Rafael Teixeira Ribeiro

S-Adenosylmethionine Promotes Oxidative Stress and Decreases Na+, K+-ATPase Activity in Cerebral Cortex Supernatants of Adolescent Rats: Implications for the Pathogenesis of S-Adenosylhomocysteine Hydrolase Deficiency

S-Adenosylmethionine (AdoMet) concentrations are highly elevated in tissues and biological fluids of patients affected by S-adenosylhomocysteine hydrolase deficiency, who are clinically characterized by cerebral symptoms whose pathogenesis is still unknown. In the present work, we investigated the effects of AdoMet on redox homeostasis and on the activity of Na+, K+-ATPase in the cerebral cortex of young rats. AdoMet caused lipid peroxidation (increase of malondialdehyde concentrations) and protein oxidation (increase of carbonyl formation and decrease of sulfhydryl content). AdoMet also reduced the antioxidant defenses (reduced glutathione, GSH) and Na+, K+-ATPase activity. Furthermore, AdoMet-induced lipid peroxidation was fully prevented by the antioxidants trolox, melatonin, and resveratrol, and the decrease of GSH concentrations was abolished by trolox, suggesting the involvement of reactive oxygen species in these effects. In this context, AdoMet induced reactive oxygen (increase of 2′,7′-dichloroflurescein-DCFH oxidation) but not nitrogen (nitrate and nitrite levels) species generation. Finally, the decrease of Na+, K+-ATPase activity provoked by AdoMet was totally prevented by trolox, implying a possible oxidation of cysteine groups of the enzyme that are critical for its function and highly susceptible to oxidative attack. It is also noted that adenosine and methionine did not alter the parameters evaluated, suggesting selective effects of AdoMet. Our data strongly indicate that disturbance of redox homeostasis caused by a major metabolite (AdoMet) accumulating in S-adenosylhomocysteine hydrolase deficiency may represent a deleterious mechanism of brain damage in this disease. Finally, reduction of Na+, K+-ATPase activity provoked by AdoMet may lead to impaired neurotransmission, but disturbance of this system should be better clarified in future studies.

cis-4-Decenoic and decanoic acids impair mitochondrial energy, redox and Ca(2+) homeostasis and induce mitochondrial permeability transition pore opening in rat brain and liver: Possible implications for the pathogenesis of MCAD deficiency.

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is biochemically characterized by tissue accumulation of octanoic (OA), decanoic (DA) and cis-4-decenoic (cDA) acids, as well as by their carnitine by-products. Untreated patients present episodic encephalopathic crises and biochemical liver alterations, whose pathophysiology is poorly known. We investigated the effects of OA, DA, cDA, octanoylcarnitine (OC) and decanoylcarnitine (DC) on critical mitochondrial functions in rat brain and liver. DA and cDA increased resting respiration and diminished ADP- and CCCP-stimulated respiration and complexes II-III and IV activities in both tissues. The data indicate that these compounds behave as uncouplers and metabolic inhibitors of oxidative phosphorylation. Noteworthy, metabolic inhibition was more evident in brain as compared to liver. DA and cDA also markedly decreased mitochondrial membrane potential, NAD(P)H content and Ca(2+) retention capacity in Ca(2+)-loaded brain and liver mitochondria. The reduction of Ca(2+) retention capacity was more pronounced in liver and totally prevented by cyclosporine A and ADP, as well as by ruthenium red, demonstrating the involvement of mitochondrial permeability transition (mPT) and Ca(2+). Furthermore, cDA induced lipid peroxidation in brain and liver mitochondria and increased hydrogen peroxide formation in brain, suggesting the participation of oxidative damage in cDA-induced alterations. Interestingly, OA, OC and DC did not alter the evaluated parameters, implying lower toxicity for these compounds. Our results suggest that DA and cDA, in contrast to OA and medium-chain acylcarnitines, disturb important mitochondrial functions in brain and liver by multiple mechanisms that are possibly involved in the neuropathology and liver alterations observed in MCAD deficiency.

