New in vitro model derived from brain-specific Mut-/- mice confirms cerebral ammonium accumulation in methylmalonic aciduria.

Methylmalonic aciduria (MMAuria) is an inborn error of metabolism leading to neurological deterioration. In this study, we used 3D organotypic brain cell cultures derived from embryos of a brain-specific Mut <sup>-/-</sup> (brain KO) mouse to investigate mechanisms leading to brain damage. We challenged our in vitro model by a catabolic stress (temperature shift). Typical metabolites for MMAuria as well as a massive NH4 <sup>+</sup> increase were found in the media of brain KO cultures. We investigated different pathways of intracerebral NH <sub>4</sub> <sup>+</sup> production and found increased expression of glutaminase 2 and diminished expression of GDH1 in Mut <sup>-/-</sup> aggregates. While all brain cell types appeared affected in their morphological development in Mut <sup>-/-</sup> aggregates, the most pronounced effects were observed on astrocytes showing swollen fibers and cell bodies. Inhibited axonal elongation and delayed myelination of oligodendrocytes were also noted. Most effects were even more pronounced after 48 h at 39 °C. Microglia activation and an increased apoptosis rate suggested degeneration of Mut <sup>-/-</sup> brain cells. NH <sub>4</sub> <sup>+</sup> accumulation might be the trigger for all observed alterations. We also found a generalized increase of chemokine concentrations in Mut <sup>-/-</sup> culture media at an early developmental stage followed by a decrease at a later stage. We proved for the first time that Mut <sup>-/-</sup> brain cells are indeed able to produce the characteristic metabolites of MMAuria. We confirmed significant NH <sub>4</sub> <sup>+</sup> accumulation in culture media of Mut <sup>-/-</sup> aggregates, suggesting that intracellular NH <sub>4</sub> <sup>+</sup> concentrations might even be higher, gave first clues on the mechanisms leading to NH <sub>4</sub> <sup>+</sup> accumulation in Mut <sup>-/-</sup> brain cells, and showed the involvement of neuroinflammatory processes in the neuropathophysiology of MMAuria