Methylmalonic Acid Impairs Cell Respiration and Glutamate Uptake in C6 Rat Glioma Cells: Implications for Methylmalonic Acidemia.

Methylmalonic acidemia is an organic acidemia caused by deficient activity of L-methylmalonyl-CoA mutase or its cofactor cyanocobalamin and it is biochemically characterized by an accumulation of methylmalonic acid (MMA) in tissue and body fluids of patients. The main clinical manifestations of this disease are neurological and observable symptoms during metabolic decompensation are encephalopathy, cerebral atrophy, coma, and seizures, which commonly appear in newborns. This study aimed to investigate the toxic effects of MMA in a glial cell line presenting astrocytic features. Astroglial C6 cells were exposed to MMA (0.1-10 mM) for 24 or 48 h and cell metabolic viability, glucose consumption, and oxygen consumption rate, as well as glutamate uptake and ATP content were analyzed. The possible preventive effects of bezafibrate were also evaluated. MMA significantly reduced cell metabolic viability after 48-h period and increased glucose consumption during the same period of incubation. Regarding the energy homeostasis, MMA significantly reduced respiratory parameters of cells after 48-h exposure, indicating that cell metabolism is compromised at resting and reserve capacity state, which might influence the cell capacity to meet energetic demands. Glutamate uptake and ATP content were also compromised after exposure to MMA, which can be influenced energy metabolism impairment, affecting the functionality of the astroglial cells. Our findings suggest that these effects could be involved in the pathophysiology of neurological dysfunction of this disease. Methylmalonic acid compromises mitochondrial functioning leading to reduced ATP production and reduces glutamate uptake by C6 astroglial cells.

Methylmalonate Induces Inflammatory and Apoptotic Potential: A Link to Glial Activation and Neurological Dysfunction.

Methylmalonic acid (MMA) accumulates in tissues in methylmalonic acidemia, a heterogeneous group of inherited childhood diseases characterized by neurological dysfunction, oxidative stress and neuroinflammation; it is associated with degeneration of striatal neurons and cerebral cortical atrophy. It is presently unknown, however, whether transient exposure to MMA in the neonatal period is sufficient to trigger inflammatory and apoptotic processes that lead to brain structural damage. Here, newborn mice were given a single intracerebroventricular dose of MMA at 12 hours after birth. Maze testing of 21- and 40-day-old mice showed that MMA-injected animals exhibited deficit in the working memory test but not in the reference test. MMA-injected mice showed increased levels of the reactive oxygen species marker 2',7'-dichlorofluorescein diacetate, tumor necrosis factor, interleukin-1ß, caspases 1, 3, and 8, and increased acetylcholinesterase activity in the cortex, hippocampus and striatum. This was associated with increased astrocyte and microglial immunoreactivity in all brain regions. These findings suggest that transient exposure to MMA may alter the redox state and cause neuroinflammatory/apoptotic processes and glial activation during critical periods of brain development. Similar processes may underlie brain dysfunction and cognitive impairment in patients with methylmalonic acidemia.

A neuronal disruption in redox homeostasis elicited by ammonia alters the glycine/glutamate (GABA) cycle and contributes to MMA-induced excitability.

Hyperammonemia is a common finding in children with methylmalonic acidemia. However, its contribution to methylmalonate-induced excitotoxicty is poorly understood. The aim of this study was to evaluate the mechanisms by which ammonia influences in the neurotoxicity induced by methylmalonate (MMA) in mice. The effects of ammonium chloride (NH4Cl 3, 6, and 12 mmol/kg; s.c.) on electroencephalographic (EEG) and behavioral convulsions induced by MMA (0.3, 0.66, and 1 µmol/2 µL, i.c.v.) were observed in mice. After, ammonia, TNF-α, IL1ß, IL-6, nitrite/nitrate (NOx) levels, mitochondrial potential (ΔΨ), reactive oxygen species (ROS) generation, Methyl-Tetrazolium (MTT) reduction, succinate dehydrogenase (SDH), and Na(+), K(+)-ATPase activity levels were measured in the cerebral cortex. The binding of [(3)H]flunitrazepam, release of glutamate-GABA; glutamate decarboxylase (GAD) and glutamine synthetase (GS) activity and neuronal damage [opening of blood brain barrier (BBB) permeability and cellular death volume] were also measured. EEG recordings showed that an intermediate dose of NH4Cl (6 mmol/kg) increased the duration of convulsive episodes induced by MMA (0.66 µmol/2 µL i.c.v). NH4Cl (6 mmol/kg) administration also induced neuronal ammonia and NOx increase, as well as mitochondrial ROS generation throughout oxidation of 2,7-dichlorofluorescein diacetate (DCFH-DA) to DCF-RS, followed by GS and GAD inhibition. The NH4Cl plus MMA administration did not alter cytokine levels, plasma fluorescein extravasation, or neuronal damage. However, it potentiated DCF-RS levels, decreased the ΔΨ potential, reduced MTT, inhibited SDH activity, and increased Na(+), K(+)-ATPase activity. NH4Cl also altered the GABA cycle characterized by GS and GAD activity inhibition, [(3)H]flunitrazepam binding, and GABA release after MMA injection. On the basis of our findings, the changes in ROS and reactive nitrogen species (RNS) levels elicited by ammonia alter the glycine/glutamate (GABA) cycle and contribute to MMA-induced excitability.

