RESUMEN
Pannexin 3 (Panx3) is a glycoprotein that forms mechanosensitive channels expressed in chondrocytes and annulus fibrosus cells of the intervertebral disc (IVD). Evidence suggests Panx3 plays contrasting roles in traumatic versus aging osteoarthritis (OA) and intervertebral disc degeneration (IDD). However, whether its deletion influences the response of joint tissue to forced use is unknown. The purpose of this study was to determine if Panx3 deletion in mice causes increased knee joint OA and IDD after forced treadmill running. Male and female wildtype (WT) and Panx3 knockout (KO) mice were randomized to either a no-exercise group (sedentary; SED) or daily forced treadmill running (forced exercise; FEX) from 24 to 30 weeks of age. Knee cartilage and IVD histopathology were evaluated by histology, while tibial secondary ossification centers were analyzed using microcomputed tomography (µCT). Both male and female Panx3 KO mice developed larger superficial defects of the tibial cartilage after forced treadmill running compared with SED WT mice. Additionally, Panx3 KO mice developed reduced bone volume, and female PANX3 KO mice had lengthening of the lateral tubercle at the intercondylar eminence. In the lower lumbar spine, both male and female Panx3 KO mice developed histopathological features of IDD after running compared to SED WT mice. These findings suggest that the combination of deleting Panx3 and forced treadmill running induces OA and causes histopathological changes associated with the degeneration of the IVDs in mice.
Asunto(s)
Conexinas , Degeneración del Disco Intervertebral , Ratones Noqueados , Osteoartritis de la Rodilla , Animales , Femenino , Osteoartritis de la Rodilla/etiología , Osteoartritis de la Rodilla/patología , Conexinas/genética , Conexinas/deficiencia , Masculino , Degeneración del Disco Intervertebral/patología , Degeneración del Disco Intervertebral/etiología , Degeneración del Disco Intervertebral/genética , Ratones Endogámicos C57BL , Carrera , Ratones , Condicionamiento Físico Animal , Cartílago Articular/patología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/deficienciaRESUMEN
GJB2 gene mutations are the most common causes of autosomal recessive non-syndromic hereditary deafness. For individuals suffering from severe to profound GJB2-related deafness, cochlear implants have emerged as the sole remedy for auditory improvement. Some previous studies have highlighted the crucial role of preserving cochlear neural components in achieving favorable outcomes after cochlear implantation. Thus, we generated a conditional knockout mouse model (Cx26-CKO) in which Cx26 was completely deleted in the cochlear supporting cells driven by the Sox2 promoter. The Cx26-CKO mice showed severe hearing loss and massive loss of hair cells and Deiter's cells, which represented the extreme form of human deafness caused by GJB2 gene mutations. In addition, multiple pathological changes in the peripheral auditory nervous system were found, including abnormal innervation, demyelination, and degeneration of spiral ganglion neurons as well as disruption of heminodes in Cx26-CKO mice. These findings provide invaluable insights into the deafness mechanism and the treatment for severe deafness in Cx26-null mice.
Asunto(s)
Conexina 26 , Conexinas , Sordera , Ratones Noqueados , Ganglio Espiral de la Cóclea , Animales , Ganglio Espiral de la Cóclea/patología , Sordera/genética , Sordera/patología , Conexinas/genética , Conexinas/deficiencia , Enfermedades Desmielinizantes/patología , Enfermedades Desmielinizantes/genética , Ratones , Neuronas/patología , Neuronas/metabolismo , Modelos Animales de Enfermedad , Degeneración Nerviosa/patología , Degeneración Nerviosa/genética , Cóclea/patologíaRESUMEN
Gap junction beta 1 (GJB1) is the pathogenic gene of X-linked Charcot-Marie-Tooth type 1 (CMTX1), a rare hereditary sensorimotor neuropathy. However, different mutations of GJB1 result in heterogeneous clinical manifestations with only some mutations leading to central nervous system involvement. We previously reported two GJB1 missense mutations: one novel mutation (c.212T > G) found in a CMTX1 family that only manifested as peripheral neuropathy, and another previously reported mutation GJB1(c.311A > C) leading to involvement of the peripheral nerves and cerebral white matter. However, the mechanism by which GJB1 mutations lead to CMTX1 has not been fully characterized. Here, we generated Schwann cells and primary cultured oligodendrocytes with these two mutations, resulting in the Cx32I71S (GJB1 c.212T > G) and Cx32K104T (GJB1 c.311A > C) mutants, to analyze the pathogenic mechanism using cytology, molecular biology, and electrophysiological methods. Both mutants showed abnormal endoplasmic reticulum aggregation, especially the Cx32K104T mutant, leading to an increase in endoplasmic reticulum stress, resulting in apoptosis. Furthermore, whole-cell patch clamp experiments in oligodendrocytes revealed that the Cx32K104T mutant reduced the cell membrane potential and inwardly rectifying potassium currents, which may be a vital element for central involvement. Therefore, our results may provide a new perspective for understanding the pathogenesis of CMTX1.