RESUMO
Systemic inflammation induces transient or permanent dysfunction in the brain by exposing it to soluble inflammatory mediators. The receptor for advanced glycation endproducts (RAGE) binds to distinct ligands mediating and increasing inflammatory processes. In this study we used an LPS-induced systemic inflammation model in rats to investigate the effect of blocking RAGE in serum, liver, cerebrospinal fluid (CSF) and brain (striatum, prefrontal cortex, ventral tegmental area and substantia nigra). Intraperitoneal injection of RAGE antibody (50µg/kg) was followed after 1h by a single LPS (5mg/kg) intraperitoneal injection. Twenty-four hours later, tissues were isolated for analysis. RAGE antibody reduced LPS-induced inflammatory effects in both serum and liver; the levels of proinflammatory cytokines (TNF-α, IL-1ß) were decreased and the phosphorylation/activation of RAGE downstream targets (ERK1/2, IκB and p65) in liver were significantly attenuated. RAGE antibody prevented LPS-induced effects on TNF-α and IL-1ß in CSF. In striatum, RAGE antibody inhibited increases in IL-1ß, Iba-1, GFAP, phospho-ERK1/2 and phospho-tau (ser202), as well as the decrease in synaptophysin levels. These effects were caused by systemic RAGE inhibition, as RAGE antibody did not cross the blood-brain barrier. RAGE antibody also prevented striatal lipoperoxidation and activation of mitochondrial complex II. In conclusion, blockade of RAGE is able to inhibit inflammatory responses induced by LPS in serum, liver, CSF and brain.
Assuntos
Anticorpos/farmacologia , Corpo Estriado/efeitos dos fármacos , Inflamação/tratamento farmacológico , Lipopolissacarídeos/farmacologia , Fígado/efeitos dos fármacos , Receptor para Produtos Finais de Glicação Avançada/imunologia , Animais , Anticorpos/uso terapêutico , Corpo Estriado/metabolismo , Citocinas/metabolismo , Inflamação/induzido quimicamente , Inflamação/metabolismo , Mediadores da Inflamação/metabolismo , Interleucina-1beta/metabolismo , Fígado/metabolismo , Masculino , Ratos , Ratos Wistar , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Human neuroblastoma SH-SY5Y cells have been used as an in vitro model for neurodegenerative disorders such as Parkinson's disease and can be induced to a mature neuronal phenotype through retinoic acid (RA) differentiation. However, mechanisms of RA-induced differentiation remain unclear. Here, we investigate the role of reactive species (RS) on SH-SY5Y neuroblastoma cells under RA differentiation, using the antioxidant Trolox® as co-treatment. We found that RA treatment for 7 days reduced the cell number and proliferative capacity and induced the expression of adult catecholaminergic/neuronal markers such as tyrosine hydroxylase (TH), ß-III tubulin, and enolase-2. Evaluation of intracellular RS production by DCFH oxidation assay and quantification of cell non-enzymatic antioxidant activity by TRAP demonstrated that RA increases RS production. Furthermore, mitochondrial NADH oxidation showed to be inhibited under differentiation with RA. Cells subjected to co-treatment with antioxidant Trolox® demonstrated a remaining proliferative capacity and a decrease in the pro-oxidant state and RS production. Besides, antioxidant treatment restores the mitochondrial NADH oxidation. Importantly, Trolox® co-treatment inhibited the appearance of morphological characteristics such as neurite extension and branching, and decreased the expression of TH, ß-III tubulin, and enolase-2 after a seven-day differentiation with RA, indicating that RS production is a necessary step in this process. Trolox® also inhibited the phosphorylation of Akt and ERK1/2, which are involved in differentiation and survival, respectively, of these cells. Altogether, these data indicate the presence of a redox-dependent mechanism in SH-SY5Y RA-differentiation process and can be a useful insight to improve understanding of neuronal differentiation signaling.