The effect of WIN 55,212-2 suggests a cannabinoid-sensitive component in the early toxicity induced by organic acids accumulating in glutaric acidemia type I and in related disorders of propionate metabolism in rat brain synaptosomes.

Several physiological processes in the CNS are regulated by the endocannabinoid system (ECS). Cannabinoid receptors (CBr) and CBr agonists have been involved in the modulation of the N-methyl-D-aspartate receptor (NMDAr) activation. Glutaric (GA), 3-hydroxyglutaric (3-OHGA), methylmalonic (MMA) and propionic (PA) acids are endogenous metabolites produced and accumulated in the brain of children affected by severe organic acidemias (OAs) with neurodegeneration. Oxidative stress and excitotoxicity have been involved in the toxic pattern exerted by these organic acids. Studying the early pattern of toxicity exerted by these metabolites is crucial to explain the extent of damage that they can produce in the brain. Herein, we investigated the effects of the synthetic CBr agonist WIN 55,212-2 (WIN) on early markers of GA-, 3-OHGA-, MMA- and PA-induced toxicity in brain synaptosomes from adult (90-day-old) and adolescent (30-day-old) rats. As pre-treatment, WIN exerted protective effects on the GA- and MMA-induced mitochondrial dysfunction, and prevented the reactive oxygen species (ROS) formation and lipid peroxidation induced by all metabolites. Our findings support a protective and modulatory role of cannabinoids in the early toxic events elicited by toxic metabolites involved in OAs.

Toxic synergism between quinolinic acid and organic acids accumulating in glutaric acidemia type I and in disorders of propionate metabolism in rat brain synaptosomes: Relevance for metabolic acidemias.

The brain of children affected by organic acidemias develop acute neurodegeneration linked to accumulation of endogenous toxic metabolites like glutaric (GA), 3-hydroxyglutaric (3-OHGA), methylmalonic (MMA) and propionic (PA) acids. Excitotoxic and oxidative events are involved in the toxic patterns elicited by these organic acids, although their single actions cannot explain the extent of brain damage observed in organic acidemias. The characterization of co-adjuvant factors involved in the magnification of early toxic processes evoked by these metabolites is essential to infer their actions in the human brain. Alterations in the kynurenine pathway (KP) - a metabolic route devoted to degrade tryptophan to form NAD(+) - produce increased levels of the excitotoxic metabolite quinolinic acid (QUIN), which has been involved in neurodegenerative disorders. Herein we investigated the effects of subtoxic concentrations of GA, 3-OHGA, MMA and PA, either alone or in combination with QUIN, on early toxic endpoints in rat brain synaptosomes. To establish specific mechanisms, we pre-incubated synaptosomes with different protective agents, including the endogenous N-methyl-d-aspartate (NMDA) receptor antagonist kynurenic acid (KA), the antioxidant S-allylcysteine (SAC) and the nitric oxide synthase (NOS) inhibitor nitro-l-arginine methyl ester (l-NAME). While the incubation of synaptosomes with toxic metabolites at subtoxic concentrations produced no effects, their co-incubation (QUIN+GA, +3-OHGA, +MMA or +PA) decreased the mitochondrial function and increased reactive oxygen species (ROS) formation and lipid peroxidation. For all cases, this effect was partially prevented by KA and l-NAME, and completely avoided by SAC. These findings suggest that early damaging events elicited by organic acids involved in metabolic acidemias can be magnified by toxic synergism with QUIN, and this process is mostly mediated by oxidative stress, and in a lesser extent by excitotoxicity and nitrosative stress. Therefore, QUIN can be hypothesized to contribute to the pathophysiology of brain degeneration in children with metabolic acidemias.