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth/genética , Conexinas/genética , Mutación Missense , Oligodendroglía/metabolismo , Potasio/metabolismo , Células de Schwann/metabolismo , Animales , Apoptosis/genética , Línea Celular , Enfermedad de Charcot-Marie-Tooth/metabolismo , Enfermedad de Charcot-Marie-Tooth/patología , Conexinas/deficiencia , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Estrés del Retículo Endoplásmico/genética , Expresión Génica , Humanos , Activación del Canal Iónico , Potenciales de la Membrana/fisiología , Modelos Biológicos , Oligodendroglía/patología , Técnicas de Placa-Clamp , Cultivo Primario de Células , Agregado de Proteínas/genética , Ratas , Ratas Sprague-Dawley , Células de Schwann/patología , Proteína beta1 de Unión ComunicanteRESUMEN
Leukotoxin (LtxA) (Trade name, Leukothera) is a protein that is secreted from the oral bacterium Aggregatibacter actinomycetemcomitans, which targets and kills activated white blood cells (WBCs) by binding to lymphocyte function associated antigen-1 (LFA-1). Interaction between LtxA and Jurkat T-cells results in cell death and is characterized by increased intracellular Ca2+, activation of caspases, clustering of LtxA and LFA-1 within lipid rafts, and involvement of the Fas death receptor. Here, we show that LtxA can kill malignant lymphocytes via apoptotic and necrotic forms of cell death. We show that LtxA causes activation of caspases and PARP, cleavage of pannexin-1 (Panx1) channels, and expulsion of ATP, ultimately leading to cell death via apoptosis and necrosis. CRISPR-Cas9 mediated knockout (K/O) of Panx1 in Jurkat cells prevented ATP expulsion and resulted in resistance to LtxA for both apoptotic and necrotic forms of death. Resistance to necrosis could only be overcome when supplementing LtxA with endogenous ATP (bzATP). The combination of LtxA and bzATP promoted only necrosis, as no Panx1 K/O cells stained positive for phosphatidylserine (PS) exposure following the combined treatment. Inhibition of LtxA/bzATP-induced necrosis was possible when pretreating Jurkat cells with oATP, a P2X7R antagonist. Similarly, blockage of P2X7Rs with oATP prevented the intracellular mobilization of Ca2+, an important early step in LtxA induced cell death. We show that LtxA is able to kill malignant lymphocytes through an apoptotic death pathway which is potentially linked to a Panx1/P2X7R mediated necrotic form of death. Thus, inhibition of ATP release appears to significantly delay the onset of LtxA induced apoptosis while completely disabling the necrotic death pathway in T-lymphocytes, demonstrating the crucial role of ATP release in LtxA-mediated cell death.
Asunto(s)
Conexinas/metabolismo , Exotoxinas/metabolismo , Linfocitos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Adenosina Trifosfato/metabolismo , Apoptosis/efectos de los fármacos , Calcio/metabolismo , Muerte Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Conexinas/deficiencia , Exotoxinas/farmacología , Técnicas de Silenciamiento del Gen , Humanos , Células Jurkat , Leucemia Linfoide/etiología , Leucemia Linfoide/metabolismo , Leucemia Linfoide/patología , Linfocitos/patología , Linfoma/etiología , Linfoma/metabolismo , Linfoma/patología , Proteínas del Tejido Nervioso/deficiencia , Receptores Purinérgicos P2X7/genética , Receptores Purinérgicos P2X7/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Microglia are brain-resident immune cells with a repertoire of functions in the brain. However, the extent of their interactions with the vasculature and potential regulation of vascular physiology has been insufficiently explored. Here, we document interactions between ramified CX3CR1 + myeloid cell somata and brain capillaries. We confirm that these cells are bona fide microglia by molecular, morphological and ultrastructural approaches. Then, we give a detailed spatio-temporal characterization of these capillary-associated microglia (CAMs) comparing them with parenchymal microglia (PCMs) in their morphological activities including during microglial depletion and repopulation. Molecularly, we identify P2RY12 receptors as a regulator of CAM interactions under the control of released purines from pannexin 1 (PANX1) channels. Furthermore, microglial elimination triggered capillary dilation, blood flow increase, and impaired vasodilation that were recapitulated in P2RY12-/- and PANX1-/- mice suggesting purines released through PANX1 channels play important roles in activating microglial P2RY12 receptors to regulate neurovascular structure and function.
