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Biochim Biophys Acta Mol Basis Dis ; 1866(1): 165587, 2020 01 01.
Article En | MEDLINE | ID: mdl-31678158

Mechanisms whereby fibrillin-1 mutations determine thoracic aorta aneurysms/dissections (TAAD) in Marfan Syndrome (MFS) are unclear. Most aortic aneurysms evolve from mechanosignaling deregulation, converging to impaired vascular smooth muscle cell (VSMC) force-generating capacity accompanied by synthetic phenotype switch. However, little is known on VSMC mechanoresponses in MFS pathophysiology. Here, we investigated traction force-generating capacity in aortic VSMC cultured from 3-month old mg∆lpn MFS mice, together with morpho-functional and proteomic data. Cultured MFS-VSMC depicted marked phenotype changes vs. wild-type (WT) VSMC, with overexpressed cell proliferation markers but either lower (calponin-1) or higher (SM alpha-actin and SM22) differentiation marker expression. In parallel, the increased cell area and its complex non-fusiform shape suggested possible transition towards a mesenchymal-like phenotype, confirmed through several markers (e.g. N-cadherin, Slug). MFS-VSMC proteomic profile diverged from that of WT-VSMC particularly regarding lower expression of actin cytoskeleton-regulatory proteins. Accordingly, MFS-VSMC displayed lower traction force-generating capacity and impaired contractile moment at physiological substrate stiffness, and markedly attenuated traction force responses to enhanced substrate rigidity. Such impaired mechanoresponses correlated with decreased number, altered morphology and delocalization of focal adhesions, as well as disorganized actin stress fiber network vs. WT-VSMC. In VSMC cultured from 6-month-old mice, phenotype changes were attenuated and both WT-VSMC and MFS-VSMC generated less traction force, presumably involving VSMC aging, but without evident senescence. In summary, MFS-VSMC display impaired force-generating capacity accompanying a mesenchymal-like phenotype switch connected to impaired cytoskeleton/focal adhesion organization. Thus, MFS-associated TAAD involves mechanoresponse impairment common to other TAAD types, but through distinct mechanisms.

Marfan Syndrome/pathology , Muscle, Smooth, Vascular/pathology , Myocytes, Smooth Muscle/pathology , Actins/metabolism , Animals , Aorta/metabolism , Aorta/pathology , Aortic Aneurysm/metabolism , Aortic Aneurysm/pathology , Biomarkers/metabolism , Cell Differentiation/physiology , Cell Proliferation/physiology , Cells, Cultured , Cytoskeleton/metabolism , Cytoskeleton/pathology , Disease Models, Animal , Female , Fibrillin-1/metabolism , Focal Adhesions/metabolism , Focal Adhesions/pathology , Male , Marfan Syndrome/metabolism , Mice , Mice, Inbred C57BL , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/metabolism , Phenotype , Proteomics/methods
Rev. Soc. Cardiol. Estado de Säo Paulo ; 28(1): 42-46, jan.-mar. 2018. ilus
Article En, Pt | LILACS | ID: biblio-906720

Estresse oxidativo é um conceito em evolução. Muito mais do que um simples desbalanço entre oxidantes e antioxidantes, representa um desequilíbrio de vias de sinalização celular redox. Sinalização redox envolve a produção de espécies reativas de oxigênio, radicalares e não radicalares, assim como vias não radicalares de oxidação por dois elétrons. Está claro que essas espécies podem ser produzidas por mecanismos enzimáticos ancestrais e ubíquos e, portanto, não representam acidentes, tendo vários efeitos fisiológicos benéficos. Novas vias de sinalização redox têm sido descritas, com ênfase no papel de peroxiredoxinas, outras tiolproteínas e estados intermediários de oxidação de tióis. Estes fatos levaram à necessidade de reavaliar a definição e a abrangência do conceito de antioxidantes. Apesar da ineficácia dos estudos clínicos com antioxidantes clássicos, há novas perspectivas, que incluem inibidores de enzimas, geradores de geram oxidantes, como NADPH oxidases e vias mitocondriais. Além disso, o uso de flavonóides e compostos relacionados, capazes de ativar vias horméticas de proteção antioxidante, é um caminho importante. Esses fatos indicam que a ciência da área deve entrar em um novo ciclo de estudos para testar novas intervenções clínicas resultantes desses avanços

Oxidative stress is an evolving concept. Much more than a simple imbalance between oxidants and antioxidants, it represents an imbalance of the cellular redox signaling pathways. Redox signaling involves the production of reactive oxygen species, both radicular and non-radicular, as well as non-radicular two-electron oxidation pathways. It is clear that these species can be produced by ancestral and ubiquitous enzymatic mechanisms, therefore they do not represent accidents, and have various beneficial physiological effects. New redox signaling pathways have been described, with emphasis on the role of peroxiredoxins, other protein thiols, and intermediary states of thiol oxidation. These facts have created a need to reassess the definition and scope of the concept of antioxidants. Despite the inefficiency of clinical studies with classic antioxidants, there are new perspectives, which include oxidant-producing enzyme inhibitors, such as NADPH oxidases and mitochondrial pathways. Furthermore, the use of flavonoids and related compounds, capable of activating hormetic pathways of antioxidant protection, is an important route. These facts indicate that the science in the area should enter a new cycle of studies, testing new clinical interventions resulting from these advances

Humans , Male , Female , Endothelium, Vascular , Cardiovascular Diseases/physiopathology , Oxidative Stress/physiology , Oxidation-Reduction , Flavonoids , Enzymes , Atherosclerosis , Inflammation , Antioxidants
Mol Neurobiol ; 54(8): 5752-5767, 2017 10.
Article En | MEDLINE | ID: mdl-27660266