Disruption of redox homeostasis and brain damage caused in vivo by methylmalonic acid and ammonia in cerebral cortex and striatum of developing rats.

Hyperammonemia is a common finding in children with methylmalonic acidemia and propionic acidemia, but its contribution to the development of the neurological symptoms in the affected patients is poorly known. Considering that methylmalonic acid (MMA) and propionic acid (PA) predominantly accumulate in these disorders, we investigated the effects of hyperammonemia induced by urease treatment in 30-day-old rats receiving an intracerebroventricular (ICV) injection of MMA or PA on important parameters of redox homeostasis in cerebral cortex and striatum. We evaluated glutathione (GSH) concentrations, sulfhydryl content, nitrate and nitrite concentrations, 2',7'-dichlorofluorescein (DCFH) oxidation, and the activity of antioxidant enzymes. MMA decreased GSH concentrations and sulfhydryl content and increased nitrate and nitrite concentrations in cerebral cortex and striatum from hyperammonemic rats, whereas MMA or ammonia per se did not alter these parameters. MMA plus hyperammonemia also decreased glutathione reductase activity in rat cerebral cortex, but did not affect catalase, superoxide dismutase and glutathione peroxidase activities, neither DCFH oxidation. Furthermore, ICV PA administration alone or combined with hyperammonemia did not alter any of the evaluated parameters. We also found that pre-treatment with antioxidants prevented GSH reduction and sulfhydryl oxidation, whereas N(ω)-nitro-L-arginine methyl ester (L-NAME) prevented the increased nitrate and nitrite concentrations provoked by MMA plus ammonia treatments. Histological alterations, including vacuolization, ischemic neurons, and pericellular edema, were observed in brain of hyperammonemic rats injected with MMA. The data indicate a synergistic effect of MMA and ammonia disturbing redox homeostasis and causing morphological brain abnormalities in rat brain.

Methylmalonic acid administration induces DNA damage in rat brain and kidney.

Accumulation of methylmalonic acid (MMA) in tissues and biological fluids is the biochemical hallmark of methylmalonic aciduria. Affected patients present renal failure and severe neurological findings. Considering that the underlying pathomechanisms of tissue damage are not yet understood, in the present work we assessed the in vivo e in vitro effects of MMA on DNA damage in brain and kidney, as well as on p53 and caspase 3 levels, in the presence or absence of gentamicin (acute renal failure model). For in vitro studies, tissue prisms were incubated in the presence of different concentrations of MMA and/or gentamicin for one hour. For in vivo studies, animals received a single injection of gentamicin (70 mg/kg) and/or three injections of MMA (1.67 µmol/g; 11 h interval between injections). The animals were killed 1 h after the last MMA injection. Controls received saline in the same volumes. DNA damage was analyzed by the comet assay. We found that MMA and gentamicin alone or combined in vitro increased DNA damage in cerebral cortex and kidney of rats. Furthermore, MMA administration increased DNA damage in both brain and kidney. Gentamicin per se induced DNA damage only in kidney, and the association of MMA plus gentamicin also caused DNA damage in cerebral cortex and kidney. On the other hand, p53 and caspase 3 levels were not altered by the administration of MMA and/or gentamicin. Our findings provide evidence that DNA damage may contribute to the neurological and renal damage found in patients affected by methylmalonic aciduria.

A Primary Study on Down-Regulated miR-9-1 and Its Biological Significances in Methylmalonic Acidemia.

Methylmalonic acidemia (MMA) is a metabolic disorder, which is caused by a deficiency of the mitochondrial enzyme methylmalonyl-CoA mutase. MMA diagnosis is dependent on the method of gas chromatography-mass spectrometry, which is expensive, complicated, and time consuming. Currently, microRNAs (miRNAs) have gained considerable interest for its function as a novel class of non-invasive and sensitive biomarkers for the diagnosis of diseases. However, there has been no related report regarding its role in MMA. Our study first detected differentially expressed microRNAs in MMA and found that the expression of miR-9-1 was significantly down-regulated and changed sensitively after VitB12 treatment. Furthermore, we confirmed that miR-9-1 was able to suppress neuronal apoptosis induced by methylmalonate. Taken together, our results suggested that miR-9-1 may act as a potential biomarker for the diagnosis and monitoring of changes in MMA and provide new insights into the pathogenesis of MMA.

