Cristiane Cecatto

Marked reduction of Na(+), K(+)-ATPase and creatine kinase activities induced by acute lysine administration in 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 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.

Reduction of Na + ,K + -ATPase activity and expression in cerebral cortex of glutaryl-CoA dehydrogenase deficient mice: A possible mechanism for brain injury in glutaric aciduria type I

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.

Ethylmalonic acid impairs brain mitochondrial succinate and malate transport

Tissue accumulation and high urinary excretion of ethylmalonic acid (EMA) occur in ethylmalonic encephalopathy (EE) and short chain acyl-CoA dehydrogenase deficiency (SCADD). Although these autosomal recessive disorders are clinically characterized by neurological abnormalities, the mechanisms underlying the brain damage are poorly known. Considering that little is known about the neurotoxicity of EMA and that hyperlacticacidemia occurs in EE and SCADD, we evaluated the effects of this metabolite on important parameters of oxidative metabolism in isolated rat brain mitochondria. EMA inhibited either ADP-stimulated or uncoupled mitochondrial respiration supported by succinate and malate, but not by glutamate plus malate. In addition, EMA mildly stimulated oxygen consumption by succinate-respiring mitochondria in resting state. Methylmalonic acid (MMA), malonic acid (MA) and butylmalonic acid (BtMA) had a similar effect on ADP-stimulated or uncoupled respiration. Furthermore, EMA-, MMA- and BtMA-induced inhibitory effects on succinate oxidation were significantly minimized by nonselective permeabilization of the mitochondrial membranes by alamethicin, whereas MA inhibitory effect was not altered. In addition, MA was the only tested compound that reduced succinate dehydrogenase activity. We also observed that EMA markedly inhibited succinate and malate transport through the mitochondrial dicarboxylate carrier. Mitochondrial membrane potential was also reduced by EMA and MA, but not by MMA, using succinate as electron donor, whereas none of these compounds was able to alter the membrane potential using glutamate plus malate as electron donors. Taken together, our results strongly indicate that EMA impairs succinate and malate uptake through the mitochondrial dicarboxylate carrier.

Uncoupling, metabolic inhibition and induction of mitochondrial permeability transition in rat liver mitochondria caused by the major long-chain hydroxyl monocarboxylic fatty acids accumulating in LCHAD deficiency.

Patients with long-chain 3-hydroxy-acyl-CoA dehydrogenase (LCHAD) deficiency commonly present liver dysfunction whose pathogenesis is unknown. We studied the effects of long-chain 3-hydroxylated fatty acids (LCHFA) that accumulate in LCHAD deficiency on liver bioenergetics using mitochondrial preparations from young rats. We provide strong evidence that 3-hydroxytetradecanoic (3HTA) and 3-hydroxypalmitic (3HPA) acids, the monocarboxylic acids that are found at the highest tissue concentrations in this disorder, act as metabolic inhibitors and uncouplers of oxidative phosphorylation. These conclusions are based on the findings that these fatty acids decreased ADP-stimulated (state 3) and uncoupled respiration, mitochondrial membrane potential and NAD(P)H content, and, in contrast, increased resting (state 4) respiration. We also verified that 3HTA and 3HPA markedly reduced Ca2+ retention capacity and induced swelling in Ca2+-loaded mitochondria. These effects were mediated by mitochondrial permeability transition (MPT) induction since they were totally prevented by the classical MPT inhibitors cyclosporin A and ADP, as well as by ruthenium red, a Ca2+ uptake blocker. Taken together, our data demonstrate that the major monocarboxylic LCHFA accumulating in LCHAD deficiency disrupt energy mitochondrial homeostasis in the liver. It is proposed that this pathomechanism may explain at least in part the hepatic alterations characteristic of the affected patients.

Disturbance of mitochondrial functions provoked by the major long-chain 3-hydroxylated fatty acids accumulating in MTP and LCHAD deficiencies in skeletal muscle.

