Inhibition of mitochondrial creatine kinase activity from rat cerebral cortex by methylmalonic acid

Accumulation of methylmalonic acid (MMA) in tissues and biological fluids is the biochemical hallmark of patients affected by the neurometabolic disorder known as methylmalonic acidemia (MMAemia). Although this disease is predominantly characterized by severe neurological findings, the underlying mechanisms of brain injury are not totally established. In the present study, we investigated the effect of MMA, as well as propionic (PA) and tiglic (TA) acids, whose concentrations are also increased but to a lesser extend in MMAemia, on total (tCK), cytosolic (Cy-CK) and mitochondrial (Mi-CK) creatine kinase (CK) activities from cerebral cortex of 30-day-old Wistar rats. Total CK activity (tCK) was measured in whole cell homogenates, whereas Cy-CK and Mi-CK were determined, respectively, in cytosolic and mitochondrial preparations from rat cerebral cortex. We verified that tCK and Mi-CK activities were significantly inhibited by MMA at concentrations as low as 1 mM, in contrast to Cy-CK which was not affected by the presence of the acid in the incubation medium. Furthermore, PA and TA, at concentrations as high as 5 mM, did not alter CK activity. We also observed that the inhibitions provoked by MMA were fully prevented by pre-incubation of the homogenates with reduced glutathione, suggesting that the inhibitory effect of MMA was possibly mediated by oxidation of essential thiol groups of the enzyme. Considering the importance of CK for brain metabolism homeostasis, our results suggest that inhibition of this enzyme by increased levels of MMA may contribute to the neurodegeneration of patients affected by MMAemia and explain previous reports showing an impairment of brain energy metabolism and a reduction of brain phosphocreatine levels caused by MMA.     

Lactate Dehydrogenase Activity is Inhibited by Methylmalonate in vitro

Methylmalonic acidemia (MMAemia) is an inherited metabolic disorder of branched amino acid and odd-chain fatty acid metabolism, involving a defect in the conversion of methylmalonyl-coenzyme A to succinyl-coenzyme A. Systemic and neurological manifestations in this disease are thought to be associated with the accumulation of methylmalonate (MMA) in tissues and biological fluids with consequent impairment of energy metabolism and oxidative stress. In the present work we studied the effect of MMA and two other inhibitors of mitochondrial respiratory chain complex II (malonate and 3-nitropropionate) on the activity of lactate dehydrogenase (LDH) in tissue homogenates from adult rats. MMA potently inhibited LDH-catalyzed conversion of lactate to pyruvate in liver and brain homogenates as well as in a purified bovine heart LDH preparation. LDH was about one order of magnitude less sensitive to inhibition by MMA when catalyzing the conversion of pyruvate to lactate. Kinetic studies on the inhibition of brain LDH indicated that MMA inhibits this enzyme competitively with lactate as a substrate (K _i=3.02±0.59 mM). Malonate and 3-nitropropionate also strongly inhibited LDH-catalyzed conversion of lactate to pyruvate in brain homogenates, while no inhibition was observed by succinate or propionate, when present in concentrations of up to 25 mM. We propose that inhibition of the lactate/pyruvate conversion by MMA contributes to lactate accumulation in blood, metabolic acidemia and inhibition of gluconeogenesis observed in patients with MMAemia. Moreover, the inhibition of LDH in the central nervous system may also impair the lactate shuttle between astrocytes and neurons, compromising neuronal energy metabolism.S. R. Mirandola and E. N. Maciel contributed equally to this work.     

Quantitative analysis of mitochondrial protein expression in methylmalonic acidemia by two-dimensional difference gel electrophoresis

Isolated methylmalonic acidemia (MMA) is a rare metabolic disease due to the deficient activity of L-methylmalonyl-CoA mutase (MCM). This mitochondrial enzyme converts L-methylmalonyl-CoA to succinyl-CoA using adenosylcobalamin (Adocbl) as cofactor. Isolated MMA is subdivided into five forms: mut MMA associated with MCM deficiency, three different defects related to mitochondrial Adocbl formation (cblA, cblB, and cblH), and cblD variant 2. We performed proteomic analysis on mitochondria from an individual with cblH/cblD disorder using 2-D DIGE to identify differentially expressed proteins in this disease. Comparative analysis of control/patient mitochondrial proteome allowed us to identify differential expression of 10 proteins. The most notable groups included proteins involved in apoptosis (cytochrome c), oxidative stress (manganese superoxide dismutase) and cell metabolism (succinyl-CoA ligase (GDP forming) and mitochondrial glycerophosphate dehydrogenase). Immunoblot analysis further validated 2-D DIGE results of two of these proteins in multiple MMA patients, suggesting that the differences in expression are a general effect in this disorder. It is feasible that the differential proteins identified in this study have a biological significance and might be related to the pathophysiology of MMA.     

