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1.
Nat Commun ; 12(1): 1955, 2021 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-33782410

RESUMO

p62/SQSTM1 is known to act as a key mediator in the selective autophagy of protein aggregates, or aggrephagy, by steering ubiquitinated protein aggregates towards the autophagy pathway. Here, we use a yeast two-hybrid screen to identify the prefoldin-like chaperone UXT as an interacting protein of p62. We show that UXT can bind to protein aggregates as well as the LB domain of p62, and, possibly by forming an oligomer, increase p62 clustering for its efficient targeting to protein aggregates, thereby promoting the formation of the p62 body and clearance of its cargo via autophagy. We also find that ectopic expression of human UXT delays SOD1(A4V)-induced degeneration of motor neurons in a Xenopus model system, and that specific disruption of the interaction between UXT and p62 suppresses UXT-mediated protection. Together, these results indicate that UXT functions as an autophagy adaptor of p62-dependent aggrephagy. Furthermore, our study illustrates a cooperative relationship between molecular chaperones and the aggrephagy machinery that efficiently removes misfolded protein aggregates.


Assuntos
Autofagia/genética , Proteínas de Ciclo Celular/genética , Chaperonas Moleculares/genética , Agregados Proteicos , Proteína Sequestossoma-1/genética , Superóxido Dismutase-1/genética , Animais , Autofagia/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células HeLa , Humanos , Leupeptinas/farmacologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Chaperonas Moleculares/metabolismo , Neurônios Motores/citologia , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Cultura Primária de Células , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Agregados Proteicos/efeitos dos fármacos , Dobramento de Proteína/efeitos dos fármacos , Proteína Sequestossoma-1/metabolismo , Transdução de Sinais , Superóxido Dismutase-1/metabolismo , Transgenes , Xenopus laevis
2.
Nat Commun ; 12(1): 1401, 2021 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-33658516

RESUMO

Effective treatments for patients suffering from heat hypersensitivity are lacking, mostly due to our limited understanding of the pathogenic mechanisms underlying this disorder. In the nervous system, activating transcription factor 4 (ATF4) is involved in the regulation of synaptic plasticity and memory formation. Here, we show that ATF4 plays an important role in heat nociception. Indeed, loss of ATF4 in mouse dorsal root ganglion (DRG) neurons selectively impairs heat sensitivity. Mechanistically, we show that ATF4 interacts with transient receptor potential cation channel subfamily M member-3 (TRPM3) and mediates the membrane trafficking of TRPM3 in DRG neurons in response to heat. Loss of ATF4 also significantly decreases the current and KIF17-mediated trafficking of TRPM3, suggesting that the KIF17/ATF4/TRPM3 complex is required for the neuronal response to heat stimuli. Our findings unveil the non-transcriptional role of ATF4 in the response to heat stimuli in DRG neurons.


Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Nociceptividade/fisiologia , Células Receptoras Sensoriais/fisiologia , Canais de Cátion TRPM/metabolismo , Fator 4 Ativador da Transcrição/genética , Animais , Membrana Celular/metabolismo , Quimiocina CXCL12/metabolismo , Gânglios Espinais/citologia , Gânglios Espinais/fisiologia , Células HEK293 , Temperatura Alta , Humanos , Injeções Espinhais , Cinesina/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Técnicas de Patch-Clamp , Transporte Proteico , Receptores CXCR4/metabolismo , Canais de Cátion TRPM/genética
3.
Nat Commun ; 12(1): 1026, 2021 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-33589589

RESUMO

Proprioceptive neurons (PNs) are essential for the proper execution of all our movements by providing muscle sensory feedback to the central motor network. Here, using deep single cell RNAseq of adult PNs coupled with virus and genetic tracings, we molecularly identify three main types of PNs (Ia, Ib and II) and find that they segregate into eight distinct subgroups. Our data unveil a highly sophisticated organization of PNs into discrete sensory input channels with distinct spatial distribution, innervation patterns and molecular profiles. Altogether, these features contribute to finely regulate proprioception during complex motor behavior. Moreover, while Ib- and II-PN subtypes are specified around birth, Ia-PN subtypes diversify later in life along with increased motor activity. We also show Ia-PNs plasticity following exercise training, suggesting Ia-PNs are important players in adaptive proprioceptive function in adult mice.


