RESUMEN
Dynamic regulation of G-protein-coupled receptor (GPCR) kinase 2 (GRK2) expression restores cellular function by protecting from overstimulation via GPCR and non-GPCR signaling. In the primary afferent neurons, GRK2 negatively regulates nociceptive tone. The present study tested the hypothesis that induction of GRK2 in the primary afferent neurons contributes to the resolution of acute pain after tissue injury. GRK2 expression in the dorsal root ganglion (DRG) was analyzed at 1 and 7 days after the incision. Intraperitoneal administration of a GRK2 inhibitor was performed 7 days post-incision in male Sprague-Dawley rats who underwent plantar incisions to analyze the pain-related behavioral effect of the GRK2 inhibitor. Separately, GRK2 expression was analyzed after injecting insulin-like growth factor 1 (IGF1) into the rat hind paw. In addition, an IGF1 receptor (IGF1R) inhibitor was administered in the plantar incision rats to determine its effect on the incision-induced hyperalgesia and GRK2 expression. Plantar incision induced an increase in GRK2 in the DRG at 7 days, but not at 1 day post-incision. Acute hyperalgesia after the plantar incision disappeared by 7 days post-incision. Intraperitoneal injection of the GRK2 inhibitor at this time reinstated mechanical hyperalgesia, although the GRK2 inhibitor did not produce hyperalgesia in naive rats. After the incision, IGF1 expression increased in the paw, but not in the DRG. Intraplantar injection of IGF1 increased GRK2 expression in the ipsilateral DRG. IGF1R inhibitor administration prevented both the induction of GRK2 and resolution of hyperalgesia after the plantar incision. These findings demonstrate that induction of GRK2 expression driven by tissue IGF1 has potent analgesic effects and produces resolution of hyperalgesia after tissue injury. Dysregulation of IGF1-GRK2 signaling could potentially lead to failure of the spontaneous resolution of acute pain and, hence, development of chronic pain after surgery.
Asunto(s)
Quinasa 2 del Receptor Acoplado a Proteína-G , Hiperalgesia , Factor I del Crecimiento Similar a la Insulina , Neuronas Aferentes , Animales , Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/enzimología , Ganglios Espinales/metabolismo , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/farmacología , Masculino , Neuronas Aferentes/efectos de los fármacos , Neuronas Aferentes/enzimología , Neuronas Aferentes/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
SARM1 is an inducible TIR-domain NAD+ hydrolase that mediates pathological axon degeneration. SARM1 is activated by an increased ratio of NMN to NAD+, which competes for binding to an allosteric activating site. When NMN binds, the TIR domain is released from autoinhibition, activating its NAD+ hydrolase activity. The discovery of this allosteric activating site led us to hypothesize that other NAD+-related metabolites might activate SARM1. Here, we show the nicotinamide analog 3-acetylpyridine (3-AP), first identified as a neurotoxin in the 1940s, is converted to 3-APMN, which activates SARM1 and induces SARM1-dependent NAD+ depletion, axon degeneration, and neuronal death. In mice, systemic treatment with 3-AP causes rapid SARM1-dependent death, while local application to the peripheral nerve induces SARM1-dependent axon degeneration. We identify 2-aminopyridine as another SARM1-dependent neurotoxin. These findings identify SARM1 as a candidate mediator of environmental neurotoxicity and suggest that SARM1 agonists could be developed into selective agents for neurolytic therapy.
Asunto(s)
Proteínas del Dominio Armadillo/metabolismo , Axones/efectos de los fármacos , Proteínas del Citoesqueleto/metabolismo , Ganglios Espinales/efectos de los fármacos , Degeneración Nerviosa , Síndromes de Neurotoxicidad/etiología , Neurotoxinas/toxicidad , Piridinas/toxicidad , Nervio Ciático/efectos de los fármacos , Activación Metabólica , Regulación Alostérica , Animales , Proteínas del Dominio Armadillo/genética , Axones/enzimología , Axones/patología , Dominio Catalítico , Muerte Celular , Citocinas/genética , Citocinas/metabolismo , Proteínas del Citoesqueleto/genética , Activación Enzimática , Femenino , Ganglios Espinales/enzimología , Ganglios Espinales/patología , Células HEK293 , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Síndromes de Neurotoxicidad/enzimología , Síndromes de Neurotoxicidad/patología , Neurotoxinas/metabolismo , Nicotinamida Fosforribosiltransferasa/genética , Nicotinamida Fosforribosiltransferasa/metabolismo , Nicotinamida-Nucleótido Adenililtransferasa/genética , Nicotinamida-Nucleótido Adenililtransferasa/metabolismo , Piridinas/metabolismo , Nervio Ciático/enzimología , Nervio Ciático/patología , Transducción de SeñalRESUMEN
High glucose (HG)-induced nucleotide-binding and oligomerization (NACHT) domain, leucine-rich repeat (LRR), and pyrin domain (PYD)-containing protein 3 (NLRP3) inflammasome activation leads to diabetic neuropathic pain. We recently showed that salidroside could suppress NLRP3 inflammasome activation in hepatocytes exposed to HG. The aim of this study was to evaluate the analgesic effect of salidroside on diabetic rats and to explore its underlying mechanisms. Rat models with diabetic neuropathic pain were induced by high-fat diet feeding combined with low dose streptozotocin injections. Doses of salidroside at 50 and 100 mg.kg-1.day-1 were administered by gavage to diabetic rats for 6 weeks. Mechanical allodynia test, thermal hyperalgesia test and biochemical analysis were performed to evaluate therapeutic effects. Primary dorsal root ganglion (DRG) cells exposed to HG at 45 mM were used to further study the effects of salidroside on the AMP-activated protein kinase (AMPK)-NLRP3 inflammasome axis and insulin sensitivity in vitro. Salidroside administration improved hyperglycemia, ameliorated insulin resistance, and alleviated neuropathic pain in diabetic rats. Moreover, salidroside induced AMPK activation and suppressed NLRP3 inflammasome activation in the DRGs of diabetic rats. In addition, salidroside treatment relieved oxidative stress, improved insulin sensitivity and regulated the AMPK-NLRP3 inflammasome axis in HG-treated DRGs in vitro. Furthermore, AMPK inhibition in vivo or AMPK silencing in vitro abolished the beneficial effects of salidroside on diabetic neuropathic pain. Together, these results indicate that salidroside alleviates diabetic neuropathic pain through its regulation of the AMPK-NLRP3 inflammasome axis in DRGs.