α-Ketoadipic Acid and α-Aminoadipic Acid Cause Disturbance of Glutamatergic Neurotransmission and Induction of Oxidative Stress In Vitro in Brain of Adolescent Rats

Tissue accumulation of α-ketoadipic (KAA) and α-aminoadipic (AAA) acids is the biochemical hallmark of α-ketoadipic aciduria. This inborn error of metabolism is currently considered a biochemical phenotype with uncertain clinical significance. Considering that KAA and AAA are structurally similar to α-ketoglutarate and glutamate, respectively, we investigated the in vitro effects of these compounds on glutamatergic neurotransmission in the brain of adolescent rats. Bioenergetics and redox homeostasis were also investigated because they represent fundamental systems for brain development and functioning. We first observed that AAA significantly decreased glutamate uptake, whereas glutamate dehydrogenase activity was markedly inhibited by KAA in a competitive fashion. In addition, AAA and more markedly KAA induced generation of reactive oxygen and nitrogen species (increase of 2′,7′-dichloroflurescein (DCFH) oxidation and nitrite/nitrate levels), lipid peroxidation (increase of malondialdehyde concentrations), and protein oxidation (increase of carbonyl formation and decrease of sulfhydryl content), besides decreasing the antioxidant defenses (reduced glutathione (GSH)) and aconitase activity. Furthermore, KAA-induced lipid peroxidation and GSH decrease were prevented by the antioxidants α-tocopherol, melatonin, and resveratrol, suggesting the involvement of reactive species in these effects. Noteworthy, the classical inhibitor of NMDA glutamate receptors MK-801 was not able to prevent KAA-induced and AAA-induced oxidative stress, determined by DCFH oxidation and GSH levels, making unlikely a secondary induction of oxidative stress through overstimulation of glutamate receptors. In contrast, KAA and AAA did not significantly change brain bioenergetic parameters. We speculate that disturbance of glutamatergic neurotransmission and redox homeostasis by KAA and AAA may play a role in those cases of α-ketoadipic aciduria that display neurological symptoms.

Mevalonolactone disrupts mitochondrial functions and induces permeability transition pore opening in rat brain mitochondria: Implications for the pathogenesis of mevalonic aciduria

&NA; Mevalonic aciduria (MVA) is caused by severe deficiency of mevalonic kinase activity leading to tissue accumulation and high urinary excretion of mevalonic acid (MA) and mevalonolactone (ML). Patients usually present severe neurologic symptoms whose pathophysiology is poorly known. Here, we tested the hypothesis that the major accumulating metabolites are toxic by investigating the in vitro effects of MA and ML on important mitochondrial functions in rat brain and liver mitochondria. ML, but not MA, markedly decreased mitochondrial membrane potential (&Dgr;&PSgr;m), NAD(P)H content and the capacity to retain Ca2+ in the brain, besides inducing mitochondrial swelling. These biochemical alterations were totally prevented by the classical inhibitors of mitochondrial permeability transition (MPT) cyclosporine A and ADP, as well as by ruthenium red in Ca2+‐loaded mitochondria, indicating the involvement of MPT and an important role for mitochondrial Ca2+ in these effects. ML also induced lipid peroxidation and markedly inhibited aconitase activity, an enzyme that is highly susceptible to free radical attack, in brain mitochondrial fractions, indicating that lipid and protein oxidative damage may underlie some of ML‐induced deleterious effects including MTP induction. In contrast, ML and MA did not compromise oxidative phosphorylation in the brain and all mitochondrial functions evaluated in the liver, evidencing a selective toxicity of ML towards the central nervous system. Our present study provides for the first time evidence that ML impairs essential brain mitochondrial functions with the involvement of MPT pore opening. It is therefore presumed that disturbance of brain mitochondrial homeostasis possibly contributes to the neurologic symptoms in MVA. HighlightsMevalonic acid (MA) and mevalonolactne (ML) accumulate in mevalonic aciduria (MVA).Patients affected by this disorder present brain dysfunction.Disruption of important mitochondrial functions and MPT are provoked by ML in brain.These pathomechanisms may be involved in MVA pathogenesis.

Experimental Evidence that In Vivo Intracerebral Administration of L-2-Hydroxyglutaric Acid to Neonatal Rats Provokes Disruption of Redox Status and Histopathological Abnormalities in the Brain