Methylmalonic acid,a biochemical hallmark of methylmalonic acidurias but no inhibitor of mitochondrial respiratory chain

Methylmalonic acidurias are biochemically characterized by an accumulation of methylmalonic acid and alternative metabolites. An impairment of energy metabolism plays a key role in the pathophysiology of this disease, resulting in neurodegeneration of the basal ganglia and renal failure. It has become the subject of intense debates whether methylmalonic acid is the major toxin, inhibiting respiratory chain complex II. To elucidate whether methylmalonic acid is a respiratory chain inhibitor, we used spectrophotometric analysis of complex II activity in submitochondrial particles from bovine heart, radiometric analysis of 14C-labeled substrates (pyruvate, malate, succinate), and analysis of ATP production in muscle from mice. Methylmalonic acid revealed no direct effects on the respiratory chain function, i.e. on single electron transferring complexes I-IV, ATPase, and mitochondrial transporters. However, we identified a variety of variables that must be carefully controlled to avoid an artificial inhibition of complex II activity. In summary, the study verifies our hypothesis that methylmalonic acid is not the major toxic metabolite in methylmalonic acidurias. Inhibition of respiratory chain and tricarboxylic acid cycle is most likely induced by synergistically acting alternative metabolites, in particular 2-methylcitric acid, malonic acid, and propionyl-CoA.     

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.     

Characterization of enzymatic deficiencies of branched chain amino-acid catabolism in human fibroblasts by genetic complementation

Leucine and Isoleucine metabolism in cultured skin fibroblasts from patients with leucinosis, beta-Ketothiolase deficiency, propionic, methylmalonic and isovaleric acidemia, was compared with that in normal fibroblasts. A simple assay was developed using (U14C) Isoleucine and (U14C) Leucine as substrates. Radioactive incorporation into protein aminoacids were measured. The (U14C) branched chain aminoacid incorporation into proteins provides an estimation of the protein synthesis and the incorporation ratio (14C) Aspartate + (14C) Glutamate/(14C) branched chain aminoacid, measures the integrity of the metabolic pathway. Complementation tests permits to characterize the genetic defect. The incorporation ratio was significantly decreased in blocked pathways, namely in leucinosis and isovaleric acidemia in the presence of (U14C) Leucine and in Leucinosis, beta-Ketothiolase deficiency, propionic and methylmalonic acidemia in the presence of (U14C) Isoleucine. There was a significant restoration of activity in mutant strains with distinct genetic defects after polyethylene-glycol fusion. This assay provides a valuable tool to screen for enzymatic deficiencies of branched chain aminoacid catabolism.     

Biomarker discovery, disease classification, and similarity query processing on high-throughput MS/MS data of inborn errors of metabolism

In newborn errors of metabolism, biomarkers are urgently needed for disease screening, diagnosis, and monitoring of therapeutic interventions. This article describes a 2-step approach to discover metabolic markers, which involves (1) the identification of marker candidates and (2) the prioritization of them based on expert knowledge of disease metabolism. For step 1, the authors developed a new algorithm, the biomarker identifier (BMI), to identify markers from quantified diseased versus normal tandem mass spectrometry data sets. BMI produces a ranked list of marker candidates and discards irrelevant metabolites based on a quality measure, taking into account the discriminatory performance, discriminatory space, and variance of metabolites' concentrations at the state of disease. To determine the ability of identified markers to classify subjects, the authors compared the discriminatory performance of several machine-learning paradigms and described a retrieval technique that searches and classifies abnormal metabolic profiles from a screening database. Seven inborn errors of metabolism-- phenylketonuria (PKU), glutaric acidemia type I (GA-I), 3-methylcrotonylglycinemia deficiency (3-MCCD), methylmalonic acidemia (MMA), propionic acidemia (PA), medium-chain acylCoAdehydrogenase deficiency (MCADD), and 3-OH long-chain acyl CoA dehydrogenase deficiency (LCHADD)-were investigated. All primarily prioritized marker candidates could be confirmed by literature. Some novel secondary candidates were identified (i.e., C16:1 and C4DC for PKU, C4DC for GA-I, and C18:1 forMCADD), which require further validation to confirm their biochemical role during health and disease.     