In the present work, we focused on mechanisms of methylmercury (MeHg) toxicity in primary astrocytes and neurons of rats. Cortical astrocytes and neurons exposed to 0.5-5 µM MeHg present a link among morphological alterations, glutathione (GSH) depletion, glutamate dyshomeostasis, and cell death. Disrupted neuronal cytoskeleton was assessed by decreased neurite length and neurite/neuron ratio. Astrocytes presented reorganization of actin and glial fibrillary acidic protein (GFAP) networks and reduced cytoplasmic area. Glutamate uptake and Na+K+ATPase activity in MeHg-treated astrocytes were preserved; however, downregulated EAAC1-mediated glutamate uptake was associated with impaired Na+K+ATPase activity in neurons. Oxidative imbalance was found in astrocytes and neurons through increased 2'7'-dichlorofluorescein (DCF) production and misregulated superoxide dismutase (SOD), catalase (CAT), and glutathione reductase (GPX) activities. Glutathione (GSH) levels were downregulated in both astrocytes and neurons. MeHg reduced neuronal viability and induced caspase 3-dependent apoptosis together with downregulated PI3K/Akt pathway. In astrocytes, necrotic death was associated with increased TNF-α and JNK/MAPK activities. Cytoskeletal remodeling and cell death were fully prevented in astrocytes and neurons by GSH, but not melatonin or Trolox supplementation. These findings support a role for depleted GSH in the cytotoxicity of MeHg leading to disruption of the cytoskeleton and cell death. Moreover, in neurons, glutamate antagonists also prevented cytoskeletal disruption and neuronal death. We propose that cytoskeleton is an end point in MeHg cytotoxicity. Oxidative imbalance and glutamate mechanisms mediate MeHg cytoskeletal disruption and apoptosis in neurons. Otherwise, redox imbalance and glutamate-independent mechanisms disrupted the cytoskeleton and induced necrosis in MeHg-exposed astrocyte.

Astrocytes/drug effects , Cytoskeleton/drug effects , Methylmercury Compounds/pharmacology , Neurons/metabolism , Animals , Animals, Newborn , Astrocytes/metabolism , Cells, Cultured , Cytoskeleton/metabolism , Female , Neurons/drug effects , Oxidative Stress/drug effects , Phosphatidylinositol 3-Kinases/metabolism , Rats, Wistar , Reactive Oxygen Species/metabolism
Mol Neurobiol ; 53(6): 3586-3595, 2016 08.
Article En | MEDLINE | ID: mdl-26099308

3-Hydroxy-3-methylglutaric aciduria (HMGA) is an inherited metabolic disorder caused by 3-hydroxy-3-methylglutaryl-CoA lyase deficiency. It is biochemically characterized by predominant tissue accumulation and high urinary excretion of 3-hydroxy-3-methylglutarate (HMG) and 3-methylglutarate (MGA). Affected patients commonly present acute symptoms during metabolic decompensation, including vomiting, seizures, and lethargy/coma accompanied by metabolic acidosis and hypoketotic hypoglycemia. Although neurological manifestations are common, the pathogenesis of brain injury in this disease is poorly known. Astrocytes are important for neuronal protection and are susceptible to damage by neurotoxins. In the present study, we investigated the effects of HMG and MGA on important parameters of redox homeostasis and cytokine production in cortical cultured astrocytes. The role of the metabolites on astrocyte mitochondrial function (thiazolyl blue tetrazolium bromide (MTT) reduction) and viability (propidium iodide incorporation) was also studied. Both organic acids decreased astrocytic mitochondrial function and the concentrations of reduced glutathione without altering cell viability. In contrast, they increased reactive species formation (2'-7'-dichlorofluorescein diacetate (DCFHDA) oxidation), as well as IL-1ß, IL-6, and TNF α release through the ERK signaling pathway. Taken together, the data indicate that the principal compounds accumulating in HMGA induce a proinflammatory response in cultured astrocytes that may possibly be involved in the neuropathology of this disease.

Acetyl-CoA C-Acetyltransferase/deficiency , Amino Acid Metabolism, Inborn Errors/metabolism , Astrocytes/metabolism , Astrocytes/pathology , Cerebral Cortex/pathology , Cytokines/metabolism , Inflammation/pathology , MAP Kinase Signaling System , Metabolome , Acetyl-CoA C-Acetyltransferase/metabolism , Amino Acid Metabolism, Inborn Errors/complications , Amino Acid Metabolism, Inborn Errors/pathology , Animals , Antioxidants/metabolism , Astrocytes/drug effects , Cell Shape/drug effects , Cells, Cultured , Flavonoids/pharmacology , Gliosis/metabolism , Gliosis/pathology , Heme Oxygenase (Decyclizing)/antagonists & inhibitors , Heme Oxygenase (Decyclizing)/metabolism , Inflammation/complications , Inflammation/metabolism , MAP Kinase Signaling System/drug effects , Male , Meglutol/analogs & derivatives , Meglutol/metabolism , Metabolome/drug effects , Mitochondria/drug effects , Mitochondria/metabolism , NF-kappa B/metabolism , Protoporphyrins/pharmacology , Rats, Wistar , Reactive Oxygen Species/metabolism
Alcohol ; 49(7): 665-74, 2015 Nov.
Article En | MEDLINE | ID: mdl-26314629

Ethanol exposure to offspring during pregnancy and lactation leads to developmental disorders, including central nervous system dysfunction. In the present work, we have studied the effect of chronic ethanol exposure during pregnancy and lactation on the phosphorylating system associated with the astrocytic and neuronal intermediate filament (IF) proteins: glial fibrillary acidic protein (GFAP), and neurofilament (NF) subunits of low, medium, and high molecular weight (NFL, NFM, and NFH, respectively) in 9- and 21-day-old pups. Female rats were fed with 20% ethanol in their drinking water during pregnancy and lactation. The homeostasis of the IF phosphorylation was not altered in the cerebral cortex, cerebellum, or hippocampus of 9-day-old pups. However, GFAP, NFL, and NFM were hyperphosphorylated in the hippocampus of 21-day-old pups. PKA had been activated in the hippocampus, and Ser55 in the N-terminal region of NFL was hyperphosphorylated. In addition, JNK/MAPK was activated and KSP repeats in the C-terminal region of NFM were hyperphosphorylated in the hippocampus of 21-day-old pups. Decreased NFH immunocontent but an unaltered total NFH/phosphoNFH ratio suggested altered stoichiometry of NFs in the hippocampus of ethanol-exposed 21-day-old pups. In contrast to the high susceptibility of hippocampal cytoskeleton in developing rats, the homeostasis of the cytoskeleton of ethanol-fed adult females was not altered. Disruption of the cytoskeletal homeostasis in neural cells supports the view that regions of the brain are differentially vulnerable to alcohol insult during pregnancy and lactation, suggesting that modulation of JNK/MAPK and PKA signaling cascades target the hippocampal cytoskeleton in a window of vulnerability in 21-day-old pups. Our findings are relevant, since disruption of the cytoskeleton in immature hippocampus could contribute to later hippocampal damage associated with ethanol toxicity.

