Ash1L ameliorates psoriasis via limiting neuronal activity-dependent release of miR-let-7b.

BACKGROUND AND PURPOSE: Psoriasis is a common autoimmune skin disease that significantly diminishes patients' quality of life. Interactions between primary afferents of the somatosensory system and the cutaneous immune system mediate the pathogenesis of psoriasis. This study aims to elucidate the molecular mechanisms of how primary sensory neurons regulate psoriasis formation. EXPERIMENTAL APPROACH: Skin and total RNA were extracted from wild-type (WT) and ASH1-like histone lysine methyltransferase (Ash1l+/- ) mice in both naive and imiquimod (IMQ)-induced psoriasis models. Immunohistochemistry, quantitative real-time polymerase chain reaction (qRT-PCR) and fluorescence-activated cell sorting (FACS) were then performed. Microfluidic chamber coculture was used to investigate the interaction between somatosensory neurons and bone marrow dendritic cells (BMDCs) ex vivo. Whole-cell patch clamp recordings were used to evaluate neuronal excitability after Ash1L haploinsufficiency in primary sensory neurons. KEY RESULTS: The haploinsufficiency of ASH1L, a histone methyltransferase, in primary sensory neurons causes both neurite hyperinnervation and increased neuronal excitability, which promote miR-let-7b release from primary afferents in the skin in a neuronal activity-dependent manner. With a 'GUUGUGU' core sequence, miR-let-7b functions as an endogenous ligand of toll-like receptor 7 (TLR7) and stimulates the activation of dermal dendritic cells (DCs) and interleukin (IL)-23/IL-17 axis, ultimately exacerbating the symptoms of psoriasis. Thus, by limiting miR-let-7b release from primary afferents, ASH1L prevents dermal DC activation and ameliorates psoriasis. CONCLUSION AND IMPLICATIONS: Somatosensory neuron ASH1L modulates the cutaneous immune system by limiting neuronal activity-dependent release of miR-let-7b, which can directly activate dermal DCs via TLR7 and ultimately lead to aggravated psoriatic lesion.

Hippo Pathway in Schwann Cells and Regeneration of Peripheral Nervous System.

Hippo pathway is an evolutionarily conserved signaling pathway comprising a series of MST/LATS kinase complexes. Its key transcriptional coactivators YAP and TAZ regulate transcription factors such as TEAD family to direct gene expression. The regulation of Hippo pathway, especially the nuclear level change of YAP and TAZ, significantly influences the cell fate switching from proliferation to differentiation, regeneration, and postinjury repair. This review outlines the main findings of Hippo pathway in peripheral nerve development, regeneration, and tumorigenesis, especially the studies in Schwann cells. We also summarize other roles of Hippo pathway in damage repair of the peripheral nerve system and discuss the potential future research which probably contributes to novel therapeutic strategies.

Somatosensory neurons express specific sets of lincRNAs, and lincRNA CLAP promotes itch sensation in mice.

Somatosensory neurons are highly heterogeneous with distinct types of neural cells responding to specific stimuli. However, the distribution and roles of cell-type-specific long intergenic noncoding RNAs (lincRNAs) in somatosensory neurons remain largely unexplored. Here, by utilizing droplet-based single-cell RNA-seq (scRNA-seq) and full-length Smart-seq2, we show that lincRNAs, but not coding mRNAs, are enriched in specific types of mouse somatosensory neurons. Profiling of lincRNAs from single neurons located in dorsal root ganglia (DRG) identifies 200 lincRNAs localized in specific types or subtypes of somatosensory neurons. Among them, the conserved cell-type-specific lincRNA CLAP associates with pruritus and is abundantly expressed in somatostatin (SST)-positive neurons. CLAP knockdown reduces histamine-induced Ca2+ influx in cultured SST-positive neurons and in vivo reduces histamine-induced scratching in mice. In vivo knockdown of CLAP also decreases the expression of neuron-type-specific and itch-related genes in somatosensory neurons, and this partially depends on the RNA binding protein MSI2. Our data reveal a cell-type-specific landscape of lincRNAs and a function for CLAP in somatosensory neurons in sensory transmission.