Chronic administration of methylmalonate on young rats alters neuroinflammatory markers and spatial memory.

The methylmalonic acidemia is an inborn error of metabolism (IEM) characterized by methylmalonic acid (MMA) accumulation in body fluids and tissues, causing neurological dysfunction, mitochondrial failure and oxidative stress. Although neurological evidence demonstrate that infection and/or inflammation mediators facilitate metabolic crises in patients, the involvement of neuroinflammatory processes in the neuropathology of this organic acidemia is not yet established. In this experimental study, we used newborn Wistar rats to induce a model of chronic acidemia via subcutaneous injections of methylmalonate (MMA, from 5th to 28th day of life, twice a day, ranged from 0.72 to 1.67 µmol/g as a function of animal age). In the following days (29th-31st) animal behavior was assessed in the object exploration test and elevated plus maze. It was performed differential cell and the number of neutrophils counting and interleukin-1 beta (IL-1ß) and tumor necrosis factor-alpha (TNF-α) levels in the blood, as well as levels of IL-1ß, TNF-α, inducible nitric oxide synthase (iNOS) and 3-nitrotyrosine (3-NT) in the cerebral cortex were measured. Behavioral tests showed that animals injected chronically with MMA have a reduction in the recognition index (R.I.) when the objects were arranged in a new configuration space, but do not exhibit anxiety-like behaviors. The blood of MMA-treated animals showed a decrease in the number of polymorphonuclear and neutrophils, and an increase in mononuclear and other cell types, as well as an increase of IL-1ß and TNF-α levels. Concomitantly, MMA increased levels of IL-1ß, TNF-α, and expression of iNOS and 3-NT in the cerebral cortex of rats. The overall results indicate that chronic administration of MMA increased pro-inflammatory markers in the cerebral cortex, reduced immune system defenses in blood, and coincide with the behavioral changes found in young rats. This leads to speculate that, through mechanisms not yet elucidated, the neuroinflammatory processes during critical periods of development may contribute to the progression of cognitive impairment in patients with methylmalonic acidemia.

Acute renal failure potentiates brain energy dysfunction elicited by methylmalonic acid.

The influence of acute renal failure induced by gentamicin administration on the effects of MMA on mitochondrial respiratory chain complexes, citrate synthase, succinate dehydrogenase and creatine kinase activities in cerebral cortex and kidney of young rats were investigated. Animals received one intraperitoneal injection of saline or gentamicin (70 mg/kg). One hour after, the animals received three consecutive subcutaneous injections of MMA (1.67 µmol/g) or saline (11 h interval between injections) and 60 min after the last injection the animals were killed. Acute MMA administration decreased creatine kinase activity in both tissues and increased complexes I-III activity in cerebral cortex. Creatine kinase activity was also inhibited by gentamicin administration. Simultaneous administration of MMA and gentamicin increased the activities of citrate synthase in cerebral cortex and kidney and complexes II-III in cerebral cortex. The other enzyme activities in cerebral cortex and kidney of animals receiving MMA plus gentamicin did not significantly differ from those observed in animals receiving only MMA. Our present data is line with the hypothesis that MMA acts as a toxin in brain and kidney of rats and suggest that renal injury potentiates the toxicity of MMA on the Krebs cycle and respiratory chain in brain and peripheral tissues.

Fish oil attenuates methylmalonate-induced seizures.

Methylmalonic acidemias are inherited metabolic disorders characterized by methylmalonate (MMA) accumulation and neurological dysfunction, including seizures. Dietary fatty acids are known as an important energy source and reduce seizure activity in selected acute animal models. This study investigated whether chronic treatment with fish oil or with oleic acid attenuates MMA-induced seizures and whether maintenance of Na(+),K(+)-ATPase activity was involved in such an effect. Adult male Wistar rats were given fish oil (85 mg/kg), oleic acid (85 mg/kg) or vehicle (0.42% aqueous Cremophor EL™, 4 mL/kg/body weight/day), p.o., for 75 days. On the 73th day a cannula was implanted in the right lateral ventricle with electrodes over the parietal cortex for EEG recording. On the 76th day the animals were injected with NaCl (2.5 µmol/2.5 µL, i.c.v.), or with MMA (2.5 µmol/2.5 µL, i.c.v.), and seizure activity was measured by electroencephagraphic (EEG) recording with concomitant behavior monitoring. The effect of prostaglandin E2 (PGE2) on Na(+),K(+)-ATPase activity of slices of cerebral cortex from NaCl-injected animals was determined. Fish oil increased the latency to MMA-induced tonic-clonic seizures, reduced the mean amplitude of ictal EEG recordings, and prevented PGE2-induced decrease of Na(+),K(+)-ATPase activity in cortical slices in vitro. Oleic acid decreased mean amplitude of ictal EEG recordings. The results support that fish oil decreases MMA-induced seizures. The decreased sensitivity of Na(+),K(+)-ATPase to the inhibitory effect of PGE2 in fish oil-treated animals may be related to the currently reported anticonvulsant activity.

