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1.
J Clin Invest ; 134(13)2024 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-38722683

RESUMO

This study reports that targeting intrinsically disordered regions of the voltage-gated sodium channel 1.7 (NaV1.7) protein facilitates discovery of sodium channel inhibitory peptide aptamers (NaViPA) for adeno-associated virus-mediated (AAV-mediated), sensory neuron-specific analgesia. A multipronged inhibition of INa1.7, INa1.6, INa1.3, and INa1.1 - but not INa1.5 and INa1.8 - was found for a prototype and named NaViPA1, which was derived from the NaV1.7 intracellular loop 1, and is conserved among the TTXs NaV subtypes. NaViPA1 expression in primary sensory neurons (PSNs) of dorsal root ganglia (DRG) produced significant inhibition of TTXs INa but not TTXr INa. DRG injection of AAV6-encoded NaViPA1 significantly attenuated evoked and spontaneous pain behaviors in both male and female rats with neuropathic pain induced by tibial nerve injury (TNI). Whole-cell current clamp of the PSNs showed that NaViPA1 expression normalized PSN excitability in TNI rats, suggesting that NaViPA1 attenuated pain by reversal of injury-induced neuronal hypersensitivity. IHC revealed efficient NaViPA1 expression restricted in PSNs and their central and peripheral terminals, indicating PSN-restricted AAV biodistribution. Inhibition of sodium channels by NaViPA1 was replicated in the human iPSC-derived sensory neurons. These results summate that NaViPA1 is a promising analgesic lead that, combined with AAV-mediated PSN-specific block of multiple TTXs NaVs, has potential as a peripheral nerve-restricted analgesic therapeutic.


Assuntos
Dependovirus , Canal de Sódio Disparado por Voltagem NAV1.7 , Células Receptoras Sensoriais , Animais , Ratos , Dependovirus/genética , Células Receptoras Sensoriais/metabolismo , Masculino , Humanos , Feminino , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.7/genética , Gânglios Espinais/metabolismo , Ratos Sprague-Dawley , Neuralgia/metabolismo , Neuralgia/genética , Neuralgia/tratamento farmacológico , Analgesia
2.
Res Sq ; 2023 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-37886453

RESUMO

We previously reported functional Piezo1 expression in Schwann cells of the peripheral nervous system. This study is designed to further investigate the role of Schwann cell Piezo1 in peripheral nociception. We first developed an adeno-associated viral (AAV) vector that has primary Schwann cell tropism after delivery into the sciatic nerve. This was achieved by packing AAV-GFP transcribed by a hybrid CMV enhancer/chicken ß-actin (CBA) promoter using a capsid AAVolig001 to generate AAVolig001-CBA-GFP. Five weeks after intrasciatic injection of AAVolig001-CBA-GFP in naïve rats, GFP expression was detected selectively in the Schwann cells of the sciatic nerve. A short hairpin RNA against rat Piezo1 (PZ1shRNA) was designed that showed efficient physical and functional knockdown of Piezo1 in NG108 neuronal cells. A dual promoter and bidirectional AAV encoding a U6-driven PZ1shRNA and CBA-transcribed GFP was packed with capsid olig001 (AAVolig001-PZ1shRNA), and AAV was injected into unilateral sciatic nerve immediately after induction of common peroneal nerve injury (CPNI). Results showed that the development of mechanical hypersensitivity in the CPNI rats injected with AAVolig001-PZ1shRNA was mitigated, compared to rats subjected with AAVolig001-scramble. Selective in vivo Schwann cell transduction and functional block of Piezo1 channel activity of primary cultured Schwann cells was confirmed. Together, our data demonstrate that 1) AAVolig001 has unique and selective primary tropism to Schwann cells via intrasciatic delivery and 2) Schwann cell Piezo1 contributes to mechanical hypersensitivity following nerve injury.

