A 3D in vitro model of rat organotypic brain cell cultures in aggregates was
used to investigate neurotoxicity mechanisms in methylmalonic aciduria.
1 mM methylmalonate (MMA), 2-methylcitrate (2-MCA) or propionate
(PA) were repeatedly added to the culture media at two different time points
of the cultures. In cultures treated with 2-MCA, we observed a significant
increase of lactate in the medium, consistent with a possible inhibition of
Krebs cycle and respiratory chain, as described earlier in the literature.
Interestingly, we further observed that 2-MCA induced an important increase
in ammonia production with concomitant decrease of glutamine
concentrations, which suggests an inhibition of the astrocytic enzyme
glutamine synthetase. These previously unreported findings may uncover
a pathogenic mechanism in this disease with deleterious effects on early
stages of brain development. By immunohistochemistry we could show that
2-MCA substantially increased the number of apoptotic cells. On the
cellular level, 2-MCA had a toxic effect (cell swelling and cell death) on
glial cells, but not on neurons. Surprisingly, MMA seemed to have a growth
stimulating effect on the cultures. We can conclude that 2-MCA was the
most toxic metabolite in our model for methylmalonic aciduria inducing
ammonia accumulation and massive apoptosis in brain cells.