RESUMO
BACKGROUND & AIMS: Cerebral oxidative stress plays an important role in the pathogenesis of hepatic encephalopathy (HE), but the underlying mechanisms are incompletely understood. Herein, we analyzed a role of heme oxygenase (HO)1, iron and NADPH oxidase 4 (Nox4) for the induction of oxidative stress and senescence in HE. METHODS: Gene and protein expression in human post-mortem brain samples was analyzed by gene array and western blot analysis. Mechanisms and functional consequences of HO1 upregulation were studied in NH4Cl-exposed astrocytes in vitro by western blot, qPCR and super-resolution microscopy. RESULTS: HO1 and the endoplasmic reticulum (ER) stress marker grp78 were upregulated, together with changes in the expression of multiple iron metabolism-related genes, in post-mortem brain samples from patients with liver cirrhosis and HE. NH4Cl elevated HO1 protein and mRNA in cultured astrocytes through glutamine synthetase (GS)-dependent upregulation of glutamine/fructose amidotransferases 1/2 (GFAT1/2), which blocked the transcription of the HO1-targeting miR326-3p in a O-GlcNAcylation dependent manner. Upregulation of HO1 by NH4Cl triggered ER stress and was associated with elevated levels of free ferrous iron and expression changes in iron metabolism-related genes, which were largely abolished after knockdown or inhibition of GS, GFAT1/2, HO1 or iron chelation. NH4Cl, glucosamine (GlcN) and inhibition of miR326-3p upregulated Nox4, while knockdown of Nox4, GS, GFAT1/2, HO1 or iron chelation prevented NH4Cl-induced RNA oxidation and astrocyte senescence. Elevated levels of grp78 and O-GlcNAcylated proteins were also found in brain samples from patients with liver cirrhosis and HE. CONCLUSION: The present study identified glucosamine synthesis-dependent protein O-GlcNAcylation as a novel mechanism in the pathogenesis of HE that triggers oxidative and ER stress, as well as senescence, through upregulation of HO1 and Nox4. LAY SUMMARY: Patients with liver cirrhosis frequently exhibit hyperammonemia and suffer from cognitive and motoric dysfunctions, which at least in part involve premature ageing of the astrocytes in the brain. This study identifies glucosamine and an O-GlcNAcylation-dependent disruption of iron homeostasis as novel triggers of oxidative stress, thereby mediating ammonia toxicity in the brain.
Assuntos
Amônia/farmacologia , Senescência Celular/genética , Heme Oxigenase (Desciclizante)/metabolismo , Heme Oxigenase-1/metabolismo , Encefalopatia Hepática/metabolismo , Proteínas de Membrana/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Regulação para Cima/genética , Adulto , Idoso , Animais , Animais Recém-Nascidos , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Células Cultivadas , Chaperona BiP do Retículo Endoplasmático , Feminino , Glucosamina/biossíntese , Heme Oxigenase-1/genética , Encefalopatia Hepática/etiologia , Humanos , Cirrose Hepática/complicações , Masculino , Glicoproteínas de Membrana/genética , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Estresse Oxidativo/genética , Ratos , Ratos Wistar , Lobo Temporal/metabolismo , Lobo Temporal/patologiaRESUMO
Urea cycle defects and acute or chronic liver failure are linked to systemic hyperammonemia and often result in cerebral dysfunction and encephalopathy. Although an important role of the liver in ammonia metabolism is widely accepted, the role of ammonia metabolizing pathways in the liver for maintenance of whole-body ammonia homeostasis in vivo remains ill-defined. Here, we show by generation of liver-specific Gln synthetase (GS)-deficient mice that GS in the liver is critically involved in systemic ammonia homeostasis in vivo. Hepatic deletion of GS triggered systemic hyperammonemia, which was associated with cerebral oxidative stress as indicated by increased levels of oxidized RNA and enhanced protein Tyr nitration. Liver-specific GS-deficient mice showed increased locomotion, impaired fear memory, and a slightly reduced life span. In conclusion, the present observations highlight the importance of hepatic GS for maintenance of ammonia homeostasis and establish the liver-specific GS KO mouse as a model with which to study effects of chronic hyperammonemia.
