A mouse DRG genetic toolkit reveals morphological and physiological diversity of somatosensory neuron subtypes.

Dorsal root ganglia (DRG) somatosensory neurons detect mechanical, thermal, and chemical stimuli acting on the body. Achieving a holistic view of how different DRG neuron subtypes relay neural signals from the periphery to the CNS has been challenging with existing tools. Here, we develop and curate a mouse genetic toolkit that allows for interrogating the properties and functions of distinct cutaneous targeting DRG neuron subtypes. These tools have enabled a broad morphological analysis, which revealed distinct cutaneous axon arborization areas and branching patterns of the transcriptionally distinct DRG neuron subtypes. Moreover, in vivo physiological analysis revealed that each subtype has a distinct threshold and range of responses to mechanical and/or thermal stimuli. These findings support a model in which morphologically and physiologically distinct cutaneous DRG sensory neuron subtypes tile mechanical and thermal stimulus space to collectively encode a wide range of natural stimuli.

DRG afferents that mediate physiologic and pathologic mechanosensation from the distal colon.

The properties of dorsal root ganglia (DRG) neurons that innervate the distal colon are poorly defined, hindering our understanding of their roles in normal physiology and gastrointestinal (GI) disease. Here, we report genetically defined subsets of colon-innervating DRG neurons with diverse morphologic and physiologic properties. Four colon-innervating DRG neuron populations are mechanosensitive and exhibit distinct force thresholds to colon distension. The highest threshold population, selectively labeled using Bmpr1b genetic tools, is necessary and sufficient for behavioral responses to high colon distension, which is partly mediated by the mechanosensory ion channel Piezo2. This Aδ-HTMR population mediates behavioral over-reactivity to colon distension caused by inflammation in a model of inflammatory bowel disease. Thus, like cutaneous DRG mechanoreceptor populations, colon-innervating mechanoreceptors exhibit distinct anatomical and physiological properties and tile force threshold space, and genetically defined colon-innervating HTMRs mediate pathophysiological responses to colon distension, revealing a target population for therapeutic intervention.

PIEZO2 in somatosensory neurons controls gastrointestinal transit.

The gastrointestinal tract is in a state of constant motion. These movements are tightly regulated by the presence of food and help digestion by mechanically breaking down and propelling gut content. Mechanical sensing in the gut is thought to be essential for regulating motility; however, the identity of the neuronal populations, the molecules involved, and the functional consequences of this sensation are unknown. Here, we show that humans lacking PIEZO2 exhibit impaired bowel sensation and motility. Piezo2 in mouse dorsal root, but not nodose ganglia is required to sense gut content, and this activity slows down food transit rates in the stomach, small intestine, and colon. Indeed, Piezo2 is directly required to detect colon distension in vivo. Our study unveils the mechanosensory mechanisms that regulate the transit of luminal contents throughout the gut, which is a critical process to ensure proper digestion, nutrient absorption, and waste removal.

Immunity to the microbiota promotes sensory neuron regeneration.

Tissue immunity and responses to injury depend on the coordinated action and communication among physiological systems. Here, we show that, upon injury, adaptive responses to the microbiota directly promote sensory neuron regeneration. At homeostasis, tissue-resident commensal-specific T cells colocalize with sensory nerve fibers within the dermis, express a transcriptional program associated with neuronal interaction and repair, and promote axon growth and local nerve regeneration following injury. Mechanistically, our data reveal that the cytokine interleukin-17A (IL-17A) released by commensal-specific Th17 cells upon injury directly signals to sensory neurons via IL-17 receptor A, the transcription of which is specifically upregulated in injured neurons. Collectively, our work reveals that in the context of tissue damage, preemptive immunity to the microbiota can rapidly bridge biological systems by directly promoting neuronal repair, while also identifying IL-17A as a major determinant of this fundamental process.

Natural Killer Cells Degenerate Intact Sensory Afferents following Nerve Injury.

