RESUMO
Astrocytes are the most abundant cells in the brain, playing vital roles in neuronal survival, growth, and function. Understanding the mechanism(s) regulating astrocyte proliferation will have important implications in brain development, response to injury, and tumorigenesis. Cyclin B1 is well known to be a critical regulator of mitotic entry via its interaction with cyclin-dependent kinase 1. In rat astrocytes, we now show that the mRNA binding protein cytoplasmic polyadenylation element binding protein 1 (CPEB1) is associated with cyclin B1 mRNA and that this interaction is enriched at the centrosome. In addition, if growth-arrested astrocytes are stimulated to divide, CPEB1 is phosphorylated and cyclin B1 mRNA is polyadenylated, both hallmarks of CPEB1 activation, resulting in an increase in cyclin B1 protein. CPEB1 binding to mRNA initially inhibits translation; therefore, removing CPEB1 from mRNA should result in an increase in translation due to derepression. Indeed, when we either knocked down CPEB1 protein with siRNA or sequestered it from endogenous mRNA by expressing RNA containing multiple CPEB1 binding sites, cyclin B1 protein was increased and cell proliferation was stimulated. Our data suggest a mechanism wherein CPEB1 is bound and represses cyclin B1 mRNA translation until a signal to proliferate phosphorylates CPEB1, resulting in an increase in cyclin B1 protein and progression into mitosis. Our results demonstrate for the first time a role for CPEB1 in regulating cell proliferation in the brain.
Assuntos
Astrócitos/citologia , Astrócitos/metabolismo , Ciclina B1/biossíntese , Ciclina B1/genética , Regulação da Expressão Gênica/fisiologia , Poliadenilação/genética , Estabilidade de RNA/genética , Proteínas de Ligação a RNA/metabolismo , Animais , Animais Recém-Nascidos , Proliferação de Células , Células Cultivadas , Centrossomo/metabolismo , Citoplasma/genética , Citoplasma/metabolismo , Feminino , Masculino , Proteínas de Ligação a RNA/genética , Ratos , Ratos Sprague-DawleyRESUMO
J. Neurochem. (2012) 123, 226-238. ABSTRACT: Fragile X syndrome (FXS), the most common single genetic cause of mental retardation and autistic spectrum disease, occurs when FMR1 gene is mutated. FMR1 encodes fragile X mental retardation protein (FMRP) which regulates translation of mRNAs playing important roles in the development of neurons as well as formation and maintenance of synapses. To examine whether FMRP regulates cell viability, we induced apoptosis in rat primary cortical neurons with glutamate in vitro and with middle cerebral artery occlusion (MCAO) in striatal neurons in vivo. Both conditions elicited a rapid, but transient FMRP expression in neurons. This up-regulated FMRP expression was abolished by pre-treatment with PI3K and Protein Kinase B (Akt) inhibitors: LY294002, Akt inhibitor IV, and VIII. Reduced FMRP expression in vitro or in vivo using small hairpin Fmr1 virus exacerbated cell death by glutamate or MCAO, presumably via hypophosphorylation of Akt and reduced expression of B-cell lymphoma-extra large (Bcl-xL). However, over-expression of FMRP using enhanced green fluorescent protein (eGFP)-FMRP constructs alleviated cell death, increased Akt activity, and enhanced Bcl-xL production. The pro-survival role of Akt-dependent up-regulation of FMRP in glutamate-stimulated cultured neuron as well as in ischemic brain may have a clinical importance in FXS as well as in neurodegenerative disorders and traumatic brain injury.
Assuntos
Retroalimentação Fisiológica/fisiologia , Proteína do X Frágil da Deficiência Intelectual/fisiologia , Neurônios/fisiologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Transdução de Sinais/fisiologia , Animais , Apoptose/fisiologia , Morte Celular/fisiologia , Sobrevivência Celular/fisiologia , Células Cultivadas , Infarto da Artéria Cerebral Média/metabolismo , Infarto da Artéria Cerebral Média/patologia , Infarto da Artéria Cerebral Média/prevenção & controle , Masculino , Neurônios/patologia , Ratos , Ratos WistarRESUMO
BACKGROUND: Fragile X syndrome (FXS), the most commonly inherited mental retardation and single gene cause of autistic spectrum disorder, occurs when the Fmr1 gene is mutated. The product of Fmr1, fragile X linked mental retardation protein (FMRP) is widely expressed in HeLa cells, however the roles of FMRP within HeLa cells were not elucidated, yet. Interacting with a diverse range of mRNAs related to cellular survival regulatory signals, understanding the functions of FMRP in cellular context would provide better insights into the role of this interesting protein in FXS. Using HeLa cells treated with etoposide as a model, we tried to determine whether FMRP could play a role in cell survival. METHODS: Apoptotic cell death was induced by etoposide treatment on Hela cells. After we transiently modulated FMRP expression (silencing or enhancing) by using molecular biotechnological methods such as small hairpin RNA virus-induced knock down and overexpression using transfection with FMRP expression vectors, cellular viability was measured using propidium iodide staining, TUNEL staining, and FACS analysis along with the level of activation of PI3K-Akt pathway by Western blot. Expression level of FMRP and apoptotic regulator BcL-xL was analyzed by Western blot, RT-PCR and immunocytochemistry. RESULTS: An increased FMRP expression was measured in etoposide-treated HeLa cells, which was induced by PI3K-Akt activation. Without FMRP expression, cellular defence mechanism via PI3K-Akt-Bcl-xL was weakened and resulted in an augmented cell death by etoposide. In addition, FMRP over-expression lead to the activation of PI3K-Akt signalling pathway as well as increased FMRP and BcL-xL expression, which culminates with the increased cell survival in etoposide-treated HeLa cells. CONCLUSIONS: Taken together, these results suggest that FMRP expression is an essential part of cellular survival mechanisms through the modulation of PI3K, Akt, and Bcl-xL signal pathways.