3.
Mol Pain ; 19: 17448069231174315, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37247618

RESUMO

Here, we present evidence showing Piezo1 protein expression in the primary sensory neurons (PSNs) and non-neuronal cells of rat peripheral nervous system. Using a knockdown/knockout validated antibody, we detected Piezo1 immunoreactivity (IR) in ∼60% of PSNs of rat dorsal root ganglia (DRG) with higher IR density in the small- and medium-sized neurons. Piezo1-IR was clearly identified in DRG perineuronal glia, including satellite glial cells (SGCs) and Schwann cells; in sciatic nerve Schwann cells surrounding the axons and cutaneous afferent endings; and in skin epidermal Merkel cells and melanocytes. Neuronal and non-neuronal Piezo1 channels were functional since various cells (dissociated PSNs and SGCs from DRGs, isolated Schwann cells, and primary human melanocytes) exhibited a robust response to Piezo1 agonist Yoda1 by an increase of intracellular Ca2+ concentration ([Ca2+]i). These responses were abolished by non-specific Piezo1 antagonist GsMTx4. Immunoblots showed elevated Piezo1 protein in DRG proximal to peripheral nerve injury-induced painful neuropathy, while PSNs and SGCs from rats with neuropathic pain showed greater Yoda1-evoked elevation of [Ca2+]i and an increased frequency of cells responding to Yoda1, compared to controls. Sciatic nerve application of GsMTx4 alleviated mechanical hypersensitivity induced by Yoda1. Overall, our data show that Piezo1 is widely expressed by the neuronal and non-neuronal cells in the peripheral sensory pathways and that painful nerve injury appeared associated with activation of Piezo1 in PSNs and peripheral glial cells.


Assuntos
Neuralgia , Neuroglia , Animais , Humanos , Ratos , Gânglios Espinais/metabolismo , Canais Iônicos/metabolismo , Neuralgia/metabolismo , Neuroglia/metabolismo , Células de Schwann/metabolismo , Nervo Isquiático/metabolismo , Células Receptoras Sensoriais/metabolismo
4.
Arthritis Res Ther ; 24(1): 168, 2022 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-35842727

RESUMO

BACKGROUND: Peripheral and central nociceptive sensitization is a critical pathogenetic component in osteoarthritis (OA) chronic pain. T-type calcium channel 3.2 (CaV3.2) regulates neuronal excitability and plays important roles in pain processing. We previously identified that enhanced T-type/CaV3.2 activity in the primary sensory neurons (PSNs) of dorsal root ganglia (DRG) is associated with neuropathic pain behavior in a rat model of monosodium iodoacetate (MIA)-induced knee OA. PSN-specific T-type/CaV3.2 may therefore represent an important mediator in OA painful neuropathy. Here, we test the hypothesis that the T-type/CaV3.2 channels in PSNs can be rationally targeted for pain relief in MIA-OA. METHODS: MIA model of knee OA was induced in male and female rats by a single injection of 2 mg MIA into intra-knee articular cavity. Two weeks after induction of knee MIA-OA pain, recombinant adeno-associated viruses (AAV)-encoding potent CaV3.2 inhibitory peptide aptamer 2 (CaV3.2iPA2) that have been characterized in our previous study were delivered into the ipsilateral lumbar 4/5 DRG. Effectiveness of DRG-CaV3.2iPA2 treatment on evoked (mechanical and thermal) and spontaneous (conditioned place preference) pain behavior, as well as weight-bearing asymmetry measured by Incapacitance tester, in the arthritic limbs of MIA rats were evaluated. AAV-mediated transgene expression in DRG was determined by immunohistochemistry. RESULTS: AAV-mediated expression of CaV3.2iPA2 selective in the DRG-PSNs produced significant and comparable mitigations of evoked and spontaneous pain behavior, as well as normalization of weight-bearing asymmetry in both male and female MIA-OA rats. Analgesia of DRG-AAV-CaV3.2iPA1, another potent CaV3.2 inhibitory peptide, was also observed. Whole-cell current-clamp recordings showed that AAV-mediated CaV3.2iPA2 expression normalized hyperexcitability of the PSNs dissociated from the DRG of MIA animals, suggesting that CaV3.2iPA2 attenuated pain behavior by reversing MIA-induced neuronal hyperexcitability. CONCLUSIONS: Together, our results add therapeutic support that T-type/CaV3.2 in primary sensory pathways contributes to MIA-OA pain pathogenesis and that CaV3.2iPAs are promising analgesic leads that, combined with AAV-targeted delivery in anatomically segmental sensory ganglia, have the potential for further development as a peripheral selective T-type/CaV3.2-targeting strategy in mitigating chronic MIA-OA pain behavior. Validation of the therapeutic potential of this strategy in other OA models may be valuable in future study.


