RESUMO
Pyramidal neurons in the medial prefrontal cortical layer 2/3 are an essential contributor to the cellular basis of working memory; thus, changes in their intrinsic excitability critically affect medial prefrontal cortex (mPFC) functional properties. Transient Receptor Potential Melastatin 4 (TRPM4), a calcium-activated nonselective cation channel (CAN), regulates the membrane potential in a calcium-dependent manner. In this study, we uncovered the role of TRPM4 in regulating the intrinsic excitability plasticity of pyramidal neurons in the mouse mPFC layer of 2/3 using a combination of conventional and nystatin perforated whole-cell recordings. Interestingly, we found that TRPM4 is open at resting membrane potential, and its inhibition increases input resistance and hyperpolarizes membrane potential. After high-frequency stimulation, pyramidal neurons increase a calcium-activated non-selective cation current, increase the action potential firing, and the amplitude of the afterdepolarization, these effects depend on intracellular calcium. Furthermore, pharmacological inhibition or genetic silencing of TRPM4 reduces the firing rate and the afterdepolarization after high frequency stimulation. Together, these results show that TRPM4 plays a significant role in the excitability of mPFC layer 2/3 pyramidal neurons by modulating neuronal excitability in a calcium-dependent manner.
Assuntos
Córtex Pré-Frontal/metabolismo , Células Piramidais/metabolismo , Canais de Cátion TRPM/metabolismo , Potenciais de Ação/fisiologia , Animais , Cálcio/metabolismo , Masculino , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Patch-Clamp , Córtex Pré-Frontal/efeitos dos fármacos , Células Piramidais/efeitos dos fármacos , Células Piramidais/patologia , Canais de Cátion TRPM/fisiologiaRESUMO
Transient receptor potential melastatin 4 (TRPM4) is a Ca2+-activated nonselective cationic channel involved in a wide variety of physiologic and pathophysiological processes. Bioinformatics analyses of the primary sequence of TRPM4 allowed us to identify a putative motif for interaction with end-binding (EB) proteins, which are microtubule plus-end tracking proteins. Here, we provide novel data suggesting that TRPM4 interacts with EB proteins. We show that mutations of the putative EB binding motif abolish the TRPM4-EB interaction, leading to a reduced expression of the mature population of the plasma membrane channel and instead display an endoplasmic reticulum-associated distribution. Furthermore, we demonstrate that EB1 and EB2 proteins are required for TRPM4 trafficking and functional activity. Finally, we demonstrated that the expression of a soluble fragment containing the EB binding motif of TRPM4 reduces the plasma membrane expression of the channel and affects TRPM4-dependent focal adhesion disassembly and cell invasion processes.-Blanco, C., Morales, D., Mogollones, I., Vergara-Jaque, A., Vargas, C., Álvarez, A., Riquelme, D., Leiva-Salcedo, E., González, W., Morales, D., Maureira, D., Aldunate, I., Cáceres, M., Varela, D., Cerda, O. EB1- and EB2-dependent anterograde trafficking of TRPM4 regulates focal adhesion turnover and cell invasion.
Assuntos
Adesões Focais/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Canais de Cátion TRPM/metabolismo , Animais , Biotinilação/fisiologia , Células COS , Adesão Celular/genética , Adesão Celular/fisiologia , Linhagem Celular , Movimento Celular/genética , Movimento Celular/fisiologia , Chlorocebus aethiops , Eletrofisiologia , Imunofluorescência , Humanos , Immunoblotting , Proteínas Associadas aos Microtúbulos/genética , Simulação de Dinâmica Molecular , Mutação/genética , Plasmídeos/genética , Canais de Cátion TRPM/genéticaRESUMO
TRPM4 is a calcium activated non-selective cation channel, impermeable to Ca2+, in neurons it has been implicated in the regulation of the excitability and in the persistent firing. Cholinergic stimulation is also implicated in changes in excitability that leads neurons to an increased firing frequency, however it is not clear whether TRPM4 is involved in the cholinergic-induced increase in firing frequency. Here using a combination of patch clamp electrophysiology, Ca2+ imaging, immunofluorescence, fluorescence recovery after photobleaching (FRAP) and pharmacological approach, we demonstrate that carbachol (Cch) increases firing frequency, intracellular Ca2+ and that TRPM4 inhibition using 9-Ph and CBA reduces firing frequency and decreases the peak in intracellular Ca2+ induced by Cch in cortical pyramidal neurons in culture. Moreover, we determined that cholinergic stimulation reduces TRPM4 recycling and stabilizes TRPM4 in the plasma membrane. Together our results indicate that cholinergic stimulation increases firing in a TRPM4 dependent manner, and also increases the TRPM4 stability in the membrane, suggesting that TRPM4 is locked in microdomains in the membrane, possibly signaling or cytoskeleton proteins complexes.
