We propose to elucidate the mechanism(s) and the nutritional and genetic determinants of deoxyuridine triphosphate (dUTP) incorporation into DNA, and its role in the etiology of neural tube closure defects (NTDs). Impairments in folate- and vitamin B12 (B12)-dependent one-carbon metabolism (OCM) are associated with common pathologies, including NTDs. Recently, we discovered that impaired folate-dependent de novo thymidylate (dTMP) biosynthesis causes NTDs in mice by generating serine hydroxymethytransferase 1 (SHMT)-deficient mice. SHMT1 is the only reported folate-dependent enzyme whose disruption causes folate-responsive NTDs, which provides evidence that de novo thymidylate (dTMP) biosynthesis and uracil accumulation in DNA underlies NTDs. Recently, others discovered that the ribonucleotide reductase (RNR)- catalyzed conversion of UDP to dUDP competes with folate dependent dTDP synthesis to regulate dUTP incorporation into DNA. The experiments described herein will test the overarching hypothesis that RNR-mediated dUDP synthesis competes with folate-dependent dTDP synthesis (via de novo dTMP biosynthesis &the enzyme dTMP kinase (TMPK)) to regulate dUTP incorporation into DNA, and that this interaction underlies folate and vitamin-B12-associated NTD pathogenesis. In support of this hypothesis, preliminary data show that maternal dietary deoxyuridine (dU) rescues NTDs in folate-deficient Shmt1+/- dams, whereas dietary uridine causes NTDs in wt mice, independent of dietary folate. This proposal integrates disparate observations in the literature, including that p53, RNR, folate and vitamin B12 are associated with NTDs, into a common mechanism and pathway. The results will establish the pathway for NTDs and inform future human and population studies for the prevention of folate- and B12-associated pathologies including NTDs.
Aim I. Determine if vitamin B12 deficiency impairs nuclear dTMP biosynthesis and modifies NTD incidence in wt and Shmt1+/- mice. These studies will establish the role of dietary folate and B12 in nuclear dTMP biosynthesis and NTD pathogenesis, and clarify the associated mechanisms.
Aim II. Determine if TMPK modifies NTD incidence in wt and Shmt1+/- mice. These studies will confirm that that disruption of de novo dTMP biosynthesis downstream of folate and B12 metabolism causes NTDs.
Aim III. Determine the role of RNR in uracil accumulation in DNA and NTD pathogenesis in mice.
This aim challenges the current dogma that uracil accumulation in DNA is caused by dUTP "misincorporation" due to impaired dTMP synthesis. These studies will determine if p53 and RNR expression affects uracil levels in DNA and NTD incidence independent of folate, and if the Shmt1 genotype modifies these outcomes.
Aim I V. Validate the genetic and metabolic mechanisms of NTD pathogenesis by dietary rescue with metabolic intermediates. We will determine the mechanism and efficacy of maternal dietary dU in preventing NTDs in Shmt1+/- mice, and the mechanism and dose of maternal dietary uridine that causes NTDs.
Folate and vitamin B12-associated pathologies are common and affect specific population subgroups across the life cycle, but the underlying mechanisms are unknown. This proposal builds upon our recent discoveries that impairments in de novo thymidylate biosynthesis play a causal role in neural tube closure defects during pregnancy. The experiments outlined in this proposal will identify new genetic, metabolic and nutritional risk factors for these common birth defects, and the results will inform medical practice and public health policy for the prevention and treatment of folate- and vitamin B12-associated pathologies.
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|Ash, J A; Jiang, X; Malysheva, O V et al. (2013) Dietary and genetic manipulations of folate metabolism differentially affect neocortical functions in mice. Neurotoxicol Teratol 38:79-91|
|Beaudin, Anna E; Abarinov, Elena V; Malysheva, Olga et al. (2012) Dietary folate, but not choline, modifies neural tube defect risk in Shmt1 knockout mice. Am J Clin Nutr 95:109-14|
|Anderson, Donald D; Woeller, Collynn F; Chiang, En-Pei et al. (2012) Serine hydroxymethyltransferase anchors de novo thymidylate synthesis pathway to nuclear lamina for DNA synthesis. J Biol Chem 287:7051-62|
|Beaudin, Anna E; Perry, Cheryll A; Stabler, Sally P et al. (2012) Maternal Mthfd1 disruption impairs fetal growth but does not cause neural tube defects in mice. Am J Clin Nutr 95:882-91|
|Beaudin, Anna E; Abarinov, Elena V; Noden, Drew M et al. (2011) Shmt1 and de novo thymidylate biosynthesis underlie folate-responsive neural tube defects in mice. Am J Clin Nutr 93:789-98|
|Beaudin, Anna E; Stover, Patrick J (2009) Insights into metabolic mechanisms underlying folate-responsive neural tube defects: a minireview. Birth Defects Res A Clin Mol Teratol 85:274-84|