Mechanosensitive Ion Channel TMEM63A Gangs Up with Local Macrophages to Modulate Chronic Post-amputation Pain.

Post-amputation pain causes great suffering to amputees, but still no effective drugs are available due to its elusive mechanisms. Our previous clinical studies found that surgical removal or radiofrequency treatment of the neuroma at the axotomized nerve stump effectively relieves the phantom pain afflicting patients after amputation. This indicated an essential role of the residual nerve stump in the formation of chronic post-amputation pain (CPAP). However, the molecular mechanism by which the residual nerve stump or neuroma is involved and regulates CPAP is still a mystery. In this study, we found that nociceptors expressed the mechanosensitive ion channel TMEM63A and macrophages infiltrated into the dorsal root ganglion (DRG) neurons worked synergistically to promote CPAP. Histology and qRT-PCR showed that TMEM63A was mainly expressed in mechanical pain-producing non-peptidergic nociceptors in the DRG, and the expression of TMEM63A increased significantly both in the neuroma from amputated patients and the DRG in a mouse model of tibial nerve transfer (TNT). Behavioral tests showed that the mechanical, heat, and cold sensitivity were not affected in the Tmem63a-/- mice in the naïve state, suggesting the basal pain was not affected. In the inflammatory and post-amputation state, the mechanical allodynia but not the heat hyperalgesia or cold allodynia was significantly decreased in Tmem63a-/- mice. Further study showed that there was severe neuronal injury and macrophage infiltration in the DRG, tibial nerve, residual stump, and the neuroma-like structure of the TNT mouse model, Consistent with this, expression of the pro-inflammatory cytokines TNF-α, IL-6, and IL-1ß all increased dramatically in the DRG. Interestingly, the deletion of Tmem63a significantly reduced the macrophage infiltration in the DRG but not in the tibial nerve stump. Furthermore, the ablation of macrophages significantly reduced both the expression of Tmem63a and the mechanical allodynia in the TNT mouse model, indicating an interaction between nociceptors and macrophages, and that these two factors gang up together to regulate the formation of CPAP. This provides a new insight into the mechanisms underlying CPAP and potential drug targets its treatment.

Mechanisms and treatments of neuropathic itch in a mouse model of lymphoma.

Our understanding of neuropathic itch is limited due to a lack of relevant animal models. Patients with cutaneous T cell lymphoma (CTCL) experience severe itching. Here, we characterize a mouse model of chronic itch with remarkable lymphoma growth, immune cell accumulation, and persistent pruritus. Intradermal CTCL inoculation produced time-dependent changes in nerve innervations in lymphoma-bearing skin. In the early phase (20 days), CTCL caused hyperinnervations in the epidermis. However, chronic itch was associated with loss of epidermal nerve fibers in the late phases (40 and 60 days). CTCL was also characterized by marked nerve innervations in mouse lymphoma. Blockade of C-fibers reduced pruritus at early and late phases, whereas blockade of A-fibers only suppressed late-phase itch. Intrathecal (i.t.) gabapentin injection reduced late-phase, but not early-phase, pruritus. IL-31 was upregulated in mouse lymphoma, whereas its receptor Il31ra was persistently upregulated in Trpv1-expressing sensory neurons in mice with CTCL. Intratumoral anti-IL-31 treatment effectively suppressed CTCL-induced scratching and alloknesis (mechanical itch). Finally, i.t. administration of a TLR4 antagonist attenuated pruritus in early and late phases and in both sexes. Collectively, we have established a mouse model of neuropathic and cancer itch with relevance to human disease. Our findings also suggest distinct mechanisms underlying acute, chronic, and neuropathic itch.

GPR177 in A-fiber sensory neurons drives diabetic neuropathic pain via WNT-mediated TRPV1 activation.

