PROJECT 2: Quantitiative Assessment of Oxidative Stress and 1-C Trafficking Defects as a Basis for NTD Risk From the broad efficacy of maternal folic acid (FA) supplementation for prevention of neural tube defects (NTDs), and knowledge that tetrahydrofolate (THF) is an essential cofactor for cellular one-carbon (1-C) transfer reactions, one may infer that NTDs largely stem from FA-reversible defects in 1-C trafficking reactions. A challenge to this view is that defective 1-C trafficking has been associated with very few of the >250 genes whose mutations cause NTD in murine models. FH4 is also an efficient antioxidant molecule, and since reactive oxygen/nitrogen species (RONS) are suggested to increase NTD risk, we hypothesized that protection against RONS is a key contributor to the NTD-preventing action of FA. Data generated during the past funding cycle provided evidence for this view, however a lack of experimental tools to date has precluded the rigorous discrimination between 1-C trafficking defects vs. RONS excess as fundamental molecular bases for gene mutation associated NTDs. Such tools are also needed to ascertain the extent to which the NTD-preventative effects of maternal FA supplementation arise from protection against 1-C trafficking defects vs. RONS-evoked abnormalities. To overcome this roadblock to discovery, we seek to establish and employ novel strategies that will allow for the first time, an unbiased/untargeted survey of both 1-C flux and redox status in ex vivo models of murine NTDs. These new tools will be applied to well-established murine NTD models, as well as new mouse models of NTD developed in Project 3, and most importantly, to investigate the 1-C trafficking and cellular redox consequences of human spina bifida candidate gene mutations, identified in Project 1 by WGS and implicated as potential drivers of NTDs. In the Program renewal, we propose the following goals: (1) establish and employ a novel untargeted stable isotope tracing technology to rigorously define 1-C trafficking defects in mouse NTD models. (2) establish and employ a novel redoxome approach to survey and quantify changes in the levels of redox-active molecules that may occur in mouse models of NTD. (3) Assess the extent to which spina bifida-associated rare SNPs in humans (identified in Project 1) contribute to aberrant 1-C trafficking and RONS. Further, we seek to identify molecules that elicit an additional attenuation in NTD prevalence by opposing oxidative stress or overcoming 1-C trafficking defects, with or without added 5- Methyl-THF in ex vivo NTD models. Our overall hypothesis: 1-C trafficking defects and RONS stress are interlinked by the mitochondrial folate/formate cycle and both contribute to the mechanistic basis for FA-preventable NTDs. A molecular understanding of these linked processes during neurulation, facilitated by new analytical tools developed herein, promise new insight into NTD causes and prevention.
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