gap towards the clinic, we generated a human pain-related integrative miRNA and mRNA molecular profile of the epidermis, the tissue hosting small nerve fibres, in a deeply phenotyped cohort of patients with sodium channel-related painful neuropathy not responding to currently available therapies. We identified four miRNAs strongly discriminating patients from healthy individuals, confirming their effect on differentially expressed gene targets driving peripheral sensory transduction, transmission, modulation and post-transcriptional modifications, with strong effects on gene targets including NEDD4. We identified a complex epidermal miRNA-mRNA network based on tissue-specific experimental data suggesting a cross-talk between epidermal cells and axons in neuropathy pain. Using immunofluorescence assay and confocal microscopy, we observed that Nav1.7 signal intensity in keratinocytes strongly inversely correlated with NEDD4 expression that was downregulated by miR-30 family, suggesting post-transcriptional fine tuning of pain-related protein expression. Our targeted molecular profiling advances the understanding of specific neuropathic pain fine signatures and may accelerate process towards personalized medicine in patients with neuropathic pain.

A severe case of primary erythromelalgia presenting as small fiber neuropathy with a novel SCN9A mutation.

Primary erythromelalgia (PEM) is a rare condition characterized by severe burning pain, erythema, and increased temperature in the extremeties. Mutations in the Nav1.7 sodium channel encoded by the SCN9A are responsible for PEM. The pathophysiology of PEM is unclear, but the involvement of neurogenic and vasogenic mechanisms has been suggested. Here we report a case of severe PEM in a 9-year-old child with a novel SCN9A mutation and examine the distribution of nerve fibers and expression of neuropeptides in the affected skin. Gene mutation analysis revealed a novel mutation p.L951I (c.2851C>A) in the heterozygous form of the SCN9A. An immunofluorescence study showed that intraepidermal nerve fibers were decreased in the affected leg, suggesting small fiber neuropathy. There was no increase in the expression of substance P (SP) or calcitonin gene-related peptide (CGRP) in the lesional skin tissue. These findings suggest SP and CGRP do not play a major role in the pathophysiology of primary erythromelalgia.

The fates of internalized NaV1.7 channels in sensory neurons: Retrograde cotransport with other ion channels, axon-specific recycling, and degradation.

Neuronal function relies on the maintenance of appropriate levels of various ion channels at the cell membrane, which is accomplished by balancing secretory, degradative, and recycling pathways. Neuronal function further depends on membrane specialization through polarized distribution of specific proteins to distinct neuronal compartments such as axons. Voltage-gated sodium channel NaV1.7, a threshold channel for firing action potentials in nociceptors, plays a major role in human pain, and its abundance in the plasma membrane is tightly regulated. We have recently characterized the anterograde axonal trafficking of NaV1.7 channels in Rab6A-positive vesicles, but the fate of internalized channels is not known. Membrane proteins that have undergone endocytosis can be directed into multiple pathways including those for degradation, recycling to the membrane, and transcytosis. Here, we demonstrate NaV1.7 endocytosis and dynein-dependent retrograde trafficking in Rab7-containing late endosomes together with other axonal membrane proteins using real-time imaging of live neurons. We show that some internalized NaV1.7 channels are delivered to lysosomes within the cell body, and that there is no evidence for NaV1.7 transcytosis. In addition, we show that NaV1.7 is recycled specifically to the axonal membrane as opposed to the soma membrane, suggesting a novel mechanism for the development of neuronal polarity. Together, these results shed light on the mechanisms by which neurons maintain excitable membranes and may inform efforts to target ion channel trafficking for the treatment of disorders of excitability.

NaV1.7 Channel Blocker [Ala5, Phe6, Leu26, Arg28]GpTx-1 Attenuates CFA-induced Inflammatory Hypersensitivity in Rats via Endogenous Enkephalin Mechanism.

