Dorsal Root Ganglion Neurons Research Articles

Fundamentals of intervertebral disc degeneration and related discogenic pain

Lumbar intervertebral disc degeneration is thought to be the main cause of low back pain, although the mechanisms by which it occurs and leads to pain remain unclear. In healthy adult discs, vessels and nerves are present only in the outer layer of the annulus fibrosus and in the bony endplate. Animal models, and histological and biomechanical studies have shown that annulus tear or endplate injury is the initiating factor for painful disc degeneration. Injury to the disc triggers a local inflammatory repair response that activates nociceptors and promotes the synthesis of neuropeptides such as substance P and calcitonin gene-related peptide, by dorsal root ganglion neurons. These neuropeptides are transported to injured discs and act as pro-inflammatory molecules, promoting the production of an “inflammatory soup” by inducing vasodilatation and plasma extravasation as well as by promoting the release of chemical mediators from disc cells and infiltrating immune cells, causing neurogenic inflammation that leads to progressive disc degeneration and discogenic pain.

Histone Methyltransferase G9a in Primary Sensory Neurons Promotes Inflammatory Pain and Transcription of Trpa1 and Trpv1 via Bivalent Histone Modifications.

Transient receptor potential ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1) channels are crucial for detecting and transmitting nociceptive stimuli. Inflammatory pain is associated with sustained increases in TRPA1 and TRPV1 expression in primary sensory neurons. However, the epigenetic mechanisms driving this upregulation remain unknown. G9a (encoded by Ehmt2) catalyzes H3K9me2 and generally represses gene transcription. In this study, we found that intrathecal administration of UNC0638, a specific G9a inhibitor, or G9a-specific siRNA, substantially reduced complete Freund's adjuvant (CFA)-induced pain hypersensitivity. Remarkably, CFA treatment did not induce persistent pain hypersensitivity in male and female mice with conditional Ehmt2 knockout in dorsal root ganglion (DRG) neurons. RNA sequencing and quantitative PCR analyses showed that CFA treatment caused a sustained increase in mRNA levels of Trpa1 and Trpv1 in the DRG. Ehmt2 knockout in DRG neurons elevated baseline Trpa1 and Trpv1 mRNA levels but notably reversed CFA-induced increases in their expression. Chromatin immunoprecipitation revealed that CFA treatment reduced G9a and H3K9me2 levels while increasing H3K9ac and H3K4me3-activating histone marks-at Trpa1 and Trpv1 promoters in the DRG. Strikingly, conditional Ehmt2 knockout in DRG neurons not only diminished H3K9me2 but also reversed CFA-induced increases in H3K9ac and H3K4me3 at Trpa1 and Trpv1 promoters. Our findings suggest that G9a in primary sensory neurons constitutively represses Trpa1 and Trpv1 transcription under normal conditions but paradoxically enhances their transcription during tissue inflammation. This latter action accounts for inflammation-induced TRPA1 and TRPV1 upregulation in the DRG. Thus, G9a could be targeted for alleviating persistent inflammatory pain.Significance statement This study demonstrates for the first time that G9a, a histone methyltransferase, in sensory neurons is crucial for the persistent pain development following inflammation. Inhibiting or knockdown of G9a at the spinal cord level reduced inflammation-induced pain hypersensitivity. Remarkably, mice lacking G9a in sensory neurons failed to develop persistent pain hypersensitivity after inflammation. Ablating G9a in sensory neurons increased baseline expression of the TRPA1 and TRPV1 ion channels but reversed their upregulation induced by inflammation. Additionally, G9a deletion blocked the inflammation-driven enrichment of activating histone marks at Trpa1 and Trpv1 promoters. These findings highlight a dual role of G9a in sensory neurons: suppressing Trpa1 and Trpv1 transcription under normal conditions while promoting their transcription during inflammation through bivalent histone modifications.

