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Using 3D organotypic rat brain cell cultures in aggregates we recently identified 2-methylcitrate (2-MCA) as the main toxic metabolite for developing brain cells in methylmalonic aciduria. Exposure to 2-MCA triggered morphological changes and apoptosis of brain cells. This was accompanied by increased ammonium and decreased glutamine levels. However, the sequence and causal relationship between these phenomena remained unclear. To understand the sequence and time course of pathogenic events, we exposed 3D rat brain cell aggregates to different concentrations of 2-MCA (0.1, 0.33 and 1.0 mM) from day in vitro (DIV) 11 to 14. Aggregates were harvested at different time points from DIV 12 to 19. We compared the effects of a single dose of 1 mM 2-MCA administered on DIV 11 to the effects of repeated doses of 1 mM 2-MCA. Pan-caspase inhibitors Z-VAD FMK or Q-VD-OPh were used to block apoptosis. Ammonium accumulation in the culture medium started within few hours after the first 2-MCA exposure. Morphological changes of the developing brain cells were already visible after 17 h. The highest rate of cleaved caspase-3 was observed after 72 h. A dose-response relationship was observed for all effects. Surprisingly, a single dose of 1 mM 2-MCA was sufficient to induce all of the biochemical and morphological changes in this model. 2-MCA-induced ammonium accumulation and morphological changes were not prevented by concomitant treatment of the cultures with pan-caspase inhibitors Z-VAD FMK or Q-VD-OPh: ammonium increased rapidly after a single 1 mM 2-MCA administration even after apoptosis blockade. We conclude that following exposure to 2-MCA, ammonium production in brain cell cultures is an early phenomenon, preceding cell degeneration and apoptosis, and may actually be the cause of the other changes observed. The fact that a single dose of 1 mM 2-MCA is sufficient to induce deleterious effects over several days highlights the potential damaging effects of even short-lasting metabolic decompensations in children affected by methylmalonic aciduria. [ABSTRACT FROM AUTHOR]