Diabetic neuropathic pain (DNP) is a common and devastating complication in patients with diabetes. The mechanisms mediating DNP are not completely elucidated, and effective treatments are lacking. A-fiber sensory neurons have been shown to mediate the development of mechanical allodynia in neuropathic pain, yet the molecular basis underlying the contribution of A-fiber neurons is still unclear. Here, we report that the orphan G protein-coupled receptor 177 (GPR177) in A-fiber neurons drives DNP via WNT5a-mediated activation of transient receptor potential vanilloid receptor-1 (TRPV1) ion channel. GPR177 is mainly expressed in large-diameter A-fiber dorsal root ganglion (DRG) neurons and required for the development of DNP in mice. Mechanistically, we found that GPR177 mediated the secretion of WNT5a from A-fiber DRG neurons into cerebrospinal fluid (CSF), which was necessary for the maintenance of DNP. Extracellular perfusion of WNT5a induced rapid currents in both TRPV1-expressing heterologous cells and nociceptive DRG neurons. Computer simulations revealed that WNT5a has the potential to bind the residues at the extracellular S5-S6 loop of TRPV1. Using a peptide able to disrupt the predicted WNT5a/TRPV1 interaction suppressed DNP- and WNT5a-induced neuropathic pain symptoms in rodents. We confirmed GPR177/WNT5A coexpression in human DRG neurons and WNT5A secretion in CSF from patients with DNP. Thus, our results reveal a role for WNT5a as an endogenous and potent TRPV1 agonist, and the GPR177-WNT5a-TRPV1 axis as a driver of DNP pathogenesis in rodents. Our findings identified a potential analgesic target that might relieve neuropathic pain in patients with diabetes.

Ultrasound-Guided Extraforaminal Thoracic Nerve Root Block Through the Midpoint of the Inferior Articular Process and the Parietal Pleura: A Clinical Application of Thoracic Paravertebral Nerve Block.

PURPOSE: Thoracic nerve root (TNR) block is performed primarily under computed tomography or X-ray fluoroscopy but is associated with radiation exposure. Ultrasound requires no radiation and distinguishes vessels, nerves, pleura, and other tissues. Few reports of ultrasound-guided TNR (US-TNR) block have been described, and the puncture end point has not been clearly defined. Herein, we evaluated the feasibility of US-TNR block using the midpoint of the inferior articular process (IAP) and parietal pleura (PP) as the puncture end point. PATIENTS AND METHODS: A prospective series of 10 patients with Herpes Zoster-associated pain underwent US-TNR-guided block performed using an in-plane technique with the midpoint of thoracic IAP and PP as the puncture end points of ultrasonography. The US-TNR block procedure was performed with ultrasound as the primary imaging tool followed by fluoroscopic confirmation. RESULTS: In all patients, the needle tips were visible at the lateral margin of the pedicle in the anteroposterior view and at the extraforaminal zone in the lateral view. The TNR and dorsal root ganglion (DRG) were delineated in all 10 patients. Furthermore, 2 mL of radiopaque agent could delineate the epidural space in 8 patients and the thoracic paravertebral (TPV) space in the other 2 patients. All patients developed numbness along the corresponding dermatome 30 min after injection of local anesthetics. The numeric rating scale (NRS) score at baseline, and at two- and four-week follow-ups were 6.50 ± 1.35, 3.50 ± 0.85 (vs NRS at baseline, P < 0.01), and 4.00 ± 0.82 (vs NRS at baseline, P < 0.01), respectively. CONCLUSION: This study demonstrated the feasibility of US-TNR block using the in-plane technique with the midpoint of thoracic IAP and PP as the puncture end point to effectively block the TNR and DRG. This technique is an accurate clinical application of TPV nerve block and provides a potential therapeutic option for the treatment of neuropathic pain.

Skin Injury Activates a Rapid TRPV1-Dependent Antiviral Protein Response.