Action potential–independent spontaneous microdomain Ca2+ transients–mediated continuous neurotransmission regulates hyperalgesia

Neurotransmitters and neuromodulators can be released via either action potential (AP)-evoked transient or AP-independent continuous neurotransmission. The elevated AP-evoked neurotransmission in the primary sensory neurons plays crucial roles in hyperalgesia. However, whether and how the AP-independent continuous neurotransmission contributes to hyperalgesia remains largely unknown. Here, we show that primary sensory dorsal root ganglion (DRG) neurons exhibit frequent spontaneous microdomain Ca2+ (smCa) activities independent of APs across the cell bodies and axons, which are mediated by the spontaneous opening of TRPA1 channels and trigger continuous neurotransmission via the cyclic adenosine monophosphate-protein kinase A signaling pathway. More importantly, the frequency of smCa activity and its triggered continuous neurotransmission in DRG neurons increased dramatically in mice experiencing inflammatory pain, inhibition of which alleviates hyperalgesia. Collectively, this work revealed the AP-independent continuous neurotransmission triggered by smCa activities in DRG neurons, which may serve as a unique mechanism underlying the nociceptive sensitization in hyperalgesia and offer a potential target for the treatment of chronic pain.

An atlas of neuropathic pain-associated molecular pathological characteristics in the mouse spinal cord

Peripheral nerve injury (PNI)-induced neuropathic pain (NP) is a severe disease with high prevalence in clinics. Gene reprogramming and tissue remodeling in the dorsal root ganglia (DRG) and spinal cord (SC) drive the development and maintenance of neuropathic pain (NP). However, our understanding of the NP-associated spatial molecular processing landscape of SC and the non-synaptic interactions between DRG neurons and SC cells remains limited. We here integrate spatial transcriptomics (ST) with single-nucleus RNA-sequencing (snRNA-seq) and bulk RNA-sequencing (bulk RNA-seq) to characterize regional pathological heterogeneity of the SC under NP conditions. First, the SC of NP mice manifests unique spatial atlases of genes, cell populations, cell-cell cross-talks, signaling pathways, and transcriptional regulatory networks compared to sham mice. We further report that injured DRG sensory neurons and the corresponding ventral horn of the SC show similar expression patterns after PNI. In addition, for the first time, we systematically exhibit “cross-talk omics” between the DRG neurons and SC dorsal horn neurons and glial cells, indicating an altered communication profile under NP conditions. Together, our findings decode the spatial and cellular heterogeneity of molecular pathological mechanisms underlying NP, providing a foundation for designing therapeutic targets for this disorder.

Piezoelectric Nanoarrays with Mechanical-Electrical Coupling Microenvironment for Innervated Bone Regeneration.

The involvement of neurons in the peripheral nervous system is crucial for bone regeneration. Mimicking extracellular matrix cues provides a more direct and effective strategy to regulate neuronal activity and enhance bone regeneration. However, the simultaneous coupling of the intrinsic mechanical-electrical microenvironment of implants to regulate innervated bone regeneration has been largely neglected. Inspired by the mechanical and bioelectric properties of the bone microenvironment, this study constructed a mechanical-electrical coupling microenvironment (M-E) model based on barium titanate piezoelectric nanoarrays, which could effectively promote innervated bone regeneration. The study found that the mechanical microenvironment provided by the nanostructure, coupled with the electrical microenvironment provided by the piezoelectric properties, created a controllable M-E. In vitro cell experiments demonstrated that this coupled microenvironment activated Piezo2 and VGCC ion channels, promoted calcium influx in DRG neurons, and activated downstream PI3K-AKT and RAS pathways. This cascade of events led to the synthesis and release of CGRP in sensory nerves, ultimately enhancing the osteogenic differentiation of BMSCs. This work not only broadens the current understanding of biomaterials that mimic the bone extracellular matrix but also provides new insights into innervated bone regeneration.

Research on Ion Channels in Dorsal Root Ganglion Neurons

Dorsal Root Ganglion (DRG) neurons serve as crucial nodes for pain signal transmission, and their excitability regulation plays a key role in various physiological and pathological processes. Ion channels, as central components in modulating DRG neuron function, are involved in biological processes such as pain conduction, neural signal integration, and plasticity. This paper systematically analyzes the structural characteristics, functional mechanisms, and roles of different types of ion channels in DRG neurons, including sodium channels, potassium channels, calcium channels, and other related channels, particularly in pathological conditions. In light of current research trends, the paper also explores the potential of ion channels as drug targets and in technological applications, while suggesting future research directions. The aim is to provide theoretical support for the diagnosis and treatment of diseases related to DRG neurons.