Inhibition by Methylmalonate of Glycine Uptake by Synaptosomes from Rat Spinal Cord

Methylmalonate is accumulated in the genetically linked deficiency of methylmalonyl-CoA mutase (methylmalonic acidemia). In this condition is also observed an elevation of the glycine levels. This communication reports the inhibition of the synaptosomal glycine uptake by methylmalonate, when present at similar concentrations to those found in methylmalonic acidemia. This inhibition could be responsible, at least in part, for the neurological damage characteristic of this disease, by increasing the glycine levels in the synaptic cleft and thus interfering with the normal function of the inhibitory glycinergic synapsis in the spinal cord.     

Methylmalonyl acidemia: from mitochondrial metabolism to defective mitophagy and disease

ABSTRACT

Methylmalonic acidemia (MMA) is an autosomal recessive inborn error of metabolism due to the deficiency of mitochondrial MMUT (methylmalonyl-CoA mutase) – an enzyme that mediates the cellular breakdown of certain amino acids and lipids. The loss of MMUT leads to the accumulation of toxic organic acids causing severe organ dysfunctions and life-threatening complications. The mechanisms linking MMUT deficiency, mitochondrial alterations and cell toxicity remain uncharacterized. Using cell and animal-based models, we recently unveiled that MMUT deficiency impedes the PINK1-induced translocation of PRKN/Parkin to MMA-damaged mitochondria, thereby halting their delivery and subsequent degradation by macroautophagy/autophagy-lysosome systems. In turn, this defective mitophagy process instigates the accumulation of dysfunctional mitochondria that spark epithelial distress and tissue damage. Correction of PINK1-directed mitophagy defects or mitochondrial dysfunctions rescues epithelial distress in MMA cells and alleviates disease-relevant phenotypes in mmut?deficient zebrafish. Our findings suggest a link between primary MMUT deficiency and diseased mitochondria, mitophagy dysfunction and cell distress, offering potential therapeutic perspectives for MMA and other metabolic diseases.     

Effect of methylmalonate on ketone body metabolism in developing rat brain

The oxidation of 3-hydroxy[3-¹⁴C]butyrate to CO₂ and its incorporation into cerebral lipids by cortex slices from one-week old rats were markedly inhibited by methylmalonate. However, methylmalonate had no effect on the metabolism of labelled aceto- acetate, glucose and acetate by brain slices. Addition of propionate in the incubation medium reduced cerebral lipogenesis from labelled 3-hydroxybutyrate and acetate. Acute methylmalonic acidemia induced in one-week old pups by injecting 3% methylmalonate solution caused a reduction in the incorporation of labelled 3-hydroxybutyrate into cerebral lipids. However, acute methylmalonic acidemia had no effect on cerebral lipogensis $\text{in vivo}$ from labelled acetate. These findings show (i) the conversion of 3-hydroxybutyrate to acetoacetate by 3-hydroxybutyrate dehydrogenase in the brain is inhibited by methylmalonate, and (ii) an inhibition of cerebral lipid synthesis by propionate, which also accumulates in patients with methylmalonic aciduria.     

Pluripotent stem cell energy metabolism: an update

Recent studies link changes in energy metabolism with the fate of pluripotent stem cells (PSCs). Safe use of PSC derivatives in regenerative medicine requires an enhanced understanding and control of factors that optimize in vitro reprogramming and differentiation protocols. Relative shifts in metabolism from naïve through “primed” pluripotent states to lineage‐directed differentiation place variable demands on mitochondrial biogenesis and function for cell types with distinct energetic and biosynthetic requirements. In this context, mitochondrial respiration, network dynamics, TCA cycle function, and turnover all have the potential to influence reprogramming and differentiation outcomes. Shifts in cellular metabolism affect enzymes that control epigenetic configuration, which impacts chromatin reorganization and gene expression changes during reprogramming and differentiation. Induced PSCs (iPSCs) may have utility for modeling metabolic diseases caused by mutations in mitochondrial DNA, for which few disease models exist. Here, we explore key features of PSC energy metabolism research in mice and man and the impact this work is starting to have on our understanding of early development, disease modeling, and potential therapeutic applications.     