Assuntos
Glutamato-Amônia Ligase/metabolismo , Hiperamonemia/enzimologia , Fígado/enzimologia , Animais , Comportamento Animal , Encéfalo/metabolismo , Encéfalo/patologia , Encéfalo/fisiopatologia , Modelos Animais de Doenças , Marcação de Genes , Glutamato-Amônia Ligase/genética , Hiperamonemia/genética , Hiperamonemia/patologia , Hiperamonemia/fisiopatologia , Fígado/metabolismo , Fígado/fisiopatologia , Locomoção , Memória , Camundongos , Camundongos Knockout , Estresse Oxidativo/genéticaRESUMO
Hepatic encephalopathy (HE) is a frequent complication of liver cirrhosis and is due to a low-grade cerebral edema associated with oxidative/nitrosative stress. Recent reports suggest that cognitive impairment in cirrhotic patients may not resolve completely after an attack of manifest HE. As astrocyte dysfunction is central to the pathogenesis of HE and astrocytes are critically involved in synaptic plasticity, we tested for sustained impairment of astrocyte function by analyzing expression levels of senescence biomarkers in ammonia-treated cultured rat astrocytes and in postmortem brain samples from cirrhotic patients with or without HE. NH4 Cl time- and dose-dependently inhibited proliferation of cultured astrocytes by up to 45% (5 mmol/L, 72 h) and strongly increased senescence-associated ß-galactosidase activity. Inhibition of astrocyte proliferation by ammonia was mediated by a l-methionine sulfoximine-, oxidative stress-, and p38(MAPK) -dependent activation of p53 associated with enhanced transcription of cell cycle inhibitory genes GADD45α and p21. Mitochondria and the nucleus were identified as sources of oxygen radical formation after prolonged NH4 Cl exposure. Concurrently, NH4 Cl (5 mmol/L) treatment inhibited both epidermal growth factor- and brain-derived neurotrophic factor (BDNF)-induced proliferation as well as BDNF-mediated astrocyte morphology changes through downregulation of the respective growth factor receptors epidermal growth factor receptor and truncated tyrosine receptor kinase B. Increased mRNA expression levels of senescence-associated genes were also found in post mortem brain samples from patients with liver cirrhosis with HE, but not in those without HE. The data suggest that ammonia toxicity and HE are associated with premature astrocyte senescence, which may impair neurotransmission and contribute to persistence of cognitive disturbances after resolution of episodes of overt HE.
Assuntos
Amônia/farmacologia , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Senescência Celular/efeitos dos fármacos , Córtex Cerebral/efeitos dos fármacos , Encefalopatia Hepática/tratamento farmacológico , Encefalopatia Hepática/metabolismo , Amônia/metabolismo , Animais , Edema Encefálico/etiologia , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Células Cultivadas , Córtex Cerebral/metabolismo , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismoRESUMO
PURPOSE: A disturbed balance between excitatory and inhibitory neurotransmission underlies epileptic activity, although reports concerning neurotransmitter systems involved remain controversial. METHODS: We quantified densities of 15 receptors in neocortical biopsies from patients with pharmacoresistant focal temporal lobe epilepsy and autopsy controls, and searched for correlations between density alterations and clinical factors or the occurrence of spontaneous synaptic potentials in vitro. KEY FINDINGS: α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainate, N-methyl-d-aspartate (NMDA), peripheral benzodiazepine, muscarinic (M)(1) , M(2) , nicotinic, α(1) , α(2h) , serotonin (5-HT)(1A) , and adenosine (A)(1) receptor densities were significantly altered in biopsies. The epileptic cohort was subdivided based on clinical (febrile seizures, hippocampal sclerosis, neocortical pathologies, surgery outcome) or electrophysiologic (spontaneous field potentials) criteria, resulting in different patterns of significantly altered receptor types when comparing a given epileptic group with controls. Only AMPA, kainate, M(2) , and 5-HT(1A) receptors were always significantly altered. γ-Aminobutyric acid (GABA)(A) , GABA(B) , and 5-HT(2) receptor alterations were never significant. Correlation patterns between receptor alterations and illness duration or seizure frequency varied depending on whether the epileptic cohort was considered as a whole or subdivided. SIGNIFICANCE: Neocortical temporal lobe epilepsy is associated with a generalized receptor imbalance resulting in a net potentiation of excitatory neurotransmission. Peripheral benzodiazepine receptor alterations highlight that astrocytes are also impaired by seizure activity.
Assuntos
Epilepsias Parciais/metabolismo , Neocórtex/metabolismo , Receptores de Superfície Celular/metabolismo , Adulto , Idoso , Estudos de Coortes , Eletroencefalografia/métodos , Epilepsias Parciais/fisiopatologia , Feminino , Humanos , Ligantes , Masculino , Pessoa de Meia-Idade , Ligação Proteica/fisiologia , Adulto JovemRESUMO
Ammonia is a major player in the pathogenesis of hepatic encephalopathy (HE) and affects astrocyte function by triggering a self-amplifying cycle between osmotic and oxidative stress. We recently demonstrated that hypoosmotic astrocyte swelling rapidly stimulates nitric oxide (NO) production and increases intracellular free Zn(2+) concentration ([Zn(2+)](i)). Here we report effects of ammonia on [Zn(2+)](i) homeostasis and NO synthesis. In cultured rat astrocytes, NH(4)Cl (5 mm) increased within 6 h both cytosolic and mitochondrial [Zn(2+)]. The [Zn(2+)](i) increase was transient and was mimicked by the nonmetabolizable CH(3)NH(3)Cl, and it was dependent on NO formation, as evidenced by the sensitivity toward the nitric oxide synthase inhibitor N(G)-monomethyl-l-arginine. The NH(4)Cl-induced NO formation was sensitive to the Ca(2+) chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester and increases in both NO and [Zn(2+)](i) were blocked by the N-methyl-d-aspartate receptor antagonist MK-801. The NH(4)Cl-triggered increase in [Zn(2+)](i) was followed by a Zn(2+)-dependent nuclear appearance of the metal response element-binding transcription factor and metallothionein messenger RNA (mRNA) induction. Metallothionein mRNA was also increased in vivo in rat cerebral cortex 6 h after an NH(4)Ac challenge. NH(4)Cl increased peripheral-type benzodiazepine receptor (PBR) protein expression, whereas PBR mRNA levels were decreased in a Zn(2+)-independent manner. The Zn(2+)-dependent upregulation of metallothionein following ammonia intoxication may reflect a cytoprotective response, whereas the increase in PBR expression may augment HE development.