Research Progress on Opioid Receptors and Ion Channels in Dorsal Root Ganglion Neurons

Dorsal root ganglion (DRG) neurons play a crucial role in the perception and modulation of pain signals, making them a critical focus in pain mechanism research. Opioid receptors, as classical analgesic targets, inhibit pain signals by regulating ion channels such as calcium channels, while ion channels are directly involved in the generation and transmission of pain signals. In recent years, the synergistic interaction between opioid receptors and ion channels has become a research hotspot, particularly under pathological pain conditions, where their complex interplay provides key insights into analgesic drug development. This paper systematically reviews the distribution and functional mechanisms of opioid receptors and ion channels in DRG neurons, explores their interaction in pain modulation, and discusses functional changes under inflammatory and neuropathic pain conditions. Additionally, it examines potential targeted therapeutic strategies based on their synergy and outlines their clinical application prospects.

Piezo Ion Channels and Their Association With Haptic Technology Use: A Narrative Review.

The recent identification of Piezo ion channels demonstrating a mechano-sensitive impact on neurons revealed distinct Piezo-1 and 2 types. While Piezo-1 predominates in neurons linked to non-sensory stimulation, such as pressure in blood vessels, Piezo-2 predominates in neurons linked to sensory stimulation, such as touch. Piezo-1 and 2 have a major bidirectional impact on transient receptor potential (TRP) ion channels, and TRPs also impact neurotransmitter release. Particularly existent in dorsal root ganglion (DRG) neurons, which are located in nerve endings, Piezo-2 is a key DRG activator. Subsequent Piezo findings have been vital to recent medical haptic technology developments, in tandem with breakthroughs in the emerging neurology subfield of connectomics plus AI developments. Included in this review are a historical Piezo overview, the interrelationship of Piezo channels with TRPs, inclusive of TRPV1/TRPV8, the impact on medical and rehab haptic technology, a focus on haptic technology use in stroke survivor rehab inclusive of pain mitigation, and the development of a haptic technology patch aimed at alleviating pain and/or anxiety. Neurogenic pain resulting from hyperalgesia/allodynia in stroke survivors is a potential target for drugs and haptics aimed at pain reduction; patients experiencing neuropathic or psychosomatic pain are other prime targets. Increased Piezo knowledge may promote more precisely targeted haptic therapeutic developments.

Gap junction intercellular communications regulates activation of SARM1 and protects against axonal degeneration

Sterile alpha and Toll/interleukin-1 receptor motif containing 1 (SARM1), a nicotinamide adenine dinucleotide (NAD)-utilizing enzyme, mediates axon degeneration (AxD) in various neurodegenerative diseases. It is activated by nicotinamide mononucleotide (NMN) to produce a calcium messenger, cyclic ADP-ribose (cADPR). This activity is blocked by elevated NAD level. Here, we verified this metabolic regulation in somatic HEK-293T cells by overexpressing NMN-adenyltransferase to elevate cellular NAD, which resulted not only in inhibition of their own SARM1 from producing cADPR but, surprisingly, also in the 5–10 neighboring wildtype cells in mixed cultures via connexin (Cx)-43. Direct visualization of gap junction intercellular communication (GJIC) was achieved by incubating cells with a permeant probe, PC11, which is converted by SARM1 into PAD11, a fluorescent NAD analog capable of traversing GJs. Extending the findings to dorsal root ganglion neurons, we further showed that CZ-48, a permeant NMN analog, or axotomy, activated SARM1 and the produced PAD11 was transferred to contacting axons via GJIC. The gap junction involved was identified as Cx36 instead. This neuronal GJIC was demonstrated to be functional, enabling healthy neurons to protect adjacent axotomized axons from degeneration. Inhibition of GJIC in mice by AAV-PHP.eB-mediated knockdown of Cx36 in brain induced neuroinflammation, which in turn activated SARM1 and resulted in axon degeneration as well as behavioral deficits. Our results demonstrate a novel intercellular regulation mechanism of SARM1 and reveal a protective role of healthy tissue against AxD induced by injury or neuroinflammation.

