RESUMO
Activation of glial cells, astrocytes and microglia, has been observed in neurodegenerative diseases including Alzheimer's disease (AD). Amyloid ß (Aß), which is aggregated and the aggregation is detected as characteristic pathology in AD brain, is known to be produced by neurons and to activate glial cells. Clearance of Aß from the brain via active transport system is important to prevent the accumulation and aggregation. Low density lipoprotein receptor-related protein 2 (LRP2/megalin) is an Aß transporter. However, expression and contribution of LRP2 in astrocytes and microglia remain to be clarified. In the present study, we examined the expression of LRP2 and its roles in cultured astrocytes prepared from rat embryonic brain cortex and mouse microglial cell line BV-2. Both cultured rat astrocytes and BV-2 cells expressed LRP2 mRNA detected by RT-PCR. When lipopolysaccharide (LPS) or all-trans retinoic acid (ATRA) were added to BV-2 cells, LRP2 mRNA expression and uptake of microbeads, Aß and insulin were increased. On the other hand, LPS decreased LRP2 expression and uptake of Aß and insulin in cultured astrocytes. Knockdown of LRP2 using siRNA attenuated the LPS- or ATRA-increased uptake of microbeads, Aß and insulin in BV-2 cells. These results suggest that LRP2 was expressed in both astrocytes and microglia and might be involved in endocytosis activities. Adequate control of LRP2 expression and function in astrocytes and microglia might regulate Aß and insulin levels in brain and would be a potential target in AD pathology.
Assuntos
Doença de Alzheimer , Insulinas , Ratos , Camundongos , Animais , Peptídeos beta-Amiloides/metabolismo , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Microglia/metabolismo , Astrócitos/metabolismo , Lipopolissacarídeos/farmacologia , Doença de Alzheimer/metabolismo , RNA Mensageiro/metabolismo , Insulinas/metabolismo , Células CultivadasRESUMO
Mutations in alpha/beta-hydrolase domain containing (ABHD) 12 gene, which encodes lysophosphatidylserine (LysoPS) lipase, cause the neurodegenerative disease PHARC (Polyneuropathy, Hearing loss, Ataxia, Retinitis pigmentosa, Cataract). Since ABHD12 is expressed by microglia in the central nervous system and is localized to the endoplasmic reticulum, accumulation of intracellular LysoPS by ABHD12 mutations is assumed to be one of the pathological mechanisms associated with microglial activation in PHARC. However, the role of microglia in the PHARC brain and the relationship between microglial function and cellular LysoPS content remains unclear. Therefore, we explored the influence of cellular LysoPS content in microglial inflammatory responses. We evaluated the effects of inhibitors of cellular LysoPS metabolism, KC01 and DO-264, on inflammatory responses using a lipopolysaccharide (LPS)-stimulated mouse microglial cell line, BV-2 and primary microglia. Treatment of DO-264, an inhibitor of cellular LysoPS degradation, enhanced LPS-induced phagocytosis concomitant with the increase in cellular LysoPS content in BV-2 cells. On the other hand, treatment with KC01, an agent had been developed as an inhibitor of LysoPS synthase, reduced phagocytosis without affecting cellular LysoPS content. Such effects of both inhibitors on phagocytosis were also confirmed using primary microglia. KC01 treatment decreased nitric oxide (NO) production, accompanied by a reduction in inducible NO synthase expression in BV-2 microglia. KC01 also suppressed LPS-induced generation of intracellular reactive oxygen species and cytokines such as interleukin-6. Our results suggest that increase in cellular LysoPS levels can exacerbate microglial inflammatory responses. Treatment to prevent the increase in cellular LysoPS in microglia may have therapeutic potential for PHARC.