Assuntos
Amônia/farmacologia , Astrócitos/efeitos dos fármacos , Metalotioneína/genética , Óxido Nítrico/metabolismo , Regulação para Cima/efeitos dos fármacos , Zinco/metabolismo , Animais , Astrócitos/metabolismo , Células Cultivadas , Masculino , RNA Mensageiro/genética , Ratos , Ratos WistarRESUMO
TGR5 (Gpbar-1) is a membrane-bound bile acid receptor in the gastrointestinal tract and immune cells with pleiotropic actions. As shown in the present study, TGR5 is also expressed in astrocytes and neurons. Here, TGR5 may act as a neurosteroid receptor, which is activated by nanomolar concentrations of 5ß-pregnan-3α-ol-20-one and micromolar concentrations of 5ß-pregnan-3α-17α-21-triol-20-one and 5α-pregnan-3α-ol-20-one (allopregnanolone). TGR5 stimulation in astrocytes and neurons is coupled to adenylate cyclase activation, elevation of intracellular Ca(2+) and the generation of reactive oxygen species. In cultured rat astrocytes, TGR5 mRNA is downregulated in the presence of neurosteroids and ammonia already at concentrations of 0.5 mmol L(-1). Furthermore, TGR5 protein levels are significantly reduced in isolated rat astrocytes after incubation with ammonia. A marked downregulation of TGR5 mRNA is also found in cerebral cortex from cirrhotic patients dying with hepatic encephalopathy (HE) when compared with brains from noncirrhotic control subjects. It is concluded that TGR5 is a novel neurosteroid receptor in brain with implications for the pathogenesis of HE.
Assuntos
Astrócitos/metabolismo , Encéfalo/metabolismo , Regulação da Expressão Gênica/fisiologia , Neurônios/metabolismo , Receptores Acoplados a Proteínas G/fisiologia , Animais , Animais Recém-Nascidos , Astrócitos/efeitos dos fármacos , Encéfalo/citologia , Proteína de Ligação a CREB/metabolismo , Cálcio/metabolismo , Células Cultivadas , Colagogos e Coleréticos/farmacologia , Técnicas de Cocultura , Colforsina/farmacologia , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Proteínas Luminescentes , Proteínas Associadas aos Microtúbulos/metabolismo , Neurônios/efeitos dos fármacos , Neurotransmissores/farmacologia , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/genética , Estatísticas não Paramétricas , Ácido Taurolitocólico/farmacologia , Transfecção/métodosRESUMO
UNLABELLED: Oxidative stress plays a major role in cerebral ammonia toxicity and the pathogenesis of hepatic encephalopathy (HE). As shown in this study, ammonia induces a rapid RNA oxidation in cultured rat astrocytes, vital mouse brain slices, and rat brain in vivo. Ammonia-induced RNA oxidation in cultured astrocytes is reversible and sensitive to MK-801, 1,2-Bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, apocynin, epigallocatechin gallate, and polyphenon 60, suggesting the involvement of N-methyl-D-aspartic acid (NMDA) receptor activation, Ca(2+), nicotinamide adenine dinucleotide phosphate, and reduced form (NADPH) oxidase-dependent oxidative stress. Also, hypo-osmolarity, tumor necrosis factor alpha (TNF-alpha), and diazepam increase RNA oxidation in cultured astrocytes, suggesting that the action of different HE-precipitating factors converges at the level of RNA oxidation. Among the oxidized RNA species, 18S-rRNA and the messenger RNA (mRNA) coding for the glutamate/aspartate transporter (GLAST) were identified. Cerebral RNA oxidation in acutely ammonia-loaded rats in vivo is reversible and predominates in neuronal soma and perivascular astrocyte processes. In neuronal dendrites, oxidized RNA colocalizes with the RNA-binding splicing protein neurooncological ventral antigen (NOVA)-2 within putative RNA transport granules, which are also found in close vicinity to postsynaptic spines. This indicates that oxidized RNA species may participate in postsynaptic protein synthesis, which is a biochemical substrate for learning and memory consolidation. Neuronal and astroglial RNA oxidation increases also in vital mouse brain slices treated with ammonia and TNF-alpha, respectively. CONCLUSION: Cerebral RNA oxidation is identified as a not yet recognized consequence of acute ammonia intoxication. RNA oxidation may affect gene expression and local protein synthesis and thereby provide another link between reactive oxygen species (ROS)/reactive nitrogen oxide species (RNOS) production and ammonia toxicity.