Electroacupuncture alleviates paclitaxel-induced peripheral neuropathy by reducing CCL2-mediated macrophage infiltration in sensory ganglia and sciatic nerve

BackgroundPaclitaxel-induced peripheral neuropathy (PIPN) is prevalent among patients receiving paclitaxel chemotherapy, which results in sensory abnormality as well as neuropathic pain. Conventional medications lack effectiveness on PIPN. Clinical trials identified beneficial effects of acupuncture on PIPN among patients receiving chemotherapy. Here we explored the mechanisms underlying how acupuncture might alleviate PIPN.MethodsA mouse model of PIPN was established by repeated paclitaxel application. Electroacupuncture (EA) was applied at ST36 and BL60 acupoints of model mice. Immunostaining, flow cytometry, behavioral assay, in vivo imaging were utilized for effects determination and mechanism exploration.ResultsEA ameliorated mechanical and cold pain hypersensitivities, reduced sensory neuron damage and improved loss in intra-epidermal nerve fibers (IENFs) in model mice. Macrophages infiltration were detected in DRG and sciatic nerve of model mice, which was reduced by EA. EA affected M1-like pro-inflammatory macrophage infiltration in DRG, whereas it did not affect M2-like macrophages. DRG neurons released chemoattractant CCL2 that recruited macrophages via CCR2 to DRG. EA reduced CCL2 overproduction by DRG neurons and reduced macrophage infiltration. Blocking CCR2 mimicked EA’s anti-allodynic effect, whereas exogenously applying recombinant CCL2 reversed the ameliorative effect of EA on macrophage infiltration and abolished EA’s anti-allodynia on model mice. EA ameliorated other signs of PIPN, including sensory neuron damage, sciatic nerve morphology impairment and IENFs loss. In mice inoculated with breast cancer cells, EA didn’t affect paclitaxel-induced antitumor effect.ConclusionsThese findings suggest EA alleviates PIPN by reducing CCL2/CCR2 mediated-pro-inflammatory macrophage infiltration into sensory ganglia as well as the sciatic nerve. Our study supports EA could be used as a potential non-pharmacological therapy for PIPN.

Protocol to study neuronal membrane proteasome function in mouse peripheral sensory neurons.

Neuronal membrane proteasomes (NMPs) are expressed on a subset of somatosensory dorsal root ganglion (DRG) neurons and influence mechanical and pain sensitivity. Here, we present a protocol for studying NMP function in mouse peripheral sensory neurons. We describe steps for procuring and culturing primary DRG neurons. We then detail biochemical and antibody feeding approaches to analyze NMP expression and localization. Finally, we include Ca2+ imaging techniques for investigating NMP function in DRG neurons. For complete details on the use and execution of this protocol, please refer to Villalón Landeros etal.1.

Maladaptive changes in the homeostasis of AEA-TRPV1/CB1R induces pain-related hyperactivity of nociceptors after spinal cord injury

BackgroundNeuropathic pain resulting from spinal cord injury (SCI) is associated with persistent hyperactivity of primary nociceptors. Anandamide (AEA) has been reported to modulate neuronal excitability and synaptic transmission through activation of cannabinoid type-1 receptors (CB1Rs) and transient receptor potential vanilloid 1 (TRPV1). However, the role of AEA and these receptors in the hyperactivity of nociceptors after SCI remains unclear.ResultsIn this study, we investigated the effects of AEA and its receptors on the hyperexcitability of mouse dorsal root ganglion (DRG) neurons after SCI. Using a whole-cell patch-clamp technique, we found that the timing of SCI-induced hyperexcitability in nociceptors paralleled an increase in the endocannabinoid AEA content. The expression of TRPV1 and CB1R was also upregulated at different time points after SCI. High-dose extracellular administration of AEA increased the excitability of naive DRG neurons, leading to the transition from a rapidly accommodating (RA) hypoexcitable state to a highly excitable non-accommodating (NA) state. These AEA-induced transitions were facilitated by increased TRPV1 transcription. Pharmacological and Ca2+ imaging experiments revealed that AEA induced hyperexcitability in nociceptors after SCI via the AEA-TRPV1-Ca2+ pathway, whereas activation of CB1Rs reduced SCI-induced hyperexcitability and maintained cytosolic Ca2+ concentration ([Ca2+]cyto) at low levels in the early stages of SCI. As the AEA and TRPV1 levels increased after SCI, adaptive neuroprotection transitioned to a maladaptive hyperactive state, leading to sustained pain.ConclusionsTaken together, this study provides new insights into how endocannabinoids regulate nociceptor activity after SCI, offering potential targets for the treatment of neuropathic pain.

