Recent research in my laboratory revealed that conditionally knocking out glutathione (GSH) synthesis from the lens results in a quasi-perfect model of age-related cataractogenesis (the LEGSKO mouse) that simulates most oxidative changes observed in human age-related cataracts. However, subsequent breeding of the mouse was associated with delayed cataractogenesis and a stunning discrepancy between total absence of mRNA and protein for the Gclc subunit of ?-glutamyl cysteine ligase and a persisting 50% (instead of the expected zero%) GSH level in the homozygous LEGSKO mouse, implying thereby the existence of an active transport system for GSH. In preliminary studies I confirmed the ability of the LEGSKO lens to take up H3-GSH against a ten-fold concentration gradient. A preliminary screen revealed that at least 143 candidate transporters from deep sequencing analysis were elevated in the LEGSKO lens out of >1500 changed genes vs. wild type lens. Together, these data provide strong support for the existence of salvage mechanisms implicated in redox and GSH homeostasis in the lens. Yet, previous attempts by others to elucidate the molecular nature of GSH transporter(s) in the lens have failed, implying the presence of a complex problem that I propose to approach using a multipronged, powerful and innovative strategy that combines yeast genetics, transcriptome RNA-seq analysis, a lens epithelial monolayer system specifically developed for transport studies, and the LEGSKO mouse itself.
In Specific Aim 1, I will use the yeast model system to perform the initial screen o the candidate transporters selected from the RNA-seq study comparing genes expressed in LEGSKO vs. WT lens.
In Specific Aim 2, I will characterize/validate the candidates from yeast screen for GSH transport in lens epithelial monolayer culture system. To achieve these goals I have enlisted the active collaboration an expert in lens genetics (Dr. David