RESUMO
The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Many countries have reported the experience of at least two contagion waves, describing associated mortality rates and population behavior. The analysis of the effect of this pandemic in different localities can provide valuable information on the key factors to consider in the face of future massive infectious diseases. This work describes the first retrospective and comparative study about behavior during the first and second waves of the COVID-19 pandemic in Chile from a primary Healthcare Center. From 19,313 real-time quantitative PCR (RT-qPCR) tests assessed, the selected 1,694 positive diagnostics showed a decrease in mortality rate in the second wave (0.6%) compared with the first (4.6%). In addition, we observed that infections in the second wave were mainly in young patients with reduced comorbidities. The population with a complete vaccination schedule shows a decrease in the duration of symptoms related to the disease, and patients with more comorbidities tend to develop severe illness. This report provides evidence to partially understand the behavior and critical factors in the severity of the COVID-19 pandemic in the population of Santiago of Chile.
Assuntos
COVID-19 , SARS-CoV-2 , COVID-19/epidemiologia , Chile/epidemiologia , Humanos , Estudos Longitudinais , Pandemias , Atenção Primária à Saúde , Estudos RetrospectivosRESUMO
TRPM4 is a non-selective cation channel activated by intracellular calcium and permeable to monovalent cations. This channel participates in the control of neuronal firing, neuronal plasticity, and neuronal death. TRPM4 depolarizes dendritic spines and is critical for the induction of NMDA receptor-dependent long-term potentiation in CA1 pyramidal neurons. Despite its functional importance, no subcellular localization or expression during postnatal development has been described in this area. To examine the localization and expression of TRPM4, we performed duplex immunofluorescence and patch-clamp in brain slices at different postnatal ages in C57BL/6J mice. At P0 we found TRPM4 is expressed with a somatic pattern. At P7, P14, and P35, TRPM4 expression extended from the soma to the apical dendrites but was excluded from the axon initial segment. Patch-clamp recordings showed a TRPM4-like current active at the resting membrane potential from P0, which increased throughout the postnatal development. This current was dependent on intracellular Ca2+ (I CAN ) and sensitive to 9-phenanthrol (9-Ph). Inhibiting TRPM4 with 9-Ph hyperpolarized the membrane potential at P14 and P35, with no effect in earlier stages. Together, these results show that TRPM4 is expressed in CA1 pyramidal neurons in the soma and apical dendrites and associated with a TRPM4-like current, which depolarizes the neurons. The expression, localization, and function of TRPM4 throughout postnatal development in the CA1 hippocampal may underlie an important mechanism of control of membrane potential and action potential firing during critical periods of neuronal development, particularly during the establishment of circuits.
RESUMO
Cell migration is critical for several physiological and pathophysiological processes. It depends on the coordinated action of kinases, phosphatases, Rho-GTPases proteins, and Ca2+ signaling. Interestingly, ubiquitination events have emerged as regulatory elements of migration. Thus, the role of proteins involved in ubiquitination processes could be relevant to a complete understanding of pro-migratory mechanisms. KCTD5 is a member of Potassium Channel Tetramerization Domain (KCTD) proteins that have been proposed as a putative adaptor for Cullin3-E3 ubiquitin ligase and a novel regulatory protein of TRPM4 channels. Here, we study whether KCTD5 participates in cell migration-associated mechanisms, such as focal adhesion dynamics and cellular spreading. Our results show that KCTD5 CRISPR/Cas9- and shRNA-based depletion in B16-F10 cells promoted an increase in cell migration and cell spreading, and a decrease in the focal adhesion area, consistent with an increased focal adhesion disassembly rate. The expression of a dominant-negative mutant of Rho-GTPases Rac1 precluded the KCTD5 depletion-induced increase in cell spreading. Additionally, KCTD5 silencing decreased the serum-induced Ca2+ response, and the reversion of this with ionomycin abolished the KCTD5 knockdown-induced decrease in focal adhesion size. Together, these data suggest that KCTD5 acts as a regulator of cell migration by modulating cell spreading and focal adhesion dynamics through Rac1 activity and Ca2+ signaling, respectively.