Sensory axons degenerate following separation from their cell body, but partial injury to peripheral nerves may leave the integrity of damaged axons preserved. We show that an endogenous ligand for the natural killer (NK) cell receptor NKG2D, Retinoic Acid Early 1 (RAE1), is re-expressed in adult dorsal root ganglion neurons following peripheral nerve injury, triggering selective degeneration of injured axons. Infiltration of cytotoxic NK cells into the sciatic nerve by extravasation occurs within 3 days following crush injury. Using a combination of genetic cell ablation and cytokine-antibody complex stimulation, we show that NK cell function correlates with loss of sensation due to degeneration of injured afferents and reduced incidence of post-injury hypersensitivity. This neuro-immune mechanism of selective NK cell-mediated degeneration of damaged but intact sensory axons complements Wallerian degeneration and suggests the therapeutic potential of modulating NK cell function to resolve painful neuropathy through the clearance of partially damaged nerves.

Retinoic acid, an essential component of the roof plate organizer, promotes the spatiotemporal segregation of dorsal neural fates.

Dorsal neural tube-derived retinoic acid promotes the end of neural crest production and transition into a definitive roof plate. Here, we analyze how this impacts the segregation of central and peripheral lineages, a process essential for tissue patterning and function. Localized in ovo inhibition in quail embryos of retinoic acid activity followed by single-cell transcriptomics unraveled a comprehensive list of differentially expressed genes relevant to these processes. Importantly, progenitors co-expressed neural crest, roof plate and dI1 interneuron markers, indicating a failure in proper lineage segregation. Furthermore, separation between roof plate and dI1 interneurons is mediated by Notch activity downstream of retinoic acid, highlighting their crucial role in establishing the roof plate-dI1 boundary. Within the peripheral branch, where absence of retinoic acid resulted in neural crest production and emigration extending into the roof plate stage, sensory progenitors failed to separate from melanocytes, leading to formation of a common glia-melanocyte cell with aberrant migratory patterns. In summary, the implementation of single-cell RNA sequencing facilitated the discovery and characterization of a molecular mechanism responsible for the segregation of dorsal neural fates during development.

Mechanism of gabapentinoid potentiation of opioid effects on cyclic AMP signaling in neuropathic pain.

Over half of spinal cord injury (SCI) patients develop opioid-resistant chronic neuropathic pain. Safer alternatives to opioids for treatment of neuropathic pain are gabapentinoids (e.g., pregabalin and gabapentin). Clinically, gabapentinoids appear to amplify opioid effects, increasing analgesia and overdose-related adverse outcomes, but in vitro proof of this amplification and its mechanism are lacking. We previously showed that after SCI, sensitivity to opioids is reduced by fourfold to sixfold in rat sensory neurons. Here, we demonstrate that after injury, gabapentinoids restore normal sensitivity of opioid inhibition of cyclic AMP (cAMP) generation, while reducing nociceptor hyperexcitability by inhibiting voltage-gated calcium channels (VGCCs). Increasing intracellular Ca2+ or activation of L-type VGCCs (L-VGCCs) suffices to mimic SCI effects on opioid sensitivity, in a manner dependent on the activity of the Raf1 proto-oncogene, serine/threonine-protein kinase C-Raf, but independent of neuronal depolarization. Together, our results provide a mechanism for potentiation of opioid effects by gabapentinoids after injury, via reduction of calcium influx through L-VGCCs, and suggest that other inhibitors targeting these channels may similarly enhance opioid treatment of neuropathic pain.

Development of an oral treatment that rescues gait ataxia and retinal degeneration in a phenotypic mouse model of familial dysautonomia.

Familial dysautonomia (FD) is a rare neurodegenerative disease caused by a splicing mutation in elongator acetyltransferase complex subunit 1 (ELP1). This mutation leads to the skipping of exon 20 and a tissue-specific reduction of ELP1, mainly in the central and peripheral nervous systems. FD is a complex neurological disorder accompanied by severe gait ataxia and retinal degeneration. There is currently no effective treatment to restore ELP1 production in individuals with FD, and the disease is ultimately fatal. After identifying kinetin as a small molecule able to correct the ELP1 splicing defect, we worked on its optimization to generate novel splicing modulator compounds (SMCs) that can be used in individuals with FD. Here, we optimize the potency, efficacy, and bio-distribution of second-generation kinetin derivatives to develop an oral treatment for FD that can efficiently pass the blood-brain barrier and correct the ELP1 splicing defect in the nervous system. We demonstrate that the novel compound PTC258 efficiently restores correct ELP1 splicing in mouse tissues, including brain, and most importantly, prevents the progressive neuronal degeneration that is characteristic of FD. Postnatal oral administration of PTC258 to the phenotypic mouse model TgFD9;Elp1Δ20/flox increases full-length ELP1 transcript in a dose-dependent manner and leads to a 2-fold increase in functional ELP1 in the brain. Remarkably, PTC258 treatment improves survival, gait ataxia, and retinal degeneration in the phenotypic FD mice. Our findings highlight the great therapeutic potential of this novel class of small molecules as an oral treatment for FD.