Inhibition of the mitochondrial respiratory chain complex activities in rat cerebral cortex by methylmalonic acid

Propionic and methylmalonic acidemic patients have severe neurologic symptoms whose etiopathogeny is still obscure. Since increase of lactic acid is detected in the urine of these patients, especially during metabolic decompensation when high concentrations of methylmalonate (MMA) and propionate (PA) are produced, it is possible that cellular respiration may be impaired in these individuals. Therefore, we investigated the effects of MMA and PA (1, 2.5 and 5 mM), the principal metabolites which accumulate in these conditions, on the mitochondrial respiratory chain complex activities succinate: 2,6-dichloroindophenol (DCIP) oxireductase (complex II); succinate: cytochrome c oxireductase (complexII+CoQ+III); NADH: cytochrome c oxireductase (complex I+CoQ+complex III); and cytochrome c oxidase (COX) (complex IV) from cerebral cortex homogenates of young rats. The effect of MMA on ubiquinol: cytochrome c oxireductase (complex III) and NADH: ubiquinone oxireductase (complex I) activities was also tested. Control groups did not contain MMA and PA in the incubation medium. MMA significantly inhibited complex I+III (32–46%), complex I (61–72%), and complex II+III (15–26%), without affecting significantly the activities of complexes II, III and IV. However, by using 1 mM succinate in the assay instead of the usual 16 mM concentration, MMA was able to significantly inhibit complex II activity in the brain homogenates. In contrast, PA did not affect any of these mitochondrial enzyme activities. The effect of MMA and PA on succinate: phenazine oxireductase (soluble succinate dehydrogenase (SDH)) was also measured in mitochondrial preparations. The results showed significant inhibition of the soluble SDH activity by MMA (11–27%) in purified mitochondrial fractions. Thus, if the in vitro inhibition of the oxidative phosphorylation system is also expressed under in vivo conditions, a deficit of brain energy production might explain some of the neurological abnormalities found in patients with methylmalonic acidemia (MMAemia) and be responsible for the lactic acidemia/aciduria identified in some of them.     

Secondary mitochondrial dysfunction in propionic aciduria: a pathogenic role for endogenous mitochondrial toxins

Mitochondrial dysfunction during acute metabolic crises is considered an important pathomechanism in inherited disorders of propionate metabolism, i.e. propionic and methylmalonic acidurias. Biochemically, these disorders are characterized by accumulation of propionyl-CoA and metabolites of alternative propionate oxidation. In the present study, we demonstrate uncompetitive inhibition of PDHc (pyruvate dehydrogenase complex) by propionyl-CoA in purified porcine enzyme and in submitochondrial particles from bovine heart being in the same range as the inhibition induced by acetyl-CoA, the physiological product and known inhibitor of PDHc. Evaluation of similar monocarboxylic CoA esters showed a chain-length specificity for PDHc inhibition. In contrast with CoA esters, non-esterified fatty acids did not inhibit PDHc activity. In addition to PDHc inhibition, analysis of respiratory chain and tricarboxylic acid cycle enzymes also revealed an inhibition by propionyl-CoA on respiratory chain complex III and alpha-ketoglutarate dehydrogenase complex. To test whether impairment of mitochondrial energy metabolism is involved in the pathogenesis of propionic aciduria, we performed a thorough bioenergetic analysis in muscle biopsy specimens of two patients. In line with the in vitro results, oxidative phosphorylation was severely compromised in both patients. Furthermore, expression of respiratory chain complexes I-IV and the amount of mitochondrial DNA were strongly decreased, and ultrastructural mitochondrial abnormalities were found, highlighting severe mitochondrial dysfunction. In conclusion, our results favour the hypothesis that toxic metabolites, in particular propionyl-CoA, are involved in the pathogenesis of inherited disorders of propionate metabolism, sharing mechanistic similarities with propionate toxicity in micro-organisms.     