Assuntos
Sinalização do Cálcio/fisiologia , Canais de Potássio/metabolismo , Animais , Cálcio/metabolismo , Adesão Celular/genética , Linhagem Celular , Movimento Celular/genética , Adesões Focais/genética , Humanos , Camundongos , Canais de Potássio/fisiologia , Transdução de Sinais , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Mammalian cells sense oxygen levels and respond to hypoxic conditions through the regulation of multiple signaling pathways and transcription factors. Here, we investigated the effects of hypoxia on the activity of two transcriptional regulators, ERK1/2 and NF-kappaB, in skeletal muscle cells in primary culture. We found that hypoxia significantly enhanced ERK1/2 phosphorylation and that it stimulated NF-kappaB-dependent gene transcription as well as nuclear translocation of a green fluorescent protein-labeled p65 NF-kappaB isoform. Phosphorylation of ERK1/2- and NF-kappaB-dependent transcription by hypoxia required calcium entry through L-type calcium channels. Calcium release from ryanodine-sensitive stores was also necessary for ERK1/2 activation but not for NF-kappaB-dependent-transcription. N-acetylcysteine, a general scavenger of reactive oxygen species, blocked hypoxia-induced ROS generation but did not affect the stimulation of ERK1/2 phosphorylation induced by hypoxia. In contrast, NF-kappaB activation was significantly inhibited by N-acetylcysteine and did not depend on ERK1/2 stimulation, as shown by the lack of effect of the upstream ERK inhibitor U-0126. These separate pathways of activation of ERK1/2 and NF-kappaB by hypoxia may contribute to muscle adaptation in response to hypoxic conditions.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Hipóxia/fisiopatologia , Fibras Musculares Esqueléticas/fisiologia , Músculo Esquelético/fisiologia , NF-kappa B/fisiologia , Transdução de Sinais/fisiologia , Animais , Western Blotting , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Sinalização do Cálcio/fisiologia , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Espaço Extracelular/metabolismo , Proteínas de Fluorescência Verde , Microscopia de Fluorescência , Músculo Esquelético/citologia , Fosforilação , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Retículo Sarcoplasmático/metabolismo , TransfecçãoRESUMO
Aging is associated with a deterioration of the acute phase response to inflammatory challenges. However, the nature of these defects remains poorly defined. We analyzed the hepatic inflammatory response after intraperitoneal administration of lipopolysaccharide (LPS) given to Fisher 344 rats aged 6, 15, and 22-23 months. Induction of the acute phase proteins (APPs), haptoglobin, alpha-1-acid glycoprotein, and T-kininogen was reduced and/or retarded with aging. Initial induction of interleukin-6 in aged rats was normal, but the later response was increased relative to younger counterparts. An exacerbated hepatic injury was observed in aged rats receiving LPS, as evidenced by the presence of multiple microabscesses in portal tracts, confluent necrosis, higher neutrophil accumulation, and elevated serum levels of alanine aminotransferase, relative to younger animals. Our results suggest that aged rats displayed a reduced expression of APPs and increased hepatic injury in response to the inflammatory insult.
Assuntos
Proteínas de Fase Aguda/metabolismo , Envelhecimento/imunologia , Hepatite Animal/imunologia , Animais , Imuno-Histoquímica , Injeções Intraperitoneais , Interleucina-6/sangue , Lipopolissacarídeos/efeitos adversos , Fígado/metabolismo , Masculino , Neutrófilos , Ratos , Ratos Endogâmicos F344RESUMO
TRPM4 is a Ca2+-activated non-selective cationic channel that conducts monovalent cations. TRPM4 has been proposed to contribute to burst firing and sustained activity in several brain regions, however, the cellular and subcellular pattern of TRPM4 expression in medial prefrontal cortex (mPFC) during postnatal development has not been elucidated. Here, we use multiplex immunofluorescence labeling of brain sections to characterize the postnatal developmental expression of TRPM4 in the mouse mPFC. We also performed electrophysiological recordings to correlate the expression of TRPM4 immunoreactivity with the presence of TRPM4-like currents. We found that TRPM4 is expressed from the first postnatal day, with expression increasing up to postnatal day 35. Additionally, in perforated patch clamp experiments, we found that TRPM4-like currents were active at resting membrane potentials at all postnatal ages studied. Moreover, TRPM4 is expressed in both pyramidal neurons and interneurons. TRPM4 expression is localized in the soma and proximal dendrites, but not in the axon initial segment of pyramidal neurons. This subcellular localization is consistent with a reduction in the basal current only when we locally perfused 9-Phenanthrol in the soma, but not upon perfusion in the medial or distal dendrites. Our results show a specific localization of TRPM4 expression in neurons in the mPFC and that a 9-Phenanthrol sensitive current is active at resting membrane potential, suggesting specific functional roles in mPFC neurons during postnatal development and in adulthood.
