Mitochondrial FAD shortage in SLC25A32 deficiency affects folate-mediated one-carbon metabolism

The SLC25A32 dysfunction is associated with neural tube defects (NTDs) and exercise intolerance, but very little is known about disease specific mechanisms due to a paucity of animal models. Here, we generated homozygous (Slc25a32Y174C/Y174C and Slc25a32K235R/K235R) and compound heterozygous (Slc25a32Y174C/K235R) knock-in mice by mimicking the missense mutations identified from our patient. A homozygous knock-out (Slc25a32-/-) mouse was also generated. The Slc25a32K235R/K235R and Slc25a32Y174C/K235R mice presented with mild motor impairment and recapitulated the biochemical disturbances of the patient. While Slc25a32-/- mice die in utero with NTDs. All Slc25a32 mutations did not hinder the mitochondrial uptake of folates but specifically blocked the uptake of flavin adenine dinucleotide (FAD). A positive correlation between SLC25A32 dysfunction and flavoenzyme deficiency was observed. Besides the flavoenzymes involved in fatty acid β-oxidation and amino acid metabolism impaired, Slc25a32-/- embryos still had a subunit of glycine cleavage system–dihydrolipoamide dehydrogenase damaged, resulting in glycine accumulation and glycine derived-formate reduction, which further disturbed folate-mediated one-carbon metabolism, leading to 5-methyltetrahydrofolate shortage and other folate intermediates accumulated. Maternal formate supplementation increased the 5-methyltetrahydrofolate levels and ameliorated the NTDs in Slc25a32-/- embryos. The Slc25a32K235R/K235R mice had no glycine oxidation defect but had another formate donor-choline metabolism interrupted and mitochondrial folates deficient. Formate supplementation increased mitochondrial folates amounts of mice, but this effect was not restricted to the Slc25a32 mutant mice. In summary, we established novel animal models, which enabled us to better understand the function of SLC25A32 and to elucidate the role of SLC25A32 dysfunction in human disease development and progression.