Impaired GSH biosynthesis disrupts eye development,lens morphogenesis and PAX6 function |
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| Institution: | 1. Department of Environmental Health Sciences, Yale School of Public Health, Yale University, 60 College Street, New Haven, CT, USA;2. 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 Medicine, Yale University School of Medicine, New Haven, CT, USA;5. Veterans Affairs Connecticut Healthcare System, West Haven, CT, USA;6. Department of Ophthalmology & Visual Science, Yale School of Medicine, New Haven, CT, USA;7. Department of Pathology, Anschutz School of Medicine, University of Colorado, Aurora, CO, USA;8. Department of Biochemistry and Mass Spectrometry Research Center, Vanderbilt University School of Medicine, Nashville, TN, USA;9. Department of Clinical Pharmacy, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Denver, Aurora, CO, USA;1. Center for Translational Ocular Immunology, USA;2. Cornea Service, Department of Ophthalmology, Tufts Medical Center, Tufts University School of Medicine Boston, MA, USA;3. Department of Ophthalmology, New England Eye Center, Tufts Medical Center, Tufts University School of Medicine, Boston, MA, USA;1. Brookline, MA, USA;2. TFOS, Boston, MA, USA;3. Department of Ophthalmology, Antioquia Ophthalmology Clinic-Clofan, Medellin, Antioquia, Colombia;4. Department of Ophthalmology and Otorhinolaryngology, Faculty of Medical Sciences, University of Campinas – UNICAMP, São Paulo, Brazil;5. Ocular Surface Center and Department of Ophthalmology, Sacco Hospital, Milan University, Milan, Italy;6. Department of Ophthalmology, New Zealand National Eye Centre, The University of Auckland, Auckland, New Zealand;7. Ophthalmology Department, Lebanese University, Beirut, Lebanon;8. Earlam and Christopher Optometrists and Contact Lens Specialists, Taunton, UK;9. Department of Ophthalmology and Visual Sciences, Federal University of São Paulo/Paulista School of Medicine, São Paulo, Brazil;10. University of Cape Town, Cape Town, South Africa;11. Sankara Nethralya, Chennai, Tamil Nadu, India;12. Cornea and Refractive Surgery, Beirut Eye and ENT Specialist Hospital, Ophthalmology Department, Lebanese University, Beirut, Lebanon, and Mediclinic Dubai Mall, Dubai, United Arab Emirates;13. Department of Frontier Medical Science and Technology for Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan;14. Department of Optometry and Visual Science, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana;15. Cabinet Ophthalmologie, Alger Centre, Algiers, Algeria;p. Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, MO, USA;q. Ophthalmology Department, Saint Joseph University, Beirut, Lebanon;r. Institute of Ophthalmology, Conde de Valenciana, National Autonomous University of Mexico, UNAM, Mexico City, Mexico;s. Department of Optometry, School of Health Sciences at the College of Health Sciences, Makerere University, Kampala, Uganda;t. University of Alexandria, Alexandria, Egypt;u. Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan;v. School of Optometry and Vision Science, UNSW Sydney, Sydney, Australia;w. Section of Academic Ophthalmology, King''s College London, London, UK, and Departments of Ophthalmology and Epidemiology, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands;x. Department of Specialized, Experimental, and Diagnostic Medicine, University of Bologna, Bologna, Italy;4. Graduate Institute of Bioelectronics and Biomedical Informatics, College of Electronic Engineering and Computer Science, National Taiwan University, Taipei, Taiwan;5. Department of Dentistry, Chung Shan Medical University and Chung Shan Medical University Hospital, Taichung, Taiwan |
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| Abstract: | PurposeThe purpose of this study was to elucidate the role and molecular consequences of impaired glutathione (GSH) biosynthesis on eye development.MethodsGSH 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/f and 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).ResultsDeletion 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.ConclusionsThese data suggest that impaired ocular GSH biosynthesis may disrupt eye development and PAX6 function. |
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