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Analgésicos/farmacología , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Neuropatías Diabéticas/prevención & control , Ganglios Espinales/efectos de los fármacos , Glucósidos/farmacología , Hipoglucemiantes/farmacología , Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Neuralgia/prevención & control , Fenoles/farmacología , Animales , Glucemia/efectos de los fármacos , Glucemia/metabolismo , Células Cultivadas , Diabetes Mellitus Experimental/sangre , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/complicaciones , Neuropatías Diabéticas/enzimología , Neuropatías Diabéticas/etiología , Neuropatías Diabéticas/fisiopatología , Ganglios Espinales/enzimología , Ganglios Espinales/fisiopatología , Resistencia a la Insulina , Masculino , Neuralgia/enzimología , Neuralgia/etiología , Neuralgia/fisiopatología , Estrés Oxidativo/efectos de los fármacos , Umbral del Dolor/efectos de los fármacos , Ratas Sprague-Dawley , Transducción de SeñalRESUMEN
The objective of study was to investigate the inhibitory effect of sinomenine on neuropathic pain on dorsal root ganglia (DRG). The DRG cell line and spinal nerve ligation (SNL) model were used in this study. The effect of sinomenine on the cell viability was examined by MTT assay. The expression of p38 MAPK, NF-κB, c-fos, SP and TNF-α was detected by using immunofluorescence and immunohistochemistry assay. We also assessed the level of p-CaMKII, COX-2, p-CREB, IL-17A, TLR4 and IL-1ß via western blotting and RT-qPCR. Compared to the controls, sinomenine showed a protective effect on TNF-α-induced apoptosis on DRG cells in a dose-dependent manner, with an increase of cell viability and a decrease of reactive oxygen species level as well as LDH release. Parallelly, sinomenine treatment significantly reduced the expression of various factors related to stress and inflammation, including p38 MAPK, NF-κB, c-fos, p-CAMKII, COX-2, p-CREB, TLR4 and IL-17A in DRG cells in vitro. Furthermore, we found that administration of sinomenine significantly reduced mechanical withdrawal threshold and thermal withdrawal latency and inhibited the inflammation and activation of p38 signaling in SNL rats. It is noting that combined therapy of sinomenine and pulsed radiofrequency exhibited higher efficacy of dorsal root ganglia inflammation than single treatment as well as the combination of oxycodone and pulsed radiofrequency. Sinomenine inhibited the apoptosis of DRG cell by regulating p38 MAPK/CREB signalling pathway, which provides evidence to alleviate neuropathic pain in clinic.
Asunto(s)
Analgésicos/farmacología , Antiinflamatorios/farmacología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Ganglios Espinales/efectos de los fármacos , Inflamación/prevención & control , Morfinanos/farmacología , Neuralgia/prevención & control , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Analgésicos Opioides/farmacología , Animales , Apoptosis/efectos de los fármacos , Conducta Animal/efectos de los fármacos , Línea Celular , Terapia Combinada , Modelos Animales de Enfermedad , Ganglios Espinales/enzimología , Ganglios Espinales/patología , Ganglios Espinales/fisiopatología , Inflamación/enzimología , Inflamación/patología , Inflamación/fisiopatología , Mediadores de Inflamación/metabolismo , Masculino , Neuralgia/enzimología , Neuralgia/patología , Neuralgia/fisiopatología , Oxicodona/farmacología , Umbral del Dolor/efectos de los fármacos , Tratamiento de Radiofrecuencia Pulsada , Ratas Sprague-Dawley , Transducción de SeñalRESUMEN
Axon degeneration is an active program of self-destruction mediated by the protein SARM1. In healthy neurons, SARM1 is autoinhibited and, upon injury autoinhibition is relieved, activating the SARM1 enzyme to deplete NAD+ and induce axon degeneration. SARM1 forms a homomultimeric octamer with each monomer composed of an N-terminal autoinhibitory ARM domain, tandem SAM domains that mediate multimerization, and a C-terminal TIR domain encoding the NADase enzyme. Here we discovered multiple intramolecular and intermolecular domain interfaces required for SARM1 autoinhibition using peptide mapping and cryo-electron microscopy (cryo-EM). We identified a candidate autoinhibitory region by screening a panel of peptides derived from the SARM1 ARM domain, identifying a peptide mediating high-affinity inhibition of the SARM1 NADase. Mutation of residues in full-length SARM1 within the region encompassed by the peptide led to loss of autoinhibition, rendering SARM1 constitutively active and inducing spontaneous NAD+ and axon loss. The cryo-EM structure of SARM1 revealed 1) a compact autoinhibited SARM1 octamer in which the TIR domains are isolated and prevented from oligomerization and enzymatic activation and 2) multiple candidate autoinhibitory interfaces among the domains. Mutational analysis demonstrated that five distinct interfaces are required for autoinhibition, including intramolecular and intermolecular ARM-SAM interfaces, an intermolecular ARM-ARM interface, and two ARM-TIR interfaces formed between a single TIR and two distinct ARM domains. These autoinhibitory regions are not redundant, as point mutants in each led to constitutively active SARM1. These studies define the structural basis for SARM1 autoinhibition and may enable the development of SARM1 inhibitors that stabilize the autoinhibited state.