Asunto(s)
Encéfalo/irrigación sanguínea , Conexinas/genética , Microglía/metabolismo , Células Mieloides/metabolismo , Proteínas del Tejido Nervioso/genética , Receptores Purinérgicos P2Y12/genética , Animales , Encéfalo/citología , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Receptor 1 de Quimiocinas CX3C/genética , Receptor 1 de Quimiocinas CX3C/metabolismo , Recuento de Células , Circulación Cerebrovascular/fisiología , Conexinas/deficiencia , Electrodos Implantados , Femenino , Regulación de la Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Masculino , Ratones , Ratones Noqueados , Microglía/citología , Células Mieloides/citología , Proteínas del Tejido Nervioso/deficiencia , Neuroimagen/instrumentación , Neuroimagen/métodos , Receptores Purinérgicos P2Y12/deficiencia , Receptores Purinérgicos P2Y12/metabolismo , Vasodilatación/fisiologíaRESUMEN
Pannexin1 (Panx1) is an ATP release channel expressed in neurons and astrocytes that plays important roles in CNS physiology and pathology. Evidence for the involvement of Panx1 in seizures includes the reduction of epileptiform activity and ictal discharges following Panx1 channel blockade or deletion. However, very little is known about the relative contribution of astrocyte and neuronal Panx1 channels to hyperexcitability. To this end, mice with global and cell type specific deletion of Panx1 were used in one in vivo and two in vitro seizure models. In the low-Mg2+in vitro model, global deletion but not cell-type specific deletion of Panx1 reduced the frequency of epileptiform discharges. This reduced frequency of discharges did not impact the overall power spectra obtained from local field potentials. In the in vitro KA model, in contrast, global or cell type specific deletion of Panx1 did not affect the frequency of discharges, but reduced the overall power spectra. EEG recordings following KA-injection in vivo revealed that although global deletion of Panx1 did not affect the onset of status epilepticus (SE), SE onset was delayed in mice lacking neuronal Panx1 and accelerated in mice lacking astrocyte Panx1. EEG power spectral analysis disclosed a Panx1-dependent cortical region effect; while in the occipital region, overall spectral power was reduced in all three Panx1 genotypes; in the frontal cortex, the overall power was not affected by deletion of Panx1. Together, our results show that the contribution of Panx1 to ictal activity is model, cell-type and brain region dependent.
Asunto(s)
Astrocitos/metabolismo , Encéfalo/metabolismo , Conexinas/deficiencia , Modelos Animales de Enfermedad , Proteínas del Tejido Nervioso/deficiencia , Neuronas/metabolismo , Convulsiones/metabolismo , Animales , Encéfalo/fisiopatología , Conexinas/genética , Electroencefalografía/métodos , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Técnicas de Cultivo de Órganos , Convulsiones/genéticaRESUMEN
Pannexin 1 (Panx1) channels export ATP and may contribute to increased concentration of the vasodilator ATP in plasma during hypoxia in vivo. We hypothesized that Panx1 channels and associated ATP export contribute to hypoxic vasodilation, a mechanism that facilitates the matching of oxygen delivery to metabolic demand of tissue. Male and female mice devoid of Panx1 (Panx1-/-) and wild-type controls (WT) were anesthetized, mechanically ventilated, and instrumented with a carotid artery catheter or femoral artery flow transducer for hemodynamic and plasma ATP monitoring during inhalation of 21% (normoxia) or 10% oxygen (hypoxia). ATP export from WT vs. Panx1-/-erythrocytes (RBC) was determined ex vivo via tonometer experimentation across progressive deoxygenation. Mean arterial pressure (MAP) was similar in Panx1-/- (n = 6) and WT (n = 6) mice in normoxia, but the decrease in MAP in hypoxia seen in WT was attenuated in Panx1-/- mice (-16 ± 9% vs. -2 ± 8%; P < 0.05). Hindlimb blood flow (HBF) was significantly lower in Panx1-/- (n = 6) vs. WT (n = 6) basally, and increased in WT but not Panx1-/- mice during hypoxia (8 ± 6% vs. -10 ± 13%; P < 0.05). Estimation of hindlimb vascular conductance using data from the MAP and HBF experiments showed an average response of 28% for WT vs. -9% for Panx1-/- mice. Mean venous plasma ATP during hypoxia was 57% lower in Panx1-/- (n = 6) vs. WT mice (n = 6; P < 0.05). Mean hypoxia-induced ATP export from RBCs from Panx1-/- mice (n = 8) was 82% lower than that from WT (n = 8; P < 0.05). Panx1 channels participate in hemodynamic responses consistent with hypoxic vasodilation by regulating hypoxia-sensitive extracellular ATP levels in blood.NEW & NOTEWORTHY Export of vasodilator ATP from red blood cells requires pannexin 1. Blood plasma ATP elevations in response to hypoxia in mice require pannexin 1. Hemodynamic responses to hypoxia are accompanied by increased plasma ATP in mice in vivo and require pannexin 1.