NT-3 contributes to chemotherapy-induced neuropathic pain through TrkC-mediated CCL2 elevation in DRG neurons.

Cancer patients undergoing treatment with antineoplastic drugs often experience chemotherapy-induced neuropathic pain (CINP), and the therapeutic options for managing CINP are limited. Here, we show that systemic paclitaxel administration upregulates the expression of neurotrophin-3 (Nt3) mRNA and NT3 protein in the neurons of dorsal root ganglia (DRG), but not in the spinal cord. Blocking NT3 upregulation attenuates paclitaxel-induced mechanical, heat, and cold nociceptive hypersensitivities and spontaneous pain without altering acute pain and locomotor activity in male and female mice. Conversely, mimicking this increase produces enhanced responses to mechanical, heat, and cold stimuli and spontaneous pain in naive male and female mice. Mechanistically, NT3 triggers tropomyosin receptor kinase C (TrkC) activation and participates in the paclitaxel-induced increases of C-C chemokine ligand 2 (Ccl2) mRNA and CCL2 protein in the DRG. Given that CCL2 is an endogenous initiator of CINP and that Nt3 mRNA co-expresses with TrkC and Ccl2 mRNAs in DRG neurons, NT3 likely contributes to CINP through TrkC-mediated activation of the Ccl2 gene in DRG neurons. NT3 may be thus a potential target for CINP treatment.

Regulation of Neuroregeneration by Long Noncoding RNAs.

In mammals, neurons in the peripheral nervous system (PNS) have regenerative capacity following injury, but it is generally absent in the CNS. This difference is attributed, at least in part, to the intrinsic ability of PNS neurons to activate a unique regenerative transcriptional program following injury. Here, we profiled gene expression following sciatic nerve crush in mice and identified long noncoding RNAs (lncRNAs) that act in the regenerating neurons and which are typically not expressed in other contexts. We show that two of these lncRNAs regulate the extent of neuronal outgrowth. We then focus on one of these, Silc1, and show that it regulates neuroregeneration in cultured cells and in vivo, through cis-acting activation of the transcription factor Sox11.

Neutrophils infiltrate sensory ganglia and mediate chronic widespread pain in fibromyalgia.

Fibromyalgia is a debilitating widespread chronic pain syndrome that occurs in 2 to 4% of the population. The prevailing view that fibromyalgia results from central nervous system dysfunction has recently been challenged with data showing changes in peripheral nervous system activity. Using a mouse model of chronic widespread pain through hyperalgesic priming of muscle, we show that neutrophils invade sensory ganglia and confer mechanical hypersensitivity on recipient mice, while adoptive transfer of immunoglobulin, serum, lymphocytes, or monocytes has no effect on pain behavior. Neutrophil depletion abolishes the establishment of chronic widespread pain in mice. Neutrophils from patients with fibromyalgia also confer pain on mice. A link between neutrophil-derived mediators and peripheral nerve sensitization is already established. Our observations suggest approaches for targeting fibromyalgia pain via mechanisms that cause altered neutrophil activity and interactions with sensory neurons.

Propionate exerts neuroprotective and neuroregenerative effects in the peripheral nervous system.