Assuntos
Biomarcadores/metabolismo , Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Neurônios/citologia , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Tretinoína/farmacologia , Regulação para Cima/efeitos dos fármacos , Antioxidantes/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fenótipo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Retinoic acid (RA) morphogenetic properties have been used in different kinds of therapies, from neurodegenerative disorders to some types of cancer such as promyelocytic leukemia and neuroblastoma. However, most of the pathways responsible for RA effects remain unknown. To investigate such pathways, we used a RA-induced differentiation model in the human neuroblastoma cells, SH-SY5Y. Our data showed that n-acetyl-cysteine (NAC) reduced cells' proliferation rate and increased cells' sensitivity to RA toxicity. Simultaneously, NAC pre-incubation attenuated nuclear factor erythroid 2-like factor 2 (NRF2) activation by RA. None of these effects were obtained with Trolox® as antioxidant, suggesting a cysteine signalization by RA. NRF2 knockdown increased cell sensibility to RA after 96 h of treatment and diminished neuroblastoma proliferation rate. Conversely, NRF2 overexpression limited RA anti-proliferative effects and increased cell proliferation. In addition, a rapid and non-genomic activation of the ERK 1/2 and PI3K/AKT pathways revealed to be equally required to promote NRF2 activation and necessary for RA-induced differentiation. Together, we provide data correlating NRF2 activity with neuroblastoma proliferation and resistance to RA treatments; thus, this pathway could be a potential target to optimize neuroblastoma chemotherapeutic response as well as in vitro neuronal differentiation protocols.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Modelos Biológicos , Fator 2 Relacionado a NF-E2/metabolismo , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Tretinoína/farmacologia , Acetilcisteína/farmacologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Müller cells constitute the main glial cell type in the retina where it interacts with virtually all cells displaying relevant functions to retinal physiology. Under appropriate stimuli, Müller cells may undergo dedifferentiation, being able to generate other neural cell types. Here, we show that purified mouse Müller cells in culture express a group of proteins related to the dopaminergic phenotype, including the nuclear receptor-related 1 protein, required for dopaminergic differentiation, as well the enzyme tyrosine hydroxylase. These dopaminergic components are active, since Müller cells are able to synthesize and release dopamine to the extracellular medium. Moreover, Müller-derived tyrosine hydroxylase can be regulated, increasing its activity because of phosphorylation of serine residues in response to agents that increase intracellular cAMP levels. These observations were extended to glial cells obtained from adult monkey retinas with essentially the same results. To address the potential use of dopaminergic Müller cells as a source of dopamine in cell therapy procedures, we used a mouse model of Parkinson's disease, in which mouse Müller cells with the dopaminergic phenotype were transplanted into the striatum of hemi-parkinsonian mice generated by unilateral injection of 6-hydroxydopamine. These cells fully decreased the apomorphine-induced rotational behavior and restored motor functions in these animals, as measured by the rotarod and the forelimb-use asymmetry (cylinder) tests. The data indicate local restoration of dopaminergic signaling in hemi-parkinsonian mice confirmed by measurement of striatal dopamine after Müller cell grafting.
Assuntos
Neurônios Dopaminérgicos/transplante , Células Ependimogliais/transplante , Transtornos Parkinsonianos/patologia , Transtornos Parkinsonianos/terapia , Animais , Cebus , Diferenciação Celular/fisiologia , Células Cultivadas , Corpo Estriado/citologia , Corpo Estriado/fisiologia , Modelos Animais de Doenças , Dopamina/metabolismo , Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/metabolismo , Células Ependimogliais/citologia , Células Ependimogliais/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Atividade Motora/fisiologia , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Transtornos Parkinsonianos/metabolismo , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Recuperação de Função Fisiológica/fisiologia , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
In a previous study we demonstrated that human neuroblastoma SH-SY5Y cells transfected with human tyrosine hydroxylase isoform 1 (SH+TH cells) were substantially more resistant to cell death induced by pro-oxidants than wild type SH-SY5Y cells (SH cells). In the present communication we used methylmercury as a model of cell stress in order to test whether SH+TH cells would behave in a similar manner in response to this stressor. Incubation with methylmercury (0.1-3 microM) for 24h caused a significant reduction in cell viability and increased apoptotic markers in both cell types. However, the effects were significantly reduced in the SH+TH cells when compared to the SH cells. Activation of p38(MAPK) was also reduced in the SH+TH compared to the SH cells after methylmercury exposure. Since p38(MAPK) is known to participate in signal transduction pathways during cell stress, our data suggest that SH+TH cells develop an increased resistance to environmental stress caused by neurotoxins such as methylmercury. In conclusion our results show that insertion of the human TH gene in cells that originally do not express this protein leads to alterations in cell homeostasis and triggers defense mechanisms against pro-oxidative insults.