Assuntos
Amônia/farmacologia , Astrócitos/metabolismo , Encéfalo/metabolismo , RNA/metabolismo , Sistema X-AG de Transporte de Aminoácidos/genética , Animais , Astrócitos/efeitos dos fármacos , Encéfalo/citologia , Células Cultivadas , Dendritos/metabolismo , Diazepam/farmacologia , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Concentração Osmolar , Oxirredução/efeitos dos fármacos , Transporte de RNA , RNA Mensageiro/metabolismo , RNA Ribossômico 18S/metabolismo , Ratos , Ratos Wistar , Distribuição Tecidual , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
PURPOSE: The astrocyte-specific glutamine synthetase (GS) plays a key role in glutamate recycling and Gamma-aminobutyric acid (GABA) metabolism. Changes in the expression or activity of GS have been proposed to contribute to epileptogenesis. The mechanisms or how and where GS may contribute to epilepsy is still a matter of discussion. Here we asked the question whether brain regions, which show an astrocytic stress response respond with alterations of GS. METHODS: Biochemical and histological alterations of GS, HSP-27, and GFAP were studied after pentylenetetrazole-induced repetitive epileptic seizures (PIRS) in rats using a topographical quantification of the GS-immunoreactivity (GSIR) in relation to the focal heat shock response (HSR). Saline-treated rats served as controls and rats treated by the GS-inhibitor, L-methionine-sulfoximine (MSO) served as a positive control. RESULTS: No changes in the amount of GSIR and GS-protein occurred during PIRS. A significant reduction of GSIR was observed by histochemistry (in situ) and in native (nonheated) protein extracts of MSO-treated rats. In rats affected by PIRS, GS-activity showed a significant, region-specific reduction in association with a nitration of the enzyme. DISCUSSION: These results show that neither PIRS nor GS-inhibition reduced the amount of GS protein, but that MSO interferes with antibody binding to native GS. PIRS resulted in a focal increase of astrocytic stress response, whereas MSO caused a widespread, homogeneous astrocytic HSR independent from quantitative changes of GS content. In rats with PIRS the regions showing a strong glial HSR, respond with reduced GS-activity and GS-nitration, which all together are clear indicators of a nitrosative stress response.
Assuntos
Epilepsia/induzido quimicamente , Epilepsia/metabolismo , Epilepsia/fisiopatologia , Glutamato-Amônia Ligase/metabolismo , Pentilenotetrazol , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Modelos Animais de Doenças , Epilepsia/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Proteína Glial Fibrilar Ácida/metabolismo , Proteínas de Choque Térmico HSP27/metabolismo , Imunoprecipitação/métodos , Masculino , Modelos Biológicos , Ratos , Ratos WistarRESUMO
The devastating consequences of hepatic failure include hepatic encephalopathy, a severe, life threatening impairment of neuronal function. Hepatic encephalopathy is caused by impaired hepatic clearance of NH4+. Cellular NH4+ uptake is accomplished mainly by the Na+,K+,2Cl- cotransporter. Here we show that hepatic clearance of NH4+ is impaired in TNFα deficient as well as TNFR1&TNFR2 double knockout mice, which both develop hyperammonemia. Despite impaired hepatic clearance of NH4+, TNFα deficient mice and TNFR1 deficient mice were protected against acute ammonia intoxication. While 54% of the wild-type mice and 60% of TNFR2 deficient mice survived an NH4+ load, virtually all TNFα deficient mice and TNFR1 deficient mice survived the treatment. Conversely, TNFα treatment of wild type mice sensitized the animals to the toxic effects of an NH4+ load. The protection of TNFα-deficient mice against an NH4+ load was paralleled by decreased cerebral expression of NKCC1. According to the present observations, inhibition of TNFα formation and/or NKCC1 may be strategies to favorably influence the clinical course of hepatic encephalopathy.
Assuntos
Hiperamonemia/genética , Receptores Tipo II do Fator de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Fator de Necrose Tumoral alfa/deficiência , Amônia/toxicidade , Animais , Encéfalo/metabolismo , Hiperamonemia/metabolismo , Fígado/química , Camundongos , Camundongos Knockout , Membro 2 da Família 12 de Carreador de Soluto/genética , Fator de Necrose Tumoral alfa/genética , Regulação para CimaRESUMO
The inducible prostaglandin synthase, cyclooxygenase-2, is upregulated in response to cerebral ischemia and contributes to potentiation of oxidative injury. Cyclooxygenase-2 expression is regulated by retinoic acid receptors, which form heterodimers with vitamin D receptors and vitamin D. In addition, vitamin D has been reported to have neuroprotective qualities. The aim of this study was to examine whether the biologically active vitamin D3-metabolite 1alpha,25-dihydroxyvitamin D3 (1,25-D3), influences the expression of inducible cyclooxygenase-2 in photothrombotically lesioned brain or is part of an independent neuroprotective mechanism. We compared groups of nonlesioned control rats and infarcted animals, which were treated with either 1,25-D3 or solvent at different times postlesion. In control animals, cyclooxygenase-2 immunoreactivity was readily evident in almost all cortical neurons of layers II/III as well as in a few pyramidal cells in layer V. Following photothrombotic infarction of the right cortical hindlimb area, there was a significant, but transient, increase in cyclooxygenase-2 labeling which was restricted to neurons of the injured hemisphere in both 1,25- D3-treated and solvent-treated rats. Highest levels of cyclooxygenase-2 immunoreactivity were seen at 12 and 24 h postlesion, followed by a gradual decrease at later time points. However, no significant differences were detected between 1,25-D3-treated and solvent-treated lesioned rats, indicating that postischemic neuronal cyclooxygenase-2 upregulation is not influenced by 1,25-D3. It is concluded that the neuroprotective effect of 1,25-D3 does not depend on modulations of neuronal COX-2 expression caused by postlesional hyperexcitation.