Asunto(s)
Proteínas del Dominio Armadillo/química , Proteínas del Citoesqueleto/química , Ganglios Espinales/enzimología , NAD/química , Neuronas/enzimología , Péptidos/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas del Dominio Armadillo/antagonistas & inhibidores , Proteínas del Dominio Armadillo/genética , Proteínas del Dominio Armadillo/metabolismo , Sitios de Unión , Microscopía por Crioelectrón , Proteínas del Citoesqueleto/antagonistas & inhibidores , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Ganglios Espinales/citología , Expresión Génica , Células HEK293 , Humanos , Ratones , Ratones Noqueados , Modelos Moleculares , Mutación , NAD/metabolismo , Neuronas/citología , Péptidos/síntesis química , Cultivo Primario de Células , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Nerve injury is an important reason of human disability and death. We studied the role of histone deacetylation in the response of the dorsal root ganglion (DRG) cells to sciatic nerve transection. Sciatic nerve transection in the rat thigh induced overexpression of histone deacetylase 1 (HDAC1) in the ipsilateral DRG at 1-4 h after axotomy. In the DRG neurons, HDAC1 initially upregulated at 1 h but then redistributed from the nuclei to the cytoplasm at 4 h after axotomy. Histone H3 was deacetylated at 24 h after axotomy. Deacetylation of histone H4, accumulation of amyloid precursor protein, a nerve injury marker, and GAP-43, an axon regeneration marker, were observed in the axotomized DRG on day 7. Neuronal injury occurred on day 7 after axotomy along with apoptosis of DRG cells, which were mostly the satellite glial cells remote from the site of sciatic nerve transection. Administration of sodium valproate significantly reduced apoptosis not only in the injured ipsilateral DRG but also in the contralateral ganglion. It also reduced the deacetylation of histones H3 and H4, prevented axotomy-induced accumulation of amyloid precursor protein, which indicated nerve injury, and overexpressed GAP-43, a nerve regeneration marker, in the axotomized DRG. Therefore, HDAC1 was involved in the axotomy-induced injury of DRG neurons and glial cells. HDAC inhibitor sodium valproate demonstrated the neuroprotective activity in the axotomized DRG.
Asunto(s)
Ganglios Espinales/enzimología , Ganglios Espinales/patología , Histona Desacetilasa 1/metabolismo , Histonas/metabolismo , Fármacos Neuroprotectores/farmacología , Nervio Ciático/lesiones , Ácido Valproico/farmacología , Acetilación , Animales , Apoptosis/efectos de los fármacos , Roturas del ADN/efectos de los fármacos , Ganglios Espinales/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , Masculino , Ratas Wistar , Nervio Ciático/efectos de los fármacos , Nervio Ciático/patología , Tubulina (Proteína)/metabolismoRESUMEN
Neuropathic pain can be generated by chronic compression of dorsal root ganglion (CCD). Stimulation of primary motor cortex can disrupt the nociceptive sensory signal at dorsal root ganglion level and reduce pain behaviors. But the mechanism behind it is still implicit. Protein kinase C gamma is known as an essential enzyme for the development of neuropathic pain, and specific inhibitor of protein kinase C gamma can disrupt the sensory signal and reduce pain behaviors. Optogenetic stimulation has been emerged as a new and promising conducive method for refractory neuropathic pain. The aim of this study was to provide evidence whether optical stimulation of primary motor cortex can modulate chronic neuropathic pain in CCD rat model. Animals were randomly divided into CCD group, sham group, and control group. Dorsal root ganglion-compressed neuropathic pain model was established in animals, and knocking down of protein kinase C gamma was also accomplished. Pain behavioral scores were significantly improved in the short hairpin Protein Kinase C gamma knockdown CCD animals during optic stimulation. Ventral posterolateral thalamic firing inhibition was also observed during light stimulation on motor cortex in CCD animal. We assessed alteration of pain behaviors in pre-light off, stimulation-light on, and post-light off state. In vivo extracellular recording of the ventral posterolateral thalamus, viral expression in the primary motor cortex, and protein kinase C gamma expression in dorsal root ganglion were investigated. So, optical cortico-thalamic inhibition by motor cortex stimulation can improve neuropathic pain behaviors in CCD animal, and knocking down of protein kinase C gamma plays a conducive role in the process. This study provides feasibility for in vivo optogenetic stimulation on primary motor cortex of dorsal root ganglion-initiated neuropathic pain.