Asunto(s)
Adenosina Trifosfato/sangre , Conexinas/sangre , Eritrocitos/metabolismo , Hemodinámica , Miembro Posterior/irrigación sanguínea , Hipoxia/sangre , Proteínas del Tejido Nervioso/sangre , Oxígeno/sangre , Animales , Presión Arterial , Conexinas/deficiencia , Conexinas/genética , Modelos Animales de Enfermedad , Femenino , Frecuencia Cardíaca , Hiperemia/sangre , Hiperemia/genética , Hiperemia/fisiopatología , Hipotensión/sangre , Hipotensión/genética , Hipotensión/fisiopatología , Hipoxia/genética , Hipoxia/fisiopatología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Flujo Sanguíneo Regional , VasodilataciónRESUMEN
Connexins oligomerise to form hexameric hemichannels in the plasma membrane that can further dock together on adjacent cells to form gap junctions and facilitate intercellular trafficking of molecules. In this study, we report the expression and function of an orphan connexin, connexin-62 (Cx62), in human and mouse (Cx57, mouse homolog) platelets. A novel mimetic peptide (62Gap27) was developed to target the second extracellular loop of Cx62, and 3-dimensional structural models predicted its interference with gap junction and hemichannel function. The ability of 62Gap27 to regulate both gap junction and hemichannel-mediated intercellular communication was observed using fluorescence recovery after photobleaching analysis and flow cytometry. Cx62 inhibition by 62Gap27 suppressed a range of agonist-stimulated platelet functions and impaired thrombosis and hemostasis. This was associated with elevated protein kinase A-dependent signaling in a cyclic adenosine monophosphate-independent manner and was not observed in Cx57-deficient mouse platelets (in which the selectivity of 62Gap27 for this connexin was also confirmed). Notably, Cx62 hemichannels were observed to function independently of Cx37 and Cx40 hemichannels. Together, our data reveal a fundamental role for a hitherto uncharacterized connexin in regulating the function of circulating cells.
Asunto(s)
Plaquetas/metabolismo , Conexinas/fisiología , Animales , Comunicación Celular/fisiología , Línea Celular , Conexinas/sangre , Conexinas/química , Conexinas/deficiencia , Conexinas/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Uniones Comunicantes/fisiología , Hemostasis/fisiología , Humanos , Integrinas/sangre , Megacariocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Moleculares , Simulación del Acoplamiento Molecular , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/farmacología , Adhesividad Plaquetaria , Agregación Plaquetaria , Conformación Proteica , Multimerización de Proteína , Relación Estructura-Actividad , Trombosis/sangreRESUMEN
OBJECTIVE: Pannexin-1 (Panx1) is suspected of having a critical role in modulating neuronal excitability and acute neurological insults. Herein, we assess the changes in behavioral and electrophysiological markers of excitability associated with Panx1 via three distinct models of epilepsy. Methods Control and Panx1 knockout C57Bl/6 mice of both sexes were monitored for their behavioral and electrographic responses to seizure-generating stimuli in three epilepsy models-(1) systemic injection of pentylenetetrazol, (2) acute electrical kindling of the hippocampus and (3) neocortical slice exposure to 4-aminopyridine. Phase-amplitude cross-frequency coupling was used to assess changes in an epileptogenic state resulting from Panx1 deletion. RESULTS: Seizure activity was suppressed in Panx1 knockouts and by application of Panx1 channel blockers, Brilliant Blue-FCF and probenecid, across all epilepsy models. In response to pentylenetetrazol, WT mice spent a greater proportion of time experiencing severe (stage 6) seizures as compared to Panx1-deficient mice. Following electrical stimulation of the hippocampal CA3 region, Panx1 knockouts had significantly shorter evoked afterdischarges and were resistant to kindling. In response to 4-aminopyridine, neocortical field recordings in slices of Panx1 knockout mice showed reduced instances of electrographic seizure-like events. Cross-frequency coupling analysis of these field potentials highlighted a reduced coupling of excitatory delta-gamma and delta-HF rhythms in the Panx1 knockout. SIGNIFICANCE: These results suggest that Panx1 plays a pivotal role in maintaining neuronal hyperexcitability in epilepsy models and that genetic or pharmacological targeting of Panx1 has anti-convulsant effects.
Asunto(s)
Conexinas/deficiencia , Epilepsia/etiología , Epilepsia/fisiopatología , Proteínas del Tejido Nervioso/deficiencia , Fenotipo , Animales , Ondas Encefálicas , Región CA3 Hipocampal/metabolismo , Región CA3 Hipocampal/fisiopatología , Modelos Animales de Enfermedad , Estimulación Eléctrica , Femenino , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Excitación Neurológica , Ratones , Ratones Noqueados , ConvulsionesRESUMEN
The optics of the ocular lens are determined by its geometry (shape and volume) and its inherent gradient of refractive index (water to protein ratio), which are in turn maintained by unique cellular physiology known as the lens internal microcirculation system. Previously, magnetic resonance imaging (MRI) has been used on ex vivo organ cultured bovine lenses to show that pharmacological perturbations to this microcirculation system disrupt ionic and fluid homeostasis and overall lens optics. In this study, we have optimised in vivo MRI protocols for use on wild-type and transgenic mouse models so that the effects of genetically perturbing the lens microcirculation system on lens properties can be studied. In vivo MRI protocols and post-analysis methods for studying the mouse lens were optimised and used to measure the lens geometry, diffusion, T1 and T2, as well as the refractive index (n) calculated from T2, in wild-type mice and the genetically modified Cx50KI46 mouse. In this animal line, gap junctional coupling in the lens is increased by knocking in the gap junction protein Cx46 into the Cx50 locus. Relative to wild-type mice, Cx50KI46 mice showed significantly reduced lens size and radius of curvature, increased T1 and T2 values, and decreased n in the lens nucleus, which was consistent with the developmental and functional changes characterised previously in this lens model. These proof of principle experiments show that in vivo MRI can be applied to transgenic mouse models to gain mechanistic insights into the relationship between lens physiology and optics, and in the future suggest that longitudinal studies can be performed to determine how this relationship is altered by age in mouse models of cataract.
