Gclc deletion in surface-ectoderm tissues induces microphthalmia

Abstract

Glutamate cysteine ligase catalytic subunit (Gclc) is the catalytic subunit for the glutamate-cysteine ligase (Gcl) enzyme. Gcl catalyzes the rate limiting step in glutathione (GSH) synthesis. Gclc is highly expressed in the developing eye. To define the regulatory role of Gclc in eye development, we developed a novel, Le-Cre transgene-driven, Gclc knockout mouse model. Gclc f/f /Le-Cre Tg/mice present with deformation of the retina, cornea, iris, and lens, consistent with a microphthalmia phenotype. Controlling for the microphthalmia phenotype of Gclc wt/wt /Le-Cre Tg/mice revealed that Gclc f/f /Le-Cre Tg/mice have a more severe microphthalmia phenotype. Thus, the loss of Gclc expression exacerbates the microphthalmia phenotype in Le-Cre mice.Gclc f/f /Le-Cre Tg/eyes present with reduced retinal and lens epithelium proliferation and increased lens cell death. Imaging mass spectrometry of ocular tissues revealed changes in the intensity and distribution of several lipid species and proteins in the retina and corneas of Gclc f/f /Le-Cre Tg/eyes. Lastly, using splice-blocking morpholinos and CRISPR/Cas9, we created two gclc knockdown zebrafish models, both of which display a microphthalmia phenotype. Combined, the mouse and zebrafish results indicate that, in chordates, Gclc has a conserved role in regulating eye development. In summary, these novel animal models are useful tools for elucidating the mechanisms involved in microphthalmia development.≈ 11% of the cases [1]. Currently, there is no cure for the severe loss of vision associated with microphthalmia. While surgical treatments exist, they only address the cosmetic abnormalities associated with microphthalmia [15]. However, recent research demonstrating the efficacy of nonsense suppression (inhibiting the effect of a nonsense mutation by pharmacologically increasing the likelihood of a near cognate aminoacyl-tRNA substitution) to rescue lens development when administered postnatally in a Pax6-deficient mouse model has provided hope that treatments for microphthalmia can be developed [16]. Clearly, a more complete understanding of the mechanisms regulating lens development should accelerate the discovery of preventative strategies and/or novel therapeutic treatments for microphthalmia. Glutathione (GSH), an intracellular antioxidant, is highly abundant in the eye, with concentrations of 20mM or more occurring in the lens cortex and epithelium [17, 18]. It is important for physiological detoxification of electrophiles and oxidants in the lens cortex and epithelium [19]. Through these functions, GSH maintains lens clarity and, as such, a decline in GSH levels is associated with cataract development [20, 21]. GSH biosynthesis involves a two-step enzymatic process [22] involving glutamate-cysteine ligase (GCL) followed by glutathione synthase (GSS). GCL, the rate limiting step in GSH synthesis, comprises a catalytic subunit (GCLC) and a modifier subunit (GCLM). Impaired GCLC or GCLM function limits GSH synthesis and reduces intracellular GSH concentrations. Th...