Maternal dietary uridine causes, and deoxyuridine prevents, neural tube closure defects in a mouse model of folate-responsive neural tube defects.

BACKGROUND: Folic acid prevents neural tube closure defects (NTDs), but the causal metabolic pathways have not been established. Serine hydroxymethyltransferase 1 (SHMT1) is an essential scaffold protein in folate-dependent de novo thymidylate synthesis in the nucleus. SHMT1-deficient mice provide a model to investigate folic acid-responsive NTDs wherein disruption of de novo thymidylate synthesis impairs neural tube closure. OBJECTIVE: We examined the effects of maternal supplementation with the pyrimidine nucleosides uridine, thymidine, or deoxyuridine with and without folate deficiency on NTD incidence in the Shmt1 mouse model. DESIGN: Shmt1(+/+) and Shmt1(-/-) female mice fed folate-replete or folate-deficient diets and supplemented with uridine, thymidine, or deoxyuridine were bred, and litters (n = 10-23 per group) were examined for the presence of NTDs. Biomarkers of impaired folate status and metabolism were measured, including plasma nucleosides, hepatic uracil content, maternal plasma folate concentrations, and incorporation of nucleoside precursors into DNA. RESULTS: Shmt1(+/-) and Shmt1(-/-) embryos from dams fed the folate-deficient diet were susceptible to NTDs. No NTDs were observed in litters from dams fed the folate-deficient diet supplemented with deoxyuridine. Surprisingly, uridine supplementation increased NTD incidence, independent of embryo genotype and dietary folic acid. These dietary nucleosides did not affect maternal hepatic uracil accumulation in DNA but did affect plasma folate concentrations. CONCLUSIONS: Maternal deoxyuridine supplementation prevented NTDs in dams fed the folate-deficient diet, whereas maternal uridine supplementation increased NTD incidence, independent of folate and embryo genotype. These findings provide new insights into the metabolic impairments and mechanisms of folate-responsive NTDs resulting from decreased Shmt1 expression.

Disruption of shmt1 impairs hippocampal neurogenesis and mnemonic function in mice.

Impaired folate-mediated one-carbon metabolism (OCM) has emerged as a risk factor for several diseases associated with age-related cognitive decline, but the underlying mechanisms remain unknown and thus hinder the identification of subpopulations most vulnerable to OCM disruption. Here we investigated the role of serine hydroxymethyltransferase 1 (SHMT1), a folate-dependent enzyme regulating de novo thymidylate biosynthesis, in influencing neuronal and cognitive function in the adult mouse. We observed Shmt1 expression in the hippocampus, including the granule cell layer of the dentate gyrus (DG), and examined hippocampal neurogenesis and hippocampal-dependent fear conditioning in mice deficient for Shmt1. We used a 3 × 3 design in which adult male Shmt1(+/+), Shmt1(+/-), and Shmt1(-/-) mice were fed folic acid control (2 mg/kg), folic acid-deficient (0 mg/kg), or folic acid-supplemented (8 mg/kg) diets from weaning through the duration of the study. Proliferation within the DG was elevated by 70% in Shmt1(+/-) mice, yet the number of newborn mature neurons was reduced by 98% compared with that in Shmt1(+/+) mice. Concomitant with these alterations, Shmt1(+/-) mice showed a 45% reduction in mnemonic recall during trace fear conditioning. Dietary folate manipulations alone did not influence neural outcomes. Together, these data identify SHMT1 as one of the first enzymes within the OCM pathway to regulate neuronal and cognitive profiles and implicate impaired thymidylate biosynthesis in the etiology of folate-related neuropathogenesis.

Maternal Mthfd1 disruption impairs fetal growth but does not cause neural tube defects in mice.