Assuntos
Lipopolissacarídeos , Doenças Neurodegenerativas , Animais , Ataxia , Catarata , Lipopolissacarídeos/toxicidade , Lisofosfolipídeos/metabolismo , Lisofosfolipídeos/farmacologia , Camundongos , Microglia/metabolismo , Monoacilglicerol Lipases/genética , Monoacilglicerol Lipases/metabolismo , Monoacilglicerol Lipases/farmacologia , Doenças Neurodegenerativas/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Polineuropatias , Retinose PigmentarRESUMO
Activation of glial cells has been observed in neurodegenerative diseases including Alzheimer's disease (AD). Aggregation of amyloid ß (Aß) is profusely observed as characteristic pathology in AD brain. In our previous study using microglial cell line BV-2, tissue-type transglutaminase (TG2) was found to be involved in phagocytosis (Kawabe et al., in Neuroimmunomodulation 22(4):243-249, 2015; Kawabe et al., Neurochem Res 2017). In the present study, we examined whether TG2 and milk fat globule EGF factor 8 protein (MFG-E8), an adaptor protein promotes macrophage to engulf apoptotic cells, were involved in Aß endocytosis. When the neuronal/glial mixed culture was stimulated freshly prepared Aß1-42 for 3 days, the incorporation of Aß was observed by immunofluorescence staining technique in Iba-1-positive microglia. Cystamine, a broad competitive inhibitor of TGs, suppressed it. When aggregated Aß was added to the mixed culture, the immunoreactivity of MFG-E8 surrounding Aß was observed, and then followed by microglial endocytosis. Using western blotting technique, MFG-E8 was detected in cell lysate of astrocyte culture, and was also detected in the medium. When microglia culture was incubated with astrocyte conditioned medium, MFG-E8 levels in microglia tended to increase. It is likely that microglia might utilize MFG-E8 released from astrocytes as well as that expressed in themselves in order to endocytose Aß aggregation. Furthermore, we confirmed that MFG-E8 could bind with TG2 in microglia culture by immunoprecipitate technique. These results suggest that microglia might uptake Aß as a complex of aggregated Aß/MFG-E8/TG2.
Assuntos
Antígenos de Superfície/metabolismo , Endocitose/fisiologia , Proteínas de Ligação ao GTP/metabolismo , Microglia/metabolismo , Proteínas do Leite/metabolismo , Transglutaminases/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Animais Recém-Nascidos , Astrócitos/metabolismo , Células Cultivadas , Glicolipídeos/metabolismo , Glicoproteínas/metabolismo , Gotículas Lipídicas , Neurônios/metabolismo , Fagocitose/fisiologia , Proteína 2 Glutamina gama-Glutamiltransferase , RatosRESUMO
Zn2+ plays a crucial role in the CNS where it accumulates in synaptic vesicles and is released during neurotransmission. Synaptically released Zn2+ is taken up by neurons and astrocytes. The majority of previous work has focused on neuronal damage caused by excess Zn2+. However, its effect on astrocyte function is not well understood. We examined the effect of extracellularly applied Zn2+ on nitric oxide (NO) production in primary cultured rat astrocytes, which were experimentally activated by lipopolysaccharide (LPS). Zn2+, at a concentration up to 125 µM, augmented LPS-induced NO production without affecting cell viability. LPS induced expression of both mRNA and protein of inducible NO synthase; this expression was enhanced by 125 µM Zn2+. Zn2+ also increased LPS-induced production of intracellular reactive oxygen species. Zn2+ enhanced the phosphorylation of p38-mitogen-activated protein kinase (MAPK) at 1-6 h after LPS treatment. The LPS-induced nuclear factor-kappaB (NFκB) activation was sustained for 6 h by Zn2+. Intracellular Zn2+ chelation with N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) or inhibition of p38-MAPK diminished the Zn2+ enhancement of LPS-induced NO production. These findings suggest that activation of MAPK and NFκB is important for mediating Zn2+enhancement of LPS-induced NO production in astrocytes. Such changes may exacerbate glial and neuronal damage during neuroinflammation.
Assuntos
Astrócitos/metabolismo , Lipopolissacarídeos/farmacologia , Neurônios/metabolismo , Óxido Nítrico/metabolismo , Zinco/metabolismo , Animais , Células Cultivadas , Microglia/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neuroglia/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismoRESUMO
Glutamate uptake is a main function of astrocytes to keep extracellular glutamate levels low and protect neurons against glutamate-induced excitotoxicity. On the other hand, astrocyte networks formed by gap junctions, which are consisted with connexins and connecting neighboring cells, are reported to play a critical role in maintaining the homeostasis in the brain. In the present study, we examined the effects of gap junction inhibitors on the glutamate uptake activity in cultured rat cortical astrocytes. At first, we confirmed the effects of gap junction inhibitors, 1-octanol and carbenoxolone, on cell-cell communication by the scrape-loading assay using a fluorescent dye Lucifer yellow. Both of 1-octanol and carbenoxolone treatments for 20 min in cultured astrocytes significantly suppressed the cell-cell communication assessed as the distance of dye-spreading. 1-octanol and carbenoxolone increased the glutamate uptake by astrocytes and glutamate aspartate transporter (GLAST) expression on the cell membrane. These results suggest that gap junction inhibitors increase the glutamate uptake activity through the increase of GLAST proteins located on the cell membrane. The regulation of gap junction in astrocytes might protect neurons against glutamate-induced excitotoxicity.