Central Nervous System Depressants/toxicity , Cytoskeleton/drug effects , Ethanol/toxicity , Hippocampus/drug effects , Lactation , Animals , Animals, Newborn , Body Weight/drug effects , Cyclic AMP-Dependent Protein Kinases/drug effects , Cyclic AMP-Dependent Protein Kinases/metabolism , Energy Intake/drug effects , Female , Glial Fibrillary Acidic Protein/metabolism , Hippocampus/embryology , Homeostasis , Intermediate Filaments/drug effects , MAP Kinase Signaling System/drug effects , Neurofilament Proteins/metabolism , Phosphorylation , Pregnancy , Rats , Rats, Wistar
Neurotox Res ; 28(3): 239-52, 2015 Oct.
Article En | MEDLINE | ID: mdl-26174040

Neurological symptoms and cerebral abnormalities are commonly observed in patients with 3-hydroxy-3-methylglutaryl-CoA lyase (HMG lyase) deficiency, which is biochemically characterized by predominant tissue accumulation of 3-hydroxy-3-methylglutaric (HMG), 3-methylglutaric (MGA), and 3-methylglutaconic (MGT) acids. Since the pathogenesis of this disease is poorly known, the present study evaluated the effects of these compounds on the cytoskeleton phosphorylating system in rat brain. HMG, MGA, and MGT caused hypophosphorylation of glial fibrillary acidic protein (GFAP) and of the neurofilament subunits NFL, NFM, and NFH. HMG-induced hypophosphorylation was mediated by inhibiting the cAMP-dependent protein kinase (PKA) on Ser55 residue of NFL and c-Jun kinase (JNK) by acting on KSP repeats of NFM and NFH subunits. We also evidenced that the subunit NR2B of NMDA receptor and Ca(2+) was involved in HMG-elicited hypophosphorylation of cytoskeletal proteins. Furthermore, the antioxidants L-NAME and TROLOX fully prevented both the hypophosphorylation and the inhibition of PKA and JNK caused by HMG, suggesting that oxidative damage may underlie these effects. These findings indicate that the main metabolites accumulating in HMG lyase deficiency provoke hypophosphorylation of cytoskeleton neural proteins with the involvement of NMDA receptors, Ca(2+), and reactive species. It is presumed that these alterations may contribute to the neuropathology of this disease.

Acetyl-CoA C-Acetyltransferase/deficiency , Amino Acid Metabolism, Inborn Errors/metabolism , Cerebral Cortex/metabolism , Corpus Striatum/metabolism , Cytoskeletal Proteins/metabolism , Oxidative Stress/physiology , Receptors, N-Methyl-D-Aspartate/metabolism , Acetyl-CoA C-Acetyltransferase/metabolism , Amino Acid Metabolism, Inborn Errors/pathology , Animals , Blotting, Western , Calcium/metabolism , Cell Survival/physiology , Cerebral Cortex/drug effects , Cerebral Cortex/growth & development , Cerebral Cortex/pathology , Corpus Striatum/drug effects , Corpus Striatum/growth & development , Corpus Striatum/pathology , Oxidative Stress/drug effects , Phosphorylation/drug effects , Phosphorylation/physiology , Rats, Wistar
Int J Dev Neurosci ; 42: 68-75, 2015 May.
Article En | MEDLINE | ID: mdl-25777939

Sulfite oxidase (SOX) deficiency is an inherited neurometabolic disorder biochemically characterized by tissue accumulation and high urinary excretion of sulfite and thiosulfate. Affected patients present severe neurological dysfunction accompanied by seizures, whose pathophysiology is poorly known. In the present study we evaluated the in vitro effects of sulfite and thiosulfate on important parameters of glutamatergic neurotransmission and redox homeostasis in rat cerebral cortex slices. We verified that sulfite, but not thiosulfate, significantly decreased glutamate uptake when cerebral cortex slices were exposed during 1h to these metabolites. We also observed that thiosulfate inhibited glutamine synthetase (GS) activity. A pronounced trend toward GS inhibition induced by sulfite was also found. Regarding redox homeostasis, sulfite, at the concentration of 10 µM, increased thiobarbituric acid-reactive substances and decreased glutathione concentrations after 1h of exposure. In contrast, thiosulfate did not alter these parameters. We also found that 500 µM sulfite increased sulfhydryl group content in rat cerebral cortex slices and increased GSH levels in a medium containing oxidized GSH (GSSG) and devoid of cortical slices, suggesting that sulfite reacts with disulfide bonds to generate sulfhydryl groups. Moreover, sulfite and thiosulfate did not alter the activities of glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST) and glucose-6-phosphate dehydrogenase (G6PDH) after 1h of incubation. However, sulfite inhibited the activities of GPx, GST and G6PDH when cortical slices were exposed for 3h to sulfite. We finally verified that sulfite did not induce cell death after 1h of incubation. Our data show that sulfite impairs glutamatergic neurotransmission and redox homeostasis in cerebral cortex. Therefore, it may be presumed that these pathomechanisms contribute, at least in part, to the seizures observed in patients affected by SOX deficiency.

Cerebral Cortex/drug effects , Glutamic Acid/metabolism , Glutathione/metabolism , Neurotransmitter Agents/metabolism , Sulfites/pharmacology , Animals , Dose-Response Relationship, Drug , Glucosephosphate Dehydrogenase/metabolism , Glutathione Peroxidase/metabolism , Glutathione Reductase/metabolism , In Vitro Techniques , L-Lactate Dehydrogenase/metabolism , Oxidative Stress/drug effects , Protein Carbonylation/drug effects , Rats , Rats, Wistar , Thiobarbituric Acid Reactive Substances/metabolism , Tritium/metabolism
J Neurol Sci ; 346(1-2): 260-7, 2014 Nov 15.
Article En | MEDLINE | ID: mdl-25241940