In inflammatory neuropathies, oxidative stress results in neuronal and Schwann cell (SC) death promoting early neurodegeneration and clinical disability. Treatment with the short-chain fatty acid propionate showed a significant immunoregulatory and neuroprotective effect in multiple sclerosis patients. Similar effects have been described for patients with chronic inflammatory demyelinating polyneuropathy (CIDP). Therefore, Schwann cell's survival and dorsal root ganglia (DRG) outgrowth were evaluated in vitro after propionate treatment and application of H2O2 or S-nitroso-N-acetyl-D-L-penicillamine (SNAP) to evaluate neuroprotection. In addition, DRG resistance was evaluated by the application of oxidative stress by SNAP ex vivo after in vivo propionate treatment. Propionate treatment secondary to SNAP application on DRG served as a neuroregeneration model. Histone acetylation as well as expression of the free fatty acid receptor (FFAR) 2 and 3, histone deacetylases, neuroregeneration markers, and antioxidative mediators were investigated. ß-hydroxybutyrate was used as a second FFAR3 ligand, and pertussis toxin was used as an FFAR3 antagonist. FFAR3, but not FFAR2, expression was evident on SC and DRG. Propionate-mediated activation of FFAR3 and histone 3 hyperacetylation resulted in increased catalase expression and increased resistance to oxidative stress. In addition, propionate treatment resulted in enhanced neuroregeneration with concomitant growth-associated protein 43 expression. We were able to demonstrate an antioxidative and neuroregenerative effect of propionate on SC and DRG mediated by FFAR3-induced histone acetylases expression. Our results describe a pathway to achieve neuroprotection/neuroregeneration relevant for patients with immune-mediated neuropathies.

Self-renewing macrophages in dorsal root ganglia contribute to promote nerve regeneration.

Sensory neurons located in dorsal root ganglia (DRG) convey sensory information from peripheral tissue to the brain. After peripheral nerve injury, sensory neurons switch to a regenerative state to enable axon regeneration and functional recovery. This process is not cell autonomous and requires glial and immune cells. Macrophages in the DRG (DRGMacs) accumulate in response to nerve injury, but their origin and function remain unclear. Here, we mapped the fate and response of DRGMacs to nerve injury using macrophage depletion, fate-mapping, and single-cell transcriptomics. We identified three subtypes of DRGMacs after nerve injury in addition to a small population of circulating bone-marrow-derived precursors. Self-renewing macrophages, which proliferate from local resident macrophages, represent the largest population of DRGMacs. The other two subtypes include microglia-like cells and macrophage-like satellite glial cells (SGCs) (Imoonglia). We show that self-renewing DRGMacs contribute to promote axon regeneration. Using single-cell transcriptomics data and CellChat to simulate intercellular communication, we reveal that macrophages express the neuroprotective and glioprotective ligand prosaposin and communicate with SGCs via the prosaposin receptor GPR37L1. These data highlight that DRGMacs have the capacity to self-renew, similarly to microglia in the Central nervous system (CNS) and contribute to promote axon regeneration. These data also reveal the heterogeneity of DRGMacs and their potential neuro- and glioprotective roles, which may inform future therapeutic approaches to treat nerve injury.

Low-Frequency Stimulation of Trpv1-Lineage Peripheral Afferents Potentiates the Excitability of Spino-Periaqueductal Gray Projection Neurons.

High-threshold dorsal root ganglion (HT DRG) neurons fire at low frequencies during inflammatory injury, and low-frequency stimulation (LFS) of HT DRG neurons selectively potentiates excitatory synapses onto spinal neurons projecting to the periaqueductal gray (spino-PAG). Here, in male and female mice, we have identified an underlying peripheral sensory population driving this plasticity and its effects on the output of spino-PAG neurons. We provide the first evidence that Trpv1-lineage sensory neurons predominantly induce burst firing, a unique mode of neuronal activity, in lamina I spino-PAG projection neurons. We modeled inflammatory injury by optogenetically stimulating Trpv1+ primary afferents at 2 Hz for 2 min (LFS), as peripheral inflammation induces 1-2 Hz firing in high-threshold C fibers. LFS of Trpv1+ afferents enhanced the synaptically evoked and intrinsic excitability of spino-PAG projection neurons, eliciting a stable increase in the number of action potentials (APs) within a Trpv1+ fiber-induced burst, while decreasing the intrinsic AP threshold and increasing the membrane resistance. Further experiments revealed that this plasticity required Trpv1+ afferent input, postsynaptic G protein-coupled signaling, and NMDA receptor activation. Intriguingly, an inflammatory injury and heat exposure in vivo also increased APs per burst, in vitro These results suggest that inflammatory injury-mediated plasticity is driven though Trpv1+ DRG neurons and amplifies the spino-PAG pathway. Spinal inputs to the PAG could play an integral role in its modulation of heat sensation during peripheral inflammation, warranting further exploration of the organization and function of these neural pathways.