Assuntos
Astrócitos/metabolismo , Junções Comunicantes/metabolismo , Ácido Glutâmico/metabolismo , Neurônios/metabolismo , Sistema X-AG de Transporte de Aminoácidos/metabolismo , Animais , Transporte Biológico/fisiologia , Comunicação Celular/fisiologia , Células Cultivadas , Conexinas/metabolismo , Ratos WistarRESUMO
In several neurodegenerative diseases such as Alzheimer's disease (AD), microglia are hyperactivated and release nitric oxide (NO) and proinflammatory cytokines, resulting its neuropathology. Mounting evidence indicates that dietary supplementation with coconut oil (CNO) reduces the cognitive deficits associated with AD; however, the precise mechanism(s) underlying the beneficial effect of CNO are unknown. In the present study, we examined the effects of lauric acid (LA), a major constituent of CNO, on microglia activated experimentally by lipopolysaccharide (LPS), using primary cultured rat microglia and the mouse microglial cell line, BV-2. LA attenuated LPS-stimulated NO production and the expression of inducible NO synthase protein without affecting cell viability. In addition, LA suppressed LPS-induced reactive oxygen species and proinflammatory cytokine production, as well as phosphorylation of p38-mitogen activated protein kinase and c-Jun N-terminal kinase. LA-induced suppression of NO production was partially but significantly reversed in the presence of GW1100, an antagonist of G protein-coupled receptor (GPR) 40, which is an LA receptor on the plasma membrane. LA also decreased LPS-induced phagocytosis, which was completely reversed by co-treatment with GW1100. Moreover, LA alleviated amyloid-ß-induced enhancement of phagocytosis. These results suggest that attenuation of microglial activation by LA may occur via the GPR40-dependent pathway. Such effects of LA may reduce glial activation and the subsequent neuronal damage in AD patients who consume CNO.
Assuntos
Ácidos Láuricos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Microglia/efeitos dos fármacos , Receptores Acoplados a Proteínas G/efeitos dos fármacos , Animais , Linhagem Celular , Óleo de Coco/farmacologia , Citocinas/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Óxido Nítrico/metabolismo , Fosforilação/efeitos dos fármacos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Mice carrying simultaneous homozygous mutations in the genes encoding citrin, the mitochondrial aspartate-glutamate carrier 2 (AGC2) protein, and mitochondrial glycerol-3-phosphate dehydrogenase (mGPD), are a phenotypically representative model of human citrin (a.k.a., AGC2) deficiency. In this study, we investigated the voluntary oral intake and preference for sucrose, glycerol or ethanol solutions by wild-type, citrin (Ctrn)-knockout (KO), mGPD-KO, and Ctrn/mGPD double-KO mice; all substances that are known or suspected precipitating factors in the pathogenesis of human citrin deficiency. The double-KO mice showed clear suppressed intake of sucrose, consuming less with progressively higher concentrations compared to the other mice. Similar observations were made when glycerol or ethanol were given. The preference of Ctrn-KO and mGPD-KO mice varied with the different treatments; essentially no differences were observed for sucrose, while an intermediate intake or similar to that of the double-KO mice was observed for glycerol and ethanol. We next examined the hepatic glycerol 3-phosphate, citrate, citrulline, lysine, glutamate and adenine nucleotide levels following forced enteral administration of these solutions. A strong correlation between the simultaneous increased hepatic glycerol 3-phosphate and decreased ATP or total adenine nucleotide content and observed aversion of the mice during evaluation of their voluntary preferences was found. Overall, our results suggest that the aversion observed in the double-KO mice to these solutions is initiated and/or mediated by hepatic metabolic perturbations, resulting in a behavioral response to increased hepatic cytosolic NADH and a decreased cellular adenine nucleotide pool. These findings may underlie the dietary predilections observed in human citrin deficient patients.