Venom-derived NaV1.7 channel blockers have promising prospects in pain management. The 34-residue tarantula peptide GpTx-1 is a potent NaV1.7 channel blocker. Its powerful analog [Ala5, Phe6, Leu26, Arg28]GpTx-1 (GpTx-1-71) displayed excellent NaV1.7 selectivity and analgesic properties in mice. The current study aimed to elucidate the anti-hyperalgesic activities of GpTx-1-71 in inflammatory pain and reveal the underlying mechanisms. Our results demonstrated that intrathecal and intraplantar injections of GpTx-1-71 dose-dependently attenuated CFA-induced inflammatory hypersensitivity in rats. Moreover, GpTx-1-71-induced anti-hyperalgesia was significantly reduced by opioid receptor antagonists and the enkephalin antibody and diminished in proenkephalin (Penk) gene knockout animals. Consistently, GpTx-1-71 treatment increased the enkephalin level in the spinal dorsal horn and promoted the Penk transcription and enkephalin release in primary dorsal root ganglion (DRG) neurons, wherein sodium played a crucial role in these processes. Mass spectrometry analysis revealed that GpTx-1-71 mainly promoted the secretion of Met-enkephalin but not Leu-enkephalin from DRG neurons. In addition, the combination of subtherapeutic Met-enkephalin and GpTx-1-71 produced synergistic anti-hyperalgesia in CFA-induced inflammatory hypersensitivity. These findings suggest that the endogenous enkephalin pathway is essential for GpTx-1-71-induced spinal and peripheral analgesia in inflammatory pain. PERSPECTIVE: This article presents a possible pharmacological mechanism underlying NaV1.7 blocker-induced analgesia in inflammatory pain, which helps us to better understand and develop venom-based painkillers for incurable pain.

Polysorbate 80 blocked a peripheral sodium channel, Nav1.7, and reduced neuronal excitability.

Polysorbate 80 is a non-ionic detergent derived from polyethoxylated sorbitan and oleic acid. It is widely used in pharmaceuticals, foods, and cosmetics as an emulsifier. Nav1.7 is a peripheral sodium channel that is highly expressed in sympathetic and sensory neurons, and it plays a critical role in determining the threshold of action potentials (APs). We found that 10 µg/mL polysorbate 80 either abolished APs or increased the threshold of the APs of dorsal root ganglions. We thus investigated whether polysorbate 80 inhibits Nav1.7 sodium current using a whole-cell patch-clamp recording technique. Polysorbate 80 decreased the Nav1.7 current in a concentration-dependent manner with a half-maximal inhibitory concentration (IC50) of 250.4 µg/mL at a holding potential of -120 mV. However, the IC50 was 1.1 µg/mL at a holding potential of -90 mV and was estimated to be 0.9 µg/mL at the resting potentials of neurons, where most channels are inactivated. The activation rate and the voltage dependency of activation of Nav1.7 were not changed by polysorbate 80. However, polysorbate 80 caused hyperpolarizing shifts in the voltage dependency of the steady-state fast inactivation curve. The blocking of Nav1.7 currents by polysorbate 80 was not reversible at a holding potential of -90 mV but was completely reversible at -120 mV, where the channels were mostly in the closed state. Polysorbate 80 also slowed recovery from inactivation and induced robust use-dependent inhibition, indicating that it is likely to bind to and stabilize the inactivated state. Our results indicate that polysorbate 80 inhibits Nav1.7 current in concentration-, state-, and use-dependent manners when used even below commercial concentrations. This suggests that polysorbate 80 may be helpful in pain medicine as an excipient. In addition, in vitro experiments using polysorbate 80 with neurons should be conducted with caution.

Reversal of Peripheral Neuropathic Pain by the Small-Molecule Natural Product Narirutin via Block of Nav1.7 Voltage-Gated Sodium Channel.

