Primary Sensory Neurons Research Articles

The Stathmin-2 membrane-targeting domain is required for axon protection and regulated degradation by DLK signaling

Axon integrity is essential for functional connectivity in the nervous system. The degeneration of stressed or damaged axons is a common and sometimes initiating event in neurodegenerative disorders. Stathmin-2 (Stmn2) is an axon maintenance factor that is depleted in Amyotrophic Lateral Sclerosis, and replenishment of Stmn2 can restore neurite outgrowth in diseased neurons. However, mechanisms responsible for Stmn2-mediated axon maintenance in injured neurons are not known. We used primary sensory neurons to interrogate the role of Stmn2 in the degeneration of severed axons. We discover that membrane association of Stmn2 is critical for its axon-protective activity. Structure-function studies revealed that axonal enrichment of Stmn2 is driven by palmitoylation as well as tubulin interaction. Using live-imaging, we discover that another Stathmin, Stmn3, co-migrates with Stmn2-containing vesicles. We also demonstrate that Stmn3 undergoes regulated degradation through DLK-JNK signaling. The Stmn2 membrane targeting domain is both necessary and sufficient for localization to a specific vesicle population and confers sensitivity to DLK-dependent degradation. Our findings reveal a broader role for DLK in tuning the local abundance of palmitoylated Stathmins in axon segments. Moreover, palmitoylation is a critical component of Stathmin-mediated axon protection, and defining the Stmn2-containing vesicle population will provide important clues toward mechanisms of axon maintenance.

Schwann cells as a target cell for the treatment of cancer pain.

Tumor erosion and metastasis can invade surrounding tissues, damage nerves, and sensitize the peripheral primary receptors, inducing pain, which can potentially worsen the suffering of patients with cancer. Reception and transmission of sensory signal receptors, abnormal activation of primary sensory neurons, and activation of glial cells are involved in cancer pain. Therefore, exploring promising therapeutic methods to suppress cancer pain is of great significance. Various studies have found that the use of functionally active cells is a potentially effective way to relieve pain. Schwann cells (SCs) act as small, biologically active pumps that secrete pain-relieving neuroactive substances. Moreover, SCs can regulate the progression of tumor cells, including proliferation and metastasis, through neuro-tumor crosstalk, which emphasizes the critical role of SCs in cancer and cancer pain. The mechanisms by which SCs repair injured nerves and exert analgesia include neuroprotection, neurotrophy, nerve regeneration, neuromodulation, immunomodulation, and enhancement of the nerve-injury microenvironment. These factors may ultimately restore the damaged or stimulated nerves and contribute to pain relief. Strategies for pain treatment using cell transplantation mainly focus on analgesia and nerve repair. Although these cells are in the initial stages of nerve repair and pain, they open new avenues for the treatment of cancer pain. Therefore, this paper discusses, for the first time, the possible mechanism of SCs and cancer pain, and new strategies and potential problems in cancer pain treatment.

NS5806 reduces carrageenan-evoked inflammation by suppressing extracellular signal-regulated kinase activation in primary sensory neurons and immune cells.

The compound NS5806 attenuates neuropathic pain via inhibiting extracellular signal-regulated kinase (ERK) activation in neuronal somata located at the dorsal root ganglion (DRG) and superficial spinal dorsal horn. NS5806 also reduces the expansion of DRG macrophages and spinal microglia several days after peripheral nerve injury, implying an anti-inflammatory effect. To test whether NS5806 inhibits inflammation, as a model we intraplantarly injected carrageenan into a hind paw of the rat. To examine whether NS5806 reduces carrageenan-evoked mechanical allodynia, thermal hyperalgesia, and edema, as well as ERK activation in the nerve fibres, mast cells, and macrophages in the hind paw skin, we used behavioural, immunohistochemical, and cytological methods. NS5806 did not impair motor function, affect basal nociception, or cause edema in naive rats. Six hours after carrageenan injection, mechanical allodynia, thermal hyperalgesia, and edema appeared in the rat's ipsilateral hind paw, and all were reduced by intraplantar co-injection of NS5806. NS5806 suppressed carrageenan-evoked ERK activation in the peripheral axons and somata of L4 DRG neurons, as well as mast cells and macrophages in the paw skin. NS5806 also reduced carrageenan-evoked mast cell degranulation and macrophage proliferation. NS5806 and the ERK pathway inhibitor PD98059 had a similar effect in inhibiting the proliferation of cultured RAW264.7 macrophages. Furthermore, all the in vivo anti-inflammatory effects of NS5806 were similar to those of PD98059. Acting like an ERK pathway inhibitor, NS5806 reduces inflammation-evoked mechanical allodynia, thermal hyperalgesia, and edema by suppressing ERK activation in primary sensory neurons, mast cells, and macrophages. Previous studies show that NS5806 only acts on neurons. This report unveils that NS5806 also acts on immune cells in the skin to exert its anti-inflammatory effects. Since NS5806 is lipid soluble for skin penetration, it suggests that NS5806 could also be developed into an anti-inflammatory drug for external use.