The role of excitotoxicity on the neuropathology of glutaric acidemia type I (GA I) is still under debate. Therefore, in the present work, we evaluated glutamate uptake by brain slices and glutamate binding to synaptic membranes, as well as glutamine synthetase activity in cerebral cortex and striatum from glutaryl-CoA dehydrogenase deficient (Gcdh(-/-)) mice along development (7, 15, 30 and 60 days of life) in the hopes of clarifying this matter. We also tested the influence of glutaric acid (GA) added exogenously on these parameters. [(3)H]Glutamate uptake was not significantly altered in cerebral cortex and striatum from Gcdh(-/-) mice, as compared to WT mice. However, GA provoked a significant decrease of [(3)H]glutamate uptake in striatum from both WT and Gcdh(-/-) mice older than 7 days. This inhibitory effect was more pronounced in Gcdh(-/-), as compared to WT mice. The use of a competitive inhibitor of glutamate astrocytic transporters indicated that the decrease of [(3)H]glutamate uptake caused by GA was due to the competition between this organic acid and glutamate for the same astrocytic transporter site. We also found that Na(+)-dependent [(3)H]glutamate binding (binding to transporters) was increased in the striatum from Gcdh(-/-) mice and that GA significantly diminished this binding both in striatum and cerebral cortex from Gcdh(-/-), but not from WT mice. Finally, we observed that glutamine synthetase activity was not changed in brain cortex and striatum from Gcdh(-/-) and WT mice and that GA was not able to alter this activity. It is therefore presumed that a disturbance of the glutamatergic neurotransmission system caused by GA may potentially be involved in the neuropathology of GA I, particularly in the striatum.

Amino Acid Metabolism, Inborn Errors/metabolism , Brain Diseases, Metabolic/metabolism , Cerebral Cortex/metabolism , Corpus Striatum/metabolism , Glutarates/pharmacology , Glutaryl-CoA Dehydrogenase/deficiency , Glutaryl-CoA Dehydrogenase/genetics , Amino Acid Metabolism, Inborn Errors/pathology , Animals , Brain Diseases, Metabolic/pathology , Cerebral Cortex/drug effects , Cerebral Cortex/pathology , Corpus Striatum/drug effects , Corpus Striatum/pathology , Disease Models, Animal , Glutamate-Ammonia Ligase/metabolism , Glutaryl-CoA Dehydrogenase/metabolism , Mice , Mice, Knockout
Mol Genet Metab ; 109(2): 144-53, 2013 Jun.
Article En | MEDLINE | ID: mdl-23611578

3-Hydroxy-3-methylglutaryl-CoA lyase (HL) deficiency is a genetic disorder biochemically characterized by predominant accumulation of 3-hydroxy-3-methylglutaric (HMG) and 3-methylglutaric (MGA) acids in tissues and biological fluids of affected individuals. Clinically, the patients present neurological symptoms and basal ganglia injury, whose pathomechanisms are partially understood. In the present study, we investigated the ex vivo effects of intrastriatal administration of HMG and MGA on important parameters of oxidative stress in striatum of developing rats. Our results demonstrate that HMG and MGA induce lipid and protein oxidative damage. HMG and MGA also increased 2',7'-dichlorofluorescein oxidation, whereas only HMG elicited nitric oxide production, indicating a role for reactive oxygen (HMG and MGA) and nitrogen (HMG) species in these effects. Regarding the enzymatic antioxidant defenses, both organic acids decreased reduced glutathione concentrations and the activities of superoxide dismutase and glutathione reductase and increased glutathione peroxidase activity. HMG also provoked an increase of catalase activity and a diminution of glucose-6-phosphate dehydrogenase activity. We finally observed that antioxidants fully prevented or attenuated HMG-induced alterations of the oxidative stress parameters, further indicating the participation of reactive species in these effects. We also observed that MK-801, a non-competitive antagonist of the N-methyl-D-aspartate (NMDA) receptor, prevented some of these effects, indicating the involvement of the NMDA receptor in HMG effects. The present data provide solid evidence that oxidative stress is induced in vivo by HMG and MGA in rat striatum and it is presumed that this pathomechanism may explain, at least in part, the cerebral alterations observed in HL deficiency.

Acetyl-CoA C-Acetyltransferase/deficiency , Amino Acid Metabolism, Inborn Errors/metabolism , Basal Ganglia/metabolism , Meglutol/analogs & derivatives , Meglutol/metabolism , Oxidative Stress , Acetyl-CoA C-Acetyltransferase/metabolism , Animals , Antioxidants/pharmacology , Basal Ganglia/growth & development , Basal Ganglia/pathology , Catalase/metabolism , Dizocilpine Maleate/pharmacology , Glutathione/metabolism , Glutathione Peroxidase/metabolism , Male , Malondialdehyde/metabolism , Protein Carbonylation , Rats , Rats, Wistar , Receptors, N-Methyl-D-Aspartate/antagonists & inhibitors , Superoxide Dismutase/metabolism , Vitamin E/pharmacology
Mol Genet Metab ; 108(1): 30-9, 2013 Jan.
Article En | MEDLINE | ID: mdl-23218171

Deficiency of glutaryl-CoA dehydrogenase (GCDH) activity or glutaric aciduria type I (GA I) is an inherited neurometabolic disorder biochemically characterized by predominant accumulation of glutaric acid and 3-hydroxyglutaric acid in the brain and other tissues. Affected patients usually present acute striatum necrosis during encephalopathic crises triggered by metabolic stress situations, as well as chronic leukodystrophy and delayed myelination. Considering that the mechanisms underlying the brain injury in this disease are not yet fully established, in the present study we investigated important parameters of oxidative stress in the brain (cerebral cortex, striatum and hippocampus), liver and heart of 30-day-old GCDH deficient knockout (Gcdh(-/-)) and wild type (WT) mice submitted to a normal lysine (Lys) (0.9% Lys), or high Lys diets (2.8% or 4.7% Lys) for 60 h. It was observed that the dietary supplementation of 2.8% and 4.7% Lys elicited noticeable oxidative stress, as verified by an increase of malondialdehyde concentrations (lipid oxidative damage) and 2-7-dihydrodichlorofluorescein (DCFH) oxidation (free radical production), as well as a decrease of reduced glutathione levels and alteration of various antioxidant enzyme activities (antioxidant defenses) in the cerebral cortex and the striatum, but not in the hippocampus, the liver and the heart of Gcdh(-/-) mice, as compared to WT mice receiving the same diets. Furthermore, alterations of oxidative stress parameters in the cerebral cortex and striatum were more accentuated in symptomatic, as compared to asymptomatic Gcdh(-/-) mice exposed to 4.7% Lys overload. Histopathological studies performed in the cerebral cortex and striatum of these animals exposed to high dietary Lys revealed increased expression of oxidative stress markers despite the absence of significant structural damage. The results indicate that a disruption of redox homeostasis in the cerebral cortex and striatum of young Gcdh(-/-) mice exposed to increased Lys diet may possibly represent an important pathomechanism of brain injury in GA I patients under metabolic stress.