Assuntos
Proteína do X Frágil da Deficiência Intelectual/biossíntese , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Estresse Fisiológico , Regulação para Cima , Antineoplásicos Fitogênicos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Etoposídeo/farmacologia , Proteína do X Frágil da Deficiência Intelectual/genética , Síndrome do Cromossomo X Frágil/genética , Síndrome do Cromossomo X Frágil/metabolismo , Células HeLa , Humanos , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMO
BACKGROUND: At the beginning of neurogenesis, massive brain cell death occurs and more than 50% of cells are eliminated by apoptosis along with neuronal differentiation. However, few studies were conducted so far regarding the regulation of neural progenitor cells (NPCs) death during development. Because of the physiological role of cell death during development, aberration of normal apoptotic cell death is detrimental to normal organogenesis.Apoptosis occurs in not only neuron but also in NPCs and neuroblast. When growth and survival signals such as EGF or LIF are removed, apoptosis is activated as well as the induction of differentiation. To investigate the regulation of cell death during developmental stage, it is essential to investigate the regulation of apoptosis of NPCs. METHODS: Neural progenitor cells were cultured from E14 embryonic brains of Sprague-Dawley rats. For in vivo VPA animal model, pregnant rats were treated with VPA (400 mg/kg S.C.) diluted with normal saline at E12. To analyze the cell death, we performed PI staining and PARP and caspase-3 cleavage assay. Expression level of proteins was investigated by Western blot and immunocytochemical assays. The level of mRNA expression was investigated by RT-PCR. Interaction of Bcl-XL gene promoter and NF-κB p65 was investigated by ChIP assay. RESULTS: In this study, FACS analysis, PI staining and PARP and caspase-3 cleavage assay showed that VPA protects cultured NPCs from cell death after growth factor withdrawal both in basal and staurosporine- or hydrogen peroxide-stimulated conditions. The protective effect of prenatally injected VPA was also observed in E16 embryonic brain. Treatment of VPA decreased the level of IκBα and increased the nuclear translocation of NF-κB, which subsequently enhanced expression of anti-apoptotic protein Bcl-XL. CONCLUSION: To the best of our knowledge, this is the first report to indicate the reduced death of NPCs by VPA at developmentally critical periods through the degradation of IκBα and the activation of NF-κB signaling. The reduced NPCs death might underlie the neurodevelopmental defects collectively called fetal valproate syndrome, which shows symptoms such as mental retardation and autism-like behavior.
Assuntos
Apoptose/efeitos dos fármacos , Citoproteção , NF-kappa B/metabolismo , Células-Tronco Neurais/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Ácido Valproico/farmacologia , Proteína bcl-X/metabolismo , Animais , Feminino , Células-Tronco Neurais/metabolismo , Gravidez , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacosRESUMO
As a member of neurotrophin family, brain derived neurotrophic factor (BDNF) plays critical roles in neuronal development, differentiation, synaptogenesis, and neural protection from the harmful stimuli. There have been reported that adenosine A2(A) receptor subtype is widely distributed in the brain regions, such as hippocampus, striatum, and cortex. Adenosine A2(A) receptor is colocalized with BDNF in brain regions and the functional interaction between A2(A) receptor stimulation and BDNF action has been suggested. In this study, we investigated the possibility that the activation of A2(A) receptor modulates BDNF production in rat primary cortical neuron. CGS21680, an adenosine A2(A) receptor agonist, induced BDNF expression and release. An antagonist against A2(A) receptor, ZM241385, prevented CGS21680-induced increase in BDNF production. A2(A) receptor stimulation induced the activation of Akt-GSK-3ß signaling pathway and the blockade of the signaling pathway with specific inhibitors abolished the increase in BDNF production, possibly via modulation of ERK1/2-CREB pathway. The physiological roles of A2(A) receptor-induced BDNF production was demonstrated by the protection of neurons from the excitotoxicity and increased neurite extension as well as synapse formation from immature and mature neurons. Taken together, activation of A2(A) receptor regulates BDNF production in rat cortical neuron, which provides neuro-protective action.
