Spatial and Temporal Protein Oxidation and Redox Signaling in Mechanisms of Structural Birth Defects
Lapehn, Samantha
2021
Abstract
Neural tube defects (NTDs) are birth defects that arise from failed neural tube closure resulting in morphological abnormality of the brain and spinal cord. NTD risk has been associated with genetic mutations, a maternal diet low in folic acid, as well as gestational exposure to anti-epileptic medications such as valproic acid (VPA). Though VPA exposure during pregnancy has been associated with a 20-fold increase in the risk of NTDs, there is not an accepted mechanism for VPA-induced NTDs. The reduction-oxidation (redox) environment is a critical component of development with evidence to suggest that redox signaling at the cellular and protein level is a key factor in developmental progression. VPA exposure has previously been associated with several biomarkers of oxidation leading to the hypothesis that VPA may act through a redox-regulated mechanism to cause NTDs. The objective of this dissertation was to identify spatial and temporal redox mechanisms of VPA teratogenesis across the period of early organogenesis in the CD-1 mouse conceptus. This was accomplished by carrying out three aims designed to identify changes in cellular redox potential, reversible oxidative post-translational modifications (PTMs) of proteins, and protein abundance that may be implicated in failed neural tube closure. Aim 1 evaluated the cellular redox environment through measurement of the glutathione (GSH) and cysteine (Cys) redox couples in four distinct compartments of the mouse conceptus over 24 hours of organogenesis following VPA exposure. This analysis demonstrated that VPA’s actions as a cellular oxidant are not universal, but are, instead, specific to time and tissue. Aim 2 utilized a systems biology proteomics approach to assess changes in the developmental mouse proteome in terms of protein oxidation and protein abundance. Measurement of whole conceptus reversible oxidative PTMs at Cys residues indicated that VPA causes an initial decrease in Cys-oxidation compared to the control with an increase in oxidation building over time. Several target pathways relevant to nervous system development, cell polarity, and cytoskeletal organization were identified as being differentially oxidized after VPA exposure. Tissue-specific assessment of protein abundance following VPA exposure identified divergent patterns of temporal sensitivity to VPA between the visceral yolk sac (VYS) and embryo (EMB). It also identified time and tissue-specific patterns of alterations in development and oxidation-reduction pathways including differentiation, growth, and Wnt signaling. Aim 3 integrated the data from previous chapters to construct a timeline of mechanistic VPA perturbation events. This timeline highlighted the time and tissue specific recurrence of developmentally relevant pathways such as differentiation, growth, Wnt signaling, cell polarity, and nervous system development that are likely to have mechanistic roles in VPA teratogenesis. Overall, this dissertation has significantly increased the breadth of knowledge related to VPA-associated redox and developmental events across mouse organogenesis. It demonstrated that both the temporality of sampling and exposure length exert meaningful effects on outcomes related to redox and protein abundance, thereby emphasizing the importance of time course evaluation of developmental toxicants. Tissue specific compartmentalization of protein oxidation-associated perturbations of developmental pathways and their related protein abundance demonstrate an important role for the VYS during early neurulation and in specific neurulation/developmental pathways throughout organogenesis. Future studies should build on these results to analyze VPA’s effects at the cell, tissue, and organ levels by focusing on the role of specific oxidative PTMs on protein form and function as it relates to neural tube closure.Deep Blue DOI
Subjects
Valproic Acid Development Redox Toxicology Neural Tube Defect Proteomics
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