The skin serves as the interface between the body and the environment and plays a fundamental role in innate antimicrobial host immunity. Antiviral proteins (AVPs) are part of the innate host defense system and provide protection against viral pathogens. How breach of the skin barrier influences innate AVP production remains largely unknown. In this study, we characterized the induction and regulation of AVPs after skin injury and identified a key role of TRPV1 in this process. Transcriptional and phenotypic profiling of cutaneous wounds revealed that skin injury induces high levels of AVPs in both mice and humans. Remarkably, pharmacologic and genetic ablation of TRPV1-mediated nociception abrogated the induction of AVPs, including Oas2, Oasl2, and Isg15 after skin injury in mice. Conversely, stimulation of TRPV1 nociceptors was sufficient to induce AVP production involving the CD301b+ cells‒IL-27‒mediated signaling pathway. Using IL-27 receptor‒knockout mice, we show that IL-27 signaling is required in the induction of AVPs after skin injury. Finally, loss of TRPV1 signaling leads to increased viral infectivity of herpes simplex virus. Together, our data indicate that TRPV1 signaling ensures skin antiviral competence on wounding.

Ultrasonography-Guided Radiofrequency Ablation for Painful Stump Neuromas to Relieve Postamputation Pain: A Pilot Study.

OBJECTIVE: Postamputation pain (PAP) is a serious problem, and thus far, there is no perfect treatment strategy. Clinically, minimally invasive treatments for peripheral neuromas are simple and feasible. This study aimed to investigate the immediate and long-term effects of ultrasonography-guided radiofrequency ablation (RFA) on PAP. METHODS: Eighteen PAP subjects with painful peripheral neuromas were treated with ultrasonography-guided RFA. RESULTS: A total of 18 PAP subjects were included in the final analyses. Fourteen of the 17 subjects with residual limb pain (RLP) (82.4%) had successful outcomes. A successful outcome was noted in 9 of the 13 subjects with phantom limb pain (PLP) (69.2%). There were no significant associations between symptom relief and sex, age, or the duration of symptoms. There were no severe complications. CONCLUSIONS: Ultrasonography-guided RFA for painful stump neuromas can effectively relieve stump pain and PLP in amputees with PAP (follow-up time was 12 months). Ultrasonography-guided RFA is easy and safe and does not involve radiation exposure, making it very suitable for clinical applications.

Anti-PD-1 treatment impairs opioid antinociception in rodents and nonhuman primates.

Emerging immunotherapies with monoclonal antibodies against programmed cell death protein-1 (PD-1) have shown success in treating cancers. However, PD-1 signaling in neurons is largely unknown. We recently reported that dorsal root ganglion (DRG) primary sensory neurons express PD-1 and activation of PD-1 inhibits neuronal excitability and pain. Opioids are mainstay treatments for cancer pain, and morphine produces antinociception via mu opioid receptor (MOR). Here, we report that morphine antinociception and MOR signaling require neuronal PD-1. Morphine-induced antinociception after systemic or intrathecal injection was compromised in Pd1 -/- mice. Morphine antinociception was also diminished in wild-type mice after intravenous or intrathecal administration of nivolumab, a clinically used anti-PD-1 monoclonal antibody. In mouse models of inflammatory, neuropathic, and cancer pain, spinal morphine antinociception was compromised in Pd1 -/- mice. MOR and PD-1 are coexpressed in sensory neurons and their axons in mouse and human DRG tissues. Morphine produced antinociception by (i) suppressing calcium currents in DRG neurons, (ii) suppressing excitatory synaptic transmission, and (iii) inducing outward currents in spinal cord neurons; all of these actions were impaired by PD-1 blockade in mice. Loss of PD-1 also enhanced opioid-induced hyperalgesia and tolerance and potentiates opioid-induced microgliosis and long-term potentiation in the spinal cord in mice. Last, intrathecal infusion of nivolumab inhibited intrathecal morphine-induced antinociception in nonhuman primates. Our findings demonstrate that PD-1 regulates opioid receptor signaling in nociceptive neurons, leading to altered opioid-induced antinociception in rodents and nonhuman primates.

Stabilization of µ-opioid receptor facilitates its cellular translocation and signaling.