Assuntos
Neurônios/metabolismo , Adenosina/análogos & derivados , Adenosina/farmacologia , Agonistas do Receptor A2 de Adenosina/farmacologia , Antagonistas do Receptor A2 de Adenosina/farmacologia , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Células Cultivadas , Córtex Cerebral/metabolismo , Quinase 3 da Glicogênio Sintase , Glicogênio Sintase Quinase 3 beta , Neurônios/efeitos dos fármacos , Fenetilaminas/farmacologia , Ratos , Ratos Sprague-Dawley , Receptor A2A de Adenosina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Regulação para CimaRESUMO
BACKGROUND: Prenatal ethanol exposure during pregnancy induces a spectrum of mental and physical disorders called fetal alcohol spectrum disorder (FASD). The central nervous system is the main organ influenced by FASD, and neurological symptoms include mental retardation, learning abnormalities, hyperactivity and seizure susceptibility in childhood along with the microcephaly. In this study, we examined whether ethanol exposure adversely affects the proliferation of NPC and de-regulates the normal ratio between glutamatergic and GABAergic neuronal differentiation using primary neural progenitor culture (NPC) and in vivo FASD models. METHODS: Neural progenitor cells were cultured from E14 embryo brain of Sprague-Dawley rat. Pregnant mice and rats were treated with ethanol (2 or 4 g/kg/day) diluted with normal saline from E7 to E16 for in vivo FASD animal models. Expression level of proteins was investigated by western blot analysis and immunocytochemical assays. MTT was used for cell viability. Proliferative activity of NPCs was identified by BrdU incorporation, immunocytochemistry and FACS analysis. RESULTS: Reduced proliferation of NPCs by ethanol was demonstrated using BrdU incorporation, immunocytochemistry and FACS analysis. In addition, ethanol induced the imbalance between glutamatergic and GABAergic neuronal differentiation via transient increase in the expression of Pax6, Ngn2 and NeuroD with concomitant decrease in the expression of Mash1. Similar pattern of expression of those transcription factors was observed using an in vivo model of FASD as well as the increased expression of PSD-95 and decreased expression of GAD67. CONCLUSIONS: These results suggest that ethanol induces hyper-differentiation of glutamatergic neuron through Pax6 pathway, which may underlie the hyper-excitability phenotype such as hyperactivity or seizure susceptibility in FASD patients.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Etanol/farmacologia , Transtornos do Espectro Alcoólico Fetal/fisiopatologia , Ácido Glutâmico/metabolismo , Células-Tronco Neurais , Neurônios/fisiologia , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Proteínas do Olho/metabolismo , Feminino , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/fisiologia , Neurogênese/efeitos dos fármacos , Neurônios/citologia , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/metabolismo , Gravidez , Ratos , Ratos Sprague-Dawley , Proteínas Repressoras/metabolismo , Ácido gama-Aminobutírico/metabolismoRESUMO
In response to brain injury, microglia migrate and accumulate in the affected sites, which is an important step in the regulation of inflammation and neuronal degeneration/regeneration. In this study, we investigated the effect of urokinase-type plasminogen activator (uPA) on the BV-2 microglial cell migration. At resting state, BV-2 microglial cells secreted uPA and the release of uPA was increased by ATP, a chemoattractant released from injured neuron. The migration of BV-2 cell was significantly induced by uPA and inhibited by uPA inhibitors. In this condition, uPA increased the activity of matrix metalloproteinase (MMP-9) and the inhibition of MMP activity with pharmacological inhibitors against either uPA (amiloride) or MMP (phenanthrolene and SB-3CT) effectively prevented BV2 cell migration. Interestingly, the level of MMP-9 protein and mRNA in the cell were not changed by uPA. These results suggest that the increase of MMP-9 activity by uPA is regulated at the post-translational level, possibly via increased activation of the enzyme. Unlike the uPA inhibitor, plasmin inhibitor PAI-1 only partially inhibited uPA-induced cell migration and MMP-9 activation. The incubation of recombinant MMP-9 with uPA resulted in the activation of MMP-9. These results suggest that uPA plays a critical role in BV-2 microglial cell migration by activating pro-MMP-9, in part by its direct action on MMP-9 and also in part by the activation of plasminogen/plasmin cascade.
Assuntos
Metaloproteinase 9 da Matriz/metabolismo , Microglia/fisiologia , Ativador de Plasminogênio Tipo Uroquinase/fisiologia , Animais , Linhagem Celular , Movimento Celular , Ativação Enzimática , Fibrinolisina/antagonistas & inibidores , Fibrinolisina/fisiologia , Metaloproteinase 9 da Matriz/genética , Inibidores de Metaloproteinases de Matriz , Camundongos , Plasminogênio/fisiologia , RNA Mensageiro/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidoresRESUMO
The role of protease-activated receptor (PARs) in the regulation of microglial activation process is increasingly evident. In the present study, we have investigated the role of PAR-2, which can be activated by trypsin-like proteases, in microglial activation and neuronal cell death. In cultured rat primary microglia, activation of PAR-2 induced nitrite production by PKC- and MAPKs-dependent mechanism. Among the three members of MAPK pathway, ERK and JNK but not p38 mediated PAR-2-induced microglial activation. The down-stream regulator of PAR-2-PKC-MAPK pathway-induced microglial activation was NF-kappaB pathway. Besides nitrite, PAR-2 activation increased production of a variety of inflammatory mediators such as ROS and pro-inflammatory cytokines including TNF-alpha and IL-1beta. The addition of culture spent media from PAR-2 activated microglia induced neuronal cell death in primary rat cortical neuron cultures with apoptotic features such as increased number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive neurons, dissipation of mitochondrial membrane potential, increased expression of pro-apoptotic Bax, decreased expression of anti-apoptotic Bcl-2, Bcl-X(L), and activation of caspase-3 in neurons. Interestingly, the increased production of cytoactive molecules as well as the neuronal cell death was normalized by PAR-2 or trypsin inhibitor or an NO synthase inhibitor, N(G)-nitro-l-arginine-methyl ester. Taken together, these results suggest that overt PAR-2 activation may induce microglial activation, which contributes to neuronal cell death.