Expression of the Transient Receptor Potential Vanilloid 1 ion channel in the supramammillary nucleus and the antidepressant effects of its antagonist AMG9810 in mice

The Transient Receptor Potential Vanilloid 1 (TRPV1) non-selective cation channel predominantly expressed in primary sensory neurons of the dorsal root and trigeminal ganglia mediates pain and neurogenic inflammation. TRPV1 mRNA and immunoreactivity were described in the central nervous system (CNS), but its precise expression pattern and function have not been clarified. Here we investigated Trpv1 mRNA expression in the mouse brain using ultrasensitive RNAScope in situ hybridization. The role of TRPV1 in anxiety, depression-like behaviors and memory functions was investigated by TRPV1-deficient mice and pharmacological antagonism by AMG9810. Trpv1 mRNA is selectively expressed in the supramammillary nucleus (SuM) co-localized with Vglut2 mRNA, but not with tyrosine hydroxylase immunopositivity demonstrating its presence in glutamatergic, but not dopaminergic neurons. TRPV1-deleted mice exhibited significantly reduced anxiety in the Light-Dark box and depression-like behaviors in the Forced Swim Test, but their performance in the Elevated Plus Maze as well as their spontaneous locomotor activity, memory and learning function in the Radial Arm Maze, Y-maze and Novel Object Recognition test were not different from WTs. AMG9810 (intraperitoneal injection 50 mg/kg) induced anti-depressant, but not anxiolytic effects. It is concluded that TRPV1 in the SuM might have functional relevance in mood regulation and TRPV1 antagonism could be a novel perspective for anti-depressant drugs.

Establishment of an Intradermal Canine IL-31-Induced Pruritus Model to Evaluate Therapeutic Candidates in Atopic Dermatitis.

Pruritic models in healthy dogs utilizing intravenous administration of interleukin 31 (IL-31) bypass the "natural" itch sensation in AD, which is initiated by pruriceptive primary afferent neurons in the skin. This study aimed to evaluate the immediate/delayed pruritus responses and the pruritic behaviors observed in an intradermal IL-31-induced pruritic model of healthy dogs and the anti-pruritic effect of oclacitinib on said model. In Phase 1, all the dogs were randomized and video-recorded for 300 min after intradermal canine recombinant IL-31 injections (1.75 µg/kg) and vehicle (phosphate-buffered saline) injections. In Phase 2, all the dogs received oral oclacitinib (0.4-0.6 mg/kg, twice daily for 4 consecutive days and once daily on day 5), with the intradermal IL-31 injection performed on day 5. Two blinded investigators reviewed the pruritic behaviors in all the video recordings. Intradermal IL-31 administration to healthy dogs caused a significant increase in the total (p = 0.0052) and local (p = 0.0003) seconds of pruritic behavior compared to the vehicle control. Oral oclacitinib administration significantly reduced the total (p = 0.0011) and local (p = 0.0156) intradermal IL-31-induced pruritic seconds; there was no significant difference in pruritic seconds between the vehicle and oclacitinib within the IL-31 groups. Significant delayed pruritic responses at 150-300 min after IL-31 injections were observed, and intradermal IL-31 failed to induce acute itch (first 30 min). Intradermal injection of IL-31 induces delayed itch responses in dogs that are diminished by the effect of oclacitinib, an oral JAK inhibitor.