Assuntos
Retroalimentação Sensorial/fisiologia , Gânglios Espinais/metabolismo , Neurônios Motores/metabolismo , Propriocepção/fisiologia , Células Receptoras Sensoriais/metabolismo , Animais , Calbindina 1/genética , Calbindina 1/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas Correpressoras/genética , Proteínas Correpressoras/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Subunidade alfa 3 de Fator de Ligação ao Core/metabolismo , Gânglios Espinais/citologia , Expressão Gênica , Proteínas com Domínio LIM/genética , Proteínas com Domínio LIM/metabolismo , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios Motores/classificação , Neurônios Motores/citologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Condicionamento Físico Animal , Células Receptoras Sensoriais/classificação , Células Receptoras Sensoriais/citologia , Análise de Célula Única , Medula Espinal/citologia , Medula Espinal/metabolismo
4.
Nat Commun ; 12(1): 426, 2021 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-33462216

RESUMO

Painful neuropathy is a frequent complication in diabetes. Proopiomelanocortin (POMC) is an endogenous opioid precursor peptide, which plays a protective role against pain. Here, we report dysfunctional POMC-mediated antinociception in sensory neurons in diabetes. In streptozotocin-induced diabetic mice the Pomc promoter is repressed due to increased binding of NF-kB p50 subunit, leading to a loss in basal POMC level in peripheral nerves. Decreased POMC levels are also observed in peripheral nervous system tissue from diabetic patients. The antinociceptive pathway mediated by POMC is further impaired due to lysosomal degradation of µ-opioid receptor (MOR). Importantly, the neuropathic phenotype of the diabetic mice is rescued upon viral overexpression of POMC and MOR in the sensory ganglia. This study identifies an antinociceptive mechanism in the sensory ganglia that paves a way for a potential therapy for diabetic neuropathic pain.


Assuntos
Diabetes Mellitus Experimental/complicações , Neuropatias Diabéticas/patologia , Nociceptividade/fisiologia , Pró-Opiomelanocortina/deficiência , Células Receptoras Sensoriais/patologia , Idoso , Idoso de 80 Anos ou mais , Animais , Diabetes Mellitus Experimental/induzido quimicamente , Neuropatias Diabéticas/etiologia , Feminino , Gânglios Espinais/citologia , Gânglios Espinais/patologia , Humanos , Lisossomos , Masculino , Camundongos , Camundongos Knockout , Pró-Opiomelanocortina/genética , Proteólise , Receptores Opioides mu/genética , Receptores Opioides mu/metabolismo , Estreptozocina/toxicidade
5.
Nat Commun ; 12(1): 435, 2021 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-33469022

RESUMO

Epidural electrical stimulation (EES) of lumbosacral sensorimotor circuits improves leg motor control in animals and humans with spinal cord injury (SCI). Upper-limb motor control involves similar circuits, located in the cervical spinal cord, suggesting that EES could also improve arm and hand movements after quadriplegia. However, the ability of cervical EES to selectively modulate specific upper-limb motor nuclei remains unclear. Here, we combined a computational model of the cervical spinal cord with experiments in macaque monkeys to explore the mechanisms of upper-limb motoneuron recruitment with EES and characterize the selectivity of cervical interfaces. We show that lateral electrodes produce a segmental recruitment of arm motoneurons mediated by the direct activation of sensory afferents, and that muscle responses to EES are modulated during movement. Intraoperative recordings suggested similar properties in humans at rest. These modelling and experimental results can be applied for the development of neurotechnologies designed for the improvement of arm and hand control in humans with quadriplegia.


Assuntos
Medula Cervical/fisiopatologia , Neurônios Motores/fisiologia , Quadriplegia/terapia , Recrutamento Neurofisiológico/fisiologia , Traumatismos da Medula Espinal/terapia , Estimulação da Medula Espinal/métodos , Vias Aferentes/fisiopatologia , Animais , Medula Cervical/citologia , Medula Cervical/diagnóstico por imagem , Medula Cervical/lesões , Simulação por Computador , Modelos Animais de Doenças , Eletrodos Implantados , Espaço Epidural , Feminino , Gânglios Espinais/citologia , Gânglios Espinais/diagnóstico por imagem , Gânglios Espinais/fisiopatologia , Humanos , Macaca fascicularis , Imagem por Ressonância Magnética , Masculino , Modelos Neurológicos , Músculo Esquelético/inervação , Quadriplegia/etiologia , Quadriplegia/fisiopatologia , Traumatismos da Medula Espinal/complicações , Traumatismos da Medula Espinal/diagnóstico , Traumatismos da Medula Espinal/fisiopatologia , Estimulação da Medula Espinal/instrumentação , Extremidade Superior/inervação
6.
Methods Mol Biol ; 2201: 71-82, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32975790

RESUMO

Immunohistochemical staining is widely used to identify opioid receptors in specific cell types throughout the nervous system. Opioid receptors are not restricted to the central nervous system, but are also present in peripheral sensory neurons, where their activation exerts analgesic effects without inducing centrally mediated side effects. Here, we describe immunohistochemical analysis of µ-opioid receptors in the peripheral sensory neuron cell bodies, along the axons and their peripheral endings in the hind paw skin, as well as in the spinal cord, under naïve and sciatic nerve damage conditions in mice. Importantly, we consider the ongoing debate on the specificity of antibodies.