Asunto(s)
Ganglios Espinales/metabolismo , Corteza Motora/metabolismo , Neuralgia/metabolismo , Optogenética/métodos , Proteína Quinasa C/metabolismo , Tálamo/metabolismo , Animales , Escala de Evaluación de la Conducta , Conducta Animal/fisiología , Femenino , Ganglios Espinales/enzimología , Ganglios Espinales/lesiones , Técnicas de Silenciamiento del Gen , Inmunohistoquímica , Corteza Motora/enzimología , Corteza Motora/efectos de la radiación , Neuralgia/genética , Fibras Ópticas , Proteína Quinasa C/genética , ARN Interferente Pequeño , Ratas , Ratas Sprague-Dawley , Tálamo/enzimologíaRESUMEN
Chemotherapy-induced peripheral neuropathy is a serious adverse effect of chemotherapeutic agents such as paclitaxel. JTC-801, a nociceptin/orphanin FQ opioid peptide (NOP) receptor antagonist, has been reported to attenuate neuropathic pain in several pain models. However, the therapeutic significance and function of JTC-801 in chemotherapy-induced peripheral neuropathy remain unclear. In this study, we determined the effect of JTC-801 on neuropathic pain induced by paclitaxel, and we explored the potential mechanism in the dorsal root ganglion (DRG). The behavioral test showed that single or multiple systemic administrations of JTC-801 significantly alleviated mechanical allodynia in paclitaxel-treated rats. Using Western blot analysis and immunohistochemistry, we found that paclitaxel increased the expression of phosphatidylinositol 3-kinase (PI3K) and phospho-Akt (p-Akt) in the DRG. Double immunofluorescence staining indicated that p-Akt was expressed in neurons in the DRG. Multiple injections of JTC-801 significantly inhibited the activation of Akt and decreased the expression of inflammatory cytokines. The data suggest that JTC-801 alleviates mechanical allodynia associated with paclitaxel-induced neuropathic pain via the PI3K/Akt pathway.
Asunto(s)
Aminoquinolinas/farmacología , Analgésicos/farmacología , Benzamidas/farmacología , Ganglios Espinales/efectos de los fármacos , Hiperalgesia/prevención & control , Antagonistas de Narcóticos/farmacología , Neuralgia/prevención & control , Umbral del Dolor/efectos de los fármacos , Fosfatidilinositol 3-Quinasa/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Conducta Animal/efectos de los fármacos , Modelos Animales de Enfermedad , Ganglios Espinales/enzimología , Ganglios Espinales/fisiopatología , Hiperalgesia/inducido químicamente , Hiperalgesia/enzimología , Hiperalgesia/fisiopatología , Mediadores de Inflamación/metabolismo , Interleucina-1beta/metabolismo , Masculino , Neuralgia/inducido químicamente , Neuralgia/enzimología , Neuralgia/fisiopatología , Paclitaxel , Fosforilación , Ratas Sprague-Dawley , Receptores Opioides/efectos de los fármacos , Receptores Opioides/metabolismo , Transducción de Señal , Factor de Necrosis Tumoral alfa/metabolismo , Receptor de NociceptinaRESUMEN
BACKGROUND: Bupivacaine (BP) is commonly used as a local anaesthetic(LA) in the clinic, but it can also cause neurotoxicity, especially in patients with diabetes. Previous studies have found that high-glucose environments can aggravate BP-induced DNA damage in nerve cells. Ku70 is subunit of the DNA damage repair enzyme DNA-PK. This study was designed to determine whether high-glucose conditions enhance BP neurotoxicity and DNA damage by inhibiting Ku70 expression. METHODS: We examined the effect of BP on apoptosis and DNA damage in murine dorsal root ganglion (DRG) neurons under hyperglycaemic conditions. Untreated DRG cells and DRG cells pretreated with NU7441, a DNA-PK inhibitor, were cultured for 3 days under normal culture conditions or with 50â¯mM glucose, and the cells were then treated with BP for 3â¯h. DNA damage was investigated via comet assays, the ratio of early to late apoptotic cells was assessed by Annexin V-FITC/PI staining, and cell viability was measured by CCK-8 assays. The protein expression levels of DNA-PK, Ku70, Bax, Bcl-2 and γH2ax were measured by immunofluorescence or Western blotting. RESULTS: Compared to its effect under normal culture conditions, BP treatment led to decreased cell viability and increased DNA damage in DRG cells grown under high-glucose conditions. The rate of DRG cell apoptosis and the expression of γH2ax, the ratio of Bax to Bcl-2 also increased under the high-glucose conditions. Furthermore, Ku70 expression was inhibited. The DNA-PK inhibitor, NU7441, could significantly inhibit DNA-PK and Ku70 expression, simultaneously further aggravating BP-induced apoptosis and DNA damage under high-glucose conditions. CONCLUSION: These data indicate that hyperglycaemia may enhance BP-induced neurotoxicity and DNA damage by inhibiting the DNA repair protein Ku70.
Asunto(s)
Anestésicos Locales/toxicidad , Apoptosis/efectos de los fármacos , Bupivacaína/toxicidad , Cromonas/toxicidad , Inhibidores Enzimáticos/toxicidad , Ganglios Espinales/efectos de los fármacos , Glucosa/toxicidad , Autoantígeno Ku/antagonistas & inhibidores , Morfolinas/toxicidad , Síndromes de Neurotoxicidad/etiología , Animales , Células Cultivadas , Daño del ADN , Ganglios Espinales/enzimología , Ganglios Espinales/patología , Autoantígeno Ku/metabolismo , Ratones , Síndromes de Neurotoxicidad/enzimología , Síndromes de Neurotoxicidad/patología , Transducción de Señal/efectos de los fármacosRESUMEN
Neurons face the challenge of maintaining cellular homeostasis through lysosomal degradation. While enzymatically active degradative lysosomes are enriched in the soma, their axonal trafficking and positioning and impact on axonal physiology remain elusive. Here, we characterized axon-targeted delivery of degradative lysosomes by applying fluorescent probes that selectively label active forms of lysosomal cathepsins D, B, L, and GCase. By time-lapse imaging of cortical neurons in microfluidic devices and standard dishes, we reveal that soma-derived degradative lysosomes rapidly influx into distal axons and target to autophagosomes and Parkinson disease-related α-synuclein cargos for local degradation. Impairing lysosome axonal delivery induces an aberrant accumulation of autophagosomes and α-synuclein cargos in distal axons. Our study demonstrates that the axon is an active compartment for local degradation and reveals fundamental aspects of axonal lysosomal delivery and maintenance. Our work establishes a foundation for investigations into axonal lysosome trafficking and functionality in neurodegenerative diseases.