Retraction: Li, Y., Wu, X. Y., & Owyang, C. (2004). Serotonin and cholecystokinin synergistically stimulate rat vagal primary afferent neurons. The Journal of Physiology, 559(2), 651-662. https://doi.org/10.1113/jphysiol.2004.064816.

The Journal of PhysiologyAccepted Articles RETRACTIONFree Access Retraction: Li, Y., Wu, X. Y., & Owyang, C., Serotonin and cholecystokinin synergistically stimulate rat vagal primary afferent neurons, Journal of Physiology, 2004, (https://doi.org/10.1113/jphysiol.2004.064816) This article retracts the following: Serotonin and cholecystokinin synergistically stimulate rat vagal primary afferent neurones Y. Li, X. Y. Wu, C. Owyang, Volume 559Issue 2The Journal of Physiology pages: 651-662 First Published online: August 25, 2004 First published: 21 March 2023 https://doi.org/10.1113/JP284694 The peer review history is available in the Supporting Information section of this article (https://doi.org/10.1113/JP284694#support-information-section). This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as https://doi.org/10.1113/JP284694 AboutPDF ToolsExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Supporting Information Filename Description tjp15508-sup-0001-PeerReview.pdf53 KB Peer Review History Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. Accepted ArticlesAccepted, unedited articles published online and citable. The final edited and typeset version of record will appear in the future. RelatedInformation

Inhibition of miR-221-3p promotes axonal regeneration and repair of primary sensory neurons via regulating p27 expression.

This study aimed to explore the key microRNA (miRNA) playing a vital role in axonal regeneration with a hostile microenvironment after spinal cord injury. Based on the theory that sciatic nerve conditioning injury (SNCI) could promote the repair of the injured dorsal column. Differentially expressed miRNAs were screened according to the microarray, revealing that 47 known miRNAs were differentially expressed after injury and perhaps involved in nerve regeneration. Among the 47 miRNAs, the expression of miR-221-3p decreased sharply in the SNCI group compared with the simple dorsal column lesion (SDCL) group. Subsequently, it was confirmed that p27 was the target gene of miR-221-3p from luciferase reporter assay. Further, we found that inhibition of miR-221-3p expression could specifically target p27 to upregulate the expression of growth-associated protein 43 (GAP-43), α-tubulin acetyltransferase (α-TAT1) together with α-tubulin, and advance the regeneration of dorsal root ganglion (DRG) neuronal axons. Chondroitin sulfate proteoglycans (CSPGs) are the main components of glial scar, which can hinder the extension and growth of damaged neuronal axons. After CSPGs were used in this study, the results demonstrated that restrained miR-221-3p expression also via p27 promoted the upregulation of GAP-43, α-TAT1, and α-tubulin and enhanced the axonal growth of DRG neurons. Hence, miR-221-3p could contribute significantly to the regeneration of DRG neurons by specifically regulating p27 in the p27/CDK2/GAP-43 and p27/α-TAT1/α-tubulin pathways even in the inhibitory environment with CSPGs.

NMDA Receptors at Primary Afferent-Excitatory Neuron Synapses Differentially Sustain Chemotherapy- and Nerve Trauma-Induced Chronic Pain.