Assuntos
Microglia/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Receptor PAR-2/metabolismo , Animais , Morte Celular , Células Cultivadas , Citocinas/biossíntese , Microglia/citologia , Microglia/enzimologia , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/biossíntese , Óxido Nítrico Sintase Tipo II/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Receptor PAR-2/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tripsina/metabolismoRESUMO
We previously demonstrated that kainic acid (KA)-mediated mitochondrial oxidative stress contributed to hippocampal degeneration and that ginsenosides attenuated KA-induced neurotoxicity and neuronal degeneration. Here, we examined whether ginsenosides affected KA-induced mitochondrial dysfunction and oxidative stress in the rat hippocampus. Treatment with ginsenosides attenuated KA-induced convulsive behavior dose-dependently. KA treatment increased lipid peroxidation and protein oxidation and decreased the reduced glutathione/oxidized glutathione (GSH/GSSG) ratio to a greater degree in the mitochondrial fraction than in the hippocampal homogenate. KA treatment resulted in decreased Mn-superoxide dismutase expression and diminished the mitochondrial membrane potential. Furthermore, KA treatment increased intramitochondrial Ca(2+) and promoted ultrastructural degeneration in hippocampal mitochondria. Treatment with ginsenosides dose-dependently attenuated convulsive behavior and the KA-induced mitochondrial effects. Protection appeared to be more evident in mitochondria than in tissue homogenates. Collectively, the results suggest that ginsenosides prevent KA-induced neurotoxicity by attenuating mitochondrial oxidative stress and mitochondrial dysfunction.
Assuntos
Ginsenosídeos/uso terapêutico , Hipocampo/efeitos dos fármacos , Ácido Caínico/toxicidade , Mitocôndrias/efeitos dos fármacos , Fármacos Neuroprotetores/uso terapêutico , Estresse Oxidativo/efeitos dos fármacos , Convulsões/tratamento farmacológico , Animais , Anticonvulsivantes/uso terapêutico , Antioxidantes/uso terapêutico , Cálcio/metabolismo , Glutationa/metabolismo , Hipocampo/fisiologia , Hipocampo/ultraestrutura , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Atividade Motora/efeitos dos fármacos , Degeneração Neural/tratamento farmacológico , Ratos , Ratos Sprague-Dawley , Convulsões/induzido quimicamente , Convulsões/patologia , Convulsões/fisiopatologia , Superóxido Dismutase/metabolismoRESUMO
Protease-activated receptors (PARs) play important roles in the regulation of brain function such as neuroinflammation by transmitting the signal from proteolytic enzymes such as thrombin and trypsin. We and others have reported that a member of the family, PAR-2 is activated by trypsin, whose involvement in the neurophysiological process is increasingly evident, and is involved in the neuroinflammatory processes including morphological changes of astrocytes. In this study, we investigated the role of PAR-2 in the production of nitric oxide (NO) in rat primary astrocytes. Treatment of PAR-2 agonist trypsin increased NO production in a dose-dependent manner, which was mediated by the induction of inducible nitric-oxide synthase. The trypsin-mediated production of NO was mimicked by PAR-2 agonist peptide and reduced by either pharmacological PAR-2 antagonist peptide or by siRNA-mediated inhibition of PAR-2 expression, which suggests the critical role of PAR-2 in this process. NO production by PAR-2 was mimicked by PMA, a PKC activator, and was attenuated by Go6976, a protein kinase C (PKC) inhibitor. PAR-2 stimulation activated three subtypes of mitogen-activated protein kinases (MAPKs): extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK. NO production by PAR-2 was blocked by inhibition of ERK, p38, and JNK pathways. PAR-2 stimulation also activated nuclear factor-kappaB (NF-kappaB) DNA binding and transcriptional activity as well as IkappaBalpha phosphorylation. Inhibitors of NF-kappaB pathway inhibited PAR-2-mediated NO production. In addition, inhibitors of MAPK pathways prevented transcriptional activation of NF-kappaB reporter constructs. These results suggest that PAR-2 activation-mediated NO production in astrocytes is transduced by the activation of MAPKs followed by NF-kappaB pathways.
Assuntos
Astrócitos/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Óxido Nítrico/biossíntese , Receptor PAR-2/metabolismo , Análise de Variância , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/enzimologia , Sobrevivência Celular/efeitos dos fármacos , Proteínas I-kappa B/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Inibidor de NF-kappaB alfa , NF-kappa B/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Nitritos/metabolismo , Proteína Quinase C/antagonistas & inibidores , Ratos , Ratos Sprague-Dawley , Tripsina/farmacologiaRESUMO
Neural progenitor cells (NPC) are self-renewing multipotent cells that generate neurons and glial cells in the brain. NPCs generate neurons and glia not only during development but also after neural injury. Recent studies have shown that endogenous NPCs are activated after brain injury and migrate toward damaged areas where astrocyte activation occurs. Considering the massive proliferation of astrocytes as well as the production of several kinds of cytoactive molecules after brain injury, such as NO, growth factors and cytokines, it is tempting to think that cytoactive molecules released by activated glial cells regulate neural progenitor differentiation and proliferation through inflammatory mediators. To test this hypothesis, we stimulated rat primary astrocytes with lipopolysaccharide (LPS) to induce the activation of astrocytes. After addition of the conditioned media from LPS-stimulated astrocytes to NPC culture, proliferation was examined by MTT assay and bromodeoxyuridine (BrdU) incorporation. The differentiation of NPC into neurons and astrocytes was examined by Western blot, ELISA and immunocytochemical staining with cell-type-specific markers. Conditioned media from LPS-stimulated astrocytes increased NPC proliferation as well as gliogenesis as compared with control conditioned media from astrocytes without LPS stimulation. In contrast, neurogenesis was decreased by LPS-conditioned media. To investigate the molecular mechanism mediating glial differentiation and proliferation of NPC by reactive astrocytes, we added inhibitors of the Erk and JNK pathways during LPS stimulation. These inhibitors - except for a p38 inhibitor - decreased NPC proliferation and glial differentiation. These results suggest that LPS stimulated astrocytes generate factors regulating NPC proliferation and gliogenesis via the Erk and JNK pathways.