The G protein-coupled µ-opioid receptor (µ-OR) mediates the majority of analgesia effects for morphine and other pain relievers. Despite extensive studies of its structure and activation mechanisms, the inherently low maturation efficiency of µ-OR represents a major hurdle to understanding its function. Here we computationally designed µ-OR mutants with altered stability to probe the relationship between cell-surface targeting, signal transduction, and agonist efficacy. The stabilizing mutation T315Y enhanced µ-OR trafficking to the plasma membrane and significantly promoted the morphine-mediated inhibition of downstream signaling. In contrast, the destabilizing mutation R165Y led to intracellular retention of µ-OR and reduced the response to morphine stimulation. These findings suggest that µ-OR stability is an important factor in regulating receptor signaling and provide a viable avenue to improve the efficacy of analgesics.

miRNA-711 Binds and Activates TRPA1 Extracellularly to Evoke Acute and Chronic Pruritus.

Increasing evidence suggests that extracellular miRNAs may serve as biomarkers of diseases, but the physiological relevance of extracellular miRNA is unclear. We find that intradermal cheek injection of miR-711 induces TRPA1-depedent itch (scratching) without pain (wiping) in naive mice. Extracellular perfusion of miR-711 induces TRPA1 currents in both Trpa1-expressing heterologous cells and native sensory neurons through the core sequence GGGACCC. Computer simulations reveal that the core sequence binds several residues at the extracellular S5-S6 loop of TRPA1, which are critical for TRPA1 activation by miR-711 but not allyl isothiocyanate. Intradermal inoculation of human Myla cells induces lymphoma and chronic itch in immune-deficient mice, associated with increased serum levels of miR-711, secreted from cancer cells. Lymphoma-induced chronic itch is suppressed by miR-711 inhibitor and a blocking peptide that disrupts the miR-711/TRPA1 interaction. Our findings demonstrated an unconventional physiological role of extracellular naked miRNAs as itch mediators and ion channel modulators.

Differential Inhibition of Nav1.7 and Neuropathic Pain by Hybridoma-Produced and Recombinant Monoclonal Antibodies that Target Nav1.7 : Differential activities of Nav1.7-targeting monoclonal antibodies.

The voltage-gated Na+ channel subtype Nav1.7 is important for pain and itch in rodents and humans. We previously showed that a Nav1.7-targeting monoclonal antibody (SVmab) reduces Na+ currents and pain and itch responses in mice. Here, we investigated whether recombinant SVmab (rSVmab) binds to and blocks Nav1.7 similar to SVmab. ELISA tests revealed that SVmab was capable of binding to Nav1.7-expressing HEK293 cells, mouse DRG neurons, human nerve tissue, and the voltage-sensor domain II of Nav1.7. In contrast, rSVmab showed no or weak binding to Nav1.7 in these tests. Patch-clamp recordings showed that SVmab, but not rSVmab, markedly inhibited Na+ currents in Nav1.7-expressing HEK293 cells. Notably, electrical field stimulation increased the blocking activity of SVmab and rSVmab in Nav1.7-expressing HEK293 cells. SVmab was more effective than rSVmab in inhibiting paclitaxel-induced mechanical allodynia. SVmab also bound to human DRG neurons and inhibited their Na+ currents. Finally, potential reasons for the differential efficacy of SVmab and rSVmab and future directions are discussed.

α-Tubulin Acetylation Restricts Axon Overbranching by Dampening Microtubule Plus-End Dynamics in Neurons.

Axon growth is tightly controlled to establish functional neural circuits during brain development. Despite the belief that cytoskeletal dynamics is critical for cell morphology, how microtubule acetylation regulates axon development in the mammalian central nervous system remains unclear. Here, we report that loss of α-tubulin acetylation by ablation of MEC-17 in mice predisposes neurons to axon overbranching and overgrowth. Introduction of MEC-17F183A lacking α-tubulin acetyltransferase activity into MEC-17-deficient neurons failed to rescue axon defects. Moreover, loss of α-tubulin acetylation led to increases in microtubule debundling, microtubule invasion into filopodia and growth cones, and microtubule plus-end dynamics along the axon. Taxol application dampened microtubule hyperdynamics and suppressed axon overbranching and overgrowth in MEC-17-deficient neurons. Thus, our study reveals that α-tubulin acetylation acts as a brake for axon overbranching and overgrowth by dampening microtubule dynamics, providing insight into the role of microtubule post-translational modifications in regulating neural development.

SHANK3 Deficiency Impairs Heat Hyperalgesia and TRPV1 Signaling in Primary Sensory Neurons.