Assuntos
Citrulinemia/metabolismo , Sacarose Alimentar/administração & dosagem , Etanol/administração & dosagem , Glicerol/administração & dosagem , Fígado/química , Trifosfato de Adenosina/metabolismo , Sistemas de Transporte de Aminoácidos Acídicos/genética , Animais , Antiporters/genética , Modelos Animais de Doenças , Glicerolfosfato Desidrogenase/genética , Glicerofosfatos/metabolismo , Humanos , Camundongos , Camundongos KnockoutRESUMO
Amphotericin B (AmB), a polyene antibiotic, is reported to cause the microglial activation to induce nitric oxide (NO) production and proinflammatory cytokines expression, and change neurotrophic factors expression in cultured microglia (Motoyoshi et al. in Neurochem Int 52:1290-1296, 2008). On the other hand, tissue-type transglutaminase (TG2) is involved in connection to phagocytes with apoptotic cells. Engulfment of neurons by activated microglia is thought to cause neurodegenerative diseases but detail is unclear, and involvement of TG2 in phagocytosis has been reported in our previous study using lipopolysaccharide-stimulated BV-2 cells (Kawabe et al. in Neuroimmunomodulation 22(4):243-249, 2015). In the present study, we examined the changes of TG2 expression, phagocytosis and pinocytosis in BV-2 cells stimulated by AmB. AmB stimulation increased TG2 expression and TG activity. Phagocytosis of dead cells and pinocytosis of fluorescent microbeads were also up-regulated by AmB stimulation in BV-2 cells. Blockade of TG activity by cystamine, an inhibitor of TGs, suppressed AmB-enhanced TG2 expression, TG activity, NO production, phagocytosis and pinocytosis. Excessive NO production from microglia and/or facilitation of phagocytosis might be involved in neuronal death. To control TG activity might make possible to protect neurons and care for CNS diseases.
Assuntos
Anfotericina B/farmacologia , Endocitose/fisiologia , Proteínas de Ligação ao GTP/biossíntese , Regulação Enzimológica da Expressão Gênica , Microglia/enzimologia , Transglutaminases/biossíntese , Regulação para Cima/fisiologia , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Endocitose/efeitos dos fármacos , Proteínas de Ligação ao GTP/genética , Camundongos , Microglia/efeitos dos fármacos , Proteína 2 Glutamina gama-Glutamiltransferase , Transglutaminases/genética , Regulação para Cima/efeitos dos fármacosRESUMO
Activation of astrocytes has been observed in neurodegenerative diseases including Alzheimer's disease (AD). Transglutaminase (TG) is a crosslinking enzyme and contributes to cell adhesion, cytoskeleton construct, extracellular matrix formation, and so on. One of the isozymes, tissue-type TG (TG2) is reported to be activated in AD. Moreover, amyloid ß1-42 (Aß), which is aggregated and the aggregation is detected as characteristic pathology in AD brain, is known to be a substrate of TG2. However, contribution and derivation of TGs in brain for Aß aggregation remain to be clarified. In the present study, we examined the effects of cultured astrocytes prepared from rat embryonic brain cortex on Aß aggregation. When freshly prepared Aß was added to cultured astrocytes for 7 days, Aß monomer decreased and Aß oligomer unchanged. On the other hand, when Aß monomer was diluted with astrocytes conditioned medium, Aß oligomer increased time-dependently, and an inhibitor of TGs, cystamine, blocked it. Furthermore, when cultured astrocytes were stimulated with aggregated Aß, TG2 expression significantly increased. These results suggest that astrocytes could uptake Aß monomer to eliminate from brain; however, TGs derived from astrocytes might accelerate Aß aggregation and the aggregated Aß might enhance TG2 in astrocytes as a vicious cycle in pathological conditions. Adequate control of TGs expression and function in astrocytes would be an important factor in AD pathology.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Astrócitos/metabolismo , Fragmentos de Peptídeos/metabolismo , Agregação Patológica de Proteínas/metabolismo , Transglutaminases/metabolismo , Peptídeos beta-Amiloides/farmacologia , Animais , Astrócitos/efeitos dos fármacos , Células Cultivadas , Feminino , Fragmentos de Peptídeos/farmacologia , Gravidez , Proteína 2 Glutamina gama-Glutamiltransferase , Ratos , Ratos Wistar , Transglutaminases/isolamento & purificaçãoRESUMO
The mitochondrial aspartate-glutamate carrier isoform 2 (citrin) and mitochondrial glycerol-3-phosphate dehydrogenase (mGPD) double-knockout mouse has been a useful model of human citrin deficiency. One of the most prominent findings has been markedly increased hepatic glycerol 3-phosphate (G3P) following oral administration of a sucrose solution. We aimed to investigate whether this change is detectable outside of the liver, and to explore the mechanism underlying the increased hepatic G3P in these mice. We measured G3P and its metabolite glycerol in plasma and urine of the mice under various conditions. Glycerol synthesis from fructose was also studied using the liver perfusion system. The citrin/mGPD double-knockout mice showed increased urine G3P and glycerol under normal, fed conditions. We also found increased plasma glycerol under fasted conditions, while oral administration of different carbohydrates or ethanol led to substantially increased plasma glycerol. Fructose infusion to the perfused liver of the double-knockout mice augmented hepatic glycerol synthesis, and was accompanied by a concomitant increase in the lactate/pyruvate (L/P) ratio. Co-infusion of either pyruvate or phenazine methosulfate, a cytosolic oxidant, with fructose corrected the high L/P ratio, leading to reduced glycerol synthesis. Overall, these findings suggest that hepatic glycerol synthesis is cytosolic NADH/NAD(+) ratio-dependent and reveal a likely regulatory mechanism for hepatic glycerol synthesis following a high carbohydrate load in citrin-deficient patients. Therefore, urine G3P and glycerol may represent potential diagnostic markers for human citrin deficiency.