Tissue accumulation of L-2-hydroxyglutaric acid (L-2-HG) is the biochemical hallmark of L-2-hydroxyglutaric aciduria (L-2-HGA), a rare neurometabolic inherited disease characterized by neurological symptoms and brain white matter abnormalities whose pathogenesis is not yet well established. L-2-HG was intracerebrally administered to rat pups at postnatal day 1 (P1) to induce a rise of L-2-HG levels in the central nervous system (CNS). Thereafter, we investigated whether L-2-HG in vivo administration could disturb redox homeostasis and induce brain histopathological alterations in the cerebral cortex and striatum of neonatal rats. L-2-HG markedly induced the generation of reactive oxygen species (increase of 2′,7′-dichloroflurescein-DCFH-oxidation), lipid peroxidation (increase of malondialdehyde concentrations), and protein oxidation (increase of carbonyl formation and decrease of sulfhydryl content), besides decreasing the antioxidant defenses (reduced glutathione-GSH) and sulfhydryl content in the cerebral cortex. Alterations of the activities of various antioxidant enzymes were also observed in the cerebral cortex and striatum following L-2-HG administration. Furthermore, L-2-HG-induced lipid peroxidation and GSH decrease in the cerebral cortex were prevented by the antioxidant melatonin and by the classical antagonist of NMDA glutamate receptor MK-801, suggesting the involvement of reactive species and of overstimulation of NMDA receptor in these effects. Finally, L-2-HG provoked significant vacuolation and edema particularly in the cerebral cortex with less intense alterations in the striatum that were possibly associated with the unbalanced redox homeostasis caused by this metabolite. Taken together, it is presumed that these pathomechanisms may underlie the neurological symptoms and brain abnormalities observed in the affected patients.

Induction of Neuroinflammatory Response and Histopathological Alterations Caused by Quinolinic Acid Administration in the Striatum of Glutaryl-CoA Dehydrogenase Deficient Mice

Glutaric acidemia type I (GA I) is an inherited neurometabolic disorder caused by a severe deficiency of the mitochondrial glutaryl-CoA dehydrogenase (GCDH) activity. Patients usually present progressive cortical leukodystrophy and commonly develop acute bilateral striatal degeneration mainly during infections that markedly worse their prognosis. A role for quinolinic acid (QA), a key metabolite of the kynurenine pathway, which is activated during inflammatory processes, on the pathogenesis of the acute striatum degeneration occurring in GA I was proposed but so far has not yet been evaluated. Therefore, we investigated whether an acute intrastriatal administration of quinolinic acid (QA) could induce histopathological alterations in the striatum of 30-day-old wild-type (WT) and GCDH knockout (Gcdh−/−) mice. Striatum morphology was evaluated by hematoxylin and eosin, T lymphocyte presence (CD3), and glial activation (GFAP and S100β) by immunohistochemistry and 3-nitrotyrosine (YNO2) by immunofluorescence. QA provoked extensive vacuolation, edema, and especially lymphocyte infiltration in the striatum of Gcdh−/−. QA also enhanced CD3 staining and the number of YNO2 positive cells in Gcdh−/− mice, relatively to WT, indicating T lymphocyte infiltration and nitrosative stress, respectively. QA-treated WT mice also showed an increase of GFAP and S100β staining, which is indicative of reactive astrogliosis, whereas the levels of these astrocytic proteins were not changed in Gcdh−/− QA-injected mice. The present data indicate that QA significantly contributes to the histopathological changes observed in the striatum of Gcdh−/− mice.

Disruption of Brain Redox Homeostasis, Microglia Activation and Neuronal Damage Induced by Intracerebroventricular Administration of S-Adenosylmethionine to Developing Rats

S-Adenosylmethionine (AdoMet) concentrations are highly elevated in tissues and biological fluids of patients affected by S-adenosylhomocysteine hydrolase deficiency. This disorder is clinically characterized by severe neurological symptoms, whose pathophysiology is not yet established. Therefore, we investigated the effects of intracerebroventricular administration of AdoMet on redox homeostasis, microglia activation, synaptophysin levels, and TAU phosphorylation in cerebral cortex and striatum of young rats. AdoMet provoked significant lipid and protein oxidation, decreased glutathione concentrations, and altered the activity of important antioxidant enzymes in cerebral cortex and striatum. AdoMet also increased reactive oxygen (2′,7′-dichlorofluorescein oxidation increase) and nitrogen (nitrate and nitrite levels increase) species generation in cerebral cortex. Furthermore, the antioxidants N-acetylcysteine and melatonin prevented most of AdoMet-induced pro-oxidant effects in both cerebral structures. Finally, we verified that AdoMet produced microglia activation by increasing Iba1 staining and TAU phosphorylation, as well as reduced synaptophysin levels in cerebral cortex. Taken together, it is presumed that impairment of redox homeostasis possibly associated with microglia activation and neuronal dysfunction caused by AdoMet may represent deleterious pathomechanisms involved in the pathophysiology of brain damage in S-adenosylhomocysteine hydrolase deficiency.