High dietary folate in pregnant mice leads to pseudo-MTHFR deficiency and altered methyl metabolism, with embryonic growth delay and short-term memory impairment in offspring.

Methylenetetrahydrofolate reductase (MTHFR) generates methyltetrahydrofolate for methylation reactions. Severe MTHFR deficiency results in homocystinuria and neurologic impairment. Mild MTHFR deficiency (677C > T polymorphism) increases risk for complex traits, including neuropsychiatric disorders. Although low dietary folate impacts brain development, recent concerns have focused on high folate intake following food fortification and increased vitamin use. Our goal was to determine whether high dietary folate during pregnancy affects brain development in murine offspring. Female mice were placed on control diet (CD) or folic acid-supplemented diet (FASD) throughout mating, pregnancy and lactation. Three-week-old male pups were evaluated for motor and cognitive function. Tissues from E17.5 embryos, pups and dams were collected for choline/methyl metabolite measurements, immunoblotting or gene expression of relevant enzymes. Brains were examined for morphology of hippocampus and cortex. Pups of FASD mothers displayed short-term memory impairment, decreased hippocampal size and decreased thickness of the dentate gyrus. MTHFR protein levels were reduced in FASD pup livers, with lower concentrations of phosphocholine and glycerophosphocholine in liver and hippocampus, respectively. FASD pup brains showed evidence of altered acetylcholine availability and Dnmt3a mRNA was reduced in cortex and hippocampus. E17.5 embryos and placentas from FASD dams were smaller. MTHFR protein and mRNA were reduced in embryonic liver, with lower concentrations of choline, betaine and phosphocholine. Embryonic brain displayed altered development of cortical layers. In summary, high folate intake during pregnancy leads to pseudo-MTHFR deficiency, disturbed choline/methyl metabolism, embryonic growth delay and memory impairment in offspring. These findings highlight the unintended negative consequences of supplemental folic acid.

Low Dietary Folate Interacts with MTHFD1 Synthetase Deficiency in Mice, a Model for the R653Q Variant, to Increase Incidence of Developmental Delays and Defects.

Background: Suboptimal folate intake, a risk factor for birth defects, is common even in areas with folate fortification. A polymorphism in methylenetetrahydrofolate dehydrogenase 1 (MTHFD1), R653Q (MTHFD1 c.1958 G > A), has also been associated with increased birth defect risk, likely through reduced purine synthesis. Objective: We aimed to determine if the interaction of MTHFD1 synthetase deficiency and low folate intake increases developmental abnormalities in a mouse model for MTHFD1 R653Q. Methods: Female Mthfd1S+/+ and Mthfd1S+/- mice were fed control or low-folate diets (2 and 0.3 mg folic acid/kg diet, respectively) before mating and during pregnancy. Embryos and placentas were examined for anomalies at embryonic day 10.5. Maternal 1-carbon metabolites were measured in plasma and liver. Results: Delays and defects doubled in litters of Mthfd1S+/- females fed low-folate diets compared to wild-type females fed either diet, or Mthfd1S+/- females fed control diets [P values (defects): diet 0.003, maternal genotype 0.012, diet × maternal genotype 0.014]. These adverse outcomes were associated with placental dysmorphology. Intrauterine growth restriction was increased by embryonic Mthfd1S+/- genotype, folate deficiency, and interaction of maternal Mthfd1S+/- genotype with folate deficiency (P values: embryonic genotype 0.045, diet 0.0081, diet × maternal genotype 0.0019). Despite a 50% increase in methylenetetrahydrofolate reductase expression in low-folate maternal liver (P diet = 0.0007), methyltetrahydrofolate concentration decreased 70% (P diet <0.0001) and homocysteine concentration doubled in plasma (P diet = 0.0001); S-adenosylmethionine decreased 40% and S-adenosylhomocysteine increased 20% in low-folate maternal liver (P diet = 0.002 and 0.0002, respectively). Conclusions: MTHFD1 synthetase-deficient mice are more sensitive to low folate intake than wild-type mice during pregnancy. Reduced purine synthesis due to synthetase deficiency and altered methylation potential due to low folate may increase pregnancy complications. Further studies and individualized intake recommendations may be required for women homozygous for the MTHFD1 R653Q variant.