Assuntos
Apoptose/efeitos dos fármacos , Compostos de Metilmercúrio/toxicidade , Neuroblastoma/enzimologia , Neurotoxinas/toxicidade , Tirosina 3-Mono-Oxigenase/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Resistência a Medicamentos/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Neuroblastoma/genética , Neuroblastoma/patologia , Fosforilação , Transfecção , Tirosina 3-Mono-Oxigenase/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
In this study, we demonstrate that human neuroblastoma SH-SY5Y cells transfected with human tyrosine hydroxylase isoform 1 (SH + TH cells) were substantially more resistant to cell death induced by hydrogen peroxide and 6-hydroxydopamine when compared to wild-type SH-SY5Y cells (SH cells). SH + TH cells exhibit increased levels of dopamine (DA) compared to SH cells. Incubation with hydrogen peroxide or 6-hydroxydopamine (10-100microM) for 24 h caused a significant reduction in cell viability and increased apoptosis in both cell types. However, these effects were significantly reduced in the SH + TH cells when compared to the SH cells. The SH + TH cells showed an improved ability to detoxify peroxide, which correlated with an increase in glutathione peroxidase and glutathione reductase activities, while catalase activity was unchanged. Our data suggest that a preconditioning-like mechanism linked to higher DA levels increased the resistance of SH + TH cells against oxidative insults, which is at least in part related to an augmentation in the activity of glutathione-related antioxidant enzymes.
Assuntos
Apoptose , Dopamina/metabolismo , Neuroblastoma/enzimologia , Estresse Oxidativo , Tirosina 3-Mono-Oxigenase/metabolismo , Apoptose/efeitos dos fármacos , Carmustina/farmacologia , Catalase/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Glutationa Peroxidase/antagonistas & inibidores , Glutationa Peroxidase/metabolismo , Glutationa Redutase/antagonistas & inibidores , Glutationa Redutase/metabolismo , Humanos , Peróxido de Hidrogênio/toxicidade , Neuroblastoma/genética , Neuroblastoma/patologia , Estresse Oxidativo/efeitos dos fármacos , Oxidopamina/toxicidade , Tiomalatos/farmacologia , Transfecção , Tirosina 3-Mono-Oxigenase/genéticaRESUMO
In this study, we investigated the involvement of glutathione peroxidase-GPx in methylmercury (MeHg)-induced toxicity using three models: (a) in mouse brain after treatment with MeHg (40 mg/L in drinking water), (b) in mouse brain mitochondrial-enriched fractions isolated from MeHg-treated animals, and (c) in cultured human neuroblastoma SH-SY5Y cells. First, adult male Swiss mice exposed to MeHg for 21 days showed a significant decrease in GPx activity in the brain and an increase in poly(ADP-ribose) polymerase cleavage, an index of apoptosis. Second, in mitochondrial-enriched fractions isolated from MeHg-treated mice, there was a significant reduction in GPx activity and a concomitant decrease in mitochondrial activity and increases in ROS formation and lipid peroxidation. Incubation of mitochondrial-enriched fractions with mercaptosuccinic acid, a GPx inhibitor, significantly augmented the toxic effects of MeHg administered in vivo. Incubation of mitochondrial-enriched fractions with exogenous GPx completely blocked MeHg-induced mitochondrial lipid peroxidation. Third, SH-SY5Y cells treated for 24 h with MeHg showed a significant reduction in GPx activity. There was a concomitant significant decrease in cell viability and increase in apoptosis. Inhibition of GPx substantially enhanced MeHg toxicity in the SH-SY5Y cells. These results suggest that GPx is an important target for MeHg-induced neurotoxicity, presumably because this enzyme is essential for counteracting the pro-oxidative effects of MeHg both in vitro and in vivo.