Assuntos
Imuno-Histoquímica/métodos , Nervos Periféricos/metabolismo , Receptores Opioides mu/imunologia , Analgésicos Opioides/metabolismo , Animais , Axônios/metabolismo , Gânglios Espinais/citologia , Humanos , Camundongos , Nervos Periféricos/imunologia , Receptores Acoplados a Proteínas-G/metabolismo , Receptores Opioides/imunologia , Receptores Opioides/metabolismo , Receptores Opioides mu/metabolismo , Nervo Isquiático/citologia , Neuropatia Ciática , Células Receptoras Sensoriais/metabolismo , Medula Espinal/metabolismo
7.
PLoS One ; 15(9): e0229475, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32915783

RESUMO

The importance of glial cells in the modulation of neuronal processes is now generally accepted. In particular, enormous progress in our understanding of astrocytes and microglia physiology in the central nervous system (CNS) has been made in recent years, due to the development of genetic and molecular toolkits. However, the roles of satellite glial cells (SGCs) and macrophages-the peripheral counterparts of astrocytes and microglia-remain poorly studied despite their involvement in debilitating conditions, such as pain. Here, we characterized in dorsal root ganglia (DRGs), different genetically-modified mouse lines previously used for studying astrocytes and microglia, with the goal to implement them for investigating DRG SGC and macrophage functions. Although SGCs and astrocytes share some molecular properties, most tested transgenic lines were found to not be suitable for studying selectively a large number of SGCs within DRGs. Nevertheless, we identified and validated two mouse lines: (i) a CreERT2 recombinase-based mouse line allowing transgene expression almost exclusively in SGCs and in the vast majority of SGCs, and (ii) a GFP-expressing line allowing the selective visualization of macrophages. In conclusion, among the tools available for exploring astrocyte functions, a few can be used for studying selectively a great proportion of SGCs. Thus, efforts remain to be made to characterize other available mouse lines as well as to develop, rigorously characterize and validate new molecular tools to investigate the roles of DRG SGCs, but also macrophages, in health and disease.


Assuntos
Gânglios Espinais/fisiologia , Macrófagos/fisiologia , Modelos Animais , Células Satélites Perineuronais/fisiologia , Animais , Astrócitos , Técnicas Biossensoriais/métodos , Células Cultivadas , Gânglios Espinais/citologia , Imuno-Histoquímica , Microscopia Intravital/métodos , Camundongos , Camundongos Transgênicos , Sondas Moleculares/química , Sondas Moleculares/genética , Imagem Óptica , Fótons , Cultura Primária de Células
8.
Neuron ; 108(1): 128-144.e9, 2020 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-32810432

RESUMO

Primary somatosensory neurons are specialized to transmit specific types of sensory information through differences in cell size, myelination, and the expression of distinct receptors and ion channels, which together define their transcriptional and functional identity. By profiling sensory ganglia at single-cell resolution, we find that all somatosensory neuronal subtypes undergo a similar transcriptional response to peripheral nerve injury that both promotes axonal regeneration and suppresses cell identity. This transcriptional reprogramming, which is not observed in non-neuronal cells, resolves over a similar time course as target reinnervation and is associated with the restoration of original cell identity. Injury-induced transcriptional reprogramming requires ATF3, a transcription factor that is induced rapidly after injury and necessary for axonal regeneration and functional recovery. Our findings suggest that transcription factors induced early after peripheral nerve injury confer the cellular plasticity required for sensory neurons to transform into a regenerative state.


