(P30-016-23) Disruption of Mitochondrial Folate Metabolism Leads to Mitochondrial DNA Leakage and Apoptosis

Objectives: Folate-mediated one-carbon metabolism (FOCM) is required for the biosynthesis of purines, thymidylate (dTMP), and methionine. FOCM is essential to maintain an adequate cellular dTMP level to preserve the integrity of the nuclear and mitochondrial genomes. Disrupting mitochondrial FOCM leads to uracil misincorporation into mitochondrial DNA (mtDNA) and impaired mitochondrial function.  However, to the mechanisms whereby uracil in mtDNA impairs mitochondrial function are uncharacterized. The release of mtDNA into the cytosol acts as a damage-associated molecular pattern (DAMP), resulting in inflammation and cell death. Previously, we reported that serine hydroxymethlytransferase 2 (Shmt2) heterozygosity impairs mitochondrial dTMP synthesis and mitochondrial function in mice by increasing uracil in mtDNA. This study aimed to investigate whether disruption of mitochondrial FOCM through Shmt2 heterozygosity leads to the release of mtDNA into the cytosol.

Methods: Shmt2^+/+ and Shmt2^+/- mouse embryonic fibroblast (MEF) cells were cultured in Minimum Essential Medium Alpha (MEM Alpha) supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum to assess the effect of decreased Shmt2 expression on the mtDNA leakage into the cytosol, DAMP activity, and apoptosis.

Results: Shmt2^+/-MEFs showed a >2-fold increase in cytosolic mtDNA leakage (p < 0.05). There was no significant difference in cellular mtDNA content between Shmt2^+/- and Shmt2^+/+ MEF cells. In addition, MEFs with decreased Shmt2 expression activated programmed cell death by increasing pro-apoptotic signals.

Conclusions: This study demonstrates that Shmt2 heterozygosity-induced disruption of mitochondrial FOCM leads to cytosolic mtDNA leakage, which activates apoptosis. These findings suggest that increased mtDNA leakage into the cytosol as a result of disrupted mitochondrial FOCM may lead to inflammation.

Funding Sources: Division of Nutritional Science, Cornell University