Assuntos
Calcitriol/farmacologia , Córtex Cerebral/enzimologia , Ciclo-Oxigenase 2/metabolismo , Trombose Intracraniana/tratamento farmacológico , Trombose Intracraniana/enzimologia , Animais , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/patologia , Modelos Animais de Doenças , Técnicas Imunoenzimáticas , Trombose Intracraniana/patologia , Masculino , Ratos , Ratos WistarRESUMO
Hepatic encephalopathy is a neuropsychiatric syndrome evolving from cerebral osmotic disturbances and oxidative/nitrosative stress. Ammonia, the main toxin of hepatic encephalopathy, triggers astrocyte senescence in an oxidative stress-dependent way. As miRNAs are critically involved in cell cycle regulation and their expression may be regulated by oxidative stress, we analysed, whether astrocyte senescence is a consequence of ammonia-induced miRNA expression changes. Using a combined miRNA and gene microarray approach, 43 miRNA species which were downregulated and 142 genes which were upregulated by NH4Cl (5 mmol/l, 48 h) in cultured rat astrocytes were found. Ammonia-induced miRNA and gene expression changes were validated by qPCR and 43 potential miRNA target genes, including HO-1, were identified by matching upregulated mRNA species with predicted targets of miRNA species downregulated by ammonia. Inhibition of HO-1 targeting miRNAs which were downregulated by NH4Cl strongly upregulated HO-1 mRNA and protein levels and inhibited astrocyte proliferation in a HO-1-dependent way. Preventing ammonia-induced upregulation of HO-1 by taurine (5 mmol/l) as well as blocking HO-1 activity by tin-protoporphyrine IX fully prevented ammonia-induced proliferation inhibition and senescence. The data suggest that ammonia induces astrocyte senescence through NADPH oxidase-dependent downregulation of HO-1 targeting miRNAs and concomitant upregulation of HO-1 at both mRNA and protein level.
Assuntos
Amônia/farmacologia , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , MicroRNAs/genética , Animais , Proliferação de Células , Células Cultivadas , Senescência Celular/efeitos dos fármacos , Senescência Celular/genética , Análise por Conglomerados , Perfilação da Expressão Gênica , Glutamina/biossíntese , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , NADPH Oxidases/metabolismo , Ratos , Reprodutibilidade dos TestesRESUMO
To date, unequivocal neuroanatomical features have been demonstrated neither for sporadic nor for familial schizophrenia. Here, we investigated the neuroanatomical changes in a transgenic rat model for a subset of sporadic chronic mental illness (CMI), which modestly overexpresses human full-length, non-mutant Disrupted-in-Schizophrenia 1 (DISC1), and for which aberrant dopamine homeostasis consistent with some schizophrenia phenotypes has previously been reported. Neuroanatomical analysis revealed a reduced density of dopaminergic neurons in the substantia nigra and reduced dopaminergic fibres in the striatum. Parvalbumin-positive interneuron occurrence in the somatosensory cortex was shifted from layers II/III to V/VI, and the number of calbindin-positive interneurons was slightly decreased. Reduced corpus callosum thickness confirmed trend-level observations from in vivo MRI and voxel-wise tensor based morphometry. These neuroanatomical changes help explain functional phenotypes of this animal model, some of which resemble changes observed in human schizophrenia post mortem brain tissues. Our findings also demonstrate how a single molecular factor, DISC1 overexpression or misassembly, can account for a variety of seemingly unrelated morphological phenotypes and thus provides a possible unifying explanation for similar findings observed in sporadic schizophrenia patients. Our anatomical investigation of a defined model for sporadic mental illness enables a clearer definition of neuroanatomical changes associated with subsets of human sporadic schizophrenia.
Assuntos
Encéfalo/patologia , Neurônios Dopaminérgicos/fisiologia , Interneurônios/fisiologia , Proteínas do Tecido Nervoso/biossíntese , Parvalbuminas/análise , Esquizofrenia/fisiopatologia , Animais , Biometria , Modelos Animais de Doenças , Expressão Gênica , Histocitoquímica , Humanos , Imageamento por Ressonância Magnética , Masculino , Ratos Sprague-Dawley , Ratos TransgênicosRESUMO
Heat shock protein-27 (HSP-27) is an inducible stress response protein. It inhibits apoptotic cell death and is a reliable marker for oxidative stress. We studied the induction of HSP-27 in rat brains on days 1, 4 and 14 after repeated, pentylenetetrazole (PTZ)-induced seizures using immunohistochemisty. Saline treated control rats showed no induction of HSP-27. HSP-27 reactive astrocytes were rarely seen 1 or 4 days after PTZ injection. When present, single astrocytes were located in the cortex and/or the hippocampus. After 14 days PTZ treatment, a bilateral distribution of HSP-27 immunoreactive glia was present in piriform and entorhinal cortices and in the dentate gyrus of most brains. Rats with most intense HSP-27 upregulation showed HSP-27 in amygdala and thalamic nuclei. Astrocytes associated with blood vessels presented strongest HSP-27 staining, but did not show upregulation of gial fibrillary acidic protein and none responded with HSP-47 expression. Additionally, HSP-27 immunoreactivity increased in the endothelial cells of blood vessels in the affected brain regions, although no neuronal induction occurred. Contrastingly, a subconvulsive dose of the glutamine synthetase inhibitor L-methionine sulfoxime, which acts directly on astrocytes, resulted in a rapid, homogeneous astrocyte-specific HSP-27 upregulation within 24 h. Thus, repeated PTZ-induced seizure activity elicits a focal "heat shock" response in endothelial cells and astrocytes of selected cerebral regions indicating that expression of HSP-27 occurred in a seizure-dependent manner within the affected cerebral circuitries. Therefore, this PTZ-model of repeated seizure activity exhibited a cortical pattern of HSP-27 expression which is most comparable to that known from patients with epilepsy.