Keratinocyte-derived extracellular vesicles in painful diabetic neuropathy.

Painful diabetic neuropathy (PDN) is a challenging complication of diabetes with patients experiencing a painful and burning sensation in their extremities. Existing treatments provide limited relief without addressing the underlying mechanisms of the disease. PDN involves the gradual degeneration of nerve fibers in the skin. Keratinocytes, the most abundant epidermal cell type, are closely positioned to cutaneous nerve terminals, suggesting the possibility of bi-directional communication. Extracellular vesicles are lipid-bilayer encapsulated nanovesicles released from many cell types that mediate cell to cell communication. The role of keratinocyte-derived extracellular vesicles (KDEVs) in influencing signaling between the skin and cutaneous nerve terminals and their contribution to the genesis of PDN has not been explored. In this study, we characterized KDEVs in a well-established high-fat diet mouse model of PDN using primary adult mouse keratinocyte cultures. We obtained highly enriched KDEVs through size-exclusion chromatography and then analyzed their molecular cargo using proteomic analysis and small RNA sequencing. We found significant differences in the protein and microRNA content of high-fat diet KDEVs compared to KDEVs obtained from control mice on a regular diet, including pathways involved in axon guidance and synaptic transmission. Additionally, using an in vivo conditional extracellular vesicle reporter mouse model, we demonstrated that epidermal-originating GFP-tagged KDEVs are retrogradely trafficked into the dorsal root ganglion (DRG) neuron cell bodies. This study presents the first comprehensive isolation and molecular characterization of the KDEV protein and microRNA cargo in RD and HFD mice. Our findings suggest a potential novel communication pathway between keratinocytes and DRG neurons in the skin, which could have implications for PDN.

Conotoxin kM-RIIIJ reveals interplay between Kv1-channels and persistent sodium currents in proprioceptive DRG neurons

Voltage-gated potassium channels (VGKCs) comprise the largest and most complex families of ion channels. Approximately 70 genes encode VGKC alpha subunits, which assemble into functional tetrameric channel complexes. These subunits can also combine to form heteromeric channels, significantly expanding the potential diversity of VGKCs. The functional expression and physiological role of heteromeric K-channels have remained largely unexplored due to the lack of tools to probe their functions. Conotoxins, from predatory cone snails, have high affinity and specificity for heteromeric combinations of K-channels and show great promise for defining their physiological roles. In this work, using conotoxin kM-RIIIJ as a pharmacological probe, we explore the expression and physiological functions of heteromeric Kv1.2 channels using constellation pharmacology platform. We report that heteromers of Kv1.2/1.1 are highly expressed in proprioceptive neurons found in the dorsal root ganglion (DRG). Inhibition of Kv1.2/1.1 heteromers leads to an influx of calcium ions, suggesting that these channels regulate neuronal excitability. We also present evidence that Kv1.2/1.1 heteromers counteract persistent sodium currents, and that inhibiting these channels leads to tonic firing of action potentials. Additionally, kM-RIIIJ impaired proprioception in mice, uncovering a previously unrecognized physiological function of heteromeric Kv1.2/1.1 channels in proprioceptive sensory neurons of the DRG.

Plasticity of Mouse Dorsal Root Ganglion Neurons by Innate Immune Activation Is Influenced by Electrophysiological Activity.

