Impaired GSH biosynthesis disrupts eye development, lens morphogenesis and PAX6 function

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 Gclc^f/f mice with hemizygous Le-Cre transgenic mice to produce Gclc^f/f/Le-Cre^Tg/- (KO) mice. Control mice included Gclc^f/fand Gclc^wt/wt/Le-Cre^Tg/- 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 Gclc^f/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.

Keywords: Glutathione; Lens development; Lens transcriptome; Microphthalmia; PAX6.