The pathogenesis of the muscular symptoms and recurrent rhabdomyolysis that are commonly manifested in patients with mitochondrial trifunctional protein (MTP) and long-chain 3-hydroxy-acyl-CoA dehydrogenase (LCHAD) deficiencies is still unknown. In this study we investigated the effects of the major long-chain monocarboxylic 3-hydroxylated fatty acids (LCHFA) accumulating in these disorders, namely 3-hydroxytetradecanoic (3HTA) and 3-hydroxypalmitic (3HPA) acids, on important mitochondrial functions in rat skeletal muscle mitochondria. 3HTA and 3HPA markedly increased resting (state 4) and decreased ADP-stimulated (state 3) and CCCP-stimulated (uncoupled) respiration. 3HPA provoked similar effects in permeabilized skeletal muscle fibers, validating the results obtained in purified mitochondria. Furthermore, 3HTA and 3HPA markedly diminished mitochondrial membrane potential, NAD(P)H content and Ca(2+) retention capacity in Ca(2+)-loaded mitochondria. Mitochondrial permeability transition (mPT) induction probably underlie these effects since they were totally prevented by cyclosporin A and ADP. In contrast, the dicarboxylic analogue of 3HTA did not alter the tested parameters. Our data strongly indicate that 3HTA and 3HPA behave as metabolic inhibitors, uncouplers of oxidative phosphorylation and mPT inducers in skeletal muscle. It is proposed that these pathomechanisms disrupting mitochondrial homeostasis may be involved in the muscle alterations characteristic of MTP and LCHAD deficiencies.

2‐Methylcitric acid impairs glutamate metabolism and induces permeability transition in brain mitochondria

Accumulation of 2‐methylcitric acid (2MCA) is observed in methylmalonic and propionic acidemias, which are clinically characterized by severe neurological symptoms. The exact pathogenetic mechanisms of brain abnormalities in these diseases are poorly established and very little has been reported on the role of 2MCA. In the present work we found that 2MCA markedly inhibited ADP‐stimulated and uncoupled respiration in mitochondria supported by glutamate, with a less significant inhibition in pyruvate plus malate respiring mitochondria. However, no alterations occurred when α‐ketoglutarate or succinate was used as respiratory substrates, suggesting a defect on glutamate oxidative metabolism. It was also observed that 2MCA decreased ATP formation in glutamate plus malate or pyruvate plus malate‐supported mitochondria. Furthermore, 2MCA inhibited glutamate dehydrogenase activity at concentrations as low as 0.5 mM. Kinetic studies revealed that this inhibitory effect was competitive in relation to glutamate. In contrast, assays of osmotic swelling in non‐respiring mitochondria suggested that 2MCA did not significantly impair mitochondrial glutamate transport. Finally, 2MCA provoked a significant decrease in mitochondrial membrane potential and induced swelling in Ca2+‐loaded mitochondria supported by different substrates. These effects were totally prevented by cyclosporine A plus ADP or ruthenium red, indicating induction of mitochondrial permeability transition. Taken together, our data strongly indicate that 2MCA behaves as a potent inhibitor of glutamate oxidation by inhibiting glutamate dehydrogenase activity and as a permeability transition inducer, disturbing mitochondrial energy homeostasis. We presume that 2MCA‐induced mitochondrial deleterious effects may contribute to the pathogenesis of brain damage in patients affected by methylmalonic and propionic acidemias.

Experimental evidence that bioenergetics disruption is not mainly involved in the brain injury of glutaryl-CoA dehydrogenase deficient mice submitted to lysine overload

Bioenergetics dysfunction has been postulated as an important pathomechanism of brain damage in glutaric aciduria type I, but this is still under debate. We investigated activities of citric acid cycle (CAC) enzymes, lactate release, respiration and membrane potential (ΔΨm) in mitochondrial preparations from cerebral cortex and striatum of 30-day-old glutaryl-CoA dehydrogenase deficient (Gcdh-/-) and wild type mice fed a baseline or a high lysine (Lys, 4.7%) chow for 60 or 96h. Brain histological analyses were performed in these animals, as well as in 90-day-old animals fed a baseline or a high Lys chow during 30 days starting at 60-day-old. A moderate reduction of citrate synthase and isocitrate dehydrogenase activities was observed only in the striatum from 30-day-old Gcdh-/- animals submitted to a high Lys chow. In contrast, the other CAC enzyme activities, lactate release, the respiratory parameters state 3, state 4, the respiratory control ratio and CCCP-stimulated (uncoupled) state, as well as ΔΨm were not altered in the striatum. Similarly, none of the evaluated parameters were changed in the cerebral cortex from these animals under baseline or Lys overload. On the other hand, histological analyses revealed the presence of intense vacuolation in the cerebral cortex of 60 and 90-day-old Gcdh-/- mice fed a baseline chow and in the striatum of 90-day-old Gcdh-/- mice submitted to Lys overload for 30 days. Taken together, the present data demonstrate mild impairment of bioenergetics homeostasis and marked histological alterations in striatum from Gcdh-/- mice under a high Lys chow, suggesting that disruption of energy metabolism is not mainly involved in the brain injury of these animals.