Assuntos
Neuralgia , Osteoartrite do Joelho , Animais , Modelos Animais de Doenças , Feminino , Gânglios Espinais/metabolismo , Ácido Iodoacético/toxicidade , Masculino , Osteoartrite do Joelho/metabolismo , Ratos , Ratos Sprague-Dawley , Células Receptoras Sensoriais/metabolismo
5.
Pain ; 163(12): 2466-2484, 2022 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-35420557

RESUMO

ABSTRACT: Ample data support a prominent role of peripheral T-type calcium channels 3.2 (Ca V 3.2) in generating pain states. Development of primary sensory neuron-specific inhibitors of Ca V 3.2 channels is an opportunity for achieving effective analgesic therapeutics, but success has been elusive. Small peptides, especially those derived from natural proteins as inhibitory peptide aptamers (iPAs), can produce highly effective and selective blockade of specific nociceptive molecular pathways to reduce pain with minimal off-target effects. In this study, we report the engineering of the potent and selective iPAs of Ca V 3.2 from the intrinsically disordered regions (IDRs) of Ca V 3.2 intracellular segments. Using established prediction algorithms, we localized the IDRs in Ca V 3.2 protein and identified several Ca V 3.2iPA candidates that significantly reduced Ca V 3.2 current in HEK293 cells stably expressing human wide-type Ca V 3.2. Two prototype Ca V 3.2iPAs (iPA1 and iPA2) derived from the IDRs of Ca V 3.2 intracellular loops 2 and 3, respectively, were expressed selectively in the primary sensory neurons of dorsal root ganglia in vivo using recombinant adeno-associated virus (AAV), which produced sustained inhibition of calcium current conducted by Ca V 3.2/T-type channels and significantly attenuated both evoked and spontaneous pain behavior in rats with neuropathic pain after tibial nerve injury. Recordings from dissociated sensory neurons showed that AAV-mediated Ca V 3.2iPA expression suppressed neuronal excitability, suggesting that Ca V 3.2iPA treatment attenuated pain by reversal of injury-induced neuronal hypersensitivity. Collectively, our results indicate that Ca V 3.2iPAs are promising analgesic leads that, combined with AAV-mediated delivery in anatomically targeted sensory ganglia, have the potential to be a selective peripheral Ca V 3.2-targeting strategy for clinical treatment of pain.


Assuntos
Analgesia , Aptâmeros de Peptídeos , Canais de Cálcio Tipo T , Neuralgia , Ratos , Humanos , Animais , Dependovirus , Manejo da Dor , Células HEK293 , Ratos Sprague-Dawley , Gânglios Espinais/metabolismo , Neuralgia/tratamento farmacológico , Células Receptoras Sensoriais/metabolismo , Analgésicos/uso terapêutico , Aptâmeros de Peptídeos/farmacologia , Peptídeos/uso terapêutico , Canais de Cálcio/metabolismo , Canais de Cálcio Tipo T/metabolismo
6.
Gene Ther ; 29(1-2): 1-15, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-32424233

RESUMO

The Sigma-1 receptor (σ1R) is highly expressed in the primary sensory neurons (PSNs) that are the critical site of initiation and maintenance of pain following peripheral nerve injury. By immunoblot and immunohistochemistry, we observed increased expression of both σ1R and σ1R-binding immunoglobulin protein (BiP) in the lumbar (L) dorsal root ganglia (DRG) ipsilateral to painful neuropathy induced by spared nerve injury (SNI). To evaluate the therapeutic potential of PSN-targeted σ1R inhibition at a selected segmental level, we designed a recombinant adeno-associated viral (AAV) vector expressing a small hairpin RNA (shRNA) against rat σ1R. Injection of this vector into the L4/L5 DRGs induced downregulation of σ1R in DRG neurons of all size groups, while expression of BiP was not affected. This was accompanied by attenuation of SNI-induced cutaneous mechanical and thermal hypersensitivity. Whole-cell current-clamp recordings of dissociated neurons showed that knockdown of σ1R suppressed neuronal excitability, suggesting that σ1R silencing attenuates pain by reversal of injury-induced neuronal hyperexcitability. These findings support a critical role of σ1R in modulating PSN nociceptive functions, and that the nerve injury-induced elevated σ1R activity in the PSNs can be a significant driver of neuropathic pain. Further understanding the role of PSN-σ1R in pain pathology may open routes to exploit this system for DRG-targeted pain therapy.


Assuntos
Neuralgia , Receptores sigma , Animais , Gânglios Espinais/metabolismo , Neuralgia/genética , Neuralgia/terapia , Ratos , Ratos Sprague-Dawley , Receptores sigma/genética , Receptores sigma/metabolismo , Células Receptoras Sensoriais/metabolismo , Receptor Sigma-1
7.
Pain ; 162(11): 2750-2768, 2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34285153