MTHFD1 formyltetrahydrofolate synthetase deficiency, a model for the MTHFD1 R653Q variant, leads to congenital heart defects in mice.

BACKGROUND: A single nucleotide polymorphism (SNP) in the synthetase domain of the trifunctional folate-dependent enzyme MTHFD1 (c.1958G>A, R653Q) has been linked to adverse pregnancy outcomes, neural tube defects, and possibly congenital heart defects. Maternal folate deficiency may also modify the risk associated with these disorders. We recently established a mouse model with a mild deficiency of 10-formyltetrahydrofolate synthetase activity in MTHFD1 (Mthfd1S(+/-) mice) to investigate disorders associated with SNPs in this gene. The effect of synthetase deficiency on embryonic heart development has not yet been examined. METHODS: Female Mthfd1S(+/+) and (+/-) mice were placed on control and folate-deficient diets for 6 weeks before mating to Mthfd1S(+/-) males. Embryos and placentae were collected at embryonic day 14.5. Embryos were evaluated for congenital heart defects by histological examination. RESULTS: Embryonic Mthfd1S(+/-) genotype was associated with an increased incidence of heart defects, primarily ventricular septal defects. Other markers of embryonic development (crown-rump length, embryonic weight, embryonic delay, placental weight, and thickness of the ventricular myocardium) were not affected by embryonic genotype. Maternal genotype and diet did not have a significant effect on these outcomes. CONCLUSION: Deficiency of the MTHFD1 10-formyltetrahydrofolate synthetase activity in embryos is associated with increased incidence of congenital heart defects.

Mouse model for deficiency of methionine synthase reductase exhibits short-term memory impairment and disturbances in brain choline metabolism.

Hyperhomocysteinaemia can contribute to cognitive impairment and brain atrophy. MTRR (methionine synthase reductase) activates methionine synthase, which catalyses homocysteine remethylation to methionine. Severe MTRR deficiency results in homocystinuria with cognitive and motor impairments. An MTRR polymorphism may influence homocysteine levels and reproductive outcomes. The goal of the present study was to determine whether mild hyperhomocysteinaemia affects neurological function in a mouse model with Mtrr deficiency. Mtrr+/+, Mtrr+/gt and Mtrrgt/gt mice (3 months old) were assessed for short-term memory, brain volumes and hippocampal morphology. We also measured DNA methylation, apoptosis, neurogenesis, choline metabolites and expression of ChAT (choline acetyltransferase) and AChE (acetylcholinesterase) in the hippocampus. Mtrrgt/gt mice exhibited short-term memory impairment on two tasks. They had global DNA hypomethylation and decreased choline, betaine and acetylcholine levels. Expression of ChAT and AChE was increased and decreased respectively. At 3 weeks of age, they showed increased neurogenesis. In the cerebellum, mutant mice had DNA hypomethylation, decreased choline and increased expression of ChAT. Our work demonstrates that mild hyperhomocysteinaemia is associated with memory impairment. We propose a mechanism whereby a deficiency in methionine synthesis leads to hypomethylation and compensatory disturbances in choline metabolism in the hippocampus. This disturbance affects the levels of acetylcholine, a critical neurotransmitter in learning and memory.

Moderate Folic Acid Supplementation in Pregnant Mice Results in Behavioral Alterations in Offspring with Sex-Specific Changes in Methyl Metabolism.