Brain/metabolism , Glutaryl-CoA Dehydrogenase/metabolism , Homeostasis , Lysine/administration & dosage , Animals , Dietary Supplements , Glutaryl-CoA Dehydrogenase/genetics , Mice , Mice, Knockout , Oxidation-Reduction , Oxidative Stress , Thiobarbituric Acid Reactive Substances/metabolism
Neurotoxicology ; 34: 175-88, 2013 Jan.
Article En | MEDLINE | ID: mdl-23182946

In the present report 15 day-old rats were injected with 0.3µmol of diphenyl ditelluride (PhTe)(2)/kg body weight and parameters of neurodegeneration were analyzed in slices from cerebellum 3 and 6 days afterwards. The earlier responses, at day 3 after injection, included hyperphosphorylation of intermediate filament (IF) proteins from astrocyte (glial fibrillary acidic protein - GFAP - and vimentin) and neuron (low-, medium- and high molecular weight neurofilament subunits: NF-L, NF-M and NF-H); increased mitogen-activated protein kinase (MAPK) (Erk and p38MAPK) and cAMP-dependent protein kinase (PKA) activities. Also, reactive astrogliosis takes part of the early responses to the insult with (PhTe)(2), evidenced by upregulated GFAP in Western blot, PCR and immunofluorescence analysis. Six days after (PhTe)(2) injection we found persistent astrogliosis, increased propidium iodide (PI) positive cells in NeuN positive population evidenced by flow cytometry and reduced immunofluorescence for NeuN, suggesting that the in vivo exposure to (PhTe)(2) progressed to neuronal death. Moreover, activated caspase 3 suggested apoptotic neuronal death. Neurodegeneration was related with decreased [(3)H]glutamate uptake and decreased Akt immunoreactivity, however phospho-GSK-3-ß (Ser9) was not altered in (PhTe)(2) injected rat. Therefore, the present results show that the earlier cerebellar responses to (PhTe)(2) include disruption of cytoskeletal homeostasis that could be related with MAPK and PKA activation and reactive astrogliosis. Akt inhibition observed at this time could also play a role in the neuronal death evidenced afterwards. The later events of the neurodegenerative process are characterized by persistent astrogliosis and activation of apoptotic neuronal death through caspase 3 mediated mechanisms, which could be related with glutamate excitotoxicity. The progression of these responses are therefore likely to be critical for the outcome of the neurodegeneration provoked by (PhTe)(2) in rat cerebellum.

Apoptosis/drug effects , Astrocytes/drug effects , Benzene Derivatives/toxicity , Cerebellum/drug effects , Cytoskeleton/drug effects , Nerve Degeneration , Organometallic Compounds/toxicity , Animals , Animals, Newborn , Astrocytes/metabolism , Astrocytes/pathology , Benzene Derivatives/administration & dosage , Blotting, Western , Caspase 3/metabolism , Cerebellum/metabolism , Cerebellum/pathology , Cyclic AMP-Dependent Protein Kinases/metabolism , Cytoskeleton/metabolism , Cytoskeleton/pathology , Female , Flow Cytometry , Fluorescent Antibody Technique , Glial Fibrillary Acidic Protein/metabolism , Homeostasis , Injections, Subcutaneous , Male , Mitogen-Activated Protein Kinases/metabolism , Neurofilament Proteins/metabolism , Organometallic Compounds/administration & dosage , Rats , Rats, Wistar , Real-Time Polymerase Chain Reaction , Signal Transduction/drug effects , Time Factors , Vimentin/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism
Mol Genet Metab ; 107(3): 375-82, 2012 Nov.
Article En | MEDLINE | ID: mdl-22999741

Mitochondrial dysfunction has been proposed to play an important role in the neuropathology of glutaric acidemia type I (GA I). However, the relevance of bioenergetics disruption and the exact mechanisms responsible for the cortical leukodystrophy and the striatum degeneration presented by GA I patients are not yet fully understood. Therefore, in the present work we measured the respiratory chain complexes activities I-IV, mitochondrial respiratory parameters state 3, state 4, the respiratory control ratio and dinitrophenol (DNP)-stimulated respiration (uncoupled state), as well as the activities of α-ketoglutarate dehydrogenase (α-KGDH), creatine kinase (CK) and Na+, K+-ATPase in cerebral cortex, striatum and hippocampus from 30-day-old Gcdh-/- and wild type (WT) mice fed with a normal or a high Lys (4.7%) diet. When a baseline (0.9% Lys) diet was given, we verified mild alterations of the activities of some respiratory chain complexes in cerebral cortex and hippocampus, but not in striatum from Gcdh-/- mice as compared to WT animals. Furthermore, the mitochondrial respiratory parameters and the activities of α-KGDH and CK were not modified in all brain structures from Gcdh-/- mice. In contrast, we found a significant reduction of Na(+), K(+)-ATPase activity associated with a lower degree of its expression in cerebral cortex from Gcdh-/- mice. Furthermore, a high Lys (4.7%) diet did not accentuate the biochemical alterations observed in Gcdh-/- mice fed with a normal diet. Since Na(+), K(+)-ATPase activity is required for cell volume regulation and to maintain the membrane potential necessary for a normal neurotransmission, it is presumed that reduction of this enzyme activity may represent a potential underlying mechanism involved in the brain swelling and cortical abnormalities (cortical atrophy with leukodystrophy) observed in patients affected by GA I.

