PROJECT 3: Embryonic Oxidative-Nitrosative Stress and NTD Risk The combinatorial effects of gene-environment interactions play a critical role in the etiology of complex diseases such as neural tube defects (NTDs). Definitive identification of disease causing interactions between environmental exposures and specific genetic variants resulting in elevated NTDs risk have been hampered by the rarity of this outcome (<1/1000 births in the U.S.), and differences in exposure assessment between studies, as well as adherence to overly simplistic etiological models. Building upon the results obtained in our original funding period, we propose to rigorously apply next generation DNA sequencing, genome editing, animal modeling, redoxome and transcriptomic approaches to further refine our efforts to better understand mitochondrial redox metabolism and its role in the generation of reactive oxidative species (ROS) that compromise neural tube closure (NTC) in genetically sensitive embryos at the cellular and molecular levels. We will rigorously test three related hypotheses using novel mouse and cellular reagents selected not only specifically test for redox stress that is mitochondrially driven-but are also reflective of the novel discoveries derived from Project 1. The first hypothesis involves determining the importance of a genetic variant in the enzyme endothelial nitric oxide synthase (NOS3) that is known to increase human NTD susceptibility by altering enzyme function to increase production of the ROS superoxide at the expense of nitric oxide (NO) production. We suspect that this variant increases the formation of the reactive nitrogen species peroxynitrite within cells secondary to the phosphorylation of NOS3 Serine 633. The second will test the hypothesis that elevated intracellular levels of ROS disrupt cell signaling pathways and cause cell damage during NTC, resulting in an increased prevalence of NTDs. Finally, we propose to test the hypothesis that mitochondria are a major source of ROS during neurulation. The results of our proposed studies, taken in the context of the results generated from Projects 1 and 2 of this Program Project Grant Proposal, will help in defining the inter-relationships among maternal environmental exposures, nutritional status as it impacts one carbon metabolism, and the underlying maternal/embryonic genetics with susceptibility to NTDs. Moreover, our unique set of biological samples from Project 1 enables us to continuously explore the genome of NTD patients that inform our mechanistically focused studies, as well as create hiPSCs on which to perform functional redoxome studies. We will also develop mutant mouse lines based on our human studies that enable us to explore ROS-induced NTDs at a molecular and cellular level to complement the human studies and obtain answers to long-standing challenging questions concerning the importance of RONS in the etiology of NTDs. Our work will significantly focus the NTD research agenda, leading to an improved understanding of the causes of NTDs, and ultimately a reduction in their prevalence.
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