Asunto(s)
Cristalino/diagnóstico por imagen , Imagen por Resonancia Magnética , Animales , Bovinos , Conexinas/deficiencia , Conexinas/genética , Difusión , Cristalino/metabolismo , Cristalino/fisiología , Ratones , Ratones NoqueadosRESUMEN
Dysferlinopathy is a genetic human disease caused by mutations in the gene that encodes the dysferlin protein (DYSF). Dysferlin is believed to play a relevant role in cell membrane repair. However, in dysferlin-deficient (blAJ) mice (a model of dysferlinopathies) the recovery of the membrane resealing function by means of the expression of a mini-dysferlin does not arrest progressive muscular damage, suggesting the participation of other unknown pathogenic mechanisms. Here, we show that proteins called connexins 39, 43 and 45 (Cx39, Cx43 and Cx45, respectively) are expressed by blAJ myofibers and form functional hemichannels (Cx HCs) in the sarcolemma. At rest, Cx HCs increased the sarcolemma permeability to small molecules and the intracellular Ca2+ signal. In addition, skeletal muscles of blAJ mice showed lipid accumulation and lack of dysferlin immunoreactivity. As sign of extensive damage and atrophy, muscles of blAJ mice presented elevated numbers of myofibers with internal nuclei, increased number of myofibers with reduced cross-sectional area and elevated creatine kinase activity in serum. In agreement with the extense muscle damage, mice also showed significantly low motor performance. We generated blAJ mice with myofibers deficient in Cx43 and Cx45 expression and found that all above muscle and systemic alterations were absent, indicating that these two Cxs play a critical role in a novel pathogenic mechanism of dysfernolophaties, which is discussed herein. Therefore, Cx HCs could constitute an attractive target for pharmacologic treatment of dyferlinopathies.
Asunto(s)
Conexina 43/genética , Conexinas/genética , Disferlina/genética , Distrofia Muscular de Cinturas/genética , Distrofia Muscular de Cinturas/prevención & control , Miofibrillas/genética , Animales , Calcio/metabolismo , Conexina 43/deficiencia , Conexinas/deficiencia , Creatina Quinasa/sangre , Creatina Quinasa/genética , Modelos Animales de Enfermedad , Disferlina/deficiencia , Expresión Génica , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Distrofia Muscular de Cinturas/metabolismo , Distrofia Muscular de Cinturas/patología , Mutación , Miofibrillas/metabolismo , Miofibrillas/patología , Permeabilidad , Condicionamiento Físico Animal , Prueba de Desempeño de Rotación con Aceleración Constante , Sarcolema/metabolismoRESUMEN
Spontaneous infra-slow brain activity (ISA) exhibits a high degree of temporal synchrony, or correlation, between distant brain regions. The spatial organization of ISA synchrony is not explained by anatomical connections alone, suggesting that active neural processes coordinate spontaneous activity. Inhibitory interneurons (IINs) form electrically coupled connections via the gap junction protein connexin 36 (Cx36) and networks of interconnected IINs are known to influence neural synchrony over short distances. However, the role of electrically coupled IIN networks in regulating spontaneous correlation over the entire brain is unknown. In this study, we performed OIS imaging on Cx36-/- mice to examine the role of this gap junction in ISA correlation across the entire cortex. We show that Cx36 deletion increased long-distance intra-hemispheric anti-correlation and inter-hemispheric correlation in spontaneous ISA. This suggests that electrically coupled IIN networks modulate ISA synchrony over long cortical distances.