The spinal dorsal horn contains vesicular glutamate transporter-2 (VGluT2)-expressing excitatory neurons and vesicular GABA transporter (VGAT)-expressing inhibitory neurons, which normally have different roles in nociceptive transmission. Spinal glutamate NMDAR hyperactivity is a crucial mechanism of chronic neuropathic pain. However, it is unclear how NMDARs regulate primary afferent input to spinal excitatory and inhibitory neurons in neuropathic pain. Also, the functional significance of presynaptic NMDARs in neuropathic pain has not been defined explicitly. Here we showed that paclitaxel treatment or spared nerve injury (SNI) similarly increased the NMDAR-mediated mEPSC frequency and dorsal root-evoked EPSCs in VGluT2 dorsal horn neurons in male and female mice. By contrast, neither paclitaxel nor SNI had any effect on mEPSCs or evoked EPSCs in VGAT neurons. In mice with conditional Grin1 (gene encoding GluN1) KO in primary sensory neurons (Grin1-cKO), paclitaxel treatment failed to induce pain hypersensitivity. Unexpectedly, SNI still caused long-lasting pain hypersensitivity in Grin1-cKO mice. SNI increased the amplitude of puff NMDA currents in VGluT2 neurons and caused similar depolarizing shifts in GABA reversal potentials in WT and Grin1-cKO mice. Concordantly, spinal Grin1 knockdown diminished SNI-induced pain hypersensitivity. Thus, presynaptic NMDARs preferentially amplify primary afferent input to spinal excitatory neurons in neuropathic pain. Although presynaptic NMDARs are required for chemotherapy-induced pain hypersensitivity, postsynaptic NMDARs in spinal excitatory neurons play a dominant role in traumatic nerve injury-induced chronic pain. Our findings reveal the divergent synaptic connectivity and functional significance of spinal presynaptic and postsynaptic NMDARs in regulating cell type-specific nociceptive input in neuropathic pain with different etiologies.SIGNIFICANCE STATEMENT Spinal excitatory neurons relay input from nociceptors, whereas inhibitory neurons repress spinal nociceptive transmission. Chronic nerve pain is associated with aberrant NMDAR activity in the spinal dorsal horn. This study demonstrates, for the first time, that chemotherapy and traumatic nerve injury preferentially enhance the NMDAR activity at primary afferent-excitatory neuron synapses but have no effect on primary afferent input to spinal inhibitory neurons. NMDARs in primary sensory neurons are essential for chemotherapy-induced chronic pain, whereas nerve trauma causes pain hypersensitivity predominantly via postsynaptic NMDARs in spinal excitatory neurons. Thus, presynaptic and postsynaptic NMDARs at primary afferent-excitatory neuron synapses are differentially engaged in chemotherapy- and nerve injury-induced chronic pain and could be targeted respectively for treating these painful conditions.

Decreased expression of synaptic genes in the vestibular ganglion of rodents following subchronic ototoxic stress.

The vestibular ganglion contains primary sensory neurons that are postsynaptic to the transducing hair cells (HC) and project to the central nervous system. Understanding the response of these neurons to HC stress or loss is of great interest as their survival and functional competence will determine the functional outcome of any intervention aiming at repair or regeneration of the HCs. We have shown that subchronic exposure to the ototoxicant 3,3′-iminodipropionitrile (IDPN) in rats and mice causes a reversible detachment and synaptic uncoupling between the HCs and the ganglion neurons. Here, we used this paradigm to study the global changes in gene expression in vestibular ganglia using RNA-seq. Comparative gene ontology and pathway analyses of the data from both model species indicated a robust downregulation of terms related to synapses, including presynaptic and postsynaptic functions. Manual analyses of the most significantly downregulated transcripts identified genes with expressions related to neuronal activity, modulators of neuronal excitability, and transcription factors and receptors that promote neurite growth and differentiation. For choice selected genes, the mRNA expression results were replicated by qRT-PCR, validated spatially by RNA-scope, or were demonstrated to be associated with decreased expression of the corresponding protein. We conjectured that decreased synaptic input or trophic support on the ganglion neurons from the HC was triggering these expression changes. To support this hypothesis, we demonstrated decreased expression of BDNF mRNA in the vestibular epithelium after subchronic ototoxicity and also downregulated expression of similarly identified genes (e.g Etv5, Camk1g, Slc17a6, Nptx2, Spp1) after HC ablation with another ototoxic compound, allylnitrile. We conclude that vestibular ganglion neurons respond to decreased input from HCs by decreasing the strength of all their synaptic contacts, both as postsynaptic and presynaptic players.

Open-Source Real-Time Closed-Loop Electrical Threshold Tracking for Translational Pain Research.