BACKGROUND: MTHFD1 encodes C1-tetrahydrofolate synthase, which is a folate-dependent enzyme that catalyzes the formation and interconversion of folate-activated one-carbon groups for nucleotide biosynthesis and cellular methylation. A polymorphism in MTHFD1 (1958G→A) impairs enzymatic activity and is associated with increased risk of adverse pregnancy outcomes, but the mechanisms are unknown. OBJECTIVE: The objective of this study was to determine whether disruption of the embryonic or maternal Mthfd1 gene or both interacts with impaired folate and choline status to affect neural tube closure, fetal growth, and fertility in mice and to investigate the underlying metabolic disruptions. DESIGN: Dams with a gene-trapped (gt) allele in Mthfd1 and wild-type dams were fed a control or folate- and choline-deficient AIN93G diet (Dyets Inc). Litters were examined for gross morphologic defects, crown-rump length, and resorptions. Folate status and amounts of folate-related metabolites were determined in pregnant dams. RESULTS: Reduced folate and choline status resulted in severe fetal growth restriction (FGR) and impaired fertility in litters harvested from Mthfd1(gt/+) dams, but embryonic Mthfd1(gt/+) genotype did not affect fetal growth. Gestational supplementation of Mthfd1(gt/+) dams with hypoxanthine increased FGR frequency and caused occasional neural tube defects (NTDs) in Mthfd1(gt/+) embryos. Mthfd1(gt/+) dams exhibited lower red blood cell folate and plasma methionine concentrations than did wild-type dams. CONCLUSIONS: Maternal Mthfd1(gt/+) genotype impairs fetal growth but does not cause NTDs when dams are maintained on a folate- and choline-deficient diet. Mthfd1(gt/+) mice exhibit a spectrum of adverse reproductive outcomes previously attributed to the human MTHFD1 1958G→A polymorphism. Mthfd1 heterozygosity impairs folate status in pregnant mice but does not significantly affect homocysteine metabolism.

Dietary folate, but not choline, modifies neural tube defect risk in Shmt1 knockout mice.

BACKGROUND: Low dietary choline intake has been proposed to increase the risk of neural tube defects (NTDs) in human populations. Mice with reduced Shmt1 expression exhibit a higher frequency of NTDs when placed on a folate- and choline-deficient diet and may represent a model of human NTDs. The individual contribution of dietary folate and choline deficiency to NTD incidence in this mouse model is not known. OBJECTIVE: To dissociate the effects of dietary folate and choline deficiency on Shmt1-related NTD sensitivity, we determined NTD incidence in embryos from Shmt1-null dams fed diets deficient in either folate or choline. DESIGN: Shmt1(+/+) and Shmt1(-/-) dams were maintained on a standard AIN93G diet (Dyets), an AIN93G diet lacking folate (FD), or an AIN93G diet lacking choline (CD). Virgin Shmt1(+/+) and Shmt1(-/-) dams were crossed with Shmt1(+/-) males, and embryos were examined for the presence of NTDs at embryonic day (E) 11.5 or E12.5. RESULTS: Exencephaly was observed only in Shmt1(-/-) embryos isolated from dams maintained on the FD diet (P = 0.004). Approximately 33% of Shmt1(-/-)embryos (n = 18) isolated from dams maintained on the FD diet exhibited exencephaly. NTDs were not observed in any embryos isolated from dams maintained on the CD (n = 100) or control (n = 152) diets or in any Shmt1(+/+) (n = 78) or Shmt1(+/-) embryos (n = 182). CONCLUSION: Maternal folate deficiency alone is sufficient to induce NTDs in response to embryonic Shmt1 disruption.

Nuclear localization of de novo thymidylate biosynthesis pathway is required to prevent uracil accumulation in DNA.

Uracil accumulates in DNA as a result of impaired folate-dependent de novo thymidylate biosynthesis, a pathway composed of the enzymes serine hydroxymethyltransferase (SHMT), thymidylate synthase (TYMS), and dihydrofolate reductase. In G1, this pathway is present in the cytoplasm and at S phase undergoes small ubiquitin-like modifier-dependent translocation to the nucleus. It is not known whether this pathway functions in the cytoplasm, nucleus, or both in vivo. SHMT1 generates 5,10-methylenetetrahydrofolate for de novo thymidylate biosynthesis, a limiting step in the pathway, but also tightly binds 5-methyltetrahydrofolate in the cytoplasm, a required cofactor for homocysteine remethylation. Overexpression of SHMT1 in cell cultures inhibits folate-dependent homocysteine remethylation and enhances thymidylate biosynthesis. In this study, the impact of increased Shmt1 expression on folate-mediated one-carbon metabolism was determined in mice that overexpress the Shmt1 cDNA (Shmt1tg+ mice). Compared with wild type mice, Shmt1tg+ mice exhibited elevated SHMT1 and TYMS protein levels in tissues and evidence for impaired homocysteine remethylation but surprisingly exhibited depressed levels of nuclear SHMT1 and TYMS, lower rates of nuclear de novo thymidylate biosynthesis, and a nearly 10-fold increase in uracil content in hepatic nuclear DNA when fed a folate- and choline-deficient diet. These results demonstrate that SHMT1 and TYMS localization to the nucleus is essential to prevent uracil accumulation in nuclear DNA and indicate that SHMT1-mediated nuclear de novo thymidylate synthesis is critical for maintaining DNA integrity.