Assuntos
Fator 3 Ativador da Transcrição/genética , Reprogramação Celular/genética , Gânglios Espinais/citologia , Regulação da Expressão Gênica/genética , Neuralgia/genética , Traumatismos dos Nervos Periféricos/genética , Células Receptoras Sensoriais/metabolismo , Animais , Axônios , Axotomia , Lesões por Esmagamento/genética , Lesões por Esmagamento/metabolismo , Vértebras Lombares , Mecanorreceptores/metabolismo , Camundongos , Regeneração Nervosa , Plasticidade Neuronal/genética , Nociceptores/metabolismo , RNA-Seq , Recuperação de Função Fisiológica , Nervo Isquiático/lesões , Nervo Isquiático/cirurgia , Análise de Célula Única , Nervos Espinhais/lesões , Nervos Espinhais/cirurgia , Transcriptoma
9.
Neuron ; 107(6): 1141-1159.e7, 2020 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-32735781

RESUMO

Diabetic peripheral neuropathy (DPN) is a highly frequent and debilitating clinical complication of diabetes that lacks therapies. Cellular oxidative stress regulates post-translational modifications, including SUMOylation. Here, using unbiased screens, we identified key enzymes in metabolic pathways and ion channels as novel molecular targets of SUMOylation that critically regulated their activity. Sensory neurons of diabetic patients and diabetic mice demonstrated changes in the SUMOylation status of metabolic enzymes and ion channels. In support of this, profound metabolic dysfunction, accelerated neuropathology, and sensory loss were observed in diabetic gene-targeted mice selectively lacking the ability to SUMOylate proteins in peripheral sensory neurons. TRPV1 function was impaired by diabetes-induced de-SUMOylation as well as by metabolic imbalance elicited by de-SUMOylation of metabolic enzymes, facilitating diabetic sensory loss. Our results unexpectedly uncover an endogenous post-translational mechanism regulating diabetic neuropathy in patients and mouse models that protects against metabolic dysfunction, nerve damage, and altered sensory perception.


Assuntos
Neuropatias Diabéticas/metabolismo , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Nociceptividade , Células Receptoras Sensoriais/metabolismo , Sumoilação , Canais de Cátion TRPV/metabolismo , Animais , Células Cultivadas , Ciclo do Ácido Cítrico , Neuropatias Diabéticas/fisiopatologia , Feminino , Gânglios Espinais/citologia , Glicólise , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL
10.
Proc Natl Acad Sci U S A ; 117(29): 17260-17268, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32632007

RESUMO

Understanding how a network of interconnected neurons receives, stores, and processes information in the human brain is one of the outstanding scientific challenges of our time. The ability to reliably detect neuroelectric activities is essential to addressing this challenge. Optical recording using voltage-sensitive fluorescent probes has provided unprecedented flexibility for choosing regions of interest in recording neuronal activities. However, when recording at a high frame rate such as 500 to 1,000 Hz, fluorescence-based voltage sensors often suffer from photobleaching and phototoxicity, which limit the recording duration. Here, we report an approach called electrochromic optical recording (ECORE) that achieves label-free optical recording of spontaneous neuroelectrical activities. ECORE utilizes the electrochromism of poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) thin films, whose optical absorption can be modulated by an applied voltage. Being based on optical reflection instead of fluorescence, ECORE offers the flexibility of an optical probe without suffering from photobleaching or phototoxicity. Using ECORE, we optically recorded spontaneous action potentials in cardiomyocytes, cultured hippocampal and dorsal root ganglion neurons, and brain slices. With minimal perturbation to cells, ECORE allows long-term optical recording over multiple days.


Assuntos
Eletrofisiologia/métodos , Neurônios/fisiologia , Poliestirenos , Tiofenos , Potenciais de Ação/fisiologia , Encéfalo/citologia , Encéfalo/fisiologia , Técnicas Eletroquímicas/métodos , Fenômenos Eletrofisiológicos , Corantes Fluorescentes , Gânglios Espinais/citologia , Gânglios Espinais/fisiologia , Humanos , Imagem Óptica , Óptica e Fotônica/métodos
11.
Sci Rep ; 10(1): 10953, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32616790

RESUMO

Anatomically incomplete spinal cord injuries can be followed by functional recovery mediated, in part, by the formation of intraspinal detour circuits. Here, we show that adult mice recover tactile and proprioceptive function following a unilateral dorsal column lesion. We therefore investigated the basis of this recovery and focused on the plasticity of the dorsal column-medial lemniscus pathway. We show that ascending dorsal root ganglion (DRG) axons branch in the spinal grey matter and substantially increase the number of these collaterals following injury. These sensory fibers exhibit synapsin-positive varicosities, indicating their integration into spinal networks. Using a monosynaptic circuit tracing with rabies viruses injected into the cuneate nucleus, we show the presence of spinal cord neurons that provide a detour pathway to the original target area of DRG axons. Notably the number of contacts between DRG collaterals and those spinal neurons increases by more than 300% after injury. We then characterized these interneurons and showed that the lesion triggers a remodeling of the connectivity pattern. Finally, using re-lesion experiments after initial remodeling of connections, we show that these detour circuits are responsible for the recovery of tactile and proprioceptive function. Taken together our study reveals that detour circuits represent a common blueprint for axonal rewiring after injury.