(1)H NMR based metabolomics of CSF and blood serum: a metabolic profile for a transgenic rat model of Huntington disease

Huntington disease (HD) is a hereditary brain disease. Although the causative gene has been found, the exact mechanisms of the pathogenesis are still unknown. Recent investigations point to metabolic and energetic dysfunctions in HD neurons. Both univariate and multivariate analyses were used to compare proton nuclear magnetic resonance spectra of serum and cerebrospinal fluid (CSF) taken from presymptomatic HD transgenic rats and their wild-type littermates. N-acetylaspartate (NAA), was found to be significantly decreased in the serum of HD rats compared to wild-type littermates. Moreover, in the serum their levels of glutamine, succinic acid, glucose and lactate are significantly increased as well. An increased concentration of lactate and glucose is also found in CSF. There is a 1:1 stoichiometry coupling glucose utilization and glutamate cycling. The observed increase in the glutamine concentration, which indicates a shutdown in the neuronal-glial glutamate-glutamine cycling, results therefore in an increased glucose concentration. The elevated succinic acid concentration might be due to an inhibition of succinate dehydrogenase, an enzyme linked to the mitochondrial respiratory chain and TCA cycle. Moreover, reduced levels of NAA may reflect an impairment of mitochondrial energy production. In addition, the observed difference in lactate supports a deficiency of oxidative energy metabolism in rats transgenic for HD as well. The observed metabolic alterations seem to be more profound in serum than in CSF in presymptomatic rats. All findings suggest that even in presymptomatic rats, a defect in energy metabolism is already apparent. These results support the hypothesis of mitochondrial energy dysfunction in HD.     

Mitochondrial function in vivo: spectroscopy provides window on cellular energetics

Mitochondria integrate the key metabolic fluxes in the cell. This role places this organelle at the center of cellular energetics and, hence, mitochondrial dysfunction underlies a growing number of human disorders and age-related degenerative diseases. Here we present novel analytical and technical methods for evaluating mitochondrial metabolism and (dys)function in human muscle in vivo. Three innovations involving advances in optical spectroscopy (OS) and magnetic resonance spectroscopy (MRS) permit quantifying key compounds in energy metabolism to yield mitochondrial oxidation and phosphorylation fluxes. The first of these uses analytical methods applied to optical spectra to measure hemoglobin (Hb) and myoglobin (Mb) oxygenation states and relative contents ([Hb]/[Mb]) to determine mitochondrial respiration (O₂ uptake) in vivo. The second uses MRS methods to quantify key high-energy compounds (creatine phosphate, PCr, and adenosine triphosphate, ATP) to determine mitochondrial phosphorylation (ATP flux) in vivo. The third involves a functional test that combines these spectroscopic approaches to determine mitochondrial energy coupling (ATP/O₂), phosphorylation capacity (ATP_max) and oxidative capacity (O_2max) of muscle. These new developments in optical and MR tools allow us to determine the function and capacity of mitochondria noninvasively in order to identify specific defects in vivo that are associated with disease in human and animal muscle. The clinical implication of this unique diagnostic probe is the insight into the nature and extent of dysfunction in metabolic and degenerative disorders, as well as the ability to follow the impact of interventions designed to reverse these disorders.     

Newborn mass screening and selective screening using electrospray tandem mass spectrometry in Japan

Electrospray tandem mass spectrometry was applied to detect a series of inherited metabolic disorders during a newborn-screening pilot study and a selective screening in Japan. In our mass screening of 102,200 newborns, five patients with propionic acidemia, two with methylmalonic acidemia, two with medium-chain acyl-CoA dehydrogenase deficiency, three with citrullinemia type II, and one with phenylketonuria were identified. In a selective screening of 164 patients with symptoms mainly related to hypoglycemia and/or hyperammonemia, 12 with fatty acid oxidation disorders and six with other disorders were found. The results indicated the importance of newborn screening using this technology in Japan.     

Congenital methylmalonic acidemia: a variant of the B12 'non-responsive' form with evidence for reduced affinity of methylmalonyl-CoA mutase for its B12-coenzyme.

Methylmalonate metabolism was investigated in fibroblasts and leukocytes of two unrelated patient with a B12-nonresponsive type of congenital methylmalonic acidemia. Intact fibroblasts from both patients showed a defective metabolism of methyl-14 c-malonate to 14CO2, whereas no such defect was found in their intact peripheral leukocytes. In disrupted fibroblasts, the conversion of methylmalonyl coenzyme A to succinyl coenzyme A was markedly reduced but was completely normalized by the addition of 5'-deoxyadenosylcobalamin (AdoCb1; 10(-5) mol/l), the specific coenzyme of methylmalonyl coenzyme A mutase. Assays with decreasing concentrations of AdoCbl (10(-5)-10(-11) mol/l) suggested a reduced affinity of the mutase apoenzyme for its coenzyme, implicating yet another variant of this heterogeneous disease.