Shmt1 heterozygosity impairs folate-dependent thymidylate synthesis capacity and modifies risk of Apc(min)-mediated intestinal cancer risk.

Folate-mediated one-carbon metabolism is required for the de novo synthesis of purines, thymidylate, and S-adenosylmethionine, the primary cellular methyl donor. Impairments in folate metabolism diminish cellular methylation potential and genome stability, which are risk factors for colorectal cancer (CRC). Cytoplasmic serine hydroxymethyltransferase (SHMT1) regulates the partitioning of folate-activated one-carbons between thymidylate and S-adenosylmethionine biosynthesis. Therefore, changes in SHMT1 expression enable the determination of the specific contributions made by thymidylate and S-adenosylmethionine biosynthesis to CRC risk. Shmt1 hemizygosity was associated with a decreased capacity for thymidylate synthesis due to downregulation of enzymes in its biosynthetic pathway, namely thymidylate synthase and cytoplasmic thymidine kinase. Significant Shmt1-dependent changes to methylation capacity, gene expression, and purine synthesis were not observed. Shmt1 hemizygosity was associated with increased risk for intestinal cancer in Apc(min)(/+) mice through a gene-by-diet interaction, indicating that the capacity for thymidylate synthesis modifies susceptibility to intestinal cancer in Apc(min)(/+) mice.

Shmt1 and de novo thymidylate biosynthesis underlie folate-responsive neural tube defects in mice.

BACKGROUND: Folic acid supplementation prevents the occurrence and recurrence of neural tube defects (NTDs), but the causal metabolic pathways underlying folic acid-responsive NTDs have not been established. Serine hydroxymethyltransferase (SHMT1) partitions folate-derived one-carbon units to thymidylate biosynthesis at the expense of cellular methylation, and therefore SHMT1-deficient mice are a model to investigate the metabolic origin of folate-associated pathologies. OBJECTIVES: We examined whether genetic disruption of the Shmt1 gene in mice induces NTDs in response to maternal folate and choline deficiency and whether a corresponding disruption in de novo thymidylate biosynthesis underlies NTD pathogenesis. DESIGN: Shmt1 wild-type, Shmt1(+/-), and Shmt1(-/-) mice fed either folate- and choline-sufficient or folate- and choline-deficient diets were bred, and litters were examined for the presence of NTDs. Biomarkers of impaired folate metabolism were measured in the dams. In addition, the effect of Shmt1 disruption on NTD incidence was investigated in Pax3(Sp) mice, an established folate-responsive NTD mouse model. RESULTS: Shmt1(+/-) and Shmt1(-/-) embryos exhibited exencephaly in response to maternal folate and choline deficiency. Shmt1 disruption on the Pax3(Sp) background exacerbated NTD frequency and severity. Pax3 disruption impaired de novo thymidylate and purine biosynthesis and altered amounts of SHMT1 and thymidylate synthase protein. CONCLUSIONS: SHMT1 is the only folate-metabolizing enzyme that has been shown to affect neural tube closure in mice by directly inhibiting folate metabolism. These results provide evidence that disruption of Shmt1 expression causes NTDs by impairing thymidylate biosynthesis and shows that changes in the expression of genes that encode folate-dependent enzymes may be key determinates of NTD risk.

Mthfd1 is a modifier of chemically induced intestinal carcinogenesis.

The causal metabolic pathways underlying associations between folate and risk for colorectal cancer (CRC) have yet to be established. Folate-mediated one-carbon metabolism is required for the de novo synthesis of purines, thymidylate and methionine. Methionine is converted to S-adenosylmethionine (AdoMet), the major one-carbon donor for cellular methylation reactions. Impairments in folate metabolism can modify DNA synthesis, genomic stability and gene expression, characteristics associated with tumorigenesis. The Mthfd1 gene product, C1-tetrahydrofolate synthase, is a trifunctional enzyme that generates one-carbon substituted tetrahydrofolate cofactors for one-carbon metabolism. In this study, we use Mthfd1(gt/+) mice, which demonstrate a 50% reduction in C1-tetrahydrofolate synthase, to determine its influence on tumor development in two mouse models of intestinal cancer, crosses between Mthfd1(gt/+) and Apc(min)(/+) mice and azoxymethane (AOM)-induced colon cancer in Mthfd1(gt/+) mice. Mthfd1 hemizygosity did not affect colon tumor incidence, number or load in Apc(min/+) mice. However, Mthfd1 deficiency increased tumor incidence 2.5-fold, tumor number 3.5-fold and tumor load 2-fold in AOM-treated mice. DNA uracil content in the colon was lower in Mthfd1(gt/+) mice, indicating that thymidylate biosynthesis capacity does not play a significant role in AOM-induced colon tumorigenesis. Mthfd1 deficiency-modified cellular methylation potential, as indicated by the AdoMet: S-adenosylhomocysteine ratio and gene expression profiles, suggesting that changes in the transcriptome and/or decreased de novo purine biosynthesis and associated mutability cause cellular transformation in the AOM CRC model. This study emphasizes the impact and complexity of gene-nutrient interactions with respect to the relationships among folate metabolism and colon cancer initiation and progression.

