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.

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.

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.

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.

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.

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.

Creatine protects against the convulsive behavior and lactate production elicited by the intrastriatal injection of methylmalonate.

Methylmalonic acidemias are metabolic disorders caused by a severe deficiency of methylmalonyl-CoA mutase activity, which are characterized by neurological dysfunction, including convulsions. It has been reported that the accumulating metabolite, L-methylmalonic acid (MMA), inhibits succinate dehydrogenase leading to ATP depletion in vitro, and that the intrastriatal injection of MMA induces convulsions through secondary NMDA receptor stimulation. In this study we investigated the effect of creatine (1.2, 3.6 and 12.0 mg/kg, (i.p.), [DOSAGE ERROR CORRECTED] succinate (1.5 micromol/striatum) and MK-801 (3 nmol/striatum) on the convulsions and on the striatal lactate increase induced by MMA (4.5 micromol/striatum) in rats. The effect of creatine on the striatal phosphocreatine content and on MMA-induced phosphocreatine depletion was also evaluated. Creatine, succinate and MK-801 pretreatment decreased the number and duration of convulsive episodes and the lactate increase elicited by MMA. Creatine, but not succinate, prevented the convulsions and the lactate increase induced by the direct stimulation of NMDA receptors. Acute creatine administration increased the total striatal phosphocreatine content and prevented MMA-induced phosphocreatine depletion. Our results suggest that MMA increases lactate production through secondary NMDA receptor activation, and it is proposed that the anticonvulsant effect of creatine against MMA-induced convulsions may be due to an increase in the phosphocreatine content available for metabolic purposes.

Methylmalonate toxicity in primary neuronal cultures.

Several inhibitors of mitochondrial complex II cause neuronal death in vivo and in vitro. The goal of the present work was to characterize in vitro the effects of malonate (a competitive blocker of the complex) which induces neuronal death in a pattern similar to that seen in striatum in Huntington's disease. Exposure of striatal and cortical cultures from embryonic rat brain for 24 h to methylmalonate, a compound which produces malonate intracellularly, led to a dose-dependent cell death. Methylmalonate (10 mM) caused >90% mortality of neurons although cortical cells were unexpectedly more vulnerable. Cell death was attenuated in a medium containing antioxidants. Further characterization revealed that DNA laddering could be detected after 3 h of treatment. Morphological observations (videomicroscopy and Hoechst staining) showed that both necrotic and apoptotic cell death occurred in parallel; apoptosis was more prevalent. A decrease in the ATP/ADP ratio was observed after 3 h of treatment with 10 mM methylmalonate. In striatal cultures it occurred concomitantly with a decline in GABA and a rise in aspartate content and the aspartate/glutamate ratio. Changes in ion concentrations were measured in similar cortical cultures from mouse brain. Neuronal [Na+]i increased while [K+]i and membrane potential decreased after 20 min of continuous incubation in 10 mM methylmalonate. These changes progressed with time, and a rise in [Ca2+]i was also observed after 1 h. The results demonstrate that malonate collapses cellular ion gradients, restoration of which imposes an additional load on the already compromised ATP-generation machinery. An early elevation in [Ca2+]i may trigger an increase in activity of proteases, lipases and endonucleases and production of free radicals and DNA damage which, ultimately, leads to cells death. The data also suggest that maturational and/or extrinsic factors are likely to be critical for the increased vulnerability of striatal neurons to mitochondrial inhibition in vivo.