Asunto(s)
Autofagosomas/enzimología , Transporte Axonal/genética , Axones/metabolismo , Lisosomas/enzimología , Lisosomas/metabolismo , Factores de Ribosilacion-ADP/genética , Factores de Ribosilacion-ADP/metabolismo , Animales , Autofagosomas/metabolismo , Autofagia/genética , Autofagia/fisiología , Transporte Axonal/fisiología , Axones/enzimología , Catepsinas/antagonistas & inhibidores , Catepsinas/metabolismo , Femenino , Ganglios Espinales/enzimología , Ganglios Espinales/metabolismo , Glucosilceramidasa/antagonistas & inhibidores , Glucosilceramidasa/metabolismo , Células HEK293 , Homeostasis/genética , Homeostasis/fisiología , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Neuronas/enzimología , Neuronas/metabolismo , Transporte de Proteínas/genética , Transporte de Proteínas/fisiología , alfa-Sinucleína/metabolismoRESUMEN
STUDY DESIGN: Animal experiment: a rat model of lumbar disc herniation (LDH) induced painful radiculopathies. OBJECTIVE: To investigate the role and mechanism of AMP-activated protein kinase (AMPK) in dorsal root ganglia (DRG) neurons in LDH-induced painful radiculopathies. SUMMARY OF BACKGROUND DATA: Overactivation of multiple pain signals in DRG neurons triggered by LDH is crucial to the development of radicular pain. AMPK is recognized as a cellular energy sensor, as well as a pain sensation modulator, but its function in LDH-induced pain hypersensitivity remains largely unknown. METHODS: The LDH rat model was established by autologous nucleus pulposus transplantation into the right lumbar 5 (L5) nerve root. At different time points after AMPK agonist metformin (250âmg/kg/d) or mammalian target of rapamycin (mTOR) inhibitor rapamycin (5âmg/kg) intraperitoneal administration, thermal and mechanical sensitivity were evaluated by measuring paw withdrawal latency (PWL) and 50% paw withdrawal thresholds (PWT). The levels of AMPK, mTOR, and p70S6K phosphorylation were determined by Western blot. We also investigated the proportion of p-AMPK positive neurons in the right L5 DRG neurons using immunofluorescence. RESULTS: LDH evoked persistent thermal hyperalgesia and mechanical allodynia on the ipsilateral paw, as indicated by the decreased PWL and 50% PWT. These pain hypersensitive behaviors were accompanied with significant inhibition of AMPK and activation of mTOR in the associated DRG neurons. Pharmacological activation of AMPK in the DRG neurons not only suppressed mTOR/p70S6K signaling, but also alleviated LDH-induced pain hypersensitive behaviors. CONCLUSION: We provide a molecular mechanism for the activation of pain signals based on AMPK-mTOR axis, as well as an intervention strategy by targeting AMPK-mTOR axis in LDH-induced painful radiculopathies. LEVEL OF EVIDENCE: N/A.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Ganglios Espinales/metabolismo , Hiperalgesia/metabolismo , Degeneración del Disco Intervertebral/metabolismo , Desplazamiento del Disco Intervertebral/metabolismo , Radiculopatía/metabolismo , Proteínas Quinasas S6 Ribosómicas 70-kDa/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Animales , Modelos Animales de Enfermedad , Ganglios Espinales/enzimología , Hiperalgesia/enzimología , Degeneración del Disco Intervertebral/enzimología , Desplazamiento del Disco Intervertebral/enzimología , Masculino , Metformina/farmacología , Neuronas/enzimología , Neuronas/metabolismo , Núcleo Pulposo/enzimología , Núcleo Pulposo/metabolismo , Dolor/enzimología , Dolor/metabolismo , Fosforilación , Radiculopatía/enzimología , Ratas , Ratas Wistar , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Transducción de Señal , Sirolimus/farmacología , Raíces Nerviosas Espinales/enzimología , Raíces Nerviosas Espinales/metabolismo , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Synergistic expression of cyclooxygenase-2 (COX-2) by interleukin-1ß (IL-1ß) and bradykinin (BK) in peri-sensory neurons results in the production of prostanoids, which affects sensory neuronal activity and responsiveness and causes hyperalgesia. To evaluate the effects of pro-inflammatory mediators on COX-2 expression, cultured rat dorsal root ganglion (DRG) cells were treated with IL-1ß and BK, which caused persistent increased COX-2 expression. Co-treatment increased COX-2 transcriptional activities in an additive manner by a COX-2 promoter luciferase assay. Immunoprecipitated HuR, an RNA-binding protein, in co-treated DRG cells contained more COX-2 mRNA than that of the control. The synergistic effects of IL-1ß and BK on COX-2 expression may be a result of RNA stabilization mediated by HuR in peri-sensory neurons. Multiple pro-inflammatory cytokines and mediators are produced during neurogenic inflammation and aberrant control of COX-2 mRNA turnover may be implicated in diseases including chronic inflammation, which results in inflammation-derived hyperalgesia around primary sensory neurons.