Assuntos
Astrócitos/metabolismo , Encéfalo/irrigação sanguínea , Encéfalo/metabolismo , Proteínas de Choque Térmico/biossíntese , Animais , Astrócitos/efeitos dos fármacos , Western Blotting , Convulsivantes/toxicidade , Modelos Animais de Doenças , Eletroencefalografia , Células Endoteliais/metabolismo , Inibidores Enzimáticos/farmacologia , Epilepsia/induzido quimicamente , Lateralidade Funcional , Proteína Glial Fibrilar Ácida/metabolismo , Glutamato-Amônia Ligase/antagonistas & inibidores , Imuno-Histoquímica , Masculino , Pentilenotetrazol/toxicidade , Ratos , Ratos Wistar , Fatores de Tempo , Regulação para CimaRESUMO
Astrocytes play a key role in the pathogenesis of ammonia-induced neurotoxicity and hepatic encephalopathy. As shown here, ammonia induces protein tyrosine nitration in cultured rat astrocytes, which is sensitive to the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801. A similar pattern of nitrated proteins is produced by NMDA. Ammonia-induced tyrosine nitration depends on a rise in [Ca2+]i, IkB degradation, and NO synthase (iNOS) induction, which are prevented by MK-801 and the intracellular Ca2+ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM). Moreover, the increase in tyrosine nitration is blunted by L-NMMA, 1400W, uric acid, Cu, Zn-superoxide dismutase/catalase treatment, and methionine-sulfoximine, which indicate the involvement of reactive nitrogen intermediates and intracellular glutamine accumulation. Such reactive nitrogen intermediates additionally mediate ammonia-induced phosphorylation of the MAP-kinases Erk-1/Erk-2 and p38MAPK. Among the proteins, which are tyrosine -nitrated by ammonia, glyceraldehyde-3-phosphate dehydrogenase, the peripheral-type benzodiazepine receptor, Erk-1, and glutamine synthetase are identified. Ammonia-induced nitration of glutamine synthetase is associated with a loss of enzymatic activity. Astroglial protein tyrosine nitration is found in brains from rats after acute ammonia-intoxication or after portacaval anastomosis, indicating the in vivo relevance of the present findings. The production of reactive nitrogen intermediates and protein tyrosine nitration may alter astrocyte function and contribute to ammonia neurotoxicity.
Assuntos
Amônia/farmacologia , Astrócitos/metabolismo , Maleato de Dizocilpina/farmacologia , Fosfoproteínas/metabolismo , Fosfotirosina/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Células Cultivadas , Sinergismo Farmacológico , Glutamato-Amônia Ligase/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Modelos Neurológicos , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase/biossíntese , Óxido Nítrico Sintase Tipo II , Fosfoproteínas/química , Fosforilação , Ratos , Receptores de GABA-A/metabolismo , Receptores de N-Metil-D-Aspartato/antagonistas & inibidoresRESUMO
In ischemic cerebral injuries a cascade of degenerative mechanisms, all participating in the development of oxidative stress, influence the condition of the tissue. The survival of viable tissue affected by secondary injury largely depends on the balance between endogenous protective mechanisms and the ongoing degenerative processes. The inducible enzyme, heme oxygenase-1 metabolizes and thus detoxifies free heme to the powerful endogenous antioxidants biliverdin and bilirubin therefore enhancing neuroprotection. The secosteroid 1alpha,25-dihydroxyvitamin D3 (1,25-D3) is a modulator of the immune system and also exhibits a strong potential for neuroprotection as recently shown in the MCAO model of cerebral ischemia. We studied the effects of 1,25-D3 treatment on heme oxygenase-1 expression following focal cortical ischemia elicited by photothrombosis. Postlesional treatment with 1,25-D3 (4 microg/kg body weight) resulted in a transient, but significant upregulation of glial heme oxygenase-1 immunoreactivity concomitant with a reduction in glial fibrillary acidic protein immunoreactivity in remote cortical regions affected by a secondary spread of injury, whereas the size of the lesion's core remained unaffected. 1,25-D3 did not produce a temporal shift or extension of injury-related heme oxygenase-1 responses, indicating that 1,25-D3 did not prolong ischemia-related heme oxygenase-1 expression. In contrast to glial heme oxygenase-1 upregulation, glial fibrillary acidic protein, a sensitive marker for reactive gliosis, was significantly reduced. These findings support an additional protective action of 1,25-D3 at the cellular level in regions affected by secondary injury-related responses.