Asunto(s)
Corteza Cerebral/metabolismo , Conexinas/deficiencia , Interneuronas/metabolismo , Red Nerviosa/metabolismo , Inhibición Neural/fisiología , Animales , Corteza Cerebral/citología , Conexinas/genética , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Red Nerviosa/citología , Distribución Aleatoria , Proteína delta-6 de Union ComunicanteRESUMEN
The astroglial gap junctional network formed by connexin (Cx) channels plays a central role in regulating neuronal activity and network synchronization. However, its involvement in the development and progression of epilepsy is not yet understood. Loss of interastrocytic gap junction (GJ) coupling has been observed in the sclerotic hippocampus of patients with mesial temporal lobe epilepsy (MTLE) and in mouse models of MTLE, leading to the suggestion that it plays a causative role in the pathogenesis. To further elucidate this clinically relevant question, we investigated consequences of astrocyte disconnection on the time course and severity of kainate-induced MTLE with hippocampal sclerosis (HS) by comparing mice deficient for astrocytic Cx proteins with wild-type mice (WT). Continuous telemetric EEG recordings and video monitoring performed over a period of 4 weeks after epilepsy induction revealed substantially higher seizure and interictal spike activity during the chronic phase in Cx deficient versus WT mice, while the severity of status epilepticus was not different. Immunohistochemical analysis showed that, despite the elevated chronic seizure activity, astrocyte disconnection did not aggravate the severity of HS. Indeed, the extent of CA1 pyramidal cell loss was similar between the experimental groups, while astrogliosis, granule cell dispersion, angiogenesis, and microglia activation were even reduced in Cx deficient as compared to WT mice. Interestingly, seizure-induced neurogenesis in the adult dentate gyrus was also independent of astrocytic Cxs. Together, our data indicate that constitutive loss of GJ coupling between astrocytes promotes neuronal hyperexcitability and attenuates seizure-induced histopathological outcomes.
Asunto(s)
Astrocitos/metabolismo , Conexinas/deficiencia , Epilepsia/inducido químicamente , Epilepsia/metabolismo , Eliminación de Gen , Ácido Kaínico/toxicidad , Animales , Astrocitos/efectos de los fármacos , Conexinas/genética , Epilepsia/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones TransgénicosRESUMEN
Hepatic ischemia/reperfusion (I/R) injury is a common complication in the clinical setting. Our previous study has shown that connexin 32 (Cx32) plays a major role in renal I/R injury; however, the role of Cx32 in hepatic I/R injury remains unknown. Liver tissue and serum samples from patients undergoing orthotopic liver transplantation (OLT) were used to evaluate the function of Cx32 in OLT post-reperfusion injury. Then, partial hepatic ischemia was established in global Cx32 knockout mice and wild-type mice followed by reperfusion. Hepatic injury markers were examined. Cx32 small interfering RNA and the p53 inhibitor, pifithrin-α, tenovin-1 were used to examine the relationship between Cx32 and the p53/puma pathways in the BRL-3A and murine primary hepatocytes hypoxia/reoxygenation (H/R) model. Corresponding to liver damage, Cx32 was significantly induced both during OLT in human patients and partial hepatic I/R in mice. Cx32 KO mice exhibited less liver injury than controls. Cx32 deficiency significantly suppressed the p53/puma pathways and hepatocyte apoptosis. Similar results were observed in the BRL-3A and murine primary hepatocytes H/R model. Propofol protected against OLT post-reperfusion injury and hepatocyte apoptosis by inhibiting Cx32. In conclusion Cx32 is a novel regulator of hepatic I/R injury through the modulation of hepatocyte apoptosis and damage, largely via the p53/puma signaling pathway.
Asunto(s)
Conexinas/antagonistas & inhibidores , Isquemia/prevención & control , Trasplante de Hígado , Hígado/irrigación sanguínea , Propofol/farmacología , Daño por Reperfusión/prevención & control , Animales , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/metabolismo , Conexinas/deficiencia , Conexinas/genética , Modelos Animales de Enfermedad , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Hepatocitos/patología , Humanos , Isquemia/metabolismo , Isquemia/patología , Poscondicionamiento Isquémico , Hígado/metabolismo , Hígado/patología , Pruebas de Función Hepática , Ratones Endogámicos C57BL , Ratones Noqueados , Propofol/administración & dosificación , Estudios Prospectivos , Ratas , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Transducción de Señal , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteína beta1 de Unión ComunicanteRESUMEN
BACKGROUND: Hepatic ischemia reperfusion (HIR) leads to a lung inflammatory response and subsequent pulmonary barrier dysfunction. The gap junction communication protein connexin 32 (Cx32), which is widely expressed in the lungs, participates in intercellular signaling. This study determined whether the communication protein Cx32 could affect pulmonary inflammation caused by HIR. METHODS: Mice were randomly allocated into four groups (n = 8/group): (i) Cx32+/+ sham group; (ii) Cx32+/+ HIR model group; (iii) Cx32-/- sham group; and (iv) Cx32-/- HIR model group. Twenty-four hours after surgery, lung tissues were collected for bright field microscopy, western blot (Cx32, JAK2, p-JAK2, STAT3, p-STAT3), and immunofluorescence (ZO-1, 8-OHDG) analyses. The collected bronchoalveolar fluid was tested for levels of interleukin-6 (IL-6), matrix metalloproteinase 12 (MMP-12), and antitrypsin (α1-AT). Lung mmu-miR-26a/b expression was detected using a PCR assay. RESULTS: Increased expression of Cx32 mRNA and protein was noted in the lungs after HIR. Cx32 deletion significantly aggravated pulmonary function from acute lung injury induced by HIR. In addition, Cx32 deletion decreased the protein level of ZO-1 (pulmonary function) and increased the level of the oxidative stress marker 8-OHDG in the lungs. Moreover, in the Cx32-/- HIR model group, the levels of IL-6 and MMP-12 in bronchoalveolar lavage fluid were significantly increased leading to activation of the JAK2/STAT3 pathway, and decreased α1-AT levels. Furthermore, we found mmu-miR-26a/b was significantly downregulated in the Cx32-/- HIR model group. CONCLUSION: HIR leads to acute lung inflammatory injury. Cx32 deletion aggravates hepatic-derived lung inflammation, partly through blocking the transferring of mmu-miR-26a/b and leading to IL-6-related JAK2/STAT3 pathway activation.