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Bradiquinina/metabolismo , Ciclooxigenasa 2/metabolismo , Proteína 1 Similar a ELAV/metabolismo , Ganglios Espinales/enzimología , Interleucina-1beta/metabolismo , Animales , Bradiquinina/administración & dosificación , Células Cultivadas , Ganglios Espinales/efectos de los fármacos , Interleucina-1beta/administración & dosificación , Masculino , ARN Mensajero/metabolismo , Ratas WistarRESUMEN
cAMP (Cyclic Adenosine monophosphate), one of the most highly studied second messengers, is regulated by a family of adenylyl cyclase (AC) enzymes. Type 3 adenylyl cyclase (abbreviated as AC3), a subtype of adenylyl cyclase, is reported to be expressed in cilia in the olfactory and central nervous system and plays an important role in many physiological functions such as olfaction, development. However, expression of AC3 in the dorsal root ganglion (DRG) is not reported. In the present study, using immunohistochemical method, we discovered that AC3 immunoreactivity (IR) is predominantly expressed in the cytoplasm of small to medium sized DRG neurons. Double labelling revealed that the majority of AC3 IR are colocalized with CGRP (a peptidergic nociceptor marker), rarely with NF200 (a myelinated neuronal marker) or IB4 (a nonpeptidergic nociceptor marker). Furthermore, dense AC3 IR exists in the superficial dorsal horn, especially in laminaâ and dorsal part of lamina II, where CGRP-positive DRG neurons terminate. The expression pattern of AC3 is similar between C57/BL6 J mouse and Sprague Dawley rat. For instance, AC3 is primarily expressed in the cell bodies of small to medium sized DRG neurons and the majority of AC3 IR is also in CGRP-containing neurons in rat. Taken together, our data suggest that AC3 is primarily expressed in the small to medium sized cell bodies and central terminals of CGRP-positive DRG neurons, implying AC3 enzyme might potentially function in nociception.
Asunto(s)
Adenilil Ciclasas/análisis , Ganglios Espinales/enzimología , Neuronas Aferentes/enzimología , Terminales Presinápticos/enzimología , Animales , Péptido Relacionado con Gen de Calcitonina/análisis , Masculino , Ratones Endogámicos C57BL , Ratas Sprague-Dawley , Especificidad de la EspecieRESUMEN
A large body of evidence indicates that nitric oxide (NO)/cGMP signaling essentially contributes to the processing of chronic pain. In general, NO-induced cGMP formation is catalyzed by 2 isoforms of guanylyl cyclase, NO-sensitive guanylyl cyclase 1 (NO-GC1) and 2 (NO-GC2). However, the specific functions of the 2 isoforms in pain processing remain elusive. Here, we investigated the distribution of NO-GC1 and NO-GC2 in the spinal cord and dorsal root ganglia, and we characterized the behavior of mice lacking either isoform in animal models of pain. Using immunohistochemistry and in situ hybridization, we demonstrate that both isoforms are localized to interneurons in the spinal dorsal horn with NO-GC1 being enriched in inhibitory interneurons. In dorsal root ganglia, the distribution of NO-GC1 and NO-GC2 is restricted to non-neuronal cells with NO-GC2 being the major isoform in satellite glial cells. Mice lacking NO-GC1 demonstrated reduced hypersensitivity in models of neuropathic pain, whereas their behavior in models of inflammatory pain was normal. By contrast, mice lacking NO-GC2 exhibited increased hypersensitivity in models of inflammatory pain, but their neuropathic pain behavior was unaltered. Cre-mediated deletion of NO-GC1 or NO-GC2 in spinal dorsal horn neurons recapitulated the behavioral phenotypes observed in the global knockout. Together, these results indicate that cGMP produced by NO-GC1 or NO-GC2 in spinal dorsal horn neurons exert distinct, and partly opposing, functions in chronic pain processing.
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Inflamación/enzimología , Neuralgia/enzimología , Isoformas de Proteínas/metabolismo , Guanilil Ciclasa Soluble/metabolismo , Animales , Modelos Animales de Enfermedad , Adyuvante de Freund/toxicidad , Ganglios Espinales/enzimología , Inflamación/inducido químicamente , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Neuralgia/etiología , Dimensión del Dolor , Isoformas de Proteínas/genética , ARN Mensajero/metabolismo , Guanilil Ciclasa Soluble/genética , Médula Espinal/enzimología , Proteína 2 de Transporte Vesicular de Glutamato/metabolismo , Proteínas del Transporte Vesicular de Aminoácidos Inhibidores/metabolismoRESUMEN
Golgi fragmentation and loss of Nicotinamide Mononucleotide Adenylyltransferase 2 (NMNAT2) are the early key features of many neurodegenerative disorders. We investigated the link between NMNAT2 loss, Golgi fragmentation and axon degeneration. Golgi fragmentation in the cultured dorsal root ganglion (DRG) neurons resulted in caspase dependent axon degeneration and neuronal cell death. NMNAT2 depletion in the DRG neurons caused Golgi fragmentation and caspase dependent axon degeneration. NMNAT2 depletion did not cause ATP loss in the axons. These results indicate that NMNAT2 is required for maintenance of Golgi structure. Loss of Golgi structure or Nmnat2 depletion causes caspase dependent neurodegeneration. cytNmnat1 overexpression inhibited the axon degeneration induced by Golgi fragmentation or NMNAT2 depletion. These results also suggest that these degeneration signals converge on a common cytNmnat1 mediated axon protective program and are distinct from the SARM1 mediated caspase independent axon degeneration.