Assuntos
Calcitriol/farmacologia , Córtex Cerebral/metabolismo , Proteína Glial Fibrilar Ácida/genética , Heme Oxigenase (Desciclizante)/genética , Trombose Intracraniana/metabolismo , Animais , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/patologia , Corantes Fluorescentes , Heme Oxigenase-1 , Trombose Intracraniana/patologia , Masculino , Ratos , Ratos Wistar , Rosa BengalaRESUMO
1alpha,25-(OH)(2)-vitamin D(3) (1,25-D(3)) and 17beta-estradiol are both known to act neuroprotectively in certain experimental in vitro and in vivo settings and it has been noted that both steroids lead to an upregulation of certain neurotrophic factors. Here, we studied the effects of 1alpha,25-(OH)(2)-vitamin D(3) or 17beta-estradiol or their combined application on heat shock protein-32 (HSP-32) distribution after focal cortical ischemia using the well established photothrombosis model. Heat shock protein-32 is a well-established marker of the cerebral oxidative stress response and contributes to neuroprotection by metabolising cytotoxic free heme to carbon monoxide, iron and biliverdin. Photothrombotically lesioned rats were injected i.p. 1h after injury with either 1 microg 1alpha,25-(OH)(2)-vitamin D(3)/kg or 7 microg 17beta-estradiol/kg or a combination of both steroids. Groups of non-lesioned steroid-treated rats and lesioned, solvent-treated rats served as controls. In contrast to non-lesioned rats, in lesioned animals a significant increase in heat shock protein-32 expression occurred which was slightly, but non-significantly altered in the groups treated either with 1alpha,25-(OH)(2)-vitamin D(3) or 17beta-estradiol alone when compared to the solvent-treated control group. Only the combined treatment with 1alpha,25-(OH)(2)-vitamin D(3) and 17beta-estradiol resulted in a significant reduction of glial heat shock protein-32 immunoreactivity within the lesion-remote cortical areas supplied by the affected middle cerebral artery (MCA), indicating that both steroids act synergistically in a protective manner.
Assuntos
Isquemia Encefálica/metabolismo , Calcitriol/farmacologia , Estradiol/farmacologia , Proteínas de Choque Térmico/metabolismo , Oxigenases , Animais , Heme Oxigenase (Desciclizante) , Imuno-Histoquímica , Masculino , Ratos , Ratos WistarRESUMO
Soluble guanylyl cylase (sGC) has been identified for being a receptor for the gaseous transmitters nitric oxide and carbon monoxide. Currently four subunits alpha1, alpha2, beta1, and beta2 have been characterized. Heterodimers of alpha and beta-subunits as well as homodimers of the beta2-subunit are known to constitute functional sGC which use GTP to form cGMP a potent signal molecule in a multitude of second messenger cascades. Since NO-cGMP signaling plays a pivotal role in neuronal development we analyzed the maturational expression pattern of the newly characterized alpha2-subunit of sGC within the brain of Wistar rats by means of RNase protection assay and immunohistochemistry. alpha2-subunit mRNA as well as immunoreactive alpha2-protein increased during postnatal cerebral development. Topographical analysis revealed a selective high expression of the alpha2-subunit in the choroid plexus and within developing sensory systems involving the olfactory and somatosensory system of the forebrain as well as parts of the auditory and visual system within the hindbrain. In cultured cortical neurons the alpha2-subunit was localized to the cell membrane, especially along neuronal processes. During the first 11 days of postnatal development several cerebral regions showed a distinct expression of the alpha2-subunit which was not paralleled by the alpha1/beta1-subunits especially within the developing thalamo-cortical circuitries of the somatosensory system. However, at later developmental stages all three subunits became more homogenously distributed among most cerebral regions, indicating that functional alpha1/beta1 and alpha2/beta1 heterodimers of sGC could be formed. Our findings indicate that the alpha2-subunit is an essential developmentally regulated constituent of cerebral sensory systems during maturation. In addition the alpha2-subunit may serve other functions than forming a functional heterodimer of sGC during the early phases of sensory pathway refinement.
Assuntos
Encéfalo/enzimologia , Encéfalo/crescimento & desenvolvimento , Vias Neurais/crescimento & desenvolvimento , Receptores Citoplasmáticos e Nucleares/biossíntese , Envelhecimento/fisiologia , Animais , Encéfalo/citologia , Núcleo Celular/enzimologia , Células Cultivadas , Guanilato Ciclase , Imuno-Histoquímica , Masculino , Vias Neurais/citologia , Neurônios/enzimologia , Neurópilo/enzimologia , Óxido Nítrico Sintase/biossíntese , Óxido Nítrico Sintase Tipo II , Ensaios de Proteção de Nucleases , Sondas RNA , RNA Mensageiro/biossíntese , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Guanilil Ciclase SolúvelRESUMO
The purpose of the present study was to determine whether a unilateral photothrombotic brain lesion induces bilateral ischemic tolerance towards a subsequent severe ischemia performed 5 days later. Severe ischemia was induced by transient (1h; t) or permanent (p) occlusion of the middle cerebral artery (MCAO). Rats were sacrificed 24h later. Preconditioning reduced the size of subsequent infarcts in both hemispheres. This effect was most prominent with tMCAO, and ipsilateral preconditioning was more effective than contralateral preconditioning (% of hemispheric volume, mean ± SD: 31.9 ± 3.7 to 19.0 ± 10.3 with ipsilateral tMCAO; 31.9 ± 3.7 to 22.9 ± 4.9 with contralateral tMCAO; 64.7 ± 4.3% to 47.2 ± 12.5% with ipsilateral pMCAO; 64.7 ± 4.3% to 53.1 ± 8.9% with contralateral pMCAO). Ischemic preconditioning was associated with a successive bilateral up-regulation of superoxide dismutases which may be involved in the development of ischemic tolerance. Our data suggest that a focal ischemic brain lesion induces neuroprotective mechanisms in extensive brain areas and thus cause bilateral ischemic tolerance within a certain time window.