The complex relationship between inflammation, its effects on neuronal excitability and the ensuing plasticity of dorsal root ganglion (DRG) sensory neurons remains to be fully explored. In this study, we have employed a system of experiments assessing the impact of inflammatory conditioned media derived from activated immune cells on the excitability and activity of DRG neurons and how this relates to subsequent growth responses of these cells. We show here that an early phase of increased neuronal activity in response to inflammatory conditioned media is critical for the engagement of plastic processes and that neuronal excitability profiles are linked through time to the structural phenotype of individual neurons. Pharmacological blockade of neuronal activity was able to abolish the growth-promoting effects of inflammatory media. Our results suggest that targeting the activity of DRG neurons may provide a novel therapeutic avenue to manipulate their growth status and potential for plasticity in response to inflammation. Importantly, the same pharmacological blockade invivo abolished pain responses in a mouse model of multiple sclerosis. While further studies are needed to fully elucidate the underlying mechanisms of the relationship between neural activity and growth status, a more complete understanding of this relationship may ultimately lead to the development of new treatments for neuropathic pain in disorders associated with heightened immune responses such as rheumatoid arthritis and multiple sclerosis.

Administration of AICAR, an AMPK Activator, Prevents and Reverses Diabetic Polyneuropathy (DPN) by Regulating Mitophagy.

Diabetic peripheral neuropathy (DPN) is a common complication of diabetes in both Type 1 (T1D) and Type 2 (T2D). While there are no specific medications to prevent or treat DPN, certain strategies can help halt its progression. In T1D, maintaining tight glycemic control through insulin therapy can effectively prevent or delay the onset of DPN. However, in T2D, overall glucose control may only have a moderate impact on DPN, although exercise is clearly beneficial. Unfortunately, optimal exercise may not be feasible for many patients with DPN because of neuropathic foot pain and poor balance. Exercise has several favorable effects on health parameters, including body weight, glycemic control, lipid profile, and blood pressure. We investigated the impact of an exercise mimetic, 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), on DPN. AICAR treatment prevented or reversed experimental DPN in mouse models of both T2D and T1D. AICAR in high-fat diet (HFD-fed) mice increased the phosphorylation of AMPK in DRG neuronal extracts, and the ratio of phosphorylated AMPK to total AMPK increased by 3-fold (HFD vs. HFD+AICAR; p < 0.001). Phospho AMP increased the levels of dynamin-related protein 1 (DRP1, a mitochondrial fission marker), increased phosphorylated autophagy activating kinase 1 (ULK1) at Serine-555, and increased microtubule-associated protein light chain 3-II (LC3-II, a marker for autophagosome assembly) by 2-fold. Mitochondria isolated from DRG neurons of HFD-fed had a decrease in ADP-stimulated state 3 respiration (120 ± 20 nmol O2/min in HFD vs. 220 ± 20 nmol O2/min in control diet (CD); p < 0.001. Mitochondria isolated from HFD+AICAR-treated mice had increased state 3 respiration (240 ± 30 nmol O2/min in HFD+AICAR). However, AICAR's protection in DPN in T2D mice was also mediated by its effects on insulin sensitivity, glucose metabolism, and lipid metabolism. Drugs that enhance AMPK phosphorylation may be beneficial in the treatment of DPN.

Genome-Wide Network Analysis of DRG-Sciatic Nerve Network-Inferred Cellular Senescence and Senescence Phenotype in Peripheral Sensory Neurons.

Accumulation of senescent neurons in the dorsal root ganglion (DRG) is an important tissue phenotype that causes age-related degeneration of peripheral sensory nerves. Senescent neurons are neurons with arrested cell cycle that have undergone cellular senescence but remain in the tissue and play various biological roles. To understand the accumulation of senescent neurons in the DRG during aging, we aimed to elucidate the mechanism that induces cellular senescence in DRG neurons and the role of senescent DRG neurons. We integrated multiple public transcriptome datasets for DRGs, which include cell bodies in neurons, and the sciatic nerve, which includes axons in neurons, using network medicine-based bioinformatics analysis. We thus inferred the molecular mechanisms involved in cellular senescence of DRG neurons, from molecular responses to senescence, in the DRG-sciatic nerve network. Network medicine-based bioinformatics analysis revealed that age-related Mapk3 decline leads to impaired cholesterol metabolism and biosynthetic function in axons, resulting in compensatory upregulation of Srebf1, a transcription factor involved in lipid and cholesterol metabolism. This in turn leads to CDKN2A-mediated cellular senescence. Furthermore, our analysis revealed that senescent DRG neurons develop a senescence phenotype characterized by activation of antigen-presenting cells via upregulation of Ctss as a hub gene. B cells were inferred as antigen-presenting cells activated by Ctss, and CD8-positive T cells were inferred as cells that receive antigen presentation from B cells.