Nociceptors are a class of primary afferent neurons that signal potentially harmful noxious stimuli. An increase in nociceptor excitability occurs in acute and chronic pain conditions. This produces abnormal ongoing activity or reduced activation thresholds to noxious stimuli. Identifying the cause of this increased excitability is required for the development and validation of mechanism-based treatments. Single-neuron electrical threshold tracking can quantify nociceptor excitability. Therefore, we have developed an application to allow such measurements and demonstrate its use in humans and rodents. APTrack provides real-time data visualization and action potential identification using a temporal raster plot. Algorithms detect action potentials by threshold crossing and monitor their latency after electrical stimulation. The plugin then modulates the electrical stimulation amplitude using an up-down method to estimate the electrical threshold of the nociceptors. The software was built upon the Open Ephys system (V0.54) and coded in C++ using the JUCE framework. It runs on Windows, Linux, and Mac operating systems. The open-source code is available (https://github.com/Microneurography/APTrack). The electrophysiological recordings were taken from nociceptors in both a mouse skin-nerve preparation using the teased fiber method in the saphenous nerve and in healthy human volunteers using microneurography in the superficial peroneal nerve. Nociceptors were classified by their response to thermal and mechanical stimuli, as well as by monitoring the activity-dependent slowing of the conduction velocity. The software facilitated the experiment by simplifying the action potential identification through the temporal raster plot. We demonstrate real-time closed-loop electrical threshold tracking of single-neuron action potentials duringin vivohuman microneurography, for the first time, and duringex vivomouse electrophysiological recordings of C-fibers and Aδ-fibers. We establish proof of principle by showing that the electrical threshold of a human heat-sensitive C-fiber nociceptor is reduced by heating the receptive field. This plugin enables the electrical threshold tracking of single-neuron action potentials and allows the quantification of changes in nociceptor excitability.

Secretome mediated interactions between sensory neurons and breast cancer cells.

The role of the nervous system in aiding cancer progression and metastasis is an important aspect of cancer pathogenesis. Interaction between cancer cells and neurons in an in vitro platform is a simple and robust method to further understand this phenomenon. In our study, we aimed to examine in vitro reciprocal effect between breast cancer cells and cancer-sensitized peripheral primary sensory neurons. Secretome obtained from either cultured DRG neurons from tumor-burdened rats, or MRMT1 breast cancer cells were used to study neuronal and cancer cell reciprocity. We utilized neurite analysis, modified cell migration assay and cell signaling pathway inhibitors to determine neurite growth patterns and cell migration in PC12/DRG neurons and MRMT1 cells, respectively. MRMT1 secretome was found to induce significant neurite outgrowth in PC12 and primary sensory neurons. Secretome-induced neurite growth in PC12 cells was partly mediated by PI3K and ERK pathways, but not by adenylyl cyclase. Conversely, secretome from tumor-sensitized sensory neuron cultures induced increased rate of migration in cultured MRMT1 cells. Results from our study provide additional support to the hypothesis that both breast cancer cells and nerve terminals secrete signaling messengers that have a reciprocal effect on each other.

Structure-Based Discovery of a New Selectivity-Enabling Motif for the FK506-Binding Protein 51.

In recent years, the selective inhibition of FKBP51 has emerged as a possible treatment for chronic pain, obesity-induced diabetes, or depression. All currently known advanced FKBP51-selective inhibitors, including the widely used SAFit2, contain a cyclohexyl residue as a key motif for enabling selectivity over the closest homologue and anti-target FKBP52. During a structure-based SAR exploration, we surprisingly discovered thiophenes as highly efficient cyclohexyl replacement moieties that retain the strong selectivity of SAFit-type inhibitors for FKBP51 over FKBP52. Cocrystal structures revealed that the thiophene-containing moieties enable selectivity by stabilizing a flipped-out conformation of Phe67 of FKBP51. Our best compound, 19b, potently binds to FKBP51 biochemically as well as in mammalian cells, desensitize TRPV1 in primary sensory neurons, and has an acceptable PK profile in mice, suggesting its use as a novel tool compound for studying FKBP51 in animal models of neuropathic pain.

Transient Receptor Potential Channel Vanilloid 1 Contributes to Facial Mechanical Hypersensitivity in a Mouse Model of Atopic Asthma