Asunto(s)
Lesión Pulmonar Aguda/etiología , Conexinas/metabolismo , Hepatopatías/complicaciones , Pulmón/metabolismo , Neumonía/etiología , Daño por Reperfusión/complicaciones , Lesión Pulmonar Aguda/genética , Lesión Pulmonar Aguda/metabolismo , Lesión Pulmonar Aguda/patología , Animales , Conexinas/deficiencia , Conexinas/genética , Modelos Animales de Enfermedad , Interleucina-6/metabolismo , Janus Quinasa 2/metabolismo , Hepatopatías/genética , Hepatopatías/metabolismo , Pulmón/patología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , MicroARNs/genética , MicroARNs/metabolismo , Neumonía/genética , Neumonía/metabolismo , Neumonía/patología , Daño por Reperfusión/genética , Daño por Reperfusión/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Proteína beta1 de Unión ComunicanteRESUMEN
Dysregulation of blood-brain barrier (BBB) transport exacerbates brain damage in acute ischemic stroke. Here, we aimed to investigate the mechanism of this BBB transport dysregulation by studying the localization and function of pannexin (Px) and connexin (Cx) hemichannels in blood-brain barrier endothelial cells of rat (TR-BBB13â¯cells) and human (hCMEC/D3 cells) under acute ischemic stroke-mimicking oxygen/glucose deprivation (OGD) and extracellular Ca2+ ([Ca2+]e)-free conditions. TR-BBB13â¯cells showed increased uptake of hemichannel-permeable sulforhodamine 101, and this increase was markedly inhibited by carbenoxolone, a hemichannel inhibitor. Transcripts of Px1 and Cx43 were detected in TR-BBB13â¯cells and freshly isolated brain microvascular endothelial cells. The basal compartment-to-cell uptake of hemichannel-permeable propidium iodide was selectively enhanced in hCMEC/D3 cells under [Ca2+]e-free conditions in the basal Transwell chamber. Immunohistochemical analysis revealed the predominant localization of Cx43 on the lateral membranes of hCMEC/D3 cells. [3H]Taurine uptake by hCMEC/D3 cells was significantly reduced in the absence of [Ca2+]e. Functional knock-down of Px1 and Cx43 with mimetic peptides significantly inhibited the increase of ATP release from hCMEC/D3 cells under [Ca2+]e-free conditions. These results suggest that polarized Px1/Cx43 hemichannel opening in brain capillary endothelial cells under acute ischemic stroke-mimicking conditions contributes to dysregulation of BBB transport function, resulting in release of intracellular taurine and ATP.
Asunto(s)
Barrera Hematoencefálica/citología , Barrera Hematoencefálica/metabolismo , Conexina 43/deficiencia , Conexinas/deficiencia , Células Endoteliales/metabolismo , Proteínas del Tejido Nervioso/deficiencia , Animales , Línea Celular Transformada , Células Cultivadas , Conexina 43/genética , Conexinas/genética , Humanos , Masculino , Ratones , Proteínas del Tejido Nervioso/genética , Transporte de Proteínas/fisiología , RatasRESUMEN
Neutrophils are the first immune cells to kill invading microbes at sites of infection using a variety of processes, including the release of proteases, phagocytosis and the production of neutrophil extracellular traps (NETs). NET formation, or NETosis, is a specific and highly efficient process, which is induced by a variety of stimuli leading to expulsion of DNA, proteases and antimicrobial peptides to the extracellular space. However, uncontrolled NETosis may lead to adverse effects and exert tissue damage in pathological conditions. Here, we show that the ATP channel pannexin1 (Panx1) is functionally expressed by bone marrow-derived neutrophils (BMDNs) of wild-type (WT) mice and that ATP contributes to NETosis induced in vitro by the calcium ionophore A23187 or phorbol 12-myristate 13-acetate (PMA). Interestingly, neutrophils isolated from Panx1-/- mice showed reduced and/or delayed induction of NETosis. Brilliant blue FCF dye (BB-FCF), a Panx1 channel inhibitor, decreased NETosis in wild-type neutrophils to the extent observed in Panx1-/- neutrophils. Thus, we demonstrate that ATP and Panx1 channels contribute to NETosis and may represent a therapeutic target.