PGE2 Potentiates Orai1-Mediated Calcium Entry Contributing to Peripheral Sensitization.

Peripheral sensitization is one of the primary mechanisms underlying the pathogenesis of chronic pain. However, candidate molecules involved in peripheral sensitization remain incompletely understood. We have shown that store-operated calcium channels (SOCs) are expressed in the dorsal root ganglion (DRG) neurons. Whether SOCs contribute to peripheral sensitization associated with chronic inflammatory pain is elusive. Here we report that global or conditional deletion of Orai1 attenuates Complete Freund's adjuvant (CFA)-induced pain hypersensitivity in both male and female mice. To further establish the role of Orai1 in inflammatory pain, we performed calcium imaging and patch-clamp recordings in wild-type (WT) and Orai1 knockout (KO) DRG neurons. We found that SOC function was significantly enhanced in WT but not in Orai1 KO DRG neurons from CFA- and carrageenan-injected mice. Interestingly, the Orai1 protein level in L3/4 DRGs was not altered under inflammatory conditions. To understand how Orai1 is modulated under inflammatory pain conditions, prostaglandin E2 (PGE2) was used to sensitize DRG neurons. PGE2-induced increase in neuronal excitability and pain hypersensitivity was significantly reduced in Orai1 KO mice. PGE2-induced potentiation of SOC entry (SOCE) was observed in WT, but not in Orai1 KO DRG neurons. This effect was attenuated by a PGE2 receptor 1 (EP1) antagonist and mimicked by an EP1 agonist. Inhibition of Gq/11, PKC, or ERK abolished PGE2-induced SOCE increase, indicating PGE2-induced SOCE enhancement is mediated by EP1-mediated downstream cascade. These findings demonstrate that Orai1 plays an important role in peripheral sensitization. Our study also provides new insight into molecular mechanisms underlying PGE2-induced modulation of inflammatory pain.Significance Statement Store-operated calcium channel (SOC) Orai1 is expressed and functional in dorsal root ganglion (DRG) neurons. Whether Orai1 contributes to peripheral sensitization is unclear. The present study demonstrates that Orai1-mediated SOC function is enhanced in DRG neurons under inflammatory conditions. Global and conditional deletion of Orai1 attenuates complete Freund's adjuvant (CFA)-induced pain hypersensitivity. We also demonstrate that prostaglandin E2 (PGE2) potentiates SOC function in DRG neurons through EP1-mediated signaling pathway. Importantly, we have found that Orai1 deficiency diminishes PGE2-induced SOC function increase and reduces PGE2-induced increase in neuronal excitability and pain hypersensitivity. These findings suggest that Orai1 plays an important role in peripheral sensitization associated with inflammatory pain. Our study reveals a novel mechanism underlying PGE2/EP1-induced peripheral sensitization. Orai1 may serve as a potential target for pathological pain.

Caldendrin Is a Repressor of PIEZO2 Channels and Touch Sensation in Mice.

The sense of touch is crucial for cognitive, emotional, and social development and relies on mechanically activated (MA) ion channels that transduce force into an electrical signal. Despite advances in the molecular characterization of these channels, the physiological factors that control their activity are poorly understood. Here, we used behavioral assays, electrophysiological recordings, and various mouse strains (males and females analyzed separately) to investigate the role of the calmodulin-like Ca2+ sensor, caldendrin, as a key regulator of MA channels and their roles in touch sensation. In mice lacking caldendrin (Cabp1 KO), heightened responses to tactile stimuli correlate with enlarged MA currents with lower mechanical thresholds in dorsal root ganglion neurons (DRGNs). The expression pattern of caldendrin in the DRG parallels that of the major MA channel required for touch sensation, PIEZO2. In transfected cells, caldendrin interacts with and inhibits the activity of PIEZO2 in a manner that requires an alternatively spliced sequence in the N-terminal domain of caldendrin. Moreover, targeted genetic deletion of caldendrin in Piezo2-expressing DRGNs phenocopies the tactile hypersensitivity of complete Cabp1 KO mice. We conclude that caldendrin is an endogenous repressor of PIEZO2 channels and their contributions to touch sensation in DRGNs.

