Loss of SHMT2 and Folate Deficiency Impair Energy Metabolism in Mouse Embryonic Fibroblasts Cells

Abstract

ObjectivesSerine hydroxymethyltransferase 2 (SHMT2) catalyzes the reversible conversion of tetrahydrofolate (THF) and serine producing THF-conjugated one-carbon units and glycine in the mitochondria. Recently biallelic SHMT2 variants were identified in humans and suggested to alter the protein’s active site, potentially disrupting enzymatic function. Indeed, patient fibroblasts exhibited impaired oxidative capacity, however, with no changes in oxidative phosphorylation protein levels. Furthermore, immortalized cells models of total SHMT2 loss or folate deficiency exhibit decreased oxidative capacity and impaired mitochondrial complex I assembly and protein levels suggesting folate-mediated one-carbon metabolism (FOCM) and the oxidative phosphorylation system are functionally coordinated. We have developed a mouse model of reduced Shmt2 expression that provides an appropriate reference model to further study the deleterious human SHMT2 variants and their connection to folate metabolism. MethodsPrimary mouse embryonic fibroblasts (MEF) were isolated from a C57Bl/6 dam crossed with a heterozygous Shmt2+/– (HET) male. MEF embryos were genotyped, and Shmt2+/+ (wild type) or Shmt2+/– MEF cells were cultured for 4 doubling in 2 nM (deficient) or 25 nM folate (sufficient) medium. Cells were examined for proliferation, total folate levels, mtDNA content, protein levels of pyruvate kinase and PGC1α, pyruvate kinase enzyme activity, and mitochondrial membrane potential and function. ResultsLoss of Shmt2 and folate deficiency significantly reduced cell proliferation and oxygen consumption rate. Total cellular folate levels and pyruvate kinase enzyme activity and protein levels were reduced with folate deficiency. While there were no changes in mitochondrial DNA content or complex 1 proteins NDUFA9 or ND3, mitochondrial membrane potential was impaired and PGC1α protein levels were elevated in Shmt2+/– MEF cells. ConclusionsOur results demonstrate that loss of SHMT2 impairs cellular proliferation and that SHMT2 expression and folate status impact cellular energy metabolism and mitochondrial health. Funding SourcesThis study was financially supported by a President’s Council of Cornell Women Award and Cornell University Division of Nutritional Sciences funds.