Neuropathic pain is a refractory chronic disease affecting millions of people worldwide. Given that present painkillers have poor efficacy or severe side effects, developing novel analgesics is badly needed. The multiplex structure of active ingredients isolated from natural products provides a new source for phytochemical compound synthesis. Here, we identified a natural product, Narirutin, a flavonoid compound isolated from the Citrus unshiu, showing antinociceptive effects in rodent models of neuropathic pain. Using calcium imaging, whole-cell electrophysiology, western blotting, and immunofluorescence, we uncovered a molecular target for Narirutin's antinociceptive actions. We found that Narirutin (i) inhibits Veratridine-triggered nociceptor activities in L4-L6 rat dorsal root ganglion (DRG) neurons, (ii) blocks voltage-gated sodium (NaV) channels subtype 1.7 in both small-diameter DRG nociceptive neurons and human embryonic kidney (HEK) 293 cell line, (iii) does not affect tetrodotoxin-resistant (TTX-R) NaV channels, and (iv) blunts the upregulation of Nav1.7 in calcitonin gene-related peptide (CGRP)-labeled DRG sensory neurons after spared nerve injury (SNI) surgery. Identifying Nav1.7 as a molecular target of Narirutin may further clarify the analgesic mechanism of natural flavonoid compounds and provide an optimal idea to produce novel selective and efficient analgesic drugs.

Derivation of nociceptive sensory neurons from hiPSCs with early patterning and temporally controlled NEUROG2 overexpression.

Despite development of protocols to differentiate human pluripotent stem cells (hPSCs), those used to produce sensory neurons remain difficult to replicate and result in heterogenous populations. There is a growing clinical burden of chronic pain conditions, highlighting the need for relevant human cellular models. This study presents a hybrid differentiation method to produce nociceptive sensory neurons from hPSCs. Lines harboring an inducible NEUROG2 construct were patterned toward precursors with small molecules followed by NEUROG2 overexpression. Neurons expressed key markers, including BRN3A and ISL1, with single-cell RNA sequencing, revealing populations of nociceptors expressing SCN9A and TRP channels. Physiological profiling with multi-electrode arrays revealed that neurons responded to noxious stimuli, including capsaicin. Finally, we modeled pain-like states to identify genes and pathways involved in pain transduction. This study presents an optimized method to efficiently produce nociceptive sensory neurons and provides a tool to aid development of chronic pain research.

Exploring the neurogenic differentiation of human dental pulp stem cells.

Human dental pulp stem cells (hDPSCs) have increasingly gained interest as a potential therapy for nerve regeneration in medicine and dentistry, however their neurogenic potential remains a matter of debate. This study aimed to characterize hDPSC neuronal differentiation in comparison with the human SH-SY5Y neuronal stem cell differentiation model. Both hDPSCs and SH-SY5Y could be differentiated to generate typical neuronal-like cells following sequential treatment with all-trans retinoic acid (ATRA) and brain-derived neurotrophic factor (BDNF), as evidenced by significant expression of neuronal proteins ßIII-tubulin (TUBB3) and neurofilament medium (NF-M). Both cell types also expressed multiple neural gene markers including growth-associated protein 43 (GAP43), enolase 2/neuron-specific enolase (ENO2/NSE), synapsin I (SYN1), nestin (NES), and peripherin (PRPH), and exhibited measurable voltage-activated Na+ and K+ currents. In hDPSCs, upregulation of acetylcholinesterase (ACHE), choline O-acetyltransferase (CHAT), sodium channel alpha subunit 9 (SCN9A), POU class 4 homeobox 1 (POU4F1/BRN3A) along with a downregulation of motor neuron and pancreas homeobox 1 (MNX1) indicated that differentiation was more guided toward a cholinergic sensory neuronal lineage. Furthermore, the Extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor U0126 significantly impaired hDPSC neuronal differentiation and was associated with reduction of the ERK1/2 phosphorylation. In conclusion, this study demonstrates that extracellular signal-regulated kinase/Mitogen-activated protein kinase (ERK/MAPK) is necessary for sensory cholinergic neuronal differentiation of hDPSCs. hDPSC-derived cholinergic sensory neuronal-like cells represent a novel model and potential source for neuronal regeneration therapies.