Assuntos
Astrócitos/metabolismo , Diferenciação Celular/fisiologia , Proliferação de Células , Citocinas/metabolismo , Neurogênese/fisiologia , Células-Tronco/fisiologia , Animais , Astrócitos/citologia , Bromodesoxiuridina , Comunicação Celular/fisiologia , Técnicas de Cultura de Células , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Inibidores Enzimáticos/farmacologia , Indicadores e Reagentes , Mediadores da Inflamação , Lipopolissacarídeos/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Neurônios/citologia , Neurônios/fisiologia , Ratos , Ratos Sprague-Dawley , Células-Tronco/citologia , Sais de Tetrazólio , Regulação para Cima/fisiologiaRESUMO
Oroxylin A is a flavonoid that is found in the roots of Scutellaria baicalensis Georgi. The aim of this study was to characterize the effects of oroxylin A on the memory impairments and pathological changes induced by Abeta(25-35) peptide in mice. The ameliorating effect of oroxylin A on memory impairment was investigated using passive avoidance and Y-maze tasks and pathological changes were identified by immunostaining and western blotting. Abeta(25-35) peptide (5nmol) was administered by intracerebroventricular injection. In the acute treatment study, a single dose of oroxylin A (5mg/kg, p.o.) treated 1h before behavioral tests was found to significantly reverse Abeta(25-35)-induced cognitive impairments based on passive avoidance and Y-maze task findings (P<0.05). Moreover, these acute effects of oroxylin A were blocked by diazepam (1mg/kg, i.p.), a GABA(A)/benzodiazepine binding site agonist (P<0.05). On the other hand, our subchronic studies revealed that oroxylin A (1 or 5mg/kg/day, p.o.) for 7 days ameliorated the memory impairment induced by Abeta(25-35) peptide. Moreover, Abeta(25-35)-induced increases in GFAP (an astroglia marker) and OX-42 (a microglia marker), and increases in iNOS positive cells in the hippocampus were found to be attenuated by subchronic oroxylin A (1 or 5mg/kg/day, i.p., P<0.05). In addition, reductions in the immunoreactivity and protein level of ChAT (a cholinergic neuronal cell marker) in the CA3 hippocampal area induced by Abeta(25-35) peptide were also attenuated by oroxylin A. Furthermore, lipid peroxidation induced by Abeta(25-35) was also reduced by oroxylin A. These results suggest that the amelioration of Abeta(25-35) peptide-induced memory impairment by oroxylin A is mediated via the GABAergic neurotransmitter system after a single administration, or by reductions in Abeta(25-35) peptide-induced astrocyte and microglia activations, iNOS expression, lipid peroxidation, and increased cholinergic neurotransmission after subchronic administration.
Assuntos
Peptídeos beta-Amiloides , Inibidores Enzimáticos/administração & dosagem , Flavonoides/administração & dosagem , Transtornos da Memória/induzido quimicamente , Transtornos da Memória/tratamento farmacológico , Fragmentos de Peptídeos , Análise de Variância , Animais , Comportamento Animal/efeitos dos fármacos , Antígeno CD11b/metabolismo , Diazepam/farmacologia , Relação Dose-Resposta a Droga , Esquema de Medicação , Comportamento Exploratório/efeitos dos fármacos , Sequestradores de Radicais Livres/metabolismo , Moduladores GABAérgicos/farmacologia , Proteína Glial Fibrilar Ácida/metabolismo , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Transtornos da Memória/patologia , Camundongos , Camundongos Endogâmicos ICR , Tempo de Reação/efeitos dos fármacosRESUMO
We previously reported that uridine blocked glucose deprivation-induced death of immunostimulated astrocytes by preserving ATP levels. Uridine phosphorylase (UPase), an enzyme catalyzing the reversible phosphorylation of uridine, was involved in this effect. Here, we tried to expand our previous findings by investigating the uridine effect on the brain and neurons using in vivo and in vitro ischemic injury models. Orally administrated uridine (50-200 mg/kg) reduced middle cerebral artery occlusion (1.5 h)/reperfusion (22 h)-induced infarct in mouse brain. Additionally, in the rat brain subjected to the same ischemic condition, UPase mRNA and protein levels were up-regulated. Next, we employed glucose deprivation-induced hypoglycemia in mixed cortical cultures of neurons and astrocytes as an in vitro model. Cells were deprived of glucose and, two hours later, supplemented with 20 mM glucose. Under this condition, a significant ATP loss followed by death was observed in neurons but not in astrocytes, which were blocked by treatment with uridine in a concentration-dependent manner. Inhibition of cellular uptake of uridine by S-(4-nitrobenzyl)-6-thioinosine blocked the uridine effect. Similar to our in vivo data, UPase expression was up-regulated by glucose deprivation in mRNA as well as protein levels. Additionally, 5-(phenylthio)acyclouridine, a specific inhibitor of UPase, prevented the uridine effect. Finally, the uridine effect was shown only in the presence of astrocytes. Taken together, the present study provides the first evidence that uridine protects neurons against ischemic insult-induced neuronal death, possibly through the action of UPase.