Fifteen to 20% of pregnant women may exceed the recommended intake of folic acid (FA) by more than four-fold. This excess could compromise neurocognitive and motor development in offspring. Here, we explored the impact of an FA-supplemented diet (5× FASD, containing five-fold higher FA than recommended) during pregnancy on brain function in murine offspring, and elucidated mechanistic changes. We placed female C57BL/6 mice for one month on control diets or 5× FASD before mating. Diets were maintained throughout pregnancy and lactation. Behavioural tests were conducted on 3-week-old pups. Pups and mothers were sacrificed at weaning. Brains and livers were collected to examine choline/methyl metabolites and immunoreactive methylenetetrahydrofolate reductase (MTHFR). 5× FASD led to hyperactivity-like behavior and memory impairment in 3-week-old pups of both sexes. Reduced MTHFR protein in the livers of FASD mothers and male pups resulted in choline/methyl metabolite disruptions in offspring liver (decreased betaine) and brain (decreased glycerophosphocholine and sphingomyelin in male pups, and decreased phosphatidylcholine in both sexes). These results indicate that moderate folate supplementation downregulates MTHFR and alters choline/methyl metabolism, contributing to neurobehavioral alterations. Our findings support the negative impact of high FA on brain development, and may lead to improved guidelines on optimal folate levels during pregnancy.

Early Manifestations of Brain Aging in Mice Due to Low Dietary Folate and Mild MTHFR Deficiency.

Folate is an important B vitamin required for methylation reactions, nucleotide and neurotransmitter synthesis, and maintenance of homocysteine at nontoxic levels. Its metabolism is tightly linked to that of choline, a precursor to acetylcholine and membrane phospholipids. Low folate intake and genetic variants in folate metabolism, such as the methylenetetrahydrofolate reductase (MTHFR) 677 C>T polymorphism, have been suggested to impact brain function and increase the risk for cognitive decline and late-onset Alzheimer's disease. Our study aimed to assess the impact of genetic and nutritional disturbances in folate metabolism, and their potential interaction, on features of cognitive decline and brain biochemistry in a mouse model. Wild-type and Mthfr+/- mice, a model for the MTHFR 677 C>T polymorphism, were fed control or folate-deficient diets from weaning until 8 and 10 months of age. We observed short-term memory impairment measured by the novel object paradigm, altered transcriptional levels of synaptic markers and epigenetic enzymes, as well as impaired choline metabolism due to the Mthfr+/- genotype in cortex or hippocampus. We also detected changes in mRNA levels of Presenillin-1, neurotrophic factors, one-carbon metabolic and epigenetic enzymes, as well as reduced levels of S-adenosylmethionine and acetylcholine, due to the folate-deficient diet. These findings shed further insights into the mechanisms by which genetic and dietary folate metabolic disturbances increase the risk for cognitive decline and suggest that these mechanisms are distinct.

Mild Methylenetetrahydrofolate Reductase Deficiency Alters Inflammatory and Lipid Pathways in Liver.

SCOPE: Dietary and genetic folate disturbances can lead to nonalcoholic fatty liver disease (NAFLD). A common variant in methylenetetrahydrofolate reductase (MTHFR 677C→T) causes mild MTHFR deficiency with lower 5-methyltetrahydrofolate for methylation reactions. The goal is to determine whether mild murine MTHFR deficiency contributes to NAFLD-related effects. METHODS AND RESULTS: Wild-type and Mthfr+/- mice, a model for the human variant, are fed control (CD) or high-fat (HFAT) diets for 8 weeks. On both diets, MTHFR deficiency results in decreased S-adenosylmethionine, increased S-adenosylhomocysteine, and decreased betaine with reduced methylation capacity, and changes in expression of several inflammatory or anti-inflammatory mediators (Saa1, Apoa1, and Pon1). On CD, MTHFR deficiency leads to microvesicular steatosis with expression changes in lipid regulators Xbp1s and Cyp7a1. The combination of MTHFR deficiency and HFAT exacerbates changes in inflammatory mediators and introduces additional effects on inflammation (Saa2) and lipid metabolism (Nr1h4, Srebf1c, Ppara, and Crot). These effects are consistent with increased expression of pro-inflammatory HDL precursors and greater lipid accumulation. MTHFR deficiency may enhance liver injury through alterations in methylation capacity, inflammatory response, and lipid metabolism. CONCLUSION: Individuals with the MTHFR variant may be at increased risk for liver disease and related complications, particularly when consuming high-fat diets.