Assuntos
Gânglios Espinais/fisiologia , Regeneração Nervosa , Vias Neurais , Neurônios/fisiologia , Recuperação de Função Fisiológica , Células Receptoras Sensoriais/fisiologia , Traumatismos da Medula Espinal/prevenção & controle , Animais , Comportamento Animal , Gânglios Espinais/citologia , Camundongos , Camundongos Endogâmicos C57BL , Plasticidade Neuronal , Neurônios/citologia , Traumatismos da Medula Espinal/etiologia , Traumatismos da Medula Espinal/patologia
12.
Immunity ; 52(5): 753-766, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32433948

RESUMO

Although the medical definition of itch has been in existence for 360 years, only in the last 20 years have we begun to understand the basic mechanisms that underlie this unique sensation. Therapeutics that specifically target chronic itch as a pathologic entity are currently still not available. Recent seminal advances in itch circuitry within the nervous system have intersected with discoveries in immunology in unexpected ways to rapidly inform emerging treatment strategies. The current review aims to introduce these basic concepts in itch biology and highlight how distinct immunologic pathways integrate with recently identified itch-sensory circuits in the nervous system to inform a major new paradigm of neuroimmunology and therapeutic development for chronic itch.


Assuntos
Gânglios Espinais/imunologia , Prurido/imunologia , Células Receptoras Sensoriais/imunologia , Pele/imunologia , Córtex Somatossensorial/imunologia , Animais , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Humanos , Modelos Imunológicos , Modelos Neurológicos , Prurido/diagnóstico , Prurido/fisiopatologia , Células Receptoras Sensoriais/metabolismo , Células Receptoras Sensoriais/fisiologia , Transdução de Sinais/imunologia , Transdução de Sinais/fisiologia , Pele/inervação , Córtex Somatossensorial/fisiopatologia
13.
Sci Rep ; 10(1): 8632, 2020 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-32451393

RESUMO

Pain evoked by visceral inflammation is often 'referred' to the somatic level. Transient receptor potential ankyrin 1 (TRPA1) has been reported to contribute to visceral pain-like behavior in dextran sulfate sodium (DSS)-evoked colitis. However, the role of TRPA1 in somatic component of hypersensitivity due to visceral inflammation is unknown. The present study investigated the role of TRPA1 in colitis-evoked mechanical hypersensitivity at the somatic level. Colitis was induced in mice by adding DSS to drinking water for one week. Control and DSS-treated mice were tested for various parameters of colitis as well as mechanical pain sensitivity in abdominal and facial regions. DSS treatment caused mechanical hypersensitivity in the abdominal and facial skin. Pharmacological blockade or genetic deletion of TRPA1 prevented the colitis-associated mechanical hypersensitivity in the abdominal and facial skin areas although the severity of colitis remained unaltered. DSS treatment increased expression of TRPA1 mRNA in cultured dorsal root ganglion (DRG) neurons, but not trigeminal ganglion neurons, and selectively enhanced currents evoked by the TRPA1 agonist, allyl isothiocyanate, in cultured DRG neurons. Our findings indicate that the TRPA1 channel contributes to colitis-associated mechanical hypersensitivity in somatic tissues, an effect associated with upregulation of TRPA1 expression and responsiveness in DRG nociceptors.


Assuntos
Colite/patologia , Dor Nociceptiva/patologia , Canal de Cátion TRPA1/metabolismo , Acetanilidas/farmacologia , Animais , Colite/induzido quimicamente , Sulfato de Dextrana/toxicidade , Potenciais Evocados/efeitos dos fármacos , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Isotiocianatos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Purinas/farmacologia , Estresse Mecânico , Canal de Cátion TRPA1/antagonistas & inibidores , Canal de Cátion TRPA1/genética , Gânglio Trigeminal/citologia , Gânglio Trigeminal/metabolismo
14.
Int J Nanomedicine ; 15: 3251-3266, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32440122