Assuntos
Hipóxia-Isquemia Encefálica/enzimologia , Degeneração Neural/enzimologia , Neurônios/enzimologia , Fármacos Neuroprotetores/farmacologia , Uridina Fosforilase/metabolismo , Uridina/farmacologia , Trifosfato de Adenosina/metabolismo , Administração Oral , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Infarto Encefálico/enzimologia , Infarto Encefálico/fisiopatologia , Infarto Encefálico/prevenção & controle , Células Cultivadas , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/enzimologia , Córtex Cerebral/fisiopatologia , Técnicas de Cocultura , Citoproteção/efeitos dos fármacos , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Glucose/deficiência , Hipóxia-Isquemia Encefálica/tratamento farmacológico , Hipóxia-Isquemia Encefálica/fisiopatologia , Infarto da Artéria Cerebral Média/tratamento farmacológico , Infarto da Artéria Cerebral Média/enzimologia , Infarto da Artéria Cerebral Média/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Degeneração Neural/fisiopatologia , Degeneração Neural/prevenção & controle , Neurônios/efeitos dos fármacos , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Uridina Fosforilase/efeitos dos fármacos , Uridina Fosforilase/genéticaRESUMO
Oxidative stress may contribute to epileptogenicity in genetic models of epilepsy. To address this, we examined the enzymatic activity of cytosolic Cu/Zn superoxide dismutase (SOD-1), mitochondrial Mn superoxide dismutase (SOD-2), and glutathione peroxidase (GPx) in the developing hippocampus of genetically epilepsy-prone rats (GEPR-9s). We also measured changes in the GSH/GSSG ratio, lipid peroxidation, and protein oxidation at post-natal days (PD) 7, 30, and 90, respectively. Compared with control Sprague-Dawley (SD) rats, GEPR-9s showed similar SOD-1 and SOD-2 activity but lower GPx activity. Epilepsy-prone rats also showed lower GSH/GSSG ratios than controls, and more lipid peroxidation (as measured by malondialdehyde levels) and protein oxidation (as measured by carbonyl levels). Treatment with kainic acid (KA) resulted in more pronounced seizures, less GPx activity, and lower GSH/GSSG ratios in GEPR-9s than in controls, but KA did not significantly affect SOD-1 or SOD-2 activity, suggesting that GEPR-9s do not compensate for reduced GPx activity by increasing SOD. Moreover, KA treatment resulted in significantly a lower GSH/GSSG ratio and GPx-like immunoreactivity and higher malondialdehyde and carbonyl levels in GEPR-9s than in controls. These findings were more evident in GEPR-9s at PD 90 than at PD 30, indicating that oxidative stress is age-dependent. Double-labeling immunocytochemical analysis demonstrated co-localization of GPx-immunoreactive glia-like cells and reactive astrocytes, as labeled by glial fibrillary acidic protein (GFAP). This suggests that mobilization of astroglial cells for synthesis of GPx protein is a response to KA insult, intended to decrease the neurotoxicity induced by peroxides. These responses were more pronounced in control SD rats than in GEPR-9s. Our results suggest that impairment of the GPx (including glutathione)-mediated antioxidant system contributed to epileptogenesis in GEPR-9s.
Assuntos
Epilepsia/enzimologia , Glutationa Peroxidase/metabolismo , Hipocampo/enzimologia , Estresse Oxidativo/genética , Envelhecimento/metabolismo , Animais , Antioxidantes/metabolismo , Resistência a Medicamentos/efeitos dos fármacos , Resistência a Medicamentos/genética , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Epilepsia/induzido quimicamente , Epilepsia/genética , Agonistas de Aminoácidos Excitatórios/toxicidade , Predisposição Genética para Doença/genética , Glutationa/metabolismo , Glutationa Peroxidase/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Hipocampo/fisiopatologia , Ácido Caínico/toxicidade , Peroxidação de Lipídeos/efeitos dos fármacos , Peroxidação de Lipídeos/genética , Masculino , Degeneração Neural/induzido quimicamente , Degeneração Neural/genética , Degeneração Neural/metabolismo , Neuroglia/metabolismo , Neurotoxinas/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Mutantes , Ratos Sprague-Dawley , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1RESUMO
We have shown that immunostimulated astrocytes produce excess nitric oxide (NO) and eventually peroxynitrite (ONOO(-)) that was closely associated with the glucose deprivation-potentiated death of astrocytes. The present study shows that activated p38 MAPK regulates ONOO(-) generation from lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma)-stimulated astrocytes. LPS+IFN-gamma-induced p38 MAPK activation and ONOO(-) generation were attenuated by SB203580 or SKF-86002, specific inhibitors of p38 MAPK. ONOO(-) generation was blocked by NADPH oxidase inhibitor, diphenyleneiodonium chloride, and nitric oxide synthase (NOS) inhibitor, N omega-nitro-L-arginine methyl ester, suggesting both enzymes are involved in ONOO(-) generation. Inhibition of p38 MAPK suppressed LPS+IFN-gamma-induced NO production through down-regulating inducible form of NOS expression. It also suppressed LPS+IFN-gamma-induced NADPH oxidase activation and eventually, the inducible form of superoxide production. Transfection with dominant negative vector of p38 alpha reduced LPS+IFN-gamma-induced ONOO(-) generation through blocking both iNOS-derived NO production and NADPH oxidase-derived O2(-) production. Our results suggest that activated p38 MAPK may serve as a potential signaling molecule in ONOO(-) generation through dual regulatory mechanisms, involving iNOS induction and NADPH oxidase activation.