Amino Acid Metabolism, Inborn Errors/genetics , Amino Acid Metabolism, Inborn Errors/pathology , Brain Diseases, Metabolic/genetics , Brain Diseases, Metabolic/pathology , Cerebral Cortex/pathology , Corpus Striatum/pathology , Glutaryl-CoA Dehydrogenase/deficiency , Hippocampus/pathology , Sodium-Potassium-Exchanging ATPase/genetics , Amino Acid Metabolism, Inborn Errors/enzymology , Animals , Brain Diseases, Metabolic/enzymology , Cerebral Cortex/enzymology , Corpus Striatum/enzymology , Creatine Kinase/genetics , Creatine Kinase/metabolism , Down-Regulation , Electron Transport/genetics , Food, Formulated , Gene Expression , Glutaryl-CoA Dehydrogenase/genetics , Hippocampus/enzymology , Humans , Ketoglutarate Dehydrogenase Complex/genetics , Ketoglutarate Dehydrogenase Complex/metabolism , Mice , Mice, Knockout , Mitochondria/genetics , Mitochondria/metabolism , Oxidative Phosphorylation , Sodium-Potassium-Exchanging ATPase/metabolism
Metab Brain Dis ; 27(4): 521-30, 2012 Dec.
Article En | MEDLINE | ID: mdl-22798168

Ornithine, ammonia and homocitrulline are the major metabolites accumulating in hyperornithinemia-hyperammonemia-homocitrullinuria syndrome, a genetic disorder characterized by neurological regression whose pathogenesis is still not understood. The present work investigated the in vivo effects of intracerebroventricular administration of ornithine and homocitrulline in the presence or absence of hyperammonemia induced by intraperitoneal urease treatment on a large spectrum of oxidative stress parameters in cerebral cortex from young rats in order to better understand the role of these metabolites on brain damage. Ornithine increased thiobarbituric acid-reactive substances (TBA-RS) levels and carbonyl formation and decreased total antioxidant status (TAS) levels. We also observed that the combination of hyperammonemia with ornithine resulted in significant decreases of sulfhydryl levels, reduced glutathione (GSH) concentrations and the activities of catalase (CAT) and glutathione peroxidase (GPx), highlighting a synergistic effect of ornithine and ammonia. Furthermore, homocitrulline caused increases of TBA-RS values and carbonyl formation, as well as decreases of GSH concentrations and GPx activity. Hcit with hyperammonemia (urease treatment) decreased TAS and CAT activity. We also showed that urease treatment per se was able to enhance TBA-RS levels. Finally, nitric oxide production was not altered by Orn and Hcit alone or in combination with hyperammonemia. Our data indicate that the major metabolites accumulating in hyperornithinemia-hyperammonemia-homocitrullinuria syndrome provoke lipid and protein oxidative damage and a reduction of the antioxidant defenses in the brain. Therefore, it is presumed that oxidative stress may represent a relevant pathomechanism involved in the brain damage found in patients affected by this disease.

Amino Acid Metabolism, Inborn Errors/metabolism , Ammonia/metabolism , Brain/metabolism , Citrulline/analogs & derivatives , Homeostasis/physiology , Ornithine/metabolism , Amino Acid Metabolism, Inborn Errors/blood , Amino Acid Metabolism, Inborn Errors/chemically induced , Ammonia/blood , Animals , Antioxidants/metabolism , Catalase/metabolism , Citrulline/metabolism , Citrulline/urine , Glutathione Peroxidase/metabolism , Injections, Intraperitoneal , Injections, Intraventricular , Male , Nitric Oxide/metabolism , Ornithine/blood , Ornithine/toxicity , Oxidation-Reduction , Oxidative Stress/drug effects , Protein Carbonylation/drug effects , Rats , Rats, Wistar , Sulfhydryl Compounds/metabolism , Thiobarbituric Acid Reactive Substances/metabolism , Urease
Mol Genet Metab ; 107(1-2): 81-6, 2012 Sep.
Article En | MEDLINE | ID: mdl-22578804

Glutaric acidemia type I (GA I) is an inherited neurometabolic disorder caused by a severe deficiency of the mitochondrial glutaryl-CoA dehydrogenase activity leading to accumulation of predominantly glutaric (GA) and 3-hydroxyglutaric (3HGA) acids in the brain and other tissues. Affected patients usually present with hypotonia and brain damage and acute encephalopathic episodes whose pathophysiology is not yet fully established. In this study we investigated important parameters of cellular bioenergetics in brain, heart and skeletal muscle from 15-day-old glutaryl-CoA dehydrogenase deficient mice (Gcdh(-/-)) submitted to a single intra-peritoneal injection of saline (Sal) or lysine (Lys - 8 µmol/g) as compared to wild type (WT) mice. We evaluated the activities of the respiratory chain complexes II, II-III and IV, α-ketoglutarate dehydrogenase (α-KGDH), creatine kinase (CK) and synaptic Na(+), K(+)-ATPase. No differences of all evaluated parameters were detected in the Gcdh(-/-) relatively to the WT mice injected at baseline (Sal). Furthermore, mild increases of the activities of some respiratory chain complexes (II-III and IV) were observed in heart and skeletal muscle of Gcdh(-/-) and WT mice after Lys administration. However, the most marked effects provoked by Lys administration were marked decreases of the activities of Na(+), K(+)-ATPase in brain and CK in brain and skeletal muscle of Gcdh(-/-) mice. In contrast, brain α-KGDH activity was not altered in WT and Gcdh(-/-) injected with Sal or Lys. Our results demonstrate that reduction of Na(+), K(+)-ATPase and CK activities may play an important role in the pathogenesis of the neurodegenerative changes in GA I.

Amino Acid Metabolism, Inborn Errors/metabolism , Brain Diseases, Metabolic/metabolism , Creatine Kinase/metabolism , Lysine/pharmacology , Sodium-Potassium-Exchanging ATPase/metabolism , Amino Acid Metabolism, Inborn Errors/drug therapy , Animals , Brain/drug effects , Brain/metabolism , Brain Diseases, Metabolic/drug therapy , Disease Models, Animal , Electron Transport/drug effects , Electron Transport/physiology , Glutaryl-CoA Dehydrogenase/deficiency , Glutaryl-CoA Dehydrogenase/metabolism , Heart/drug effects , Ketoglutarate Dehydrogenase Complex/metabolism , Lysine/administration & dosage , Mice , Mice, Knockout , Muscle, Skeletal/drug effects , Muscle, Skeletal/metabolism , Myocardium/metabolism
Mol Genet Metab ; 106(1): 31-8, 2012 May.
Article En | MEDLINE | ID: mdl-22445450