Assuntos
Isquemia Encefálica/fisiopatologia , Encéfalo/fisiopatologia , Lateralidade Funcional/fisiologia , Infarto da Artéria Cerebral Média/fisiopatologia , Precondicionamento Isquêmico/métodos , Animais , Encéfalo/patologia , Isquemia Encefálica/patologia , Modelos Animais de Doenças , Imuno-Histoquímica , Infarto da Artéria Cerebral Média/patologia , Masculino , Estimulação Luminosa , Distribuição Aleatória , Ratos Wistar , Índice de Gravidade de Doença , Superóxido Dismutase/metabolismo , Trombose , Regulação para Cima/fisiologia , Redução de PesoRESUMO
The role of NADPH oxidase (NOX) and the regulatory subunit p47(phox) for hypoosmotic ROS generation was studied in cultured rat astrocytes and brain slices of wilde type and p47(phox) knock-out mice. Cultured rat astrocytes express mRNAs encoding for the regulatory subunit p47(phox), NOX1, 2, and 4, and the dual oxidases (DUOX)1 and 2, but not NOX3. Hypoosmotic (205 mosmol/L) swelling of cultured astrocytes induced a rapid generation of ROS that was accompanied by serine phosphorylation of p47(phox) and prevented by the NADPH oxidase inhibitor apocynin. Apocynin also impaired the hypoosmotic tyrosine phosphorylation of Src. Both, hypoosmotic ROS generation and p47(phox) serine phosphorylation were sensitive to the acidic sphingomyelinase inhibitors AY9944 and desipramine, the protein kinase C (PKC)zeta-inhibitory pseudosubstrate peptide, the NMDA receptor antagonist MK-801 and the intracellular Ca(2+) chelator BAPTA-AM. Also hypoosmotic exposure of wilde type mouse cortical brain slices increased ROS generation, which was allocated in part to the astrocytes and which was absent in presence of apocynin and in cortical brain slices from p47(phox) knock-out mice. Also ammonia induced a rapid ROS production in cultured astrocytes and brain slices, which was sensitive to apocynin. The data suggest that astrocyte swelling triggers a p47(phox)-dependent NADPH oxidase-catalyzed ROS production. The findings further support a close interrelation between osmotic and oxidative stress in astrocytes, which may be relevant to different brain pathologies including hepatic encephalopathy.
Assuntos
Edema Encefálico/metabolismo , Encefalopatia Hepática/metabolismo , Hiperamonemia/metabolismo , NADPH Oxidases/metabolismo , Estresse Oxidativo , Amônia/metabolismo , Amônia/toxicidade , Animais , Animais Recém-Nascidos , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/fisiopatologia , Edema Encefálico/fisiopatologia , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Encefalopatia Hepática/fisiopatologia , Hiperamonemia/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADPH Oxidases/genética , Técnicas de Cultura de Órgãos , Pressão Osmótica , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Equilíbrio Hidroeletrolítico/efeitos dos fármacos , Equilíbrio Hidroeletrolítico/fisiologiaRESUMO
Nitric-oxide-sensitive guanylyl cyclase (NO-sGC) plays a pivotal role in many second messenger cascades. Neurotransmission- and neuropathology-related changes in NO-sGC have been suggested. However, the cellular localization of NO-sGC in primate brains, including humans, remains unknown. Biochemical evidence has linked the alpha(2)-subunit of NO-sGC directly to neurotransmission in rodents. Here, we have used a recently characterized subunit-specific antibody for the localization of the alpha(2)-subunit on sections from the cerebelli of the common marmoset (Callithrix jacchus; New World monkey) and macaque monkeys (Macaca mulatta, M. fascicularis; Old World monkeys). In contrast to the more ubiquitous cytoplasmic presence of subunit-beta(1), the alpha(2)-subunit is mainly confined to the somato-dendritic membrane including the spines of the Purkinje cells. Only limited colocalization with presynaptically localized synaptophysin has been seen under our staining conditions, indicating a higher abundance of subunit-alpha(2) at the postsynaptic site. This localization indicates that subunit-alpha(2) links NO-sGC to neurotransmission, whereas subunit-beta(1) may act as a cytoplasmic regulator/activator by contributing to active heterodimer formation via translocation from the cytoplasm to the cell membrane. The last-mentioned action may be a prerequisite for generating nitric-oxide-dependent, subcellular, and postsynaptically localized cGMP signals along neuronal processes.