Sensitive skin, a common pathophysiological feature of allergic diseases, is defined as an unpleasant sensation in response to stimuli that normally should not provoke such sensations. However, the relationship between allergic inflammation and hypersensitive skin in the trigeminal system remains to be elucidated. To explore whether bronchial allergic inflammation affects facial skin and primary sensory neurons, we used an ovalbumin (OVA)–induced asthma mouse model. Significant mechanical hypersensitivity was observed in the facial skin of mice with pulmonary inflammation induced by OVA sensitization compared to mice treated with adjuvant or vehicle as controls. The skin of OVA-treated mice showed an increased number of nerve fibers, especially rich intraepithelial nerves, compared to controls. Transient receptor potential channel vanilloid 1 (TRPV1)–immunoreactive nerves were enriched in the skin of OVA-treated mice. Moreover, epithelial TRPV1 expression was higher in OVA-treated mice than in controls. Trigeminal ganglia of OVA-treated mice displayed larger numbers of activated microglia/macrophages and satellite glia. In addition, more TRPV1 immunoreactive neurons were found in the trigeminal ganglia of OVA-treated mice than in controls. Mechanical hypersensitivity was suppressed in OVA-treated Trpv1-deficient mice, while topical skin application of a TRPV1 antagonist before behavioral testing reduced the reaction induced by mechanical stimulation. Our findings reveal that mice with allergic inflammation of the bronchi had mechanical hypersensitivity in the facial skin that may have resulted from TRPV1-mediated neuronal plasticity and glial activation in the trigeminal ganglion.

Rapid injection of lumbar dorsal root ganglia under direct vision: Relevant anatomy, protocol, and behaviors.

Dorsal root ganglia (DRG) are anatomically well-defined structures that contain all primary sensory neurons and are distension nodules of the dorsal root in the spinal cord near the medial surface of each foramen. Therefore, DRG is considered to be a desirable target for injection to manage chronic pain. But it presents a limitation in probing deep into it without in vivo injection technology. Here, we described a technique for administering intraganglionic injections of lumbar DRG under direct vision. We use partial osteotomy rather than laminectomy, which removes more bone, to preserve spinal structures while gaining adequate DRG access. To monitor the intraoperative progress of the DRG injection, a non-toxic dye was utilized. The effectiveness of the injection on the diffusion of AAV (adeno-associated virus) within the ganglion was assessed by histopathology at postoperative day 21. Behavioral tests showed that neither motor nor sensory abilities were affected by saline or AAV injections. Meanwhile, the decreased pain threshold of SNI (spared nerve injury) was considerably restored by pharmacological inhibition of DRG neurons. Our research achieved a new minimally invasive and intuitive intra-ganglionic injection in mice. In addition, the present protocol may serve as a valuable resource for planning preclinical studies of DRG injection.

In situ delivery of a curcumin-loaded dynamic hydrogel for the treatment of chronic peripheral neuropathy

Patients with peripheral nerve injuries would highly likely suffer from chronic neuropathic pain even after surgical intervention. The primary reasons for this involve sustained neuroinflammatory and dysfunctional changes in the nervous system after the nerve injury. We previously reported an injectable boronic ester-based hydrogel with inherent antioxidative and nerve protective properties. Herein, we first explored the anti-neuroinflammatory effects of Curcumin on primary sensory neurons and activated macrophages in vitro. Next, we incorporated thiolated Curcumin-Pluronic F-127 micelles (Cur-M) into our boronic ester-based hydrogel to develop an injectable hydrogel that serves as sustained curcumin release system (Gel-Cur-M). By orthotopically injecting the Gel-Cur-M to sciatic nerves of mice with chronic constriction injuries, we found that the bioactive components could remain on the nerves for at least 21 days. In addition, the Gel-Cur-M exhibited superior functions compared to Gel and Cur-M alone, which includes ameliorating hyperalgesia while simultaneously improving locomotor and muscular functions after the nerve injury. This could stem from in situ anti-inflammation, antioxidation, and nerve protection. Furthermore, the Gel-Cur-M also showed extended beneficial effects for preventing the overexpression of TRPV1 as well as microglial activation in the lumbar dorsal root ganglion and spinal cord, respectively, which also contributed to its analgesic effects. The underlying mechanism may involve the suppression of CC chemokine ligand-2 and colony-stimulating factor-1 in the injured sensory neurons. Overall, this study suggests that orthotopic injection of the Gel-Cur-M is a promising therapeutic strategy that especially benefits patients with peripheral neuropathy who require surgical interventions.

A sensory neuron-specific long non-coding RNA reduces neuropathic pain by rescuing KCNN1 expression.