RESUMO

ABSTRACT: Piezo2 mechanotransduction channel is a crucial mediator of sensory neurons for sensing and transducing touch, vibration, and proprioception. We here characterized Piezo2 expression and cell specificity in rat peripheral sensory pathway using a validated Piezo2 antibody. Immunohistochemistry using this antibody revealed Piezo2 expression in pan primary sensory neurons of dorsal root ganglia in naïve rats, which was actively transported along afferent axons to both central presynaptic terminals innervating the spinal dorsal horn (DH) and peripheral afferent terminals in the skin. Piezo2 immunoreactivity (IR) was also detected in the postsynaptic neurons of the DH and in the motor neurons of the ventral horn, but not in spinal glial fibrillary acidic protein-positive and Iba1-positive glia. Notably, Piezo2-IR was clearly identified in peripheral nonneuronal cells, including perineuronal glia, Schwann cells in the sciatic nerve and surrounding cutaneous afferent endings, as well as in skin epidermal Merkel cells and melanocytes. Immunoblots showed increased Piezo2 in dorsal root ganglia ipsilateral to plantar injection of complete Freund's adjuvant, and immunostaining revealed increased Piezo2-IR intensity in the DH ipsilateral to complete Freund's adjuvant injection. This elevation of DH Piezo2-IR was also evident in various neuropathic pain models and monosodium iodoacetate knee osteoarthritis pain model, compared with controls. We conclude that (1) the pan neuronal profile of Piezo2 expression suggests that Piezo2 may function extend beyond simply touch or proprioception mediated by large-sized low-threshold mechanosensitive primary sensory neurons; (2) Piezo2 may have functional roles involving sensory processing in the spinal cord, Schwann cells, and skin melanocytes; and (3) aberrant Piezo2 expression may contribute pain pathogenesis.


Assuntos
Mecanotransdução Celular , Neuralgia , Animais , Gânglios Espinais/metabolismo , Canais Iônicos/metabolismo , Neuroglia/metabolismo , Neurônios Aferentes/metabolismo , Ratos , Células Receptoras Sensoriais/metabolismo
8.
Mol Pain ; 16: 1744806920963807, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33054557

RESUMO

The monosodium iodoacetate knee osteoarthritis model has been widely used for the evaluation of osteoarthritis pain, but the pathogenesis of associated chronic pain is not fully understood. The T-type calcium channel 3.2 (CaV3.2) is abundantly expressed in the primary sensory neurons, in which it regulates neuronal excitability at both the somata and peripheral terminals and facilitates spontaneous neurotransmitter release at the spinal terminals. In this study, we investigated the involvement of primary sensory neuron-CaV3.2 activation in monosodium iodoacetate osteoarthritis pain. Knee joint osteoarthritis pain was induced by intra-articular injection of monosodium iodoacetate (2 mg) in rats, and sensory behavior was evaluated for 35 days. At that time, knee joint structural histology, primary sensory neuron injury, and inflammatory gliosis in lumbar dorsal root ganglia, and spinal dorsal horn were examined. Primary sensory neuron-T-type calcium channel current by patch-clamp recording and CaV3.2 expression by immunohistochemistry and immunoblots were determined. In a subset of animals, pain relief by CaV3.2 inhibition after delivery of CaV3.2 inhibitor TTA-P2 into sciatic nerve was investigated. Knee injection of monosodium iodoacetate resulted in osteoarthritis histopathology, weight-bearing asymmetry, sensory hypersensitivity of the ipsilateral hindpaw, and inflammatory gliosis in the ipsilateral dorsal root ganglia, sciatic nerve, and spinal dorsal horn. Neuronal injury marker ATF-3 was extensively upregulated in primary sensory neurons, suggesting that neuronal damage was beyond merely knee-innervating primary sensory neurons. T-type current in dissociated primary sensory neurons from lumbar dorsal root ganglia of monosodium iodoacetate rats was significantly increased, and CaV3.2 protein levels in the dorsal root ganglia and spinal dorsal horn ipsilateral to monosodium iodoacetate by immunoblots were significantly increased, compared to controls. Perineural application of TTA-P2 into the ipsilateral sciatic nerve alleviated mechanical hypersensitivity and weight-bearing asymmetry in monosodium iodoacetate osteoarthritis rats. Overall, our findings demonstrate an elevated CaV3.2 expression and enhanced function of primary sensory neuron-T channels in the monosodium iodoacetate osteoarthritis pain. Further study is needed to delineate the importance of dysfunctional primary sensory neuron-CaV3.2 in osteoarthritis pain.