RESUMO
Cerebral ischemia-reperfusion injury triggers a deleterious process ending in neuronal death. This process has two components, a glutamate-dependent and a glutamate-independent mechanism. In the glutamate-independent mechanism, neurons undergo a slow depolarization eventually leading to neuronal death. However, little is known about the molecules that take part in this process. Here we show by using mice cortical neurons in culture and ischemia-reperfusion protocols that TRPM4 is fundamental for the glutamate-independent neuronal damage. Thus, by blocking excitotoxicity, we reveal a slow activating, glibenclamide- and 9-phenanthrol-sensitive current, which is activated within 5 min upon ischemia-reperfusion onset. TRPM4 shRNA-based silenced neurons show a reduced ischemia-reperfusion induced current and depolarization. Neurons were protected from neuronal death up to 3 hours after the ischemia-reperfusion challenge. The activation of TRPM4 during ischemia-reperfusion injury involves the increase in both, intracellular calcium and H2O2, which may act together to produce a sustained activation of the channel.
Assuntos
Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Neurônios/metabolismo , Neurônios/patologia , Oxigênio/metabolismo , Traumatismo por Reperfusão/patologia , Canais de Cátion TRPM/metabolismo , Animais , Morte Celular , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Traumatismo por Reperfusão/metabolismoRESUMO
Antigen cross-presentation is a crucial step in the assembly of an antitumor immune response leading to activation of naïve CD8 T cells. This process has been extensively used in clinical trials, in which dendritic cells generated in vitro are loaded with tumor antigens and then autotransplanted to the patients. Recently, the use of autologous transplant of dendritic cells fused with dying tumor cells has demonstrated good results in clinical studies. In this work, we generated a similar process in vivo by treating mice with dead tumor cells [cell bodies (CBs)] expressing the fusogenic protein of the infectious salmon anemia virus (ISAV). ISAV fusion protein retains its fusogenic capability when is expressed on mammalian cells in vitro and the CBs expressing it facilitates DCs maturation, antigen transfer by antigen-presenting cells, and increase cross-presentation by DCs in vitro. Additionally, we observed in the melanoma model that CBs with or without ISAV fusion protein reduce tumor growth in prophylactic treatment; however, only ISAV expressing CBs showed an increase CD4 and CD8 cells in spleen. Overall, our results suggest that CBs could be used as a complement with other type of strategies to amplify antitumor immune response.
RESUMO
Membrane association of estrogen receptors (ER) depends on cysteine palmitoylation and two leucines in the ligand binding domain (LBD), conserved in most steroid receptors. The role of this region, corresponding to helix 8 of the glucocorticoid receptor (GR) LBD, on membrane association of GR was studied in 4B cells, expressing endogenous GR, and Cos-7 cells transfected EGFP-GR constructs. 4B cells preloaded with radiolabeled palmitic acid showed no radioactivity incorporation into immunoprecipitated GR. Moreover, mutation C683A (corresponding to ER palmitoylation site) did not affect corticosterone-induced membrane association of GR. Mutations L687-690A, L682A, E680G and K685G prevented membrane and also nuclear localization through reduced ligand binding. L687-690A mutation decreased association of GR with heat shock protein 90 and transcriptional activity, without overt effects on receptor protein stability. The data demonstrate that palmitoylation does not mediate membrane association of GR, but that the region 680-690 (helix 8) is critical for ligand binding and receptor function.