Assuntos
Encéfalo/metabolismo , Regulação Enzimológica da Expressão Gênica , Glutationa Peroxidase/metabolismo , Mitocôndrias/metabolismo , Neuroblastoma/metabolismo , Animais , Apoptose/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Regulação para Baixo/efeitos dos fármacos , Glutationa Peroxidase/genética , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Compostos de Metilmercúrio/toxicidade , Camundongos , Mitocôndrias/efeitos dos fármacos , Neuroblastoma/patologia , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Tiomalatos/farmacologiaRESUMO
Cadmium (Cd(2+)) is a common environmental pollutant, which is widely used in industry and is a constituent of tobacco smoke. Exposure to this heavy metal has been linked to a wide range of detrimental effects on mammalian cells. In this study, the action of Cd(2+) on protein phosphorylation in bovine adrenal chromaffin cells (BACCs) was examined. Cells were incubated with (32)Pi in the presence of Cd(2+) (1-50 microM) and proteins were separated by one- or two-dimensional electrophoresis. An increase in the phosphorylation of BACCs proteins, without changing cell viability, was observed in response to Cd(2+) (5-50 microM). Particularly at three spots, with molecular weight of 25kDa and isoeletric point range 4.0-4.5, which were identified as phosphorylated isoforms of the heat shock protein of 27kDa (Hsp27). Phosphorylation of the p38(MAPK), a member of mitogen-activated protein kinase (MAPK) family, was stimulated by Cd(2+) over the same concentration range and it was the major upstream protein kinase involved in the phosphorylation of all three spots of Hsp27. Cd(2+) also stimulated the phosphorylation of other MAPK family member, the extracellular signal-regulated kinase (ERK)-1/2. Therefore, primary adrenal chromaffin cells are a target for Cd(2+) and both the ERK1/2 and the p38(MAPK) are activated. Additionally, Hsp27 is highly phosphorylated in response to the metal exposure, due to p38(MAPK) activation. These biochemical effects of Cd(2+) might disrupt the normal secretory function of these cells.
Assuntos
Glândulas Suprarrenais/efeitos dos fármacos , Cloreto de Cádmio/toxicidade , Células Cromafins/efeitos dos fármacos , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Glândulas Suprarrenais/citologia , Glândulas Suprarrenais/metabolismo , Animais , Bovinos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Células Cromafins/citologia , Células Cromafins/metabolismo , Relação Dose-Resposta a Droga , Eletroforese em Gel Bidimensional , Inibidores Enzimáticos/farmacologia , Imidazóis/farmacologia , Immunoblotting , Isótopos de Fósforo , Fosforilação/efeitos dos fármacos , Piridinas/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
S100B belongs to a family of calcium-binding proteins involved in cell cycle and cytoskeleton regulation. We observed an inhibitory effect of S100B on glial fibrillary acidic protein (GFAP) phosphorylation, when stimulated by cAMP or Ca2+/calmodulin, in a cytoskeletal fraction from primary astrocyte cultures. We found that S100B has no direct effect on CaM KII activity, the major kinase in this cytoskeletal fraction able to phosphorylate GFAP. The inhibition of GFAP phosphorylation is most likely due to the binding of S100B to the phosphorylation sites on this protein and blocking the access of these sites to the protein kinases. This inhibition was dependent on Ca2+. However, Zn2+ could substitute for Ca2+. The inhibitory effect of S100B was prevented by TRTK-12, a peptide that blocks S100B interaction with several target proteins including glial fibrillary acidic protein. These data suggest a role for S100B in the assembly of intermediate filaments in astrocytes.
Assuntos
Proteína Glial Fibrilar Ácida/metabolismo , Fatores de Crescimento Neural/fisiologia , Oligopeptídeos/farmacologia , Proteínas S100/fisiologia , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteína de Capeamento de Actina CapZ , Fatores de Crescimento Neural/antagonistas & inibidores , Fragmentos de Peptídeos , Fosforilação , Ratos , Ratos Wistar , Subunidade beta da Proteína Ligante de Cálcio S100 , Proteínas S100/antagonistas & inibidoresRESUMO
S100B is a calcium binding protein from astrocytes that regulates protein phosphorylation by binding to substrates and protein kinases. S100B might also regulate protein phosphatases and this was investigated for protein phosphatase 2B (calcineurin). The results indicate that S100B (5-10 microM) increased the activity of both purified and cytoskeletal calcineurin in a Ca-dependent manner. This effect was blocked by a specific inhibitor of calcineurin activity, but not by TRTK-12 (an inhibitor of S100B binding to other protein targets). The present results and the known co-localization of S100B and calcineurin in the astrocyte cytoskeleton suggest that S100B may play a role in the phosphorylation state of cytoskeletal proteins.