RESUMO
The biomolecule acetate can be utilized for energy production, lipid synthesis, and several metabolic processes. Acetate supplementation reduces neuroglial activation in a model of neuroinflammation induced by intraventricular injection of lipopolysaccharide (LPS). To investigate the mechanisms underlying the anti-inflammatory effect of acetate on glial cells, we examined the effect of acetate on nitric oxide (NO) production, which was experimentally activated by LPS, in cultured primary rat astrocytes. Acetate attenuated the LPS-induced NO production in a dose-dependent manner, although cell viability was not affected. Acetate suppressed the phosphorylation of p38-mitogen-activated protein kinase 24 h after LPS treatment. Acetate decreased the LPS-induced production of intracellular reactive oxygen species (ROS) at 4-24 h concomitant with an increase in glutathione. Acetate rescued astrocytes from the hydrogen peroxide-induced cell death by reducing ROS levels. These findings suggest that attenuation of NO production by acetate may alleviate glial cell damage during neuroinflammation. Acetate may offer a glioprotective effect through an anti-oxidative mechanism.
Assuntos
Astrócitos/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Microglia/efeitos dos fármacos , Óxido Nítrico/biossíntese , Animais , Astrócitos/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Inflamação/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neuroglia/metabolismo , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismoRESUMO
OBJECTIVES: In peripheral macrophages, tissue-type transglutaminase (TG2) is reported to be involved in phagocytosis of apoptotic cells. However, the contribution of TG2 to microglial phagocytosis has not been investigated. In this study, using a microglial cell line, BV-2, we examined the changes in TG2 expression, phagocytosis and pinocytosis in cells stimulated by lipopolysaccharide (LPS). METHODS: Cells of the mouse microglial cell line BV-2 were stimulated by LPS with or without cystamine, an inhibitor of TG enzyme activity, for 24 h. TG2 expression was measured by real-time RT-PCR and Western blotting. TG activity was evaluated using biotinylated pentylamine as a substrate. Pinocytosis was determined by uptake of 1-µm fluorescent microbeads. Phagocytosis was assessed by uptake of dead cells, human neuroblastoma SH-SY5Y cells, which were pretreated with H2O2 for 24 h. RESULTS: Phagocytosis of dead cells and pinocytosis of fluorescent microbeads were up-regulated by LPS stimulation together with TG2 expression. Blockade of TG enzyme activity by cystamine suppressed TG2 expression, phagocytosis and pinocytosis. CONCLUSIONS: These results suggested that LPS-induced TG2 was involved in the mechanism of pinocytosis and phagocytosis in microglia.
Assuntos
Endocitose/fisiologia , Proteínas de Ligação ao GTP/metabolismo , Lipopolissacarídeos/farmacologia , Microglia/metabolismo , Transglutaminases/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Camundongos , Proteína 2 Glutamina gama-GlutamiltransferaseRESUMO
Elucidation of the functions of astrocytes is important for understanding of the pathogenic mechanism of various neurodegenerative diseases. Theophylline is a common drug for bronchial asthma and occasionally develops side-effects, such as acute encephalopathy; although the pathogenic mechanism of the side-effects is unknown. The lipopolysaccharide (LPS)-induced nitricoxide (NO) production is generally used for an index of the activation of astrocyte in vitro. In this study, in order to elucidate the effect of theophylline on the astrocytic functions, we examined the LPS-induced NO production and the expression of iNOS in cultured rat cortex astrocytes.Theophylline alone could not induce the NO production; however, NO production induced by LPS was enhanced by theophylline in a dose-dependent manner; and by isobutylmethylxanthine, a phosphodiesterase inhibitor. The theophylline enhancement of LPS-induced NO production was further increased by dibutyryl cyclic AMP, a membrane-permeable cAMP analog; and by forskolin, an adenylate cyclase activator. When the cells were preincubated with Rp-8-Br-cAMP, an inhibitor of protein kinase A, the theophylline enhancement of LPS-induced NO production was decreased. The extent of iNOS protein expression induced by LPS was also enhanced by theophylline.It is likely that phosphodiesterase inhibition is a major action mechanism for the theophylline enhancement of LPS-induced NO production in astrocytes. Theophylline-induced acute encephalopathy might be due to the hyper-activation of astrocytes via cAMP signaling to produce excess amount of NO.