Assuntos
Astrócitos/enzimologia , Encefalite/enzimologia , NADPH Oxidases/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Ácido Peroxinitroso/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Animais Recém-Nascidos , Astrócitos/efeitos dos fármacos , Astrócitos/imunologia , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Encefalite/imunologia , Encefalite/fisiopatologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Inibidores Enzimáticos/farmacologia , Interferon gama/imunologia , Interferon gama/farmacologia , Lipopolissacarídeos/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Óxido Nítrico/biossíntese , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Ratos , Ratos Sprague-DawleyRESUMO
We investigated the effect of the cAMP system on lipopolysaccharide (LPS)-induced changes in the activity of matrix metalloproteinases (MMPs) and tissue plasminogen activator (tPA) in rat primary astrocytes. LPS stimulation increased MMP-9 and decreased tPA activity in rat primary astrocytes. Co-treatment with a cAMP analog, dibutyryl-cAMP (db-cAMP), or the cAMP elevating beta-adrenergic agonist, isoproterenol, concentration-dependently inhibited LPS-induced MMP-9 activity. In contrast, db-cAMP concentration-dependently increased tPA activity in both basal and LPS-stimulated rat primary astrocytes. To confirm the effect of cAMP on MMP-9 and tPA activity, we treated LPS-stimulated astrocytes with cAMP phosphodiesterase inhibitors, IBMX or rolipram, and they exhibited similar effects to db-cAMP, namely decreasing MMP-9 activity and increasing tPA activity. RT-PCR analysis of MMP-9 mRNA expression and MMP-9 promoter luciferase reporter assays revealed transcriptional upregulation by LPS stimulation and downregulation by db-cAMP. In contrast, the level of tPA mRNA expression was increased both by LPS and by cAMP treatment. Consistent with RT-PCR analysis, tPA promoter reporter assays showed increased activity by both LPS and cAMP stimulation. Interestingly, the level of mRNA encoding plasminogen activator inhibitor-1 (PAI-1) was increased by LPS stimulation and decreased back to control level after co-treatment with db-cAMP, suggesting that PAI-1 expression plays a major role in the regulation of tPA activity. To examine PKA involvement in the effects of db-cAMP on MMP-9 and tPA activity, we added the PKA inhibitors, H89 or rp-cAMP, along with db-cAMP, and they inhibited db-cAMP-mediated changes in tPA activity without affecting MMP-9 activity. These data suggest that cAMP differentially modulates MMP-9 and tPA activity through a mechanism related to PKA activation. The differential regulation of MMP-9 and tPA by the cAMP system may confer more sophisticated regulation of physiological processes, such as extracellular matrix remodeling and cell migration, by activated astrocytes.
Assuntos
Astrócitos/efeitos dos fármacos , AMP Cíclico/metabolismo , Lipopolissacarídeos/farmacologia , Metaloproteinase 9 da Matriz/metabolismo , Ativador de Plasminogênio Tecidual/metabolismo , Animais , Astrócitos/enzimologia , Astrócitos/metabolismo , Sequência de Bases , Células Cultivadas , Primers do DNA , Ativação Enzimática , Regiões Promotoras Genéticas , Processamento Pós-Transcricional do RNA , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ativador de Plasminogênio Tecidual/genéticaRESUMO
Chronic hypoperfusion-induced changes in blood-brain barrier (BBB) tight junction components have not been well studied. In the present study, we investigated the temporal profiles of claudin-3 (a BBB tight junction element) and myleoperoxidase (MPO, a marker of neutrophil infiltration) in the cortical and thalamic regions of rat brain subjected to chronic cerebral hypoperfusion. Chronic cerebral hypoperfusion was induced by an occlusion of two common carotid arteries and the immunoreactivity of claudin-3 or MPO was determined at 1, 2, 3, or 6 weeks after the occlusion. A typical pattern of BBB breakdown was observed from 2 weeks of the occlusion in cortical and thalamic regions based on Evans Blue leakage. Claudin-3 immunoreactivity was increased only in cortical regions after 2 weeks of occlusion. However, after 3 weeks of occlusion, marked increases in claudin-3 immunoreactivity were observed in both cortical and thalamic regions (P<0.05), which persisted for at least 6 weeks after the occlusion despite a slight reduction. In contrast, MPO immunoreactivity was increased only in the thalamic regions after 2 weeks of occlusion. But the pattern of MPO immunoreactivity at 3 and 6 weeks after the occlusion was same as claudin-3. At these time points, MPO immunoreactivity was significantly increased in both cortical and thalamic regions (P<0.05). These results show that chronic cerebral hypoperfusion increases the immunoreactivity of claudin-3 and neutrophil infiltration in cortical and thalamic regions of the brain, and demonstrate changes in BBB tight junction status during chronic cerebral hypoperfusion.
