Neural tube defects (NTDs) are among the most common birth defects in humans. The causes of NTDs are multifactorial, including genetic, environmental, and nutritional factors. Maternal folic acid (FA) status is one of the strongest links to NTD susceptibility. Numerous studies have shown that supplemental FA can reduce NTD prevalence by as much as 70% in some populations. Despite more than 40 years of intensive effort, we still do not understand the mechanisms that underlie these folate-dependent processes. We have begun to address this existing data gap utilizing a new mouse NTD model (Mthfd1l KO) that closely replicates the human NTD phenotype, and does not require additional nutritional intervention to express the NTD phenotype. In this new mouse model, loss of a specific folate-dependent enzyme (mitochondrial MTHFD1L) leads to NTDs. This is the most specific metabolic defect yet associated with NTD susceptibility/etiology, and suggests that FA provides essential one-carbon units for nucleotide and methyl group biosynthesis. These biosynthetic pathways are especially active in the rapidly growing embryo, where they support cell proliferation and death, migration, and differentiation during neural tube closure (NTC). We will test the following specific hypotheses using this mouse model: (1) Maternal supplementation with methionine, purines, thymidylate and S-adenosylmethionine can protect against NTDs in nullizygous Mthfd1l KO (Mthfd1lz/z) embryos, (2) Depakote (Valproic Acid; VPA), the leading cause of pharmaceutical-induced NTDs, inhibits mitochondrial 1C metabolism, thus it is possible that formate can prevent NTDs caused by this teratogen, and (3) cell proliferation and apoptosis, cell migration, and differentiation programs are disrupted in Mthfd1lz/z embryos, leading to neural tube and orofacial defects. We demonstrated that maternal supplementation of MTHFD1L dams with formate, the product of the MTHFD1L enzymatic reaction, decreases the incidence of NTDs and partially rescues the growth deficit in embryos lacking a functional Mthfd1l.
In Specific Aim 1 we will determine which supplements downstream of the MTHFD1L reaction can rescue the NTD phenotype. This will be explored in a number of FA responsive and non-responsive NTD mutant strains, and in VPA-sensitive mouse strains. We will identify which cellular processes are dysregulated in Mthfd1lz/z embryos and VPA-sensitive mouse strains, leading to improper NTC. Metabolomic and epigenetic studies will be pursued to fully characterize the mutant mice.
Specific Aim 2 will focus on the requirement for formate in neural stem cells and neural crest stem cells using neurosphere growth and differentiation assays, as well as additional epigenetic investigations.
Specific Aim 3 will involve DNA resequencing of the human MTHFD1L gene and functional analyses of identified variants in a spina bifida cohort. This research program offers hope for developing the first effective intervention for non-FA responsive NTDs by illuminating the underlying mechanisms by which formate prevents NTDs. Developing interventions that benefit non-folate responsive NTDs is crucial for preventing these preventable birth defects.
Neural tube defects (NTDs) arise when the neural tube fails to close during neurulation. Both genetic and non- genetic factors are implicated in the etiology of NTDs, with up to 70% of the variance in NTD prevalence attributable to genetic factors. NTDs stand out as a preventable birth defect. Although it is established that periconceptional use of folic acid (FA) prevents a significant percentage of the population burden of NTD, the mechanisms underlying those processes by which FA reduces NTD risk remains unknown. Importantly, are significant numbers of NTDs that are not preventable by FA supplementation. NTDs remain a substantial public health problem whose etiology remains far from being fully understood. Our proposed strategy is designed to provide a proof of principle validation of our working hypothesis that the non-FA responsive NTDs may be prevented by intervention with formate.
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