Assuntos
Astrócitos/efeitos dos fármacos , Óxido Nítrico Sintase Tipo II/biossíntese , Óxido Nítrico/biossíntese , Teofilina/farmacologia , 8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Animais , Astrócitos/metabolismo , Bucladesina/farmacologia , Células Cultivadas , Colforsina/farmacologia , AMP Cíclico/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Sinergismo Farmacológico , Lipopolissacarídeos/farmacologia , NF-kappa B/metabolismo , Ratos , Tionucleotídeos/farmacologiaRESUMO
In Alzheimer's disease (AD) pathology, the accumulation of amyloid-beta (Aß), a main component of senile plaques, activates glial cells and causes neuroinflammation. Excessive neuroinflammation results in neuronal dropouts and finally produces the symptoms of AD. Recent studies suggest that disorder in sphingosine-1-phosphate (S1P) metabolism, especially the decreased expression of sphingosine kinase (SK)1, followed by the reduction in the amount of S1P, can be a promotive factor in AD onset. Thus, we explored the possibility that dysregulated S1P metabolism affects AD through the altered function in glial cells. We evaluated the effect of PF-543, a pharmacological inhibitor of SK1, on the inflammatory responses by lipopolysaccharide (LPS)-activated glial cells, microglia, and astrocytes. The treatment with PF-543 decreased the intracellular S1P content in glial cells. The PF-543 treatment enhanced the nitric oxide (NO) production in the LPS-treated neuron/glia mixed culture. Furthermore, we found that the augmented production of NO and reactive oxygen species (ROS) in the PF-543-treated astrocytes affected the microglial inflammatory responses through humoral factors in the experiment using an astrocyte-conditioned medium. The PF-543 treatment also decreased the microglial Aß uptake and increased the number of injured neurons in the Aß-treated neuron/glia mixed culture. These results suggest that a decrease in the glial S1P content can exacerbate neuroinflammation and neurodegeneration through altered glial cell functions.
RESUMO
Under some pathological conditions in brain, a large amount of superoxide anion (O(2)(-)) is produced, causing various cellular damages. Among three isozymes of superoxide dismutase (SOD), extracellular (EC)-SOD should play a role to detoxify O(2)(-) in extracellular space; however, a little is known about EC-SOD in brain. Although dopamine (DA) stored in the synaptic vesicle is stable, the excess leaked DA is spontaneously oxidized to yield O(2)(-) and reactive DA quinones, causing damages of dopaminergic neurons. In the present study, we examined the effects of DA on SOD expression in cultured rat cortical astrocytes. By means of RT-PCR, all mRNA of three isozymes of SOD could be detected; however, only EC-SOD was increased by DA exposure for 24 h, dose-dependently. The expression of EC-SOD protein and the cell-surface SOD activity in astrocytes also increased with 100 µM DA exposure. The increase of EC-SOD mRNA by DA was inhibited by a DA transporter inhibitor, GBR12909, whereas it was not changed by DA receptor antagonists, SKF-83566 (D1) and haloperidol (D2). Furthermore, a monoamine oxidase inhibitor, pargyline, and antioxidants, N-acetyl-L-cysteine and glutathione, also did not affect the DA-induced expression of EC-SOD mRNA. On the other hand, an inhibitor of nuclear factor kappaB (NF-κB), ammonium pyrrolidine-1-carbodithioate, suppressed the DA-induced expression of EC-SOD mRNA. These results suggest that DA incorporated into the cells caused the induction of EC-SOD mRNA followed by the enhancements of EC-SOD protein level and the enzyme activity, and that NF-κB activation is involved in the mechanisms of the EC-SOD induction. The regulation of EC-SOD in astrocytes surrounding dopaminergic neurons may contribute to the defensive mechanism against oxidative stress in brain.