In the present work we evaluated a variety of indicators of oxidative stress in distinct brain regions (striatum, cerebral cortex and hippocampus), the liver, and heart of 30-day-old glutaryl-CoA dehydrogenase deficient (Gcdh(-/-)) mice. The parameters evaluated included thiobarbituric acid-reactive substances (TBA-RS), 2-7-dihydrodichlorofluorescein (DCFH) oxidation, sulfhydryl content, and reduced glutathione (GSH) concentrations. We also measured the activities of the antioxidant enzymes glutathione peroxidase (GPx), glutathione reductase (GR), catalase (CAT), superoxide dismutase (SOD) and glucose-6-phosphate dehydrogenase (G6PD). Under basal conditions glutaric (GA) and 3-OH-glutaric (3OHGA) acids were elevated in all tissues of the Gcdh(-/-) mice, but were essentially absent in WT animals. In contrast there were no differences between WT and Gcdh(-/-) mice in any of the indicators or oxidative stress under basal conditions. Following a single intra-peritoneal (IP) injection of lysine (Lys) there was a moderate increase of brain GA concentration in Gcdh(-/-) mice, but no change in WT. Lys injection had no effect on brain 3OHGA in either WT or Gcdh(-/-) mice. The levels of GA and 3OHGA were approximately 40% higher in striatum compared to cerebral cortex in Lys-treated mice. In the striatum, Lys administration provoked a marked increase of lipid peroxidation, DCFH oxidation, SOD and GR activities, as well as significant reductions of GSH levels and GPx activity, with no alteration of sulfhydryl content, CAT and G6PD activities. There was also evidence of increased lipid peroxidation and SOD activity in the cerebral cortex, along with a decrease of GSH levels, but to a lesser extent than in the striatum. In the hippocampus only mild increases of SOD activity and DCFH oxidation were observed. In contrast, Lys injection had no effect on any of the parameters of oxidative stress in the liver or heart of Gcdh(-/-) or WT animals. These results indicate that in Gcdh(-/-) mice cerebral tissue, particularly the striatum, is at greater risk for oxidative stress than peripheral tissues following Lys administration.

Antioxidants/metabolism , Brain/metabolism , Glutaryl-CoA Dehydrogenase , Lysine/administration & dosage , Oxidative Stress , Animals , Brain/enzymology , Glutaryl-CoA Dehydrogenase/deficiency , Glutaryl-CoA Dehydrogenase/genetics , Lipid Peroxidation , Liver/enzymology , Liver/metabolism , Lysine/adverse effects , Mice , Mice, Transgenic , Myocardium/enzymology , Myocardium/metabolism , Tissue Distribution
Life Sci ; 89(7-8): 276-81, 2011 Aug 15.
Article En | MEDLINE | ID: mdl-21762704

AIMS: We investigated the effects of in vivo intrastriatal administration of glycine (Gly), which is found at high concentrations in the brain of patients affected by nonketotic hyperglycinemia (NKH), on important parameters of oxidative stress. MAIN METHODS: Thiobarbituric acid-reactive substances values (TBA-RS, lipid peroxidation), carbonyl formation (protein oxidative damage), sulfhydryl content, reduced glutathione concentrations, nitric oxide production and the activities of the antioxidant enzymes glutathione peroxidase, glutathione reductase, catalase, superoxide dismutase and glucose-6-phosphate dehydrogenase (antioxidant defenses) were measured in striatum from 30-day-old rats after Gly injection. KEY FINDINGS: Gly administration significantly increased TBA-RS values, implying lipid oxidative damage. Furthermore, Gly-induced increase of TBA-RS was fully prevented by the NMDA receptor antagonist MK-801, indicating the involvement of the NMDA glutamate receptor in this effect. Gly injection also induced protein carbonyl formation, as well as elevation of the activities of glutathione peroxidase, glutathione reductase, catalase and superoxide dismutase. In contrast, glutathione levels, sulfhydryl content, nitric oxide production and the activity of glucose-6-phosphate dehydrogenase were not modified by Gly. SIGNIFICANCE: The data shows that Gly in vivo administration causes lipid peroxidation, probably secondary to NMDA stimulation, induces protein oxidation and modulates the activities of important antioxidant enzymes in the striatum. In case these findings can be extrapolated to the human NKH, it is feasible that oxidative stress may be involved in the pathophysiology of the brain injury observed in patients with this neurometabolic disease.

Brain/drug effects , Corpus Striatum/drug effects , Glycine Agents/administration & dosage , Glycine/administration & dosage , Lipid Peroxidation/drug effects , Protein Carbonylation/drug effects , Animals , Brain/metabolism , Corpus Striatum/metabolism , Disease Models, Animal , Dizocilpine Maleate/pharmacology , Hyperglycinemia, Nonketotic/metabolism , Hyperglycinemia, Nonketotic/prevention & control , Microinjections , Neuroprotective Agents/pharmacology , Oxidoreductases/metabolism , Rats , Rats, Wistar , Reactive Oxygen Species/metabolism , Thiobarbituric Acid Reactive Substances/metabolism
Cell Mol Neurobiol ; 31(5): 775-85, 2011 Jul.
Article En | MEDLINE | ID: mdl-21424830

Methylmalonic acidemia and propionic acidemia are organic acidemias biochemically characterized by predominant tissue accumulation of methylmalonic acid (MMA) and propionic acid (PA), respectively. Affected patients present predominantly neurological symptoms, whose pathogenesis is not yet fully established. In the present study we investigated the in vitro effects of MMA and PA on important parameters of lipid and protein oxidative damage and on the production of reactive species in synaptosomes from cerebrum of developing rats. Synaptosomes correspond to nerve terminals that have been used to investigate toxic properties of compounds on neuronal cells. The in vivo effects of intrastriatal injection of MMA and PA on the same parameters and on enzymatic antioxidant defenses, were also studied. MMA-induced in vitro and in vivo lipid peroxidation and protein oxidative damage. Furthermore, the lipid oxidative damage was attenuated or prevented, pending on the doses utilized, by the free radical scavengers α-tocopherol, melatonin and by the NMDA glutamate receptor antagonist MK-801, implying the involvement of reactive species and glutamate receptor activation in these effects. In addition, 2',7'-dichlorofluorescein diacetate oxidation was significantly increased in synaptosomes by MMA, reinforcing that reactive species generation is elicited by this organic acid. We also verified that glutathione peroxidase activity was inhibited by intrastriatal MMA injection. In contrast, PA did not induce any significant effect on all parameters examined in vitro and in vivo, implying a selective action for MMA. The present data demonstrate that oxidative stress is induced by MMA in vitro in nerve terminals and in vivo in striatum, suggesting the participation of neuronal cells in MMA-elicited oxidative damage.