Moderate folic acid supplementation and MTHFD1-synthetase deficiency in mice, a model for the R653Q variant, result in embryonic defects and abnormal placental development.

BACKGROUND: Moderately high folic acid intake in pregnant women has led to concerns about deleterious effects on the mother and fetus. Common polymorphisms in folate genes, such as methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase (MTHFD1) R653Q, may modulate the effects of elevated folic acid intake. OBJECTIVES: We investigated the effects of moderate folic acid supplementation on reproductive outcomes and assessed the potential interaction of the supplemented diet with MTHFD1-synthetase (Mthfd1S) deficiency in mice, which is a model for the R653Q variant. DESIGN: Female Mthfd1S+/+ and Mthfd1S+/- mice were fed a folic acid-supplemented diet (FASD) (5-fold higher than recommended) or control diets before mating and during pregnancy. Embryos and placentas were assessed for developmental defects at embryonic day 10.5 (E10.5). Maternal folate and choline metabolites and gene expression in folate-related pathways were examined. RESULTS: The combination of FASD and maternal MTHFD1-synthetase deficiency led to a greater incidence of defects in E10.5 embryos (diet × maternal genotype, P = 0.0016; diet × embryonic genotype, P = 0.054). The methylenetetrahydrofolate reductase (MTHFR) protein and methylation potential [ratio of S-adenosylmethionine (major methyl donor):S-adenosylhomocysteine) were reduced in maternal liver. Although 5-methyltetrahydrofolate (methylTHF) was higher in maternal circulation, the methylation potential was lower in embryos. The presence of developmental delays and defects in Mthfd1S+/- embryos was associated with placental defects (P = 0.003). The labyrinth layer failed to form properly in the majority of abnormal placentas, which compromised the integration of the maternal and fetal circulation and presumably the transfer of methylTHF and other nutrients. CONCLUSIONS: Moderately higher folate intake and MTHFD1-synthetase deficiency in pregnant mice result in a lower methylation potential in maternal liver and embryos and a greater incidence of defects in embryos. Although maternal circulating methylTHF was higher, it may not have reached the embryos because of abnormal placental development; abnormal placentas were observed predominantly in abnormally developed embryos. These findings have implications for women with high folate intakes, particularly if they are polymorphic for MTHFD1 R653Q.

High Dietary Folate in Mice Alters Immune Response and Reduces Survival after Malarial Infection.

Malaria is a significant global health issue, with nearly 200 million cases in 2013 alone. Parasites obtain folate from the host or synthesize it de novo. Folate consumption has increased in many populations, prompting concerns regarding potential deleterious consequences of higher intake. The impact of high dietary folate on the host's immune function and response to malaria has not been examined. Our goal was to determine whether high dietary folate would affect response to malarial infection in a murine model of cerebral malaria. Mice were fed control diets (CD, recommended folate level for rodents) or folic acid-supplemented diets (FASD, 10x recommended level) for 5 weeks before infection with Plasmodium berghei ANKA. Survival, parasitemia, numbers of immune cells and other infection parameters were assessed. FASD mice had reduced survival (p<0.01, Cox proportional hazards) and higher parasitemia (p< 0.01, joint model of parasitemia and survival) compared with CD mice. FASD mice had lower numbers of splenocytes, total T cells, and lower numbers of specific T and NK cell sub-populations, compared with CD mice (p<0.05, linear mixed effects). Increased brain TNFα immunoreactive protein (p<0.01, t-test) and increased liver Abca1 mRNA (p<0.01, t-test), a modulator of TNFα, were observed in FASD mice; these variables correlated positively (rs = 0.63, p = 0.01). Bcl-xl/Bak mRNA was increased in liver of FASD mice (p<0.01, t-test), suggesting reduced apoptotic potential. We conclude that high dietary folate increases parasite replication, disturbs the immune response and reduces resistance to malaria in mice. These findings have relevance for malaria-endemic regions, when considering anti-folate anti-malarials, food fortification or vitamin supplementation programs.