Ethylmalonic Acid Induces Permeability Transition in Isolated Brain Mitochondria

Predominant accumulation of ethylmalonic acid (EMA) in tissues and biological fluids is a characteristic of patients affected by short chain acyl-CoA dehydrogenase deficiency and ethylmalonic encephalopathy. Neurological abnormalities are frequently found in these disorders, but the mechanisms underlying the brain injury are still obscure. Since hyperlacticacidemia is also found in many affected patients indicating a mitochondrial dysfunction; in the present work, we evaluated the in vitro and ex vivo effects of EMA plus Ca2+ on mitochondrial integrity and redox balance in succinate-supported brain organelles. We verified that the evaluated parameters were disturbed only when EMA was associated with exogenous micromolar Ca2+ concentrations. Thus, we found that this short chain organic acid plus Ca2+ dissipated the membrane potential and provoked mitochondrial swelling, as well as impaired the mitochondrial Ca2+ retention capacity, resulting in a rapid Ca2+ release and decreased NAD(P)H matrix content. In contrast, EMA was not able to stimulate mitochondrial hydrogen peroxide generation. We also observed that all these effects were prevented by the mitochondrial Ca2+ uptake inhibitor ruthenium red and the mitochondrial permeability transition (MPT) inhibitors cyclosporin A (CsA) and ADP. Furthermore, mitochondria isolated from rat brains after in vivo intrastriatal administration of EMA was more susceptible to Ca2+-induced swelling, which was fully prevented by CsA and ADP. Finally, EMA significantly decreased striatal slice viability, which was attenuated by CsA. The data strongly indicate that EMA reduced the mitochondrial threshold for Ca2+-induced MPT reinforcing the role of this cation in EMA-induced disruption of mitochondrial bioenergetics. It is, therefore, presumed that EMA acting synergistically with Ca2+ compromises mitochondrial energy homeostasis in the central nervous system that may explain at least in part the neurologic alterations presented by patients with abnormal levels of this organic acid.

Deregulation of mitochondrial functions provoked by long‐chain fatty acid accumulating in long‐chain 3‐hydroxyacyl‐CoA dehydrogenase and mitochondrial permeability transition deficiencies in rat heart – mitochondrial permeability transition pore opening as a potential contributing pathomechanism of cardiac alterations in these disorders

Mitochondrial trifunctional protein and long‐chain 3‐hydroxyacyl‐CoA dehydrogenase deficiencies are fatty acid oxidation disorders biochemically characterized by tissue accumulation of long‐chain fatty acids and derivatives, including the monocarboxylic long‐chain 3‐hydroxy fatty acids (LCHFAs) 3‐hydroxytetradecanoic acid (3HTA) and 3‐hydroxypalmitic acid (3HPA). Patients commonly present severe cardiomyopathy for which the pathogenesis is still poorly established. We investigated the effects of 3HTA and 3HPA, the major metabolites accumulating in these disorders, on important parameters of mitochondrial homeostasis in Ca2+‐loaded heart mitochondria. 3HTA and 3HPA significantly decreased mitochondrial membrane potential, the matrix NAD(P)H pool and Ca2+ retention capacity, and also induced mitochondrial swelling. These fatty acids also provoked a marked decrease of ATP production reflecting severe energy dysfunction. Furthermore, 3HTA‐induced mitochondrial alterations were completely prevented by the classical mitochondrial permeability transition (mPT) inhibitors cyclosporin A and ADP, as well as by ruthenium red, a Ca2+ uptake blocker, indicating that LCHFAs induced Ca2+‐dependent mPT pore opening. Milder effects only achieved at higher doses of LCHFAs were observed in brain mitochondria, implying a higher vulnerability of heart to these fatty acids. By contrast, 3HTA and docosanoic acids did not change mitochondrial homeostasis, indicating selective effects for monocarboxylic LCHFAs. The present data indicate that the major LCHFAs accumulating in mitochondrial trifunctional protein and long‐chain 3‐hydroxyacyl‐CoA dehydrogenase deficiencies induce mPT pore opening, compromising Ca2+ homeostasis and oxidative phosphorylation more intensely in the heart. It is proposed that these pathomechanisms may contribute at least in part to the severe cardiac alterations characteristic of patients affected by these diseases.

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.