Characterization of MTHFD2L expression and alternative splicing and loss of MTHFD1L activity in murine embryos and adults




Bryant, Joshua Dale

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In Eukaryotes, folate-dependent one-carbon (1C) metabolism is a highly compartmentalized process in which mitochondria play a central role. Defects in folate metabolism are associated with diseases such as cancer, Alzheimer's disease, and neural tube defects (NTDs). 1C units are attached to tetrahydrofolate (THF) and carried in various oxidation states between folate-dependent enzymes. There is an exchange of 1C units across the mitochondrial membrane, with 1C donors such as serine and glycine being oxidized to formate in the mitochondria, which is then released into the cytoplasm. 1C units in the cytoplasm can be used for the synthesis of purines, thymidylate, and methionine for the methyl cycle. The core of the pathway in both compartments is catalyzed by the methylene-tetrahydrofolate (MTHFD) gene family. These enzymes catalyze the reversible interconversion between CH₂-THF, CH⁺-THF, CHO-THF, and formate. The cytoplasmic protein MTHFD1 is trifunctional and carries the CH₂-THF dehydrogenase, CH⁺-THF cyclohydrolase, and 10-CHO-THF synthetase activities necessary to carry out these interconversions. In the mitochondria, two bifunctional isozymes, MTHFD2 and MTHFD2L, carry the dehydrogenase/cyclohydrolase (D/C) activities. The monofunctional enzyme MTHFD1L is responsible for the synthetase activity. MTHFD2 is only expressed in embryos and transformed cells, and the enzyme responsible for the D/C activity in adults was unknown until the recent discovery of MTHFD2L. In this work, characterization of the expression of MTHFD2L in mouse embryos and adults is described. Expression of MTHFD2L in embryos was found to be switched on between embryonic days 8.5-10.5, and remains high throughout development. MTHFD2L is also widely expressed in adults, with highest expression in brain and lung. A splice variant of MTHFD2L lacking exon 8 was found to be abundant in embryos but was not catalytically active in vitro or in vivo. MTHFD1L is an essential protein, and SNPs in MTHFD1L are associated with increased risk for Alzheimer's disease and NTDs in humans. Loss of MTHFD1L activity in adult mice with and without a folate deficient diet was investigated. Indications of sex-dependent behavioral anomalies were found, with evidence for genotype-dependent hyperactivity in male mice and diet-dependent anxiety in female mice, but further investigation of these findings is warranted. Finally, metabolic defects associated with NTDs and growth restriction in MTHFD1L-null (Mthfd1l [superscript z/z]) embryos were identified. Glycolysis, the TCA cycle, and the metabolism of methionine, purines, and multiple amino acids were found to be disrupted in Mthfd1l [superscript z/z] embryos. These altered metabolic pathways suggest potential future therapies for preventing NTDs in humans.



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