Expression of proinflammatory factors in renal cortex induced by methylmalonic acid.

BACKGROUND: Methylmalonic aciduria is an inborn error of metabolism that causes renal failure and tubulointerstitial (TI) nephritis as complications. This study aimed to examine the levels of expression of several genes related to inflammation, oxidative stress, and mitochondrial function in the renal cortex of rats receiving methylmalonic acid (MMA). METHODS: Rats received MMA subcutaneously for a month. Tumor necrosis factor alpha (TNFα), nuclear factor-kappa B, interleukin 1 beta (IL-1ß), and cyclooxygenase 2 (COX-2) genes were examined by real-time polymerase chain reaction. We also examined transforming growth factor beta (TGF-ß) related to TI fibrosis, c-FOS, belonging to the immediate early gene family of transcription factors, and expression of SIRT1, related to energy production. RESULTS: There was significantly higher expression of TNFα and a trend toward a higher level of TGF-ß transcripts in the methylmalonic model group compared with the controls. However, SIRT1 expression was not different among the groups. Urinary MMA excretion correlated positively with mRNA level of TGF-ß. The expression of COX-2 was positively associated with the expression of c-FOS and inversely related to the expression of IL-1ß. CONCLUSIONS: The higher levels of TNFα and TGF-ß transcripts suggest inflammation and differentiation processes in the renal cortex in rats because of MMA. After 1 month of MMA injections, expression levels of SIRT1 were not affected, suggesting mitochondrial preservation in early stages of the disease.

Experimental evidence that methylmalonic acid provokes oxidative damage and compromises antioxidant defenses in nerve terminal and striatum of young rats.

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.

Mitochondrial energy metabolism in neurodegeneration associated with methylmalonic acidemia.

Methylmalonic acidemia is one of the most prevalent inherited metabolic disorders involving neurological deficits. In vitro experiments, animal model studies and tissue analyses from human patients suggest extensive impairment of mitochondrial energy metabolism in this disease. This review summarizes changes in mitochondrial energy metabolism occurring in methylmalonic acidemia, focusing mainly on the effects of accumulated methylmalonic acid, and gives an overview of the results found in different experimental models. Overall, experiments to date suggest that mitochondrial impairment in this disease occurs through a combination of the inhibition of specific enzymes and transporters, limitation in the availability of substrates for mitochondrial metabolic pathways and oxidative damage.

Cobalamin C defect: natural history, pathophysiology, and treatment.

Cobalamin C (Cbl-C) defect is the most common inborn cobalamin metabolism error; it causes impaired conversion of dietary vitamin B12 into its two metabolically active forms, methylcobalamin and adenosylcobalamin. Cbl-C defect causes the accumulation of methylmalonic acid and homocysteine and decreased methionine synthesis. The gene responsible for the Cbl-C defect has been recently identified, and more than 40 mutations have been reported. MMACHC gene is located on chromosome 1p and catalyzes the reductive decyanation of CNCbl. Cbl-C patients present with a heterogeneous clinical picture and, based on their age at onset, can be categorized into two distinct clinical forms. Early-onset patients, presenting symptoms within the first year, show a multisystem disease with severe neurological, ocular, haematological, renal, gastrointestinal, cardiac, and pulmonary manifestations. Late-onset patients present a milder clinical phenotype with acute or slowly progressive neurological symptoms and behavioral disturbances. To improve clinical course and metabolic abnormalities, treatment of Cbl-C defect usually consists of a combined approach that utilizes vitamin B12 to increase intracellular cobalamin and to maximize deficient enzyme activities, betaine to provide a substrate for the conversion of homocysteine into methionine through betaine-homocysteine methyltransferase, and folic acid to enhance remethylation pathway. No proven efficacy has been demonstrated for carnitine and dietary protein restriction. Despite these measures, the long-term follow-up is unsatisfactory especially in patients with early onset, with frequent progression of neurological and ocular impairment. The unfavorable outcome suggests that better understanding of the pathophysiology of the disease is needed to improve treatment protocols and to develop new therapeutic approaches.