RESUMO

Background: Peripheral neuropathy is a common and painful side effect that occurs in patients with cancer induced by Oxaliplatin (OXL). The neurotoxicity correlates with the damage of dorsal root ganglion (DRG) neurons and Schwann cells (SCs). Hydroxysafflor yellow A (HSYA), icariin, epimedin B and 3, 4-dihydroxybenzoic acid (DA) are the main neuroprotective ingredients identified in Wen-Luo-Tong (WLT), a traditional Chinese medicinal topical compound. The purpose of this study was to prepare and evaluate the efficacy of an ethosomes gel formulation loaded with a combination of HSYA, icariin, epimedin B and DA. However, the low LogP value, poor solubility and macromolecule are several challenges for topical delivery of these drugs. Methods: Ethosomes were prepared by the single-step injection technique. Particle size, entrapment efficiency and in vitro drug deposition studies were determined to select the optimum ethosomes. The optimized ethosomes were further incorporated into carbopol to obtain a gel. The rheological properties, morphology, in vitro drug release, in vitro gel application and skin distribution of the ethosomes gels were studied. A rat model of oxaliplatin-induced neuropathy was established to assess the therapeutic efficacy of the ethosomes gel. Results: Seventy percent (v/v) ethanol, cinnamaldehyde and Phospholipon 90G were employed to develop ethosomes a carrier system. This system had a high entrapment efficiency, carried large amounts of HSYA, epimedin B, DA and icarrin, and penetrated deep into the epidermis and dermis. The optimized ethosomes had the maximum deposition of icariin, HSYA, epimedin B and relative higher amount of DA in epidermis (2.00±0.13 µg/cm2, 5.72±0.75 µg/cm2, 1.97±0.27 µg/cm2 and 9.25±1.21 µg/cm2, respectively). 0.5% carbopol 980 was selected to develop the ethosomes gel with desirable viscoelasticity and spreadability, which was suitable for topical application. The mechanical allodynia and hyperalgesia induced by OXL in rats were significantly reduced after the new ethosomes gel was applied to rats compared to model group. Conclusion: Based on our findings, the ethosomes gel delivery system provided a new formulation for the topical delivery of HSYA, icariin, epimedin B and DA to counteract OXL-induced peripheral neuropathy.


Assuntos
Géis/química , Fármacos Neuroprotetores/uso terapêutico , Oxaliplatina/efeitos adversos , Doenças do Sistema Nervoso Periférico/induzido quimicamente , Doenças do Sistema Nervoso Periférico/tratamento farmacológico , Acroleína/análogos & derivados , Acroleína/química , Administração Tópica , Animais , Comportamento Animal , Liberação Controlada de Fármacos , Sinergismo Farmacológico , Gânglios Espinais/citologia , Lipossomos , Masculino , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/administração & dosagem , Fármacos Neuroprotetores/farmacologia , Oxaliplatina/administração & dosagem , Tamanho da Partícula , Ratos Wistar , Reologia , Pele/metabolismo , Absorção Cutânea/efeitos dos fármacos
15.
Sci Rep ; 10(1): 4932, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-32188875

RESUMO

Acoustic manipulation is an emerging non-invasive method enabling precise spatial control of cells in their native environment. Applying this method for organizing neurons is invaluable for neural tissue engineering applications. Here, we used surface and bulk standing acoustic waves for large-scale patterning of Dorsal Root Ganglia neurons and PC12 cells forming neuronal cluster networks, organized biomimetically. We showed that by changing parameters such as voltage intensity or cell concentration we were able to affect cluster properties. We examined the effects of acoustic arrangement on cells atop 3D hydrogels for up to 6 days and showed that assembled cells spontaneously grew branches in a directed manner towards adjacent clusters, infiltrating the matrix. These findings have great relevance for tissue engineering applications as well as for mimicking architectures and properties of native tissues.


Assuntos
Neurônios/fisiologia , Som , Animais , Técnicas de Cultura de Células , Células Cultivadas , Fenômenos Eletrofisiológicos , Gânglios Espinais/citologia , Neuritos/fisiologia , Células PC12 , Ratos , Engenharia Tecidual
16.
Proc Natl Acad Sci U S A ; 117(10): 5494-5501, 2020 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-32079727

