Brian Thompson1, Ying Chen1, Emily A Davidson2, Rolando Garcia-Milian3, Jaya Prakash Golla4, Nicholas Apostolopoulos5, David J Orlicky6, Kevin Schey7, David C Thompson8, Vasilis Vasiliou9. 1. Department of Environmental Health Sciences, Yale School of Public Health, Yale University, 60 College Street, New Haven, CT, USA. 2. Department of Environmental Health Sciences, Yale School of Public Health, Yale University, 60 College Street, New Haven, CT, USA; Department of Cellular & Molecular Physiology, Yale School of Medicine, Yale University, New Haven, CT, USA. 3. Bioinformatics Support Program, Cushing/Whitney Medical Library, Yale School of Medicine, New Haven, CT, USA. 4. Department of Environmental Health Sciences, Yale School of Public Health, Yale University, 60 College Street, New Haven, CT, USA; Department of Medicine, Yale University School of Medicine, New Haven, CT, USA; Veterans Affairs Connecticut Healthcare System, West Haven, CT, USA. 5. Department of Ophthalmology & Visual Science, Yale School of Medicine, New Haven, CT, USA. 6. Department of Pathology, Anschutz School of Medicine, University of Colorado, Aurora, CO, USA. 7. Department of Biochemistry and Mass Spectrometry Research Center, Vanderbilt University School of Medicine, Nashville, TN, USA. 8. Department of Clinical Pharmacy, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Denver, Aurora, CO, USA. 9. Department of Environmental Health Sciences, Yale School of Public Health, Yale University, 60 College Street, New Haven, CT, USA. Electronic address: vasilis.vasiliou@yale.edu.
Abstract
PURPOSE: The purpose of this study was to elucidate the role and molecular consequences of impaired glutathione (GSH) biosynthesis on eye development. METHODS: GSH biosynthesis was impaired in surface ectoderm-derived ocular tissues by crossing Gclcf/f mice with hemizygous Le-Cre transgenic mice to produce Gclcf/f/Le-CreTg/- (KO) mice. Control mice included Gclcf/fand Gclcwt/wt/Le-CreTg/- mice (CRE). Eyes from all mice (at various stages of eye development) were subjected to histological, immunohistochemical, Western blot, RT-qPCR, RNA-seq, and subsequent Gene Ontology, Ingenuity Pathway Analysis and TRANSFAC analyses. PAX6 transactivation activity was studied using a luciferase reporter assay in HEK293T cells depleted of GSH using buthionine sulfoximine (BSO). RESULTS: Deletion of Gclc diminished GSH levels, increased reactive oxygen species (ROS), and caused an overt microphthalmia phenotype characterized by malformation of the cornea, iris, lens, and retina that is distinct from and much more profound than the one observed in CRE mice. In addition, only the lenses of KO mice displayed reduced crystallin (α, β), PITX3 and Foxe3 expression. RNA-seq analyses at postnatal day 1 revealed 1552 differentially expressed genes (DEGs) in the lenses of KO mice relative to those from Gclcf/f mice, with Crystallin and lens fiber cell identity genes being downregulated while lens epithelial cell identity and immune response genes were upregulated. Bioinformatic analysis of the DEGs implicated PAX6 as a key upstream regulator. PAX6 transactivation activity was impaired in BSO-treated HEK293T cells. CONCLUSIONS: These data suggest that impaired ocular GSH biosynthesis may disrupt eye development and PAX6 function.
PURPOSE: The purpose of this study was to elucidate the role and molecular consequences of impaired glutathione (GSH) biosynthesis on eye development. METHODS: GSH biosynthesis was impaired in surface ectoderm-derived ocular tissues by crossing Gclcf/f mice with hemizygous Le-Cre transgenic mice to produce Gclcf/f/Le-CreTg/- (KO) mice. Control mice included Gclcf/fand Gclcwt/wt/Le-CreTg/- mice (CRE). Eyes from all mice (at various stages of eye development) were subjected to histological, immunohistochemical, Western blot, RT-qPCR, RNA-seq, and subsequent Gene Ontology, Ingenuity Pathway Analysis and TRANSFAC analyses. PAX6 transactivation activity was studied using a luciferase reporter assay in HEK293T cells depleted of GSH using buthionine sulfoximine (BSO). RESULTS: Deletion of Gclc diminished GSH levels, increased reactive oxygen species (ROS), and caused an overt microphthalmia phenotype characterized by malformation of the cornea, iris, lens, and retina that is distinct from and much more profound than the one observed in CRE mice. In addition, only the lenses of KO mice displayed reduced crystallin (α, β), PITX3 and Foxe3 expression. RNA-seq analyses at postnatal day 1 revealed 1552 differentially expressed genes (DEGs) in the lenses of KO mice relative to those from Gclcf/f mice, with Crystallin and lens fiber cell identity genes being downregulated while lens epithelial cell identity and immune response genes were upregulated. Bioinformatic analysis of the DEGs implicated PAX6 as a key upstream regulator. PAX6 transactivation activity was impaired in BSO-treated HEK293T cells. CONCLUSIONS: These data suggest that impaired ocular GSH biosynthesis may disrupt eye development and PAX6 function.
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