Antioxidants/metabolism , Methylmalonic Acid/toxicity , Neostriatum/drug effects , Neostriatum/pathology , Nerve Endings/drug effects , Nerve Endings/pathology , Oxidative Stress/drug effects , Animals , Fluoresceins/metabolism , Glutathione Peroxidase/metabolism , Lipid Peroxidation/drug effects , Methylmalonic Acid/administration & dosage , Neostriatum/enzymology , Oxidation-Reduction/drug effects , Protein Carbonylation/drug effects , Rats , Rats, Wistar , Reactive Oxygen Species/metabolism , Synaptosomes/drug effects , Synaptosomes/metabolism , Thiobarbituric Acid Reactive Substances/metabolism
Brain Res ; 1382: 259-65, 2011 Mar 25.
Article En | MEDLINE | ID: mdl-21241675

Pristanic acid (Prist) is accumulated in various peroxisomal disorders characterized by severe neurological dysfunction whose pathogenesis is poorly understood. Since oxidative damage has been demonstrated in brain of patients affected by neurodegenerative disorders, in the present work we investigated the in vitro effects of Prist on important parameters of oxidative stress in cerebral cortex from young rats. Prist significantly increased malondialdehyde levels, reflecting an increase of lipid peroxidation. This effect was totally prevented by the free radical scavenger melatonin, suggesting the involvement of reactive species. Prist also provoked protein oxidative damage, as determined by increased carbonyl formation and sulfhydryl oxidation. Otherwise, it did not alter nitric oxide production, indicating that nitrogen reactive species were not implicated in the lipid and oxidative damage provoked by Prist. Furthermore, the concentration of glutathione (GSH), the major brain non-enzymatic antioxidant defense, was significantly decreased by Prist and this decrease was fully prevented by melatonin and attenuated by α-tocopherol. It is therefore presumed that Prist elicits oxidative stress in the brain probably via reactive oxygen species formation and that this pathomechanism may possibly be involved in the brain damage found in patients affected by peroxisomal disorders where Prist accumulates.

Brain Chemistry/drug effects , Brain Damage, Chronic/chemically induced , Cerebral Cortex/drug effects , Fatty Acids/toxicity , Oxidative Stress/drug effects , Peroxisomal Disorders/chemically induced , Animals , Brain Chemistry/physiology , Brain Damage, Chronic/metabolism , Brain Damage, Chronic/physiopathology , Cerebral Cortex/growth & development , Cerebral Cortex/physiopathology , Disease Models, Animal , Fatty Acids/metabolism , Lipid Peroxidation/drug effects , Lipid Peroxidation/physiology , Male , Oxidative Stress/physiology , Peroxisomal Disorders/metabolism , Peroxisomal Disorders/physiopathology , Rats , Rats, Wistar
Neurochem Res ; 36(2): 205-14, 2011 Feb.
Article En | MEDLINE | ID: mdl-20976553

Lysine (Lys) accumulation in tissues and biological fluids is the biochemical hallmark of patients affected by familial hyperlysinemia (FH) and other inherited metabolic disorders. In the present study we investigated the effects of acute administration of Lys on relevant parameters of energy metabolism and oxidative stress in striatum of young rats. We verified that Lys in vivo intrastriatal injection did not change the citric acid cycle function and creatine kinase activity, but, in contrast, significantly inhibited synaptic Na(+),K(+)-ATPase activity in striatum prepared 2 and 12 h after injection. Moreover, Lys induced lipid peroxidation and diminished the concentrations of glutathione 2 h after injection. These effects were prevented by the antioxidant scavengers melatonin and the combination of α-tocopherol and ascorbic acid. Lys also inhibited glutathione peroxidase activity 12 h after injection. Therefore it is assumed that inhibition of synaptic Na(+),K(+)-ATPase and oxidative damage caused by brain Lys accumulation may possibly contribute to the neurological manifestations of FH and other neurometabolic conditions with high concentrations of this amino acid.

Corpus Striatum/drug effects , Corpus Striatum/metabolism , Corpus Striatum/pathology , Lysine/pharmacology , Oxidative Stress/drug effects , Sodium-Potassium-Exchanging ATPase/antagonists & inhibitors , Animals , Oxidation-Reduction , Rats , Rats, Wistar , Synapses/metabolism , Thiobarbituric Acid Reactive Substances/metabolism
Int J Dev Neurosci ; 28(4): 297-301, 2010 Jun.
Article En | MEDLINE | ID: mdl-20307643

The present work investigated the effects of intrastriatal administration of d-serine on relevant parameters of oxidative stress in striatum of young rats. d-Serine significantly induced lipid peroxidation, reflected by the significant increase of thiobarbituric acid-reactive substances, and significantly diminished the striatum antioxidant defenses, as verified by a decrease of the levels of reduced glutathione and total antioxidant status. Finally, d-serine inhibited superoxide dismutase activity, without altering the activities of glutathione peroxidase and catalase. In contrast, this d-amino acid did not alter sulfhydryl oxidation, a measure of protein oxidative damage. The present data indicate that d-serine in vivo administration induces lipid oxidative damage and decreases the antioxidant defenses in the striatum of young rats. Therefore, it is presumed that this oxidative stress may be a pathomechanism involved at least in part in the neurological damage found in patients affected by disorders in which d-serine metabolism is compromised, leading to altered concentrations of this d-amino acid.

Corpus Striatum/drug effects , Corpus Striatum/metabolism , Lipid Peroxidation/drug effects , Oxidative Stress/drug effects , Serine/pharmacology , Animals , Catalase/metabolism , Glutathione/metabolism , Glutathione Peroxidase/metabolism , Male , Oxidation-Reduction/drug effects , Rats , Rats, Wistar , Superoxide Dismutase/metabolism , Thiobarbituric Acid Reactive Substances/metabolism