Asunto(s)
Adenosina Trifosfato/farmacología , Trampas Extracelulares/metabolismo , NADP/metabolismo , Animales , Células de la Médula Ósea/citología , Calcimicina/farmacología , Conexinas/antagonistas & inhibidores , Conexinas/deficiencia , Conexinas/metabolismo , Trampas Extracelulares/efectos de los fármacos , Cinética , Ratones , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/metabolismo , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Renal ischemia/reperfusion injury (IRI) is a significant challenge in perioperative medicine and is related to oxidative programmed cell death. However, the role of ferroptosis, a newly discovered form of oxidative cell death, has not been evaluated widely. Pannexin 1 (PANX1), an ATP-releasing pathway family protein, has pro-apoptotic effects during kidney injury. Here, we demonstrate that PANX1 deletion protects against renal IRI by regulating ferroptotic cell death. Panx1 knockout mice subjected to renal IRI had decreased plasma creatinine, malondialdehyde (MDA) levels in kidney tissues, and tubular cell death (visible as decreased TUNEL-positive renal tubular cells) compared with WT mice. In cultured human kidney 2 (HK-2) cells, silenced Panx1 expression significantly attenuated ferroptotic lipid peroxidation and iron accumulation induced by the ferroptosis inducer erastin. Moreover, the Panx1 silencing significantly modulated ferroptosis-related protein expression. Furthermore, Panx1 deletion induced the expression of a cytoprotective chaperone, heme oxygenase-1 (HO-1), and inhibited ferroptinophagy via the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway. In summary, Panx1 deletion protects against renal IRI by attenuating MAPK/ERK activation in a ferroptotic pathway. Our findings provide critical insights into the role of PANX1 in ferroptotic cell death and highlight a potential therapeutic target for the management of acute kidney injury (AKI) during the perioperative period.
Asunto(s)
Conexinas/metabolismo , Ferroptosis , Riñón/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Daño por Reperfusión/metabolismo , Animales , Supervivencia Celular , Células Cultivadas , Conexinas/deficiencia , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas del Tejido Nervioso/deficienciaRESUMEN
Since cost-effective osteoanabolic treatment options remain to be established, it is relevant to identify specific molecules physiologically regulating osteoblast differentiation and/or activity that are principally accessible as drug targets. Specific or predominant gene expression in a given cell type often predicts a relevant function in the respective tissue. Thus, we aimed to identify genes encoding membrane-associated proteins with selective expression in differentiated osteoblasts. We therefore applied an unbiased approach, i.e. Affymetrix Gene Chip hybridization, to compare global gene expression in primary murine osteoblasts at two stages of differentiation. For the most strongly induced genes we analyzed their expression pattern in different tissues, which led us to identify known and unknown osteoblast differentiation markers with predominant expression in bone. One of these genes was Panx3, encoding a transmembrane hemichannel with ill-defined function in skeletal remodeling. To decipher the role of Panx3 in osteoblasts we first generated Panx3-fl/fl mice carrying a Runx2-Cre transgene. Using undecalcified histology followed by bone-specific histomorphometry we did not observe any significant difference between 24â¯weeks old Cre-negative and Cre-positive littermates. We additionally generated and analyzed mice with ubiquitous Panx3 deletion, where a delay of endochondral ossification did not translate into a detectable skeletal phenotype after weaning, possibly explained by compensatory induction of Panx1. Of note, newborn Panx3-deficient mice displayed significantly reduced serum glucose levels, which was not the case in older animals. Our findings demonstrate that Panx3 expression in osteoblasts is not required for postnatal bone remodeling, which essentially rules out its suitability as a target protein for osteoanabolic medication.
Asunto(s)
Remodelación Ósea , Conexinas/metabolismo , Osteoblastos/metabolismo , Animales , Animales Recién Nacidos , Diferenciación Celular/genética , Conexinas/deficiencia , Regulación de la Expresión Génica , Ratones Endogámicos C57BL , Especificidad de Órganos , Osteoblastos/citología , FenotipoRESUMEN
Pannexin-3 (Panx3) is a gap junction protein that is required for regulating cell cycle exit and the differentiation of osteoblasts and chondrocytes during skeletal development. However, the role of Panx3 in skin tissue regeneration remains unclear. After dorsal skin punch biopsies, Panx3-knockout mice exhibited a significant delay in wound healing with insufficient re-epithelialization, decreased inflammatory reaction, and reduced collagen remodeling. Panx3 expression coincided with inflammatory reactions both in vivo and in vitro. By applying exogenous tumor necrosis factor-α to mimic inflammation in vitro, Panx3 expression was induced in HaCaT cells. In addition, Panx3 depletion reduced epithelial-mesenchymal transition during skin wound healing. A protein essential for signaling in epithelial-mesenchymal transition, transforming growth factor-ß interacted with Panx3 by modulating intracellular adenosine triphosphate levels and thereby enhanced HaCaT cell migration ability with Panx3 overexpression. In conclusion, Panx3 plays a key role in the skin wound healing process by controlling keratinocytes and keratinocyte-mesenchyme cross-talk via hemichannel and endoplasmic reticulum Ca2+ channel functions, which differs from another gap junction, connexin 43 (Cx43), during skin wound healing.