Assuntos
Benzamidas/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo T/metabolismo , Neuralgia/metabolismo , Osteoartrite do Joelho/metabolismo , Piperidinas/farmacologia , Células Receptoras Sensoriais/metabolismo , Fator 3 Ativador da Transcrição/metabolismo , Animais , Escala de Avaliação Comportamental , Benzamidas/uso terapêutico , Bloqueadores dos Canais de Cálcio/uso terapêutico , Difosfatos/toxicidade , Modelos Animais de Doenças , Gânglios Espinais/metabolismo , Imidazóis/toxicidade , Imuno-Histoquímica , Inflamação/metabolismo , Masculino , Nociceptores/metabolismo , Osteoartrite do Joelho/induzido quimicamente , Osteoartrite do Joelho/patologia , Osteoartrite do Joelho/fisiopatologia , Piperidinas/uso terapêutico , Ratos , Ratos Sprague-Dawley , Nervo Isquiático/efeitos dos fármacos , Células Receptoras Sensoriais/patologia , Regulação para Cima
9.
Mol Pain ; 16: 1744806920925425, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32484015

RESUMO

Transient receptor potential ankyrin 1 (TRPA1) is well documented as an important molecule in pain hypersensitivity following inflammation and nerve injury and in many other cellular biological processes. Here, we show that TRPA1 is expressed not only by sensory neurons of the dorsal root ganglia (DRG) but also in their adjacent satellite glial cells (SGCs), as well as nonmyelinating Schwann cells. TRPA1 immunoreactivity is also detected in various cutaneous structures of sensory neuronal terminals, including small and large caliber cutaneous sensory fibers and endings. The SGC-expressed TRPA1 is functional. Like DRG neurons, dissociated SGCs exhibit a robust response to the TRPA1-selective agonist allyl isothiocyanate (AITC) by an increase of intracellular Ca2+ concentration ([Ca2+]i). These responses are abolished by the TRPA1 antagonist HC030031 and are absent in SGCs and neurons from global TRPA1 null mice. SGCs and neurons harvested from DRG proximal to painful tissue inflammation induced by plantar injection of complete Freund's adjuvant show greater AITC-evoked elevation of [Ca2+]i and slower recovery compared to sham controls. Similar TRPA1 sensitization occurs in both SGCs and neurons during neuropathic pain induced by spared nerve injury. Together, these results show that functional TRPA1 is expressed by sensory ganglia SGCs, and TRPA1 function in SGCs is enhanced after both peripheral inflammation and nerve injury, and suggest that TRPA1 in SGCs may contribute to inflammatory and neuropathic pain.


Assuntos
Inflamação/patologia , Neuralgia/metabolismo , Neuralgia/patologia , Neuroglia/patologia , Células Receptoras Sensoriais/patologia , Canal de Cátion TRPA1/metabolismo , Animais , Tamanho Celular , Gânglios Espinais/metabolismo , Gânglios Espinais/patologia , Isotiocianatos , Masculino , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Ratos Sprague-Dawley , Células de Schwann/metabolismo , Células Receptoras Sensoriais/metabolismo
10.
Pain Rep ; 4(1): e703, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30801043

RESUMO

INTRODUCTION: Tmem100 modulates interactions between TRPA1 and TRPV1. The cell specificity of Tmem100 expression in dorsal root ganglia (DRGs) is not well defined, nor is the effect of peripheral nerve injury on Tmem100 expression. OBJECTIVE: This study was designed to determine the cell specificity of Tmem100 expression in DRG and its subcellular localization, and to examine how Tmem100 expression may be altered in painful conditions. METHODS: Dorsal root ganglion Tmem100 expression was determined by immunohistochemistry, immunoblot, and quantitative real-time PCR, and compared between various experimental rat pain models and controls. RESULTS: Tmem100 is expressed in both neurons and perineuronal glial cells in the rat DRG. The plasma membrane and intracellular localization of Tmem100 are identified in 83% ± 6% of IB4-positive and 48% ± 6% of calcitonin gene-related peptide-positive neurons, as well as in medium- and large-sized neurons, with its immunopositivity colocalized to TRPV1 (94% ± 5%) and TRPA1 (96% ± 3%). Tmem100 is also detected in the perineuronal satellite glial cells and in some microglia. Tmem100 protein is significantly increased in the lumbar DRGs in the complete Freund adjuvant inflammatory pain. By contrast, peripheral nerve injury by spinal nerve ligation diminishes Tmem100 expression in the injured DRG, with immunoblot and immunohistochemistry experiments showing reduced Tmem100 protein levels in both neurons and satellite glial cells of DRGs proximal to injury, whereas Tmem100 is unchanged in adjacent DRGs. The spared nerve injury model also reduces Tmem100 protein in the injured DRGs. CONCLUSION: Our data demonstrate a pain pathology-dependent alteration of DRG Tmem100 protein expression, upregulated during CFA inflammatory pain but downregulated during neuropathic pain.

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