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

Abstract

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 Ca(2+)-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. We propose that brain glutamate oxidation is disturbed by 2-methylcitric acid (2MCA), which accumulates in tissues from patients with propionic and methylmalonic acidemias because of a competitive inhibition of glutamate dehydrogenase (GDH) activity. 2MCA also induced mitochondrial permeability transition (PT) and decreased ATP generation in brain mitochondria. We believe that these pathomechanisms may be involved in the neurological dysfunction of these diseases.

Keywords: 2-methylcitric acid; glutamate dehydrogenase; glutamate oxidative metabolism; mitochondrial permeability transition; mitochondrial respiration.