Nerve injury to peripheral somatosensory system causes refractory neuropathic pain. Maladaptive changes of gene expression in primary sensory neurons are considered molecular basis of this disorder. Long non-coding RNAs (lncRNAs) are key regulators of gene transcription; however, their significance in neuropathic pain remains largely elusive.Here, we reported a novel lncRNA, named sensory neuron-specific lncRNA (SS-lncRNA), for its expression exclusively in dorsal root ganglion (DRG) and trigeminal ganglion. SS-lncRNA was predominantly expressed in small DRG neurons and significantly downregulated due to a reduction of early B cell transcription factor 1 in injured DRG after nerve injury. Rescuing this downregulation reversed a decrease of the calcium-activated potassium channel subfamily N member 1 (KCNN1) in injured DRG and alleviated nerve injury-induced nociceptive hypersensitivity. Conversely, DRG downregulation of SS-lncRNA reduced the expression of KCNN1, decreased total potassium currents and afterhyperpolarization currents and increased excitability in DRG neurons and produced neuropathic pain symptoms.Mechanistically, downregulated SS-lncRNA resulted in the reductions of its binding to Kcnn1 promoter and heterogeneous nuclear ribonucleoprotein M (hnRNPM), consequent recruitment of less hnRNPM to the Kcnn1 promoter and silence of Kcnn1 gene transcription in injured DRG.These findings indicate that SS-lncRNA may relieve neuropathic pain through hnRNPM-mediated KCNN1 rescue in injured DRG and offer a novel therapeutic strategy specific for this disorder.

Abstract 1193: Sensory innervation directly promotes gastric cancer and modulates the tumor microenvironment

Abstract Tumor innervation contributes directly to cancer progression, but there appears to be organ-specific variation in the type of innervation. While our previous studies showed cholinergic signaling promoted gastric cancer and vagal denervation inhibited gastric cancer, it is noteworthy that sensory nerves comprise 80% of the vagal axons. To investigate the role of sensory innervation in gastric carcinogenesis, we performed retrograde neural tracing and identified CGRP+ peptidergic neurons in nodose ganglia and dorsal root ganglia as primary gastric sensory neurons. While VAChT+ parasympathetic nerves are the main nerves innervating the normal stomach, CGRP+ sensory nerves show the greatest degree of expansion in mouse gastric tumors. Indeed, mouse gastric tumors show upregulation of NGF, a growth factor essential for sensory nerve survival, and gastric NGF overexpression in an NGF knock-in mouse also resulted in increased sensory innervation. Inhibition of NGF/Trk signaling by Entrectinib markedly attenuates NGF-dependent sensory nerve expansion. In addition to overall increased axonogenesis of sensory nerves, mouse models of gastric cancer showed upregulation of αCGRP peptide in gastric tumors and Calca mRNA in gastric sensory neurons, indicating increased activation of sensory neurons during gastric carcinogenesis. To study the effect of such activation, sensory neurons were then directly activated by treatment of Trpv1-Cre;hM3Dq mice with clozapine-N-oxide (CNO), which led to marked gastric hyperplasia with increased proliferation. Interestingly, in an acetic acid-induced ulcer model, activation of sensory neurons reduced the ulcer size and strongly promoted gland regeneration. Activation of sensory neurons, in a syngeneic, orthotopic model of gastric cancer in the Trpv1-Cre;hM3Dq mice, led to a significant increase in tumor volume, Ki-67+ cancer cells, CD31+ vessels and M2 macrophage polarization, as well as decreased CD8+ T cells. Analysis of available single-cell transcriptome data from mouse and human stomach showed that Calcrl/Ramp1 (CGRP receptors) are expressed in gastric epithelial cells and most stromal cells, while the expression levels of Tacr1 (receptor for Substance P) and Sstr1-5 (receptors for Somatostatin) were much lower. Importantly, some Calcrl/Ramp1+ cells are proliferative cells with strong Ki-67 expression. Our data show that Calcrl is also overexpressed in mouse gastric tumors, while TCGA data reveals significant upregulation of CALCRL in human gastric cancers and that high expression of CALCRL is correlated with poor overall survival. Finally, we established a co-culture model using DRGs and gastric cancer organoids, and found that sensory nerves can generate synaptic-like terminals on cancer organoids. In conclusion, our data indicate that sensory innervation directly promotes gastric cancer growth and also modulates the tumor microenvironment. Citation Format: Xiaofei Zhi, Feijing Wu, Jin Qian, Yosuke Ochiai, Guodong Lian, Ermanno Malagola, Timothy C. Wang. Sensory innervation directly promotes gastric cancer and modulates the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1193.