Assuntos
Astrócitos/enzimologia , Dopamina/farmacologia , Espaço Extracelular/enzimologia , Superóxido Dismutase/biossíntese , 2,3,4,5-Tetra-Hidro-7,8-Di-Hidroxi-1-Fenil-1H-3-Benzazepina/análogos & derivados , 2,3,4,5-Tetra-Hidro-7,8-Di-Hidroxi-1-Fenil-1H-3-Benzazepina/farmacologia , Animais , Astrócitos/efeitos dos fármacos , Western Blotting , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Relação Dose-Resposta a Droga , NF-kappa B/biossíntese , Proteínas do Tecido Nervoso/biossíntese , Piperazinas/farmacologia , RNA Mensageiro/biossíntese , Ratos , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Estimulação QuímicaRESUMO
BACKGROUND: Activation of microglia and astrocytes has been observed in Alzheimer's disease (AD). Transglutaminase 2 (TG2) is reported to be activated in AD and involved in cell proliferation, differentiation, and inflammation. Moreover, amyloid ß (Aß) aggregation is detected as a characteristic pathology in the AD brain, and is known to be a substrate of TG2. All-trans retinoic acid (ATRA) can modify cell proliferation and differentiation, and is reported to have therapeutic effects on AD pathology. OBJECTIVE: We aimed to assess the effects of ATRA in microglia and astrocytes on TG2 expression and glial functions. METHODS: After treatment with ATRA, TG2 expression and TG activity were assayed in both murine microglia BV-2 cells and cultured rat brain astrocytes. Endocytosis activity in BV-2 cells and Aß aggregation by astrocytes conditioned medium were also assessed. RESULTS: In both BV-2 cells and cultured astrocytes, ATRA increased TG2 expression and TG activity. The increase was blocked by AGN194310, an RA receptor antagonist. ATRA enhanced the endocytosis activity in BV-2 cells, and the addition of AGN194310 reversed it. The addition of cystamine, a competitive TG inhibitor, also reduced ATRA-enhanced endocytosis activity. On the other hand, Aß aggregation was potentiated by ATRA-treated astrocytes conditioned medium compared to control astrocytes conditioned medium. CONCLUSION: These results suggest that ATRA increased TG2 expression and TG activity via RA receptor in microglia and astrocytes. ATRA-enhanced TGs might be involved in phagocytosis and Aß aggregation. Adequate control of TGs expression and function in microglia and astrocytes can be an important factor in AD pathology.
RESUMO
Under pathological conditions such as ischemia/reperfusion, a large amount of superoxide anion (O(2) (-)) is produced and released in brain. Among three isozymes of superoxide dismutase (SOD), extracellular (EC)-SOD, known to be excreted outside cells and bound to extracellular matrix, should play a role to detoxify O(2) (-) in extracellular space; however, a little is known about EC-SOD in brain. In order to evaluate the SOD activity in extracellular space of CNS as direct as possible, we attempted to measure the cell-surface SOD activity on primary cultured rat brain cells by the inhibition of color development of a water-soluble tetrazolium due to O(2) (-) generation by xanthine oxidase/hypoxanthine added into extracellular medium of intact cells. The cell-surface SOD activity on cultured neuron and microglia was below the detection limit; however, that on cultured astrocyte was high enough to measure. By means of RT-PCR, all mRNA of three isozymes of SOD could be detected in the three types of the cells examined; however, the semi-quantitative analysis revealed that the level of EC-SOD mRNA in astrocytes was significantly higher than that in neurons and microglia. When astrocytes were stimulated with lipopolysaccharide (LPS) for 12-24 h, the cell-surface SOD activity decreased to a half, whereas the activity recovered after 36-48 h. The decrease in the activity was dependent on the LPS concentration. On the other hand, the SOD activity in the medium increased by the LPS-stimulation in a dose dependent manner; suggesting that the SOD protein localized on cell-surface, probably EC-SOD, was released into the medium. These results suggest that EC-SOD of astrocyte play a role for detoxification of extracellular O(2) (-) and the regulation of EC-SOD in astrocytes may contribute to the defensive mechanism against oxidative stress in brain.
Assuntos
Astrócitos/metabolismo , Lipopolissacarídeos/farmacologia , Superóxido Dismutase/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Sequência de Bases , Células Cultivadas , Meios de Cultura , Primers do DNA , Espaço Extracelular/enzimologia , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , Ratos , Ratos Wistar , Superóxido Dismutase/genéticaRESUMO
Depression is one of the most prevalent and livelihood-threatening forms of mental illnesses and the neural circuitry underlying depression remains incompletely understood. Recent studies suggest that the neuronal plasticity involved with brain-derived neurotrophic factor (BDNF) plays an important role in the recovery from depression. Some antidepressants are reported to induce BDNF expression in vivo; however, the mechanisms have been considered solely in neurons and not fully elucidated. In the present study, we evaluated the effects of imipramine, a classic tricyclic antidepressant drug, on BDNF expression in cultured rat brain astrocytes. Imipramine dose-dependently increased BDNF mRNA expression in astrocytes. The imipramine-induced BDNF increase was suppressed with inhibitors for protein kinase A (PKA) or MEK/ERK. Moreover, imipramine exposure activated transcription factor cAMP response element binding protein (CREB) in a dose-dependent manner. These results suggested that imipramine induced BDNF expression through CREB activation via PKA and/or ERK pathways. Imipramine treatment in depression might exert antidepressant action through BDNF production from astrocytes, and glial BDNF expression might be a target of developing novel antidepressants.