Abnormal pain sensitivity is commonly associated with autism spectrum disorders (ASDs) and affects the life quality of ASD individuals. SHANK3 deficiency was implicated in ASD and pain dysregulation. Here, we report functional expression of SHANK3 in mouse dorsal root ganglion (DRG) sensory neurons and spinal cord presynaptic terminals. Homozygous and heterozygous Shank3 complete knockout (Δe4-22) results in impaired heat hyperalgesia in inflammatory and neuropathic pain. Specific deletion of Shank3 in Nav1.8-expressing sensory neurons also impairs heat hyperalgesia in homozygous and heterozygous mice. SHANK3 interacts with transient receptor potential subtype V1 (TRPV1) via Proline-rich region and regulates TRPV1 surface expression. Furthermore, capsaicin-induced spontaneous pain, inward currents in DRG neurons, and synaptic currents in spinal cord neurons are all reduced after Shank3 haploinsufficiency. Finally, partial knockdown of SHANK3 expression in human DRG neurons abrogates TRPV1 function. Our findings reveal a peripheral mechanism of SHANK3, which may underlie pain deficits in SHANK3-related ASDs.

Toll-like receptor 4 contributes to chronic itch, alloknesis, and spinal astrocyte activation in male mice.

Increasing evidence suggests that Toll-like receptor 4 (TLR4) contributes importantly to spinal cord glial activation and chronic pain sensitization; however, its unique role in acute and chronic itch is unclear. In this study, we investigated the involvement of TLR4 in acute and chronic itch models in male mice using both transgenic and pharmacological approaches. Tlr4 mice exhibited normal acute itch induced by compound 48/80 and chloroquine, but these mice showed substantial reductions in scratching in chronic itch models of dry skin, induced by acetone and diethylether followed by water (AEW), contact dermatitis, and allergic contact dermatitis on the neck. Intrathecal (spinal) inhibition of TLR4 with lipopolysaccharide Rhodobacter sphaeroides did not affect acute itch but suppressed AEW-induced chronic itch. Compound 48/80 and AEW also produced robust alloknesis, a touch-elicited itch in wild-type mice, which was suppressed by intrathecal lipopolysaccharide R sphaeroides and Tlr4 deletion. Acetone and diethylether followed by water induced persistent upregulation of Tlr4 mRNA and increased TLR4 expression in GFAP-expressing astrocytes in spinal cord dorsal horn. Acetone and diethylether followed by water also induced TLR4-dependent astrogliosis (GFAP upregulation) in spinal cord. Intrathecal injection of astroglial inhibitor L-α-aminoadipate reduced AEW-induced chronic itch and alloknesis without affecting acute itch. Spinal TLR4 was also necessary for AEW-induced chronic itch in the cheek model. Interestingly, scratching plays an essential role in spinal astrogliosis because AEW-induced astrogliosis was abrogated by putting Elizabethan collars on the neck to prevent scratching the itchy skin. Our findings suggest that spinal TLR4 signaling is important for spinal astrocyte activation and astrogliosis that may underlie alloknesis and chronic itch.

A monoclonal antibody that targets a NaV1.7 channel voltage sensor for pain and itch relief.

Voltage-gated sodium (NaV) channels control the upstroke of the action potentials in excitable cells. Multiple studies have shown distinct roles of NaV channel subtypes in human physiology and diseases, but subtype-specific therapeutics are lacking and the current efforts have been limited to small molecules. Here, we present a monoclonal antibody that targets the voltage-sensor paddle of NaV1.7, the subtype critical for pain sensation. This antibody not only inhibits NaV1.7 with high selectivity, but also effectively suppresses inflammatory and neuropathic pain in mice. Interestingly, the antibody inhibits acute and chronic itch despite well-documented differences in pain and itch modulation. Using this antibody, we discovered that NaV1.7 plays a key role in spinal cord nociceptive and pruriceptive synaptic transmission. Our studies reveal that NaV1.7 is a target for itch management, and the antibody has therapeutic potential for suppressing pain and itch. Our antibody strategy may have broad applications for voltage-gated cation channels.