Mechanisms of impaired mitochondrial one-carbon metabolism and drug exposures to valproic acid or dolutegravir in neural tube defects

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.