Assuntos
Receptores de Glucocorticoides/química , Receptores de Glucocorticoides/metabolismo , Sequência de Aminoácidos , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Sequência Conservada , Corticosterona/farmacologia , Cisteína/metabolismo , Dexametasona/metabolismo , Relação Dose-Resposta a Droga , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Ligantes , Lipoilação/efeitos dos fármacos , Luciferases/metabolismo , Dados de Sequência Molecular , Proteínas Mutantes/química , Mutação/genética , Ácido Palmítico/metabolismo , Ligação Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , Ratos , Sequências Repetitivas de Aminoácidos , Alinhamento de Sequência , Relação Estrutura-Atividade , Transcrição Gênica/efeitos dos fármacos , Trítio/metabolismoRESUMO
The hypothesis that rapid glucocorticoid inhibition of pituitary ACTH secretion mediates a feedforward/feedback mechanism responsible for the hourly glucocorticoid pulsatility was tested in cultured pituitary cells. Perifusion with 30 pM CRH caused sustained the elevation of ACTH secretion. Superimposed corticosterone pulses inhibited CRH-stimulated ACTH release, depending on prior glucocorticoid clearance. When CRH perifusion started after 2 hours of glucocorticoid-free medium, corticosterone levels in the stress range (1 µM) caused a delayed (25 min) and prolonged inhibition of CRH-stimulated ACTH secretion, up to 60 minutes after corticosterone withdrawal. In contrast, after 6 hours of glucocorticoid-free medium, basal corticosterone levels inhibited CRH-stimulated ACTH within 5 minutes, after rapid recovery 5 minutes after corticosterone withdrawal. The latter effect was insensitive to actinomycin D but was prevented by the glucocorticoid receptor antagonist, RU486, suggesting nongenomic effects of the classical glucocorticoid receptor. In hypothalamic-derived 4B cells, 10 nM corticosterone increased immunoreactive glucocorticoid receptor content in membrane fractions, with association and clearance rates paralleling the effects on ACTH secretion from corticotrophs. Corticosterone did not affect CRH-stimulated calcium influx, but in AtT-20 cells, it had biphasic effects on CRH-stimulated Src phosphorylation, with early inhibition and late stimulation, suggesting a role for Src phosphorylation on the rapid glucocorticoid feedback. The data suggest that the nongenomic/membrane effects of classical GR mediate rapid and reversible glucocorticoid feedback inhibition at the pituitary corticotrophs downstream of calcium influx. The sensitivity and kinetics of these effects is consistent with the hypothesis that pituitary glucocorticoid feedback is part of the mechanism for adrenocortical ultradian pulse generation.
Assuntos
Hormônio Adrenocorticotrópico/metabolismo , Corticosterona/administração & dosagem , Corticotrofos/metabolismo , Glucocorticoides/metabolismo , Receptores de Glucocorticoides/metabolismo , Animais , Sinalização do Cálcio , Células Cultivadas , Hormônio Liberador da Corticotropina , Retroalimentação Fisiológica , Feminino , Ligantes , Masculino , Fosforilação , Ratos Sprague-Dawley , Quinases da Família src/metabolismoRESUMO
Neuronal electrical activity increases intracellular Ca(2+) concentration and generates reactive oxygen species. Here, we show that high frequency field stimulation of primary hippocampal neurons generated Ca(2+) signals with an early and a late component, and promoted hydrogen peroxide generation via a neuronal NADPH oxidase. Hydrogen peroxide generation required both Ca(2+) entry through N-methyl-D-aspartate receptors and Ca(2+) release mediated by ryanodine receptors (RyR). Field stimulation also enhanced nuclear translocation of the NF-κB p65 protein and NF-κB -dependent transcription, and increased c-fos mRNA and type-2 RyR protein content. Preincubation with inhibitory ryanodine or with the antioxidant N-acetyl L-cysteine abolished the increase in hydrogen peroxide generation and the late Ca(2+) signal component induced by electrical stimulation. Primary cortical cells behaved similarly as primary hippocampal cells. Exogenous hydrogen peroxide also activated NF-κB-dependent transcription in hippocampal neurons; inhibitory ryanodine prevented this effect. Selective inhibition of the NADPH oxidase or N-acetyl L-cysteine also prevented the enhanced translocation of p65 in hippocampal cells, while N-acetyl L-cysteine abolished the increase in RyR2 protein content induced by high frequency stimulation. In conclusion, the present results show that electrical stimulation induced reciprocal activation of ryanodine receptor-mediated Ca(2+) signals and hydrogen peroxide generation, which stimulated jointly NF-κB activity.