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Ganglios Espinales/enzimología , Aparato de Golgi/enzimología , Neuronas/enzimología , Nicotinamida-Nucleótido Adenililtransferasa/deficiencia , Animales , Apoptosis/fisiología , Células Cultivadas , Ganglios Espinales/patología , Aparato de Golgi/patología , Ratones , Neuronas/patología , Nicotinamida-Nucleótido Adenililtransferasa/antagonistas & inhibidores , Nicotinamida-Nucleótido Adenililtransferasa/genética , ARN Interferente Pequeño/administración & dosificaciónRESUMEN
OBJECTIVE: To investigate the role of p38MAPK signal pathway in spinal cord and dorsal root ganglion (DRG) in rats with phantom limb pain and the effects of specific inhibitors.â© Methods: Healthy adult male SD rats (n=48) were cut off one side of the sciatic under anesthesia to establish a model of phantom limb pain. In addition, the healthy rats were taken as a sham group (group S, n=24). The animals were scored by observing the action of chewing (0=no chewing, 13=the worst chewing) after the operation and were sacrificed on the following day after the operation. The successful model of phantom limb pain were randomly divided into 2 groups: a phantom limb pain group (group P, n=24) and a phantom limb pain plus inhibitor group (group P+I, n=24). SB203580 was given to the rat at 0.8 mg/kg on every Monday until the rats were sacrificed, the rest of the rats received an equal amount of saline. Eight rats from each group were randomly taken for the determination of levels of P-p38MAPK in spinal cord and DRG before administration and on the 4th, 6th, 8th weekend following the administration, respectively.â© Results: In the sham group, no animal developed chewing. Meanwhile, rats in successful model of phantom limb pain group began chewing from the 2nd day after operation with scores at eight to eleven. The chewing scores in the P+I group were reduced after the treatment. Compared with group S, P-p38MAPK levels were elevated in groups of P and P+I (P<0.05 or P<0.01). Compared with group P, P-p38MAPK level was decreased in the group P+I (P<0.05 or P<0.01).â© Conclusion: P38MAPK signal pathway involves in the development of phantom limb pain.
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Ganglios Espinales/enzimología , Masticación/fisiología , Miembro Fantasma/enzimología , Automutilación/enzimología , Médula Espinal/enzimología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacología , Imidazoles/farmacología , Masculino , Miembro Fantasma/etiología , Miembro Fantasma/fisiopatología , Piridinas/farmacología , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Nervio Ciático/lesiones , Automutilación/fisiopatología , Transducción de Señal , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
Background Intense nociceptive signaling arising from ongoing injury activates primary afferent nociceptive systems to generate peripheral sensitization. ERK1/2 phosphorylation in dorsal root ganglion can be used to visualize intracellular signal activity immediately after noxious stimulation. The aim of this study was to investigate spatiotemporal characteristics of ERK1/2 phosphorylation against tissue injury in the primary afferent neurons. Methods Plantar incisions were made in the hind paws of Sprague-Dawley rats (n =150). Levobupivacaine was injected into the plantar aspect of the paws and ankles, Mitogen-activated protein kinase kinase (MEK) inhibitor was injected into the paw, and carbenoxolone, dual inhibitor of the gap junction and pannexin channel, was intraperitoneally injected. Pain hypersensitivity was investigated by a behavioral study, while phosphorylated ERK1/2 was detected in dorsal root ganglion and hind paw using immunohistochemistry and Western blot. Results Phosphorylated ERK1/2 was induced in dorsal root ganglion (26.8 ± 2.9% at baseline, 65.6 ± 3.6% at 2 min, and 26.3 ± 3.4% at 2 h) after the incision. NF-200 positive A-fiber neurons and satellite glial cells were positive for phosphorylated ERK1/2. Injury-induced pain hypersensitivity was abolished by MEK inhibitor. Levobupivacaine treatment inhibited phosphorylated ERK1/2 induction, carbenoxolone treatment inhibited glial phosphorylated ERK1/2 at 2 min after the injury, and carbenoxolone inhibited pain hypersensitivity and neuronal phosphorylated ERK1/2 at 1 h after the injury. Conclusion ERK1/2 phosphorylation in A-fiber neurons and satellite glial cells immediately after injury contributes to the generation of pain hypersensitivity. Signal communication between neurons and satellite glial cells expands the duration of neuronal ERK1/2 phosphorylation and pain hypersensitivity at 1 h after tissue injury.
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Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Extremidades/patología , Ganglios Espinales/enzimología , Ganglios Espinales/patología , Neuroglía/enzimología , Neuronas/enzimología , Dolor/enzimología , Analgésicos/farmacología , Animales , Bupivacaína/farmacología , Bupivacaína/uso terapéutico , Activación Enzimática , Extremidades/cirugía , Hipersensibilidad/enzimología , Hipersensibilidad/patología , Masculino , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Dolor/tratamiento farmacológico , Dolor/patología , Inhibidores de Proteínas Quinasas/farmacología , Ratas Sprague-DawleyRESUMEN
Chemotherapy-induced peripheral neuropathy (CIPN) is a dose-limiting complication which develops as a consequence of treatment with chemotherapeutic agents like oxaliplatin and is a mainstay of therapy for colorectal cancer. Ever since CIPN was identified, understanding its exact pathomechanisms remains a clinical challenge. The role of mitochondrial dysfunction and glial cell activation has surfaced in the etiology of CIPN. Rosmarinic acid (RA), a known mitoprotectant exerts neuroprotection against the oxidative stress and neuroinflammation in various disease conditions. Hence, in the present study, we investigated the effect using rosmarinic acid (25 and 50 mg/kg, po) in the experimental model of oxaliplatin-induced peripheral neuropathy (OIPN) in rats. Results showed that RA significantly (p < 0.001) prevented the functional deficits, reversed oxaliplatin-induced mechanical allodynia and cold hyperalgesia in rats. It reduced the oxidative stress, improved the mitochondrial function, and prevented the oxaliplatin-induced loss of ATP levels. RA significantly (p < 0.01) inhibited the spinal glial cell activation and suppressed the expression of inflammatory markers. RA treatment also resulted in the activation of adenosine monophosphate-activated protein kinase (AMPK) in the peripheral nerves and dorsal root ganglion (DRG) which also might have contributed to its neuroprotective actions. In vitro screening also revealed that RA did not compromise the anti-cancer activity of oxaliplatin in colon cancer cells (HT-29). Taken together, the above results demonstrate the therapeutic activity of RA against the oxaliplatin-induced mitochondrial dysfunction and neuroinflammation and thus, suggest its potential for the management of OIPN. Graphical Abstract Schematic representation of neuroprotective mechanisms of rosmarinic acid via AMPK activation in oxaliplatin-evoked peripheral neuropathy.