Scalable generation of sensory neurons from human pluripotent stem cells.

Development of new non-addictive analgesics requires advanced strategies to differentiate human pluripotent stem cells (hPSCs) into relevant cell types. Following principles of developmental biology and translational applicability, here we developed an efficient stepwise differentiation method for peptidergic and non-peptidergic nociceptors. By modulating specific cell signaling pathways, hPSCs were first converted into SOX10+ neural crest, followed by differentiation into sensory neurons. Detailed characterization, including ultrastructural analysis, confirmed that the hPSC-derived nociceptors displayed cellular and molecular features comparable to native dorsal root ganglion (DRG) neurons, and expressed high-threshold primary sensory neuron markers, transcription factors, neuropeptides, and over 150 ion channels and receptors relevant for pain research and axonal growth/regeneration studies (e.g., TRPV1, NAV1.7, NAV1.8, TAC1, CALCA, GAP43, DPYSL2, NMNAT2). Moreover, after confirming robust functional activities and differential response to noxious stimuli and specific drugs, a robotic cell culture system was employed to produce large quantities of human sensory neurons, which can be used to develop nociceptor-selective analgesics.

Synchrony-Division Neural Multiplexing: An Encoding Model

Cortical neurons receive mixed information from the collective spiking activities of primary sensory neurons in response to a sensory stimulus. A recent study demonstrated an abrupt increase or decrease in stimulus intensity and the stimulus intensity itself can be respectively represented by the synchronous and asynchronous spikes of S1 neurons in rats. This evidence capitalized on the ability of an ensemble of homogeneous neurons to multiplex, a coding strategy that was referred to as synchrony-division multiplexing (SDM). Although neural multiplexing can be conceived by distinct functions of individual neurons in a heterogeneous neural ensemble, the extent to which nearly identical neurons in a homogeneous neural ensemble encode multiple features of a mixed stimulus remains unknown. Here, we present a computational framework to provide a system-level understanding on how an ensemble of homogeneous neurons enable SDM. First, we simulate SDM with an ensemble of homogeneous conductance-based model neurons receiving a mixed stimulus comprising slow and fast features. Using feature-estimation techniques, we show that both features of the stimulus can be inferred from the generated spikes. Second, we utilize linear nonlinear (LNL) cascade models and calculate temporal filters and static nonlinearities of differentially synchronized spikes. We demonstrate that these filters and nonlinearities are distinct for synchronous and asynchronous spikes. Finally, we develop an augmented LNL cascade model as an encoding model for the SDM by combining individual LNLs calculated for each type of spike. The augmented LNL model reveals that a homogeneous neural ensemble model can perform two different functions, namely, temporal- and rate-coding, simultaneously.

Rac1/PAK1 signaling contributes to bone cancer pain by Regulation dendritic spine remodeling in rats.

Bone cancer pain (BCP) is severe chronic pain caused by tumor metastasis to the bones, often resulting in significant skeletal remodeling and fractures. Currently, there is no curative treatment. Therefore, insight into the underlying mechanisms could guide the development of mechanism-based therapeutic strategies for BCP. We speculated that Rac1/PAK1 signaling plays a critical role in the development of BCP. Tumor cells implantation (TCI) into the tibial cavity resulted in bone cancer-associated mechanical allodynia. Golgi staining revealed changes in the excitatory synaptic structure of WDR (Wide-dynamic range) neurons in the spinal cord, including increased postsynaptic density (PSD) length and thickness, and width of the cleft. Behavioral and western blotting test revealed that the development and persistence of pain correlated with Rac1/PAK1 signaling activation in primary sensory neurons. Intrathecal injection of NSC23766, a Rac1 inhibitor, reduced the persistence of BCP as well as reversed the remodeling of dendrites. Therefore, we concluded that activation of the Rac1/PAK1 signaling pathway in the spinal cord plays an important role in the development of BCP through remodeling of dendritic spines. Modulation of the Rac1/PAK1 pathway may be a potential strategy for BCP treatment.