RESUMO

Somatosensory neurons have historically been classified by a variety of approaches, including structural, anatomical, and genetic markers; electrophysiological properties; pharmacological sensitivities; and more recently, transcriptional profile differentiation. These methodologies, used separately, have yielded inconsistent classification schemes. Here, we describe phenotypic differences in response to pharmacological agents as measured by changes in cytosolic calcium concentration for the rapid classification of neurons in vitro; further analysis with genetic markers, whole-cell recordings, and single-cell transcriptomics validated these findings in a functional context. Using this general approach, which we refer to as tripartite constellation analysis (TCA), we focused on large-diameter dorsal-root ganglion (L-DRG) neurons with myelinated axons. Divergent responses to the K-channel antagonist, κM-conopeptide RIIIJ (RIIIJ), reliably identified six discrete functional cell classes. In two neuronal subclasses (L1 and L2), block with RIIIJ led to an increase in [Ca] i Simultaneous electrophysiology and calcium imaging showed that the RIIIJ-elicited increase in [Ca] i corresponded to different patterns of action potentials (APs), a train of APs in L1 neurons, and sporadic firing in L2 neurons. Genetically labeled mice established that L1 neurons are proprioceptors. The single-cell transcriptomes of L1 and L2 neurons showed that L2 neurons are Aδ-low-threshold mechanoreceptors. RIIIJ effects were replicated by application of the Kv1.1 selective antagonist, Dendrotoxin-K, in several L-DRG subclasses (L1, L2, L3, and L5), suggesting the presence of functional Kv1.1/Kv1.2 heteromeric channels. Using this approach on other neuronal subclasses should ultimately accelerate the comprehensive classification and characterization of individual somatosensory neuronal subclasses within a mixed population.


Assuntos
Gânglios Espinais/citologia , Células Receptoras Sensoriais/classificação , Células Receptoras Sensoriais/fisiologia , Animais , Cálcio/metabolismo , Conotoxinas/farmacologia , Citosol/metabolismo , Gânglios Espinais/efeitos dos fármacos , Canal de Potássio Kv1.1/antagonistas & inibidores , Camundongos , Camundongos Transgênicos , Peptídeos/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Células Receptoras Sensoriais/efeitos dos fármacos , Análise de Célula Única , Transcriptoma
17.
J Neurosci ; 40(11): 2189-2199, 2020 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-32019828

RESUMO

The interaction between the immune system and the nervous system has been at the center of multiple research studies in recent years. Whereas the role played by cytokines as neuronal mediators is no longer contested, the mechanisms by which cytokines modulate pain processing remain to be elucidated. In this study, we have analyzed the involvement of granulocyte-macrophage colony stimulating factor (GM-CSF) in nociceptor activation in male and female mice. Previous studies have suggested GM-CSF might directly activate neurons. However, here we established the absence of a functional GM-CSF receptor in murine nociceptors, and suggest an indirect mechanism of action, via immune cells. We report that GM-CSF applied directly to magnetically purified nociceptors does not induce any transcriptional changes in nociceptive genes. In contrast, conditioned medium from GM-CSF-treated murine macrophages was able to drive nociceptor transcription. We also found that conditioned medium from nociceptors treated with the well established pain mediator, nerve growth factor, could also modify macrophage gene transcription, providing further evidence for a bidirectional crosstalk.SIGNIFICANCE STATEMENT The interaction of the immune system and the nervous system is known to play an important role in the development and maintenance of chronic pain disorders. Elucidating the mechanisms of these interactions is an important step toward understanding, and therefore treating, chronic pain disorders. This study provides evidence for a two-way crosstalk between macrophages and nociceptors in the peripheral nervous system, which may contribute to the sensitization of nociceptors by cytokines in pain development.


Assuntos
Dor Crônica/fisiopatologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/fisiologia , Nociceptores/efeitos dos fármacos , Animais , Sinalização do Cálcio/efeitos dos fármacos , Comunicação Celular , Células Cultivadas , Dor Crônica/induzido quimicamente , Meios de Cultivo Condicionados/farmacologia , Feminino , Gânglios Espinais/citologia , Regulação da Expressão Gênica/efeitos dos fármacos , Inflamação/induzido quimicamente , Inflamação/fisiopatologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fator de Crescimento Neural/farmacologia , Nociceptores/fisiologia , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/efeitos dos fármacos , Fator de Transcrição STAT5/fisiologia , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/metabolismo , Transcrição Genética/efeitos dos fármacos
18.
Sci Rep ; 10(1): 1880, 2020 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-32024965