Prevention by L-carnitine of DNA damage induced by propionic and L-methylmalonic acids in human peripheral leukocytes in vitro.

Propionic acidemia (PAemia) and methylmalonic acidemia (MMAemia) are inborn errors of propionate metabolism characterized by the accumulation of, respectively, propionic and l-methylmalonic acids (and their metabolites) in the blood and tissues of affected patients. The conditions lead to severe metabolic complications in the neonatal period and to long-term neurological manifestations. Treatment for these disorders consists of a protein-restricted diet, supplemented with synthetic formulas of amino acids, but excluding isoleucine, threonine, valine and methionine; and l-carnitine, to promote detoxication. In vitro and in vivo studies have demonstrated that lipid and protein oxidative damage may be involved in the pathophysiology of these diseases, but DNA damage has not been fully investigated. In this work, we evaluated in vitro the effects of PA and MMA, in the presence or absence of l-carnitine, on DNA damage in peripheral leukocytes, as determined by the alkaline comet assay, using silver staining and visual scoring. PA and MMA induced a DNA damage index (DI) significantly higher than that of the control group. l-Carnitine significantly reduced PA- and MMA-induced DNA damage, in a concentration-dependent manner. Our findings indicate that PA and MMA induce DNA damage and l-carnitine is able to prevent this damage.

Methylmalonate-induced seizures are attenuated in inducible nitric oxide synthase knockout mice.

Methylmalonic acidemias consist of a group of inherited neurometabolic disorders caused by deficiency of methylmalonyl-CoA mutase activity clinically and biochemically characterized by neurological dysfunction, methylmalonic acid (MMA) accumulation, mitochondrial failure and increased reactive species production. Although previous studies have suggested that nitric oxide (NO) plays a role in the neurotoxicity of MMA, the involvement of NO-induced nitrosative damage from inducible nitric oxide synthase (iNOS) in MMA-induced seizures are poorly understood. In the present study, we showed a decrease of time spent convulsing induced by intracerebroventricular administration of MMA (2 micromol/2 microL; i.c.v.) in iNOS knockout (iNOS(-/-)) mice when compared with wild-type (iNOS(+/+)) littermates. Visual analysis of electroencephalographic recordings (EEG) showed that MMA injection induced the appearance of high-voltage synchronic spike activity in the ipsilateral cortex which spreads to the contralateral cortex while quantitative electroencephalographic analysis showed larger wave amplitude during MMA-induced seizures in wild-type mice when compared with iNOS knockout mice. We also report that administration of MMA increases NOx (NO(2) plus NO(3) content) and 3-nitrotyrosine (3-NT) levels in a greater extend in iNOS(+/+) mice than in iNOS(-/-) mice, indicating that NO overproduction and NO-mediated damage to proteins are attenuated in iNOS knockout mice. In addition, the MMA-induced decrease in Na(+), K(+)-ATPase activity, but not in succinate dehydrogenase (SDH) activity, was less pronounced in iNOS(-/-) when compared with iNOS(+/+) mice. These results reinforce the assumption that metabolic collapse contributes for the secondary toxicity elicited by MMA and suggest that oxidative attack by NO derived from iNOS on selected target such as Na(+), K(+)-ATPase enzyme might represent an important role in this excitotoxicity induced by MMA. Therefore, these results may be of value in understating the pathophysiology of the neurological features observed in patients with methylmalonic acidemia and in the development of new strategies for treatment of these patients.

Methylene blue prevents methylmalonate-induced seizures and oxidative damage in rat striatum.