Constitutive KCC2 Cell- and Synapse-Specifically Regulates NMDA Receptor Activity in the Spinal Cord.

K+-Cl- cotransporter-2 (KCC2) critically controls neuronal chloride homeostasis and maintains normal synaptic inhibition by GABA and glycine. Nerve injury diminishes synaptic inhibition in the spinal cord via KCC2 impairment. However, how KCC2 regulates nociceptive input to spinal excitatory and inhibitory neurons remains elusive. Here, we show that basal GABA reversal potentials were significantly more depolarized in vesicular GABA transporter (VGAT)-expressing inhibitory neurons than those in vesicular glutamate transporter-2 (VGluT2)-expressing excitatory neurons in spinal cords of male and female mice. Strikingly, inhibiting KCC2 with VU0463271 increased currents elicited by puff NMDA and the NMDAR-mediated frequency of mEPSCs in VGluT2, but not in VGAT, dorsal horn neurons. Notably, VU0463271 had no effect on EPSCs monosynaptically evoked from the dorsal root in VGluT2 neurons. Furthermore, VU0463271 augmented α2δ-1-NMDAR interactions and their protein levels in spinal cord synaptosomes. In Cacna2d1 KO mice, VU0463271 had no effect on puff NMDA currents or the mEPSC frequency in dorsal horn neurons. Disrupting α2δ-1-NMDAR interactions with α2δ-1 C-terminus mimicking peptide diminished VU0463271-induced potentiation in the mEPSC frequency and puff NMDA currents in VGluT2 neurons. Additionally, intrathecal injection of VU0463271 reduced mechanical and thermal thresholds in wild-type mice, but not in Cacna2d1 KO mice. VU0463271-induced pain hypersensitivity in mice was abrogated by co-treatment with the NMDAR antagonist, pregabalin (an α2δ-1 inhibitory ligand), or α2δ-1 C-terminus mimicking peptide. Our findings suggest that KCC2 controls presynaptic and postsynaptic NMDAR activity specifically in excitatory dorsal horn neurons. KCC2 impairment preferentially potentiates nociceptive transmission between spinal excitatory interneurons via α2δ-1-bound NMDARs.Significance statementImpaired function of potassium-chloride cotransporter-2 (KCC2), a key regulator of neuronal inhibition, in the spinal cord plays a major role in neuropathic pain. This study unveils that KCC2 controls spinal nociceptive synaptic strength via NMDA receptors in a cell type- and synapse type-specific manner. KCC2 inhibition preferentially augments presynaptic and postsynaptic NMDA receptor activity in spinal excitatory interneurons via α2δ-1 (previously known as a calcium channel subunit). Importantly, spinal KCC2 impairment triggers pain hypersensitivity through α2δ-1-coupled NMDA receptors. These findings pinpoint the cell and molecular substrates for the reciprocal relationship between spinal synaptic inhibition and excitation in chronic neuropathic pain. Targeting both KCC2 and α2δ-1­NMDA receptor complexes could be an effective strategy in managing neuropathic pain conditions.

Complete persistence of the primary somatosensory system in zebrafish.

The somatosensory system detects peripheral stimuli that are translated into behaviors necessary for survival. Fishes and amphibians possess two somatosensory systems in the trunk: the primary somatosensory system, formed by the Rohon-Beard neurons, and the secondary somatosensory system, formed by the neural crest cell-derived neurons of the Dorsal Root Ganglia. Rohon-Beard neurons have been characterized as a transient population that mostly disappears during the first days of life and is functionally replaced by the Dorsal Root Ganglia. Here, I follow Rohon-Beard neurons in vivo and show that the entire repertoire remains present in zebrafish from 1-day post-fertilization until the juvenile stage, 15-days post-fertilization. These data indicate that zebrafish retain two complete somatosensory systems until at least a developmental stage when the animals display complex behavioral repertoires.

