Browsing by Subject "One-carbon metabolism"

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Characterization of the role of the mitochondrial one-carbon metabolism during embryonic development
(2016-12) Shin, Minhye; Appling, Dean Ramsay; Finnell, Richard; Hoffman, David; Fast, Walter; Vokes, Steven
Neural tube defects (NTDs) are one of serious structural birth defects resulting from failure of neural tube closure. Folic acid supplementation is the essential factor for prevention of NTDs. Folate-dependent one-carbon metabolism is a central metabolic pathway participating in a diverse range of metabolic reactions. Mitochondrial one-carbon metabolism is crucial for production of formate as a major 1C donor to the cytoplasm and regeneration of redox cofactors. Mitochondrial MTHFD family enzymes, MTHFD2, MTHFD2L, and MTHFD1L, are major contributors for formate production in mammalian mitochondria. The MTHFD2 and MTHFD2L isozymes possess both CH₂-THF dehydrogenase and CH⁺-THF cyclohydrolase activities, catalyzing the reaction of 10-CHO-THF production in mitochondria. The dehydrogenase activity of these bifunctional enzymes can use either NAD⁺ or NADP⁺ with dual redox cofactor specificity, but requires both phosphate and Mg²⁺ when using NAD⁺. The NADP⁺-dependent dehydrogenase activity is inhibited by inorganic phosphate. With polyglutamylated THF substrate, both of MTHFD2 and MTHFD2L show higher NADP⁺-dependent activity than the monoglutamylated substrate. Phylogenetic analysis indicates that MTHFD2L may be evolved from invertebrate MTHFD2 which is homologous to a primitive fungal MTHFD1. MTHFD1L is expressed ubiquitously throughout the embryogenesis during neural tube closure, and significantly detected at the basal surface of the dorsal neuroepithelium. Lacking Mthfd1l causes retardation in growth and developmental progression. Mthfd1l knockout mouse embryos show defects in proliferation during late neural tube closure and head mesenchyme development during early neural tube closure. However, proliferation during early neural tube closure, apoptosis, and neural crest cell migration were not affected by the loss of Mthfd1l. Finally, we show that maternal formate supplementation significantly improves the dysregulated cellular processes in Mthfd1l [superscript z/z] embryos. This study elucidates the specific metabolic mechanisms underlying folate-associated birth defects, including NTDs.
Dietary and genetic influences on neural tube defects
(2014-08) Fathe, Kristin Renee; Finnell, Richard H.
Neural tube defects (NTDs) are a world health issue, affecting approximately 1 in every 1000 live births. These congenital defects arise from the improper closure of the neural tube during development, resulting in significant, life-threatening malformations of the central nervous system. Although it has been observed that supplementing women of child-bearing age with folates greatly decreases the chances of having an NTD affected baby, unfortunately these defects still occur. It is accepted that these complex disorders arise from a combination of genetic, environmental, and dietary influences. One such dietary influence is the one-carbon metabolism metabolite, homocysteine. Homocysteine is a byproduct of methylation reactions in the cell that exists in an inverse homeostasis with folate. Homocysteine can also undergo a transformation that allows it to then react with exposed lysine or cysteine residues on proteins, in a process known as N-homocysteinylation or S-homocysteinylation respectively. High levels of homocysteine have been long correlated with many disease states, including NTDs. One potential mechanism by which homocysteine confers its negative effects is through protein N-homocysteinylation. Here, a novel and high-throughput assay for N-homocysteinylation determination is described. This assay is shown to be accurate with mass spectrometry then shown to be biologically relevant using known hyperhomocysteinemia mouse models. This assay was then applied to a cohort of neural tube closure staged mouse embryos with two different genetic mutations that have previously been shown to predispose mice to NTDs. The genotypes explored here are mutations to the LRP6 gene and the Folr1 gene, both of which have been described as folate-responsive NTD mouse models. It was seen that maternal diet and embryonic genotype had the largest influence on the developmental outcome of these embryos; however, the inverse relationship between folate and homocysteine seemed to be established at this early time point, emphasizing the importance of the balance in one-carbon metabolism. One of these genes, LRP6, was then explored in a human cohort of spina bifida cases. Four novel mutations to the LRP6 gene were found and compared to the mouse model used in the previous study. One of the mutations found in the human population was seen to mimic that of the LRP6 mouse model, therefore expanding the potential of this NTD model.
Mechanisms of impaired mitochondrial one-carbon metabolism and drug exposures to valproic acid or dolutegravir in neural tube defects
(2020-05-14) Steele, John William; Finnell, Richard H.; Appling, Dean Ramsay; Agarwala, Seema; Wallingford, John B; Gross, Steven S
Neural tube defects (NTDs) are among the most severe and prevalent human congenital malformations. Their etiology is complex and multifactorial, influenced by dynamically interacting genetic and environmental factors. It is well known that maternal dietary folate status is the greatest modifying factor associated with risk for NTD-affected pregnancies, and that dietary fortification of folic acid (FA) can prevent a significant proportion of NTDs. However, many NTDs have proven to be FA-resistant, presenting a need to understand mechanisms underlying these FA-resistant defects and develop novel intervention strategies targeting this population. A class of FA-resistant NTD mouse models have been developed by inactivating genes associated with mitochondrial one-carbon metabolism (mOCM). Thus, Part One of this work sought to elucidate mechanisms by which impaired mOCM results in FA-resistant NTDs. By crossing mice heterozygous for loss of Slc25a32, a gene coding for the mitochondrial folate transporter, with mice heterozygous for the Crooked Tail (Cd) allele of Lrp6, a gene coding for a Wnt co-receptor, it was discovered that a proportion of resulting co-heterozygous offspring presented with NTDs, suggesting a novel digenic interaction between Lrp6 and mOCM. Further experiments demonstrated that Lrp6 regulates expression of mOCM genes in mouse embryos, while CHO cells lacking Slc25a32 demonstrated impaired Wnt signaling rescued by the one-carbon donor, glycine. Building on those data, it was discovered that maternal glycine supplementation could prevent NTDs in Slc25a32 null embryos, and that glycine or serine supplementation may reduce NTDs in Lrp6 Cd mice. Part One experiments also demonstrated that embryonic stem cells lacking Mthfd1l, another mOCM gene, have proliferation defects and are sensitive to hypoxia. Environmental exposure to certain pharmaceutical compounds also increases risk for NTDs. Part Two of this work sought to identify mechanisms of NTD pathology associated with two common pharmaceuticals, the anticonvulsant, valproic acid (VPA), and the HIV integrase inhibitor, dolutegravir (DTG). Untargeted metabolic profiling was performed on VPA-treated mouse embryos, and predictive biomarkers of VPA sensitivity were identified by comparing VPA-affected and unaffected embryos. Other experiments identified a novel, calcium-enhanced interaction between DTG, folate, and folate receptor, suggesting a plausible mechanism by which DTG may enhance NTD risk.