Assuntos
Inibidores da Captação Adrenérgica/farmacologia , Antidepressivos Tricíclicos/farmacologia , Astrócitos/efeitos dos fármacos , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Hipocampo/efeitos dos fármacos , Imipramina/farmacologia , Regulação para Cima/efeitos dos fármacos , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Fator Neurotrófico Derivado do Encéfalo/genética , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/agonistas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulação para Baixo/efeitos dos fármacos , Embrião de Mamíferos , Hipocampo/citologia , Hipocampo/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas do Tecido Nervoso/agonistas , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/metabolismo , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Ratos WistarRESUMO
Brain edema and associated increased intracranial pressure are major consequences of traumatic brain injury (TBI). An important early component of the edema associated with TBI is astrocyte swelling (cytotoxic edema). Mechanisms for such swelling, however, are poorly understood. Ion channels/transporters/exchangers play a major role in cell volume regulation, and a disturbance in one or more of these systems may result in cell swelling. To examine potential mechanisms in TBI-mediated brain edema, we employed a fluid percussion model of in vitro barotrauma and examined the role of the ion transporter Na(+)-K(+)-2Cl(-)-cotransporter 1 (NKCC1) in trauma-induced astrocyte swelling as this transporter has been strongly implicated in the mechanism of cell swelling in various neurological conditions. Cultures exposed to trauma (3, 4, 5 atm pressure) caused a significant increase in NKCC1 activity (21%, 42%, 110%, respectively) at 3 h. At 5 atm pressure, trauma significantly increased NKCC1 activity at 1 h and it remained increased for up to 3 h. Trauma also increased the phosphorylation (activation) of NKCC1 at 1 and 3 h. Inhibition of MAPKs and oxidative/nitrosative stress diminished the trauma-induced NKCC1 phosphorylation as well as its activity. Bumetanide, an inhibitor of NKCC1, significantly reduced the trauma-induced astrocyte swelling (61%). Silencing NKCC1 with siRNA led to a reduction in trauma-induced NKCC1 activity as well as in cell swelling. These findings demonstrate the critical involvement of NKCC1 in the astrocyte swelling following in vitro trauma, and suggest that blocking NKCC1 activity may represent a useful therapeutic strategy for the cytotoxic brain edema associated with the early phase of TBI.
Assuntos
Astrócitos/metabolismo , Edema Encefálico/patologia , Simportadores/metabolismo , Análise de Variância , Animais , Animais Recém-Nascidos , Astrócitos/efeitos dos fármacos , Edema Encefálico/etiologia , Lesões Encefálicas/complicações , Lesões Encefálicas/patologia , Células Cultivadas , Óxidos N-Cíclicos/farmacologia , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/fisiologia , NG-Nitroarginina Metil Éster/farmacologia , Percussão/efeitos adversos , RNA Mensageiro , RNA Interferente Pequeno/metabolismo , Ratos , Simportadores/genética , Fatores de Tempo , Transfecção , Cotransportadores de K e Cl-RESUMO
AIMS: To show that acetate attenuates neuroinflammatory responses in activated microglia. BACKGROUND: Dietary acetate supplementation alleviates neuroglial activation in a rat model of neuroinflammation induced by intraventricular administration of lipopolysaccharide (LPS). However, the precise mechanism(s) underlying the anti-inflammatory effect of acetate, is not fully understood. OBJECTIVE: To determine whether acetate has inhibitory effects on LPS-induced neuroinflammatory responses in microglia. METHODS: We examined LPS-stimulated nitric oxide (NO) production in primary rat microglia and BV-2 cells. Protein expression of inducible NO synthase (iNOS) was determined by western blot analysis. The intracellular generation of reactive oxygen species (ROS) and glutathione (GSH) were also evaluated. RESULTS: In primary microglia, acetate decreased LPS-stimulated NO production in a dose-dependent manner, reaching significance at greater than 10 mM, and cell viability was not affected. Acetate suppressed LPS-induced expression of iNOS protein concomitantly with the decrease in NO. The LPS-induced increase in intracellular ROS production was attenuated by acetate. In addition, acetate prevented LPS-induced reduction of GSH. Notably, such suppressive effects of acetate on NO and ROS production were not observed in BV-2 cells. CONCLUSION: These findings suggest that acetate may alleviate neuroinflammatory responses by attenuating NO and ROS production in primary microglia but not in BV-2 cells. Other: All animals received humane care, and the animal protocols used in this study were approved by the Ethics Committees for Animal Experimentation.