Downregulation of Chloride voltage-gated channel 7 contributes to hyperalgesia following spared nerve injury.

Alterations in anion balance potential, along with the involvement of cation-chloride cotransporters, play pivotal roles in the development of hyperalgesia after peripheral nerve injury (PNI). Chloride voltage-gated channel 7 (CLCN7) is the predominant member of the CLC protein family. Investigations on CLCN7 have focused primarily on its involvement in osteosclerosis and lysosomal storage disorders; nevertheless, its contribution to neuropathic pain (NP) has not been determined. In this investigation, we noted high expression of CLCN7 in neurons situated within the spinal dorsal horns (SDHs) and dorsal root ganglions (DRGs). Immunofluorescence analysis revealed that CLCN7 was predominantly distributed among IB4-positive and CGRP-positive neurons. Furthermore, the expression of CLCN7 was observed to be mainly reduced in neurons within the SDHs and in small and medium-sized neurons located in the DRGs of spared nerve injury (SNI) mice. Knockdown of CLCN7 via siRNA in the DRGs resulted in increased mechanical and thermal hyperalgesia in naïve mice. Furthermore, the excitability of cultured DRG neurons in vitro was augmented upon treatment with CLCN7 siRNA. These findings suggested that CLCN7 downregulation following SNI was crucial for the manifestation of mechanical and thermal hyperalgesia, highlighting potential targeting strategies for treating NP.

Piezo1-Mediated Neurogenic Inflammatory Cascade Exacerbates Ventricular Remodeling After Myocardial Infarction.

BACKGROUND: Heart failure is associated with a high rate of mortality and morbidity, and ventricular remodeling invariably precedes heart failure. Ventricular remodeling is fundamentally driven by mechanotransduction that is regulated by both the nervous system and the immune system. However, it remains unknown which key molecular factors govern the neuro/immune/cardio axis that underlies mechanotransduction during ventricular remodeling. Here, we investigated whether the mechanosensitive Piezo cation channel-mediated neurogenic inflammatory cascade underlies ventricular remodeling-related mechanotransduction. METHODS: By ligating the left coronary artery of rats to establish an in vivo model of chronic myocardial infarction (MI), lentivirus-mediated thoracic dorsal root ganglion (TDRG)-specific Piezo1 knockdown rats and adeno-associated virus-PHP.S-mediated TDRG neuron-specific Piezo1 knockout mice were used to investigate whether Piezo1 in the TDRG plays a functional role during ventricular remodeling. Subsequently, neutralizing antibody-mediated TDRG IL-6 (interleukin-6) inhibition rats and adeno-associated virus-PHP.S-mediated TDRG neuron-specific IL-6 knockdown mice were used to determine the mechanism underlying neurogenic inflammation. Primary TDRG neurons were used to evaluate Piezo1 function in vitro. RESULTS: Expression of Piezo1 and IL-6 was increased, and these factors were functionally activated in TDRG neurons at 4 weeks after MI. Both knockdown of TDRG-specific Piezo1 and deletion of TDRG neuron-specific Piezo1 lessened the severity of ventricular remodeling at 4 weeks after MI and decreased the level of IL-6 in the TDRG or heart. Furthermore, inhibition of TDRG IL-6 or knockdown of TDRG neuron-specific IL-6 also ameliorated ventricular remodeling and suppressed the IL-6 cascade in the heart, whereas the Piezo1 level in the TDRG was not affected. In addition, enhanced Piezo1 function, as reflected by abundant calcium influx induced by Yoda1 (a selective agonist of Piezo1), led to increased release of IL-6 from TDRG neurons in mice 4 weeks after MI. CONCLUSIONS: Our findings point to a critical role for Piezo1 in ventricular remodeling at 4 weeks after MI and reveal a neurogenic inflammatory cascade as a previously unknown facet of the neuronal immune signaling axis underlying mechanotransduction.