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Cinamatos/uso terapéutico , Depsidos/uso terapéutico , Mitocondrias/metabolismo , Neuralgia/tratamiento farmacológico , Neuroglía/patología , Oxaliplatino/efectos adversos , Enfermedades del Sistema Nervioso Periférico/inducido químicamente , Enfermedades del Sistema Nervioso Periférico/tratamiento farmacológico , Médula Espinal/patología , Adenilato Quinasa/metabolismo , Animales , Antineoplásicos/efectos adversos , Antioxidantes/farmacología , Conducta Animal/efectos de los fármacos , Línea Celular , Cinamatos/farmacología , Fragmentación del ADN/efectos de los fármacos , Depsidos/farmacología , Activación Enzimática/efectos de los fármacos , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/enzimología , Ganglios Espinales/patología , Homeostasis , Humanos , Inflamación/patología , Peroxidación de Lípido/efectos de los fármacos , Masculino , Malondialdehído/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Neuralgia/complicaciones , Neuralgia/patología , Neuritas/efectos de los fármacos , Neuritas/metabolismo , Neurogénesis/efectos de los fármacos , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Nitritos/metabolismo , Enfermedades del Sistema Nervioso Periférico/complicaciones , Enfermedades del Sistema Nervioso Periférico/patología , Ratas Sprague-Dawley , Nervio Ciático/efectos de los fármacos , Nervio Ciático/enzimología , Nervio Ciático/patología , Ácido RosmarínicoRESUMEN
Reactive oxygen species (ROS) contribute to tissue damage and remodelling mediated by the inflammatory response after injury. Here we show that ROS, which promote axonal dieback and degeneration after injury, are also required for axonal regeneration and functional recovery after spinal injury. We find that ROS production in the injured sciatic nerve and dorsal root ganglia requires CX3CR1-dependent recruitment of inflammatory cells. Next, exosomes containing functional NADPH oxidase 2 complexes are released from macrophages and incorporated into injured axons via endocytosis. Once in axonal endosomes, active NOX2 is retrogradely transported to the cell body through an importin-ß1-dynein-dependent mechanism. Endosomal NOX2 oxidizes PTEN, which leads to its inactivation, thus stimulating PI3K-phosporylated (p-)Akt signalling and regenerative outgrowth. Challenging the view that ROS are exclusively involved in nerve degeneration, we propose a previously unrecognized role of ROS in mammalian axonal regeneration through a NOX2-PI3K-p-Akt signalling pathway.
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Axones/enzimología , Exosomas/enzimología , Ganglios Espinales/enzimología , NADPH Oxidasa 2/metabolismo , Degeneración Nerviosa , Regeneración Nerviosa , Traumatismos de los Nervios Periféricos/enzimología , Especies Reactivas de Oxígeno/metabolismo , Nervio Ciático/enzimología , Traumatismos de la Médula Espinal/enzimología , Animales , Axones/patología , Receptor 1 de Quimiocinas CX3C/metabolismo , Línea Celular , Modelos Animales de Enfermedad , Dineínas/metabolismo , Endocitosis , Endosomas/enzimología , Endosomas/patología , Exosomas/patología , Ganglios Espinales/lesiones , Ganglios Espinales/patología , Macrófagos/enzimología , Macrófagos/patología , Ratones Endogámicos C57BL , Ratones Noqueados , NADPH Oxidasa 2/deficiencia , NADPH Oxidasa 2/genética , Proteínas Nucleares/metabolismo , Fosfohidrolasa PTEN/metabolismo , Traumatismos de los Nervios Periféricos/genética , Traumatismos de los Nervios Periféricos/patología , Traumatismos de los Nervios Periféricos/fisiopatología , Fosfatidilinositol 3-Quinasa/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Nervio Ciático/lesiones , Nervio Ciático/patología , Nervio Ciático/fisiopatología , Transducción de Señal , Traumatismos de la Médula Espinal/genética , Traumatismos de la Médula Espinal/patología , Traumatismos de la Médula Espinal/fisiopatología , beta CarioferinasRESUMEN
The progress of axonal degeneration (AxD) following injury or insult impacts both recovery from axonal transection and protection of axons from diverse insults, or axonopathy. Here we provide evidence that increases in capase-6 (Casp6) expression and action contribute to the progression of AxD. The expression of Casp6 protein and mRNA in distal branches of sensory axons undergoing AxD was confirmed. We developed and utilized a new model of axonopathy in live mice by serially visualizing the viability of cutaneous axons in the ear pinna that expressed an axonal YFP transgene, in response to capasaicin-induced AxD. Both specific pharmacological inhibition of caspase-6 and local knockdown offered early but subtle and mild attenuation of axonopathy. To evaluate an axon autonomous role of Casp6, we examined axon integrity following transection ex vivo, and analyzed the serial morphological fragmentation of neurofilament expression as a structural index of AxD. Adding a specific Casp6 inhibitor to the preparation delayed neurofilament fragmentation. Intact motor axons of Casp6 null mice had normal electrophysiological properties but, as tested serially during AxD, there was attenuated loss of excitability. Following transection, morphological features of AxD were evident in both wild type and Casp6-/- mice but the latter had evidence of slowed progression. Taken together, our findings suggest a subtle but dispensable enabling role of local Casp6 expression in axons undergoing AxD. Serial analysis of cutaneous ear pinna axons in live mice provides a useful and novel model of axonal integrity.