Assuntos
Astrócitos/enzimologia , Calcineurina/metabolismo , Proteínas do Citoesqueleto/metabolismo , Fatores de Crescimento Neural/metabolismo , Proteínas S100/metabolismo , Animais , Animais Recém-Nascidos , Astrócitos/efeitos dos fármacos , Astrócitos/ultraestrutura , Calcineurina/efeitos dos fármacos , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Células Cultivadas , Proteínas do Citoesqueleto/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Fatores de Crescimento Neural/farmacologia , Fosforilação/efeitos dos fármacos , Ratos , Ratos Wistar , Subunidade beta da Proteína Ligante de Cálcio S100 , Proteínas S100/farmacologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
Lead (Pb(2+)) is widely recognized as a neurotoxicant whose mechanisms of action are not completely established. We have previously demonstrated that Pb(2+) can activate the p38(MAPK) pathway and increase the phosphorylation of Hsp27 in bovine adrenal chromaffin cells and human SH SY5Y cells over a short incubation period (1 h). In the present work we analyzed the effects of Pb(2+) administered in vivo on the level and the phosphorylation state of ERK1/2 and p38(MAPK) in the hippocampus of immature rats. Rats were treated with lead acetate (2, 8 or 12 mg/kg, i.p.) or saline (control) over the 8th to 12th postnatal days, and hippocampal slices were prepared on the 14th day. The Pb(2+) level in the lead-treated animals increased 2.5-6-fold in the blood (3.0-6.0 microg/dl) and 2.0-3.0-fold in the forebrain (78-103 ng/g wet weight), compared to control (saline). The phosphorylation of both ERK1/2 and p38(MAPK) was significantly increased by prior exposure to Pb(2+) in vivo. In in vitro experiments, hippocampal slices from 14-day-old rats were exposed to Pb(2+) (1-10 microM) for 1 and 3 h. There were no changes in the phosphorylation state of ERK and p38(MAPK) for 1-h incubation, whereas a significant increase of ERK1/2 and p38(MAPK) phosphorylation by Pb(2+) (5 microM) was observed for the 3-h incubation. Cell viability measured using MTT was not modified in any of the conditions tested. These results indicate that the phosphorylation of hippocampal ERK1/2 and p38(MAPK) is stimulated by lead in a period of rapid brain development, an effect that may underlie, at least in part, the neurotoxicty elicited by this metal.
Assuntos
Hipocampo/efeitos dos fármacos , Chumbo/toxicidade , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Análise de Variância , Animais , Animais Recém-Nascidos , Western Blotting , Sobrevivência Celular/efeitos dos fármacos , Feminino , Hipocampo/metabolismo , Técnicas In Vitro , Chumbo/metabolismo , Masculino , Dose Máxima Tolerável , Proteína Quinase 3 Ativada por Mitógeno , Fosforilação/efeitos dos fármacos , Prosencéfalo/efeitos dos fármacos , Prosencéfalo/metabolismo , Ratos , Ratos Wistar , Fatores de Tempo , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Lead (Pb2+) is a cytotoxic metal ion whose mechanism of action is not established. However, Pb2+ is known to interact with a wide variety of molecules involved in signal transduction. In this study the effect of Pb2+ on protein phosphorylation in bovine adrenal chromaffin cells and human SH SY5Y cells was examined. Cells were incubated with 32P(i) for 1 h in the presence of Pb2+ (1-10 microM) and the proteins were separated by two-dimensional PAGE. An increase in the phosphorylation of a number of proteins was observed in response to Pb2+, including three spots, MW 25 kDa, and pI's in the range 4.0-4.5. These proteins were immunoidentified as three isoforms of the heat-shock protein 27 kDa (Hsp27), and the identity of the most basic spot was confirmed by amino acid sequencing. Phosphorylation of p38MAPK was increased by Pb2+ and the effect of Pb2+ on Hsp27 phosphorylation was blocked by the p38MAPK inhibitor SB203580 (1 microM). The results were similar for bovine chromaffin cells and human SH SY5Y cells. This is the first report showing that Pb2+ can modulate the phosphorylation state of Hsp27 via activation of the p38MAPK pathway.