Mthfd1 is an essential gene in mice and alters biomarkers of impaired one-carbon metabolism.

Cytoplasmic folate-mediated one carbon (1C) metabolism functions to carry and activate single carbons for the de novo synthesis of purines, thymidylate, and for the remethylation of homocysteine to methionine. C1 tetrahydrofolate (THF) synthase, encoded by Mthfd1, is an entry point of 1Cs into folate metabolism through its formyl-THF synthetase (FTHFS) activity that catalyzes the ATP-dependent conversion of formate and THF to 10-formyl-THF. Disruption of FTHFS activity by the insertion of a gene trap vector into the Mthfd1 gene results in embryonic lethality in mice. Mthfd1gt/+ mice demonstrated lower hepatic adenosylmethionine levels, which is consistent with formate serving as a source of 1Cs for cellular methylation reactions. Surprisingly, Mthfd1gt/+ mice exhibited decreased levels of uracil in nuclear DNA, indicating enhanced de novo thymidylate synthesis, and suggesting that serine hydroxymethyltransferase and FTHFS compete for a limiting pool of unsubstituted THF. This study demonstrates the essentiality of the Mthfd1 gene and indicates that formate-derived 1Cs are utilized for de novo purine synthesis and the remethylation of homocysteine in liver. Further, the depletion of cytoplasmic FTHFS activity enhances thymidylate synthesis, affirming the competition between thymidylate synthesis and homocysteine remethylation for THF cofactors.

Cytoplasmic serine hydroxymethyltransferase regulates the metabolic partitioning of methylenetetrahydrofolate but is not essential in mice.

The hydroxymethyl group of serine is a primary source of tetrahydrofolate (THF)-activated one-carbon units that are required for the synthesis of purines and thymidylate and for S-adenosylmethionine (AdoMet)-dependent methylation reactions. Serine hydroxymethyltransferase (SHMT) catalyzes the reversible and THF-dependent conversion of serine to glycine and 5,10-methylene-THF. SHMT is present in eukaryotic cells as mitochondrial SHMT and cytoplasmic (cSHMT) isozymes that are encoded by distinct genes. In this study, the essentiality of cSHMT-derived THF-activated one-carbons was investigated by gene disruption in the mouse germ line. Mice lacking cSHMT are viable and fertile, demonstrating that cSHMT is not an essential source of THF-activated one-carbon units. cSHMT-deficient mice exhibit altered hepatic AdoMet levels and uracil content in DNA, validating previous in vitro studies that indicated this enzyme regulates the partitioning of methylenetetrahydrofolate between the thymidylate and homocysteine remethylation pathways. This study suggests that mitochondrial SHMT-derived one-carbon units are essential for folate-mediated one-carbon metabolism in the cytoplasm.

Inhibition of 5,10-methenyltetrahydrofolate synthetase.

The interaction of 5-formyltetrahydrofolate analogs with murine methenyltetrahydrofolate synthetase (MTHFS) was investigated using steady-state kinetics, molecular modeling, and site-directed mutagenesis. MTHFS catalyzes the irreversible cyclization of 5-formyltetrahydrofolate to 5,10-methenyltetrahydrofolate. Folate analogs that cannot undergo the rate-limiting step in catalysis were inhibitors of murine MTHFS. 5-Formyltetrahydrohomofolate was an effective inhibitor of murine MTHFS (K(i)=0.7 microM), whereas 5-formyl,10-methyltetrahydrofolate was a weak inhibitor (K(i)=10 microM). The former, but not the latter, was slowly phosphorylated by MTHFS. 5-Formyltetrahydrohomofolate was not a substrate for murine MTHFS, but was metabolized when the MTHFS active site Y151 was mutated to Ala. MTHFS active site residues do not directly facilitate N10 attack on the on the N5-iminium phosphate intermediate, but rather restrict N10 motion around N5. Inhibitors specifically designed to block N10 attack appear to be less effective than the natural 10-formyltetrahydrofolate polyglutamate inhibitors.