Methylene blue (MB) is a thiazine dye with cationic and lipophilic properties that acts as an electron transfer mediator in the mitochondria. Due to this metabolic improving activity and free radicals scavenging effects, MB has been used in the treatment of methemoglobinemia and ifosfamide-induced encephalopathy. Considering that methylmalonic acidemia consists of a group of inherited metabolic disorders biochemically characterized by impaired mitochondrial oxidative metabolism and reactive species production, we decided to investigate whether MB, protects against the behavioral and neurochemical alterations elicited by the intrastriatal injection of methylmalonate (MMA). In the present study we showed that intrastriatal injection of MB (0.015-1.5nmol/0.5microl) protected against seizures (evidenced by electrographic recording), protein carbonylation and Na(+),K(+)-ATPase inhibition ex vivo induced by MMA (4.5micromol/1.5microl). Furthermore, we investigated whether convulsions elicited by intrastriatal MMA administration are accompanied by striatal protein carbonyl content increase and changes in Na(+),K(+)-ATPase activity in rat striatum. The effect of MB (0.015-1.5nmol/0.5microl) and MMA (4.5micromol/0.5microl) on striatal NO(x) (NO(2) plus NO(3)) content was also evaluated. Statistical analysis revealed that the MMA-induced NO(x) content increase was attenuated by intrastriatal injection of MB and the duration of convulsive episodes correlated with Na(+),K(+)-ATPase inhibition, but not with MMA-induced total protein carbonylation. In view of that MB decreases MMA-induced neurotoxicity assessed by behavioral and neurochemical parameters, the authors suggest that MB may be of value to attenuate neurological deficits of methylmalonic acidemic patients.

Diazoxide protects against methylmalonate-induced neuronal toxicity.

Methylmalonic acidemia is an inherited metabolic disorder that leads to brain damage associated to the accumulation of methylmalonic acid (MMA) and impairment of energy metabolism. We demonstrate here that treatment with diazoxide, an agonist of mitochondrial ATP-sensitive K(+) channels (mitoK(ATP)), can prevent death promoted by treatment with MMA in PC12 cells and freshly prepared rat brain slices. This diazoxide effect was reversed by 5-hydroxydecanoate, a mitoK(ATP) antagonist, confirming it occurs due to the activity of this channel. Diazoxide was not capable of preventing inner membrane potential loss promoted by MMA and Ca(2+) in isolated mitochondria, indicating it does not directly prevent mitochondrial damage. Furthermore, diazoxide did not prevent respiratory inhibition in cells treated with MMA. Interestingly, we found that the mitochondrial inner membrane potential within intact cells treated with MMA was maintained in part by the reverse activity of ATP synthase (ATP hydrolysis) and that diazoxide prevented the formation of the membrane potential in the presence of MMA, in a manner sensitive to 5-hydroxydecanoate. Furthermore, the effects of diazoxide on cell survival after treatment with MMA were similar to those of ATP synthase inhibitor oligomycin and adenine nucleotide translocator inhibitor atractyloside. These results indicate that diazoxide prevents PC12 cell death promoted by MMA by decreasing mitochondrial ATP hydrolysis. These results uncover new potential neuroprotective effects of mitoK(ATP) agonists under situations in which oxidative phosphorylation is inhibited.

Ammonia potentiates methylmalonic acid-induced convulsions and TBARS production.

Hyperammonemia is a common finding in children with methylmalonic acidemia, an inherited metabolic disease characterized by mental retardation, convulsions, and accumulation of methylmalonic acid (MMA). Although it has been suggested that MMA induces convulsions through succinate dehydrogenase (SDH) inhibition, very little is known about the contribution of hyperammonemia to the development of convulsions in these patients. In the present study we investigated the effects of ammonium ions on the convulsant action of MMA, MMA-induced inhibition of striatal succinate dehydrogenase, and the striatal content of thiobarbituric acid-reactive substances (TBARS). Adult rats were injected with ammonium acetate (1.5 mmol/kg, sc) or sodium acetate (1.5 mmol/kg, sc), followed 5 min later by buffered MMA (3 micromol/microl) or NaCl (4.5 micromol/microl) injected into the striatum. The animals were observed in an open field for the appearance of convulsive episodes. After 30 min of behavioral evaluation, the animals were sacrificed and had their striatal TBARS content measured. Ammonium acetate pretreatment caused no behavioral effects per se, but potentiated MMA-induced convulsions and increased basal TBARS content and MMA-induced TBARS production in the striatum. Ammonium chloride had no effect on basal succinate dehydrogenase activity and did not alter MMA-induced inhibition of SDH in vitro. These results suggest that ammonia potentiates MMA-induced behavioral effects through a mechanism that does not involve further succinate dehydrogenase inhibition, but may involve facilitation of MMA-induced oxidative damage and provide evidence that ammonia and MMA may have mutually additive toxicity.