Human birth tissue products as a non-opioid medicine to inhibit post-surgical pain.

Pain after surgery causes significant suffering. Opioid analgesics cause severe side effects and accidental death. Therefore, there is an urgent need to develop non-opioid therapies for managing post-surgical pain. Local application of Clarix Flo (FLO), a human amniotic membrane (AM) product, attenuated established post-surgical pain hypersensitivity without exhibiting known side effects of opioid use in mice. This effect was achieved through direct inhibition of nociceptive dorsal root ganglion (DRG) neurons via CD44-dependent pathways. We further purified the major matrix component, the heavy chain-hyaluronic acid/pentraxin 3 (HC-HA/PTX3) from human AM that has greater purity and water solubility than FLO. HC-HA/PTX3 replicated FLO-induced neuronal and pain inhibition. Mechanistically, HC-HA/PTX3-induced cytoskeleton rearrangements to inhibit sodium current and high-voltage activated calcium current on nociceptive DRG neurons, suggesting it is a key bioactive component mediating pain relief. Collectively, our findings highlight the potential of naturally derived biologics from human birth tissues as an effective non-opioid treatment for post-surgical pain. Moreover, we unravel the underlying neuronal mechanisms of pain inhibition induced by FLO and HC-HA/PTX3.

C2230, a preferential use- and state-dependent CaV2.2 channel blocker, mitigates pain behaviors across multiple pain models.

Antagonists (e.g., Ziconotide, Gabapentin) of the CaV2.2 (N-type) calcium channels are used clinically as analgesics for chronic pain. However, their use is limited by narrow therapeutic windows, difficult dosing routes (Ziconotide), misuse and overdoses (Gabapentin), as well as a litany of adverse effects. Expansion of novel pain therapeutics may emerge from mechanism-based interrogation of CaV2.2. Here we report the identification of C2230, an aryloxy-hydroxypropylamine, as a CaV2.2 blocker. C2230 trapped and stabilized inactivated CaV2.2 in a slow-recovering state and accelerated the open-state inactivation of the channel, conferring an advantageous use-dependent inhibition profile. C2230 inhibited CaV2.2 during high-frequency stimulation, while sparing other voltage-gated ion channels. C2230 inhibited CaV2.2 in dorsal root and trigeminal ganglia neurons from rats, marmosets, and humans in a G-protein-coupled receptor-independent manner. Further, C2230 reduced evoked excitatory postsynaptic currents and excitatory neurotransmitter release in the spinal cord, leading to relief of neuropathic, orofacial, and osteoarthritic pain-like behaviors via three different routes of administration. C2230 also decreased fiber photometry-based calcium responses in the parabrachial nucleus, mitigated aversive behavioral responses to mechanical stimuli after neuropathic injury, and preserved protective pain responses, all without affecting motor or cardiovascular function. Finally, site-directed mutation analysis demonstrated that C2230 binds differently than other known CaV2.2 blockers, making it a promising lead compound for analgesic development.

GFRα1 Promotes Axon Regeneration after Peripheral Nerve Injury by Functioning as a Ligand.

The neurotrophic factor, Glial cell line derived neurotrophi factor (GDNF), exerts a variety of biological effects through binding to its receptors, GDNF family receptor alpha-1 (GFRα1), and RET. However, the existence of cells expressing GFRα1 but not RET raises the possibility that GFRα1 can function independently from RET. Here, it is shown that GFRα1 released from repair Schwann cells (RSCs) functions as a ligand in a GDNF-RET-independent manner to promote axon regeneration after peripheral nerve injury (PNI). Local administration of GFRα1 into injured nerve promoted axon regeneration, even more when combined with GDNF blockade. GFRα1 bound to a receptor complex consisting of NCAM and integrin α7β1 of dorsal root ganglion neurons in a GDNF-RET independent manner. This is further confirmed by the Ret Y1062F knock-in mice, which cannot transmit most of GDNF-RET signaling. Finally, local administration of GFRα1 into injured sciatic nerve promoted functional recovery. These findings reveal a novel role of GFRα1 as a ligand, the molecular mechanism supporting axon regeneration by RSCs, and a novel therapy for peripheral nerve repair.