RESUMO

FRMD6, a member of the group of FERM-domain proteins, is involved both in communication between cells, interactions with extracellular matrix, cellular apoptotic and regenerative mechanisms. FRMD6 was first discovered in the rodent sciatic nerve, and in the present immunohistochemical study we investigated the distribution of FRMD6 in the dorsal root ganglia (DRGs), sciatic nerve and spinal cord following sciatic nerve injury. FRMD6-immunoreactivity was found in the cytoplasm, nucleus or both, and in a majority of DRG neurons. FRMD6-immunoreactivity co-existed with several well-known neuronal markers, including calcitonin gene-related peptide, isolectin B4 and neurofilament 200 in mouse DRGs. After peripheral nerve injury, the FRMD6 mRNA levels and the overall percentage of FRMD6-positive neuron profiles (NPs) were decreased in ipsilateral lumbar DRGs, the latter mainly affecting small size neurons with cytoplasmic localization. Conversely, the proportion of NPs with nuclear FRMD6-immunoreactivity was significantly increased. In the sciatic nerve, FRMD6-immunoreactivity was observed in non-neuronal cells and in axons, and accumulated proximally to a ligation of the nerve. In the spinal cord FRMD6-immunoreactivity was detected in neurons in both dorsal and ventral horns, and was upregulated in ipsilateral dorsal horn after peripheral nerve axotomy. Our results demonstrate that FRMD6 is strictly regulated by peripheral nerve injury at the spinal level.


Assuntos
Gânglios Espinais/patologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Traumatismos dos Nervos Periféricos/patologia , Medula Espinal/patologia , Animais , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Modelos Animais de Doenças , Gânglios Espinais/citologia , Células HEK293 , Humanos , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Células NIH 3T3 , Neurônios , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Nervo Isquiático/lesões , Regulação para Cima
19.
FASEB J ; 34(1): 287-302, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31914619

RESUMO

Unique features of sensory neuron subtypes are manifest by their distinct physiological and pathophysiological functions. Using patch-clamp electrophysiology, Ca2+ imaging, calcitonin gene-related peptide release assay from tissues, protein biochemistry approaches, and behavioral physiology on pain models, this study demonstrates the diversity of sensory neuron pathophysiology is due in part to subtype-dependent sensitization of TRPV1 and TRPA1. Differential sensitization is influenced by distinct expression of inflammatory mediators, such as prostaglandin E2 (PGE2), bradykinin (BK), and nerve growth factor (NGF) as well as multiple kinases, including protein kinase A (PKA) and C (PKC). However, the co-expression and interaction of TRPA1 with TRPV1 proved to be the most critical for differential sensitization of sensory neurons. We identified N- and C-terminal domains on TRPV1 responsible for TRPA1-TRPV1 (A1-V1) complex formation. Ablation of A1-V1 complex with dominant-negative peptides against these domains substantially reduced the sensitization of TRPA1, as well as BK- and CFA-induced hypersensitivity. These data indicate that often occurring TRP channel complexes regulate diversity in neuronal sensitization and may provide a therapeutic target for many neuroinflammatory pain conditions.


Assuntos
Cálcio/metabolismo , Gânglios Espinais/fisiologia , Hipersensibilidade/patologia , Dor/patologia , Células Receptoras Sensoriais/fisiologia , Canal de Cátion TRPA1/fisiologia , Canais de Cátion TRPV/fisiologia , Animais , Gânglios Espinais/citologia , Hipersensibilidade/metabolismo , Masculino , Camundongos , Camundongos Knockout , Nociceptividade , Dor/metabolismo , Células Receptoras Sensoriais/citologia
20.
Sci Rep ; 10(1): 1300, 2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-31992767

RESUMO

The dorsal root ganglia (DRG) house the primary afferent neurons responsible for somatosensation, including pain. We previously identified Jedi-1 (PEAR1/MEGF12) as a phagocytic receptor expressed by satellite glia in the DRG involved in clearing apoptotic neurons during development. Here, we further investigated the function of this receptor in vivo using Jedi-1 null mice. In addition to satellite glia, we found Jedi-1 expression in perineurial glia and endothelial cells, but not in sensory neurons. We did not detect any morphological or functional changes in the glial cells or vasculature of Jedi-1 knockout mice. Surprisingly, we did observe changes in DRG neuron activity. In neurons from Jedi-1 knockout (KO) mice, there was an increase in the fraction of capsaicin-sensitive cells relative to wild type (WT) controls. Patch-clamp electrophysiology revealed an increase in excitability, with a shift from phasic to tonic action potential firing patterns in KO neurons. We also found alterations in the properties of voltage-gated sodium channel currents in Jedi-1 null neurons. These results provide new insight into the expression pattern of Jedi-1 in the peripheral nervous system and indicate that loss of Jedi-1 alters DRG neuron activity indirectly through an intercellular interaction between non-neuronal cells and sensory neurons.


Assuntos
Potenciais de Ação , Receptores de Superfície Celular/deficiência , Células Receptoras Sensoriais/metabolismo , Animais , Biomarcadores , Linhagem Celular , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Neuroglia/metabolismo , Neuroglia/ultraestrutura , Técnicas de Patch-Clamp , Células Receptoras Sensoriais/ultraestrutura
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