Assuntos
Doenças das Artérias Carótidas/patologia , Córtex Cerebral/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteínas de Membrana/metabolismo , Tálamo/metabolismo , Animais , Claudina-3 , Modelos Animais de Doenças , Fator Estimulador de Colônias de Granulócitos/metabolismo , Interleucina-3/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes , Fatores de TempoRESUMO
Previously, we and others have demonstrated that wogonin, an active component from the root of Scutellaria baicalensis Georgi, has a neuroprotective effect in cerebral ischemic insult. The neuroprotective effect of wogonin may at least in part be due to its anti-inflammatory properties. Microglial cells, well-known residential macrophages in the central nervous system, migrate to the ischemic lesion and play a pivotal role in the development of chronic inflammation. In the present study, we observed that wogonin potently inhibited microglial migration toward a chemokine, monocyte chemoattractant protein-1 (MCP-1). The anti-migratory effect of wogonin was provoked at nanomolar concentrations, at which wogonin did not significantly inhibit the production of cytokines and chemokines. NF-kappaB has previously shown to regulate microglial cell migration, and activation of cAMP-signaling pathway has also been associated with inhibition of microglial cell motility. In the present study, wogonin at low micromolar concentrations completely suppressed the activity of NF-kappaB in MCP-1-stimulated microglia, and NF-kappaB inhibitors such as N-acetyl cysteine and pyrrolidinedithiocarbamate inhibited the MCP-1-induced migration of microglial cells. However, wogonin did not stimulate the production of cAMP in microglial cells. Our results indicate that the anti-inflammatory activity of wogonin is exerted at least in part by suppressing microglial cell motility via inhibition of NF-kappaB activity.
Assuntos
Medicamentos de Ervas Chinesas/farmacologia , Flavanonas/farmacologia , Microglia/efeitos dos fármacos , NF-kappa B/antagonistas & inibidores , Scutellaria/química , Animais , Movimento Celular/efeitos dos fármacos , Quimiocina CCL2/antagonistas & inibidores , Quimiocina CCL5/antagonistas & inibidores , AMP Cíclico/biossíntese , Microglia/fisiologia , Ratos , Ratos Sprague-DawleyRESUMO
Glycogen synthase kinase (GSK)-3beta and extracellular signal-regulated kinase (ERK) regulate several cellular signaling pathways in common, including embryonic development, cell differentiation and apoptosis. In this study, we investigated whether GSK-3beta inhibition is involved in ERK activation, which affects the activation of NF-kappaB and induction of MMP-9 in cultured rat primary astrocytes. Here, we found that GSK-3beta inhibition using GSK-3beta inhibitor TDZD-8 increased the phosphorylation of GSK-3beta at Ser9 site as well as the phosphorylation of ERK1/2 and Akt at Ser473 site. In this condition, GSK-3beta inhibition increased MMP-9 but not MMP-2 activity in a concentration-dependent manner. In RT-PCR analysis, MMP-9 mRNA level was increased by GSK-3beta inhibition in a concentration-dependent manner. MMP-9 promoter reporter activity was similarly increased by GSK-3beta inhibition. Pretreatment of U-0126 (MEK1/2 inhibitor) completely abolished the GSK-3beta inhibition-induced phosphorylation of ERK1/2. U-0126 prevented GSK-3beta inhibition-mediated induction of MMP-9 reporter activity as well as the MMP-9 gene expression. The transcriptional activity of NF-kappaB was significantly increased by GSK-3beta inhibition, which was determined by nuclear translocation of NF-kappaB. Inhibition of ERK1/2 activity by U-0126 also completely blocked the nuclear translocation of NF-kappaB. Transfection of dominant negative plasmid (S9A) of GSK-3beta significantly decreased phosphorylation of ERK, MMP-9 expression and nuclear translocation of NF-kappaB by GSK-3beta inhibition as compared to wild type GSK-3beta. These data suggest that GSK-3beta inhibition mediates ERK1/2 activation followed by NF-kappaB activation, which directly regulates the induction of MMP-9 in rat primary astrocytes.
Assuntos
Astrócitos/enzimologia , Quinase 3 da Glicogênio Sintase/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , NF-kappa B/metabolismo , Animais , Astrócitos/citologia , Núcleo Celular/metabolismo , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Quinase 3 da Glicogênio Sintase/efeitos dos fármacos , Glicogênio Sintase Quinase 3 beta , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Fosforilação , Transporte Proteico/fisiologia , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/fisiologia , Tiadiazóis/farmacologiaRESUMO
In previous studies, we identified sedative effects of Scutellaria baicalensis extracts and found that these extracts or their constituents may also have anticonvulsive effects. Wogonin is a natural product isolated from S. baicalensis, which possesses central nervous system effects such as anxiolytic and neuroprotective activities. In this study, we investigated the effects of wogonin on convulsion related behaviors, such as myorelaxation, motor coordination, and anticonvulsant effects of wogonin on chemical induced seizure and electroshock seizure in mice or rats. The effect of wogonin on membrane potential was also observed. Wogonin was intraperitoneally injected into mice or rats 30 min prior to testing. Animals treated with wogonin did not change locomotor activities as well as endurance times on the rota-rod, which indicates that wogonin did not cause a sedative and myorelaxation effect. Wogonin significantly blocked convulsion induced by pentylenetetrazole and electroshock but not convulsion induced by strychnine. Wogonin also significantly reduced the electrogenic response score, but flumazenil treatment reversed this decrease to the level of the control group. The wogonin treatment increased Cl(-)influx into the intracellular area as dose increased. Flumazenil and bicuculline treatment, however, inhibited the Cl(-) influx induced by wogonin. These results indicate that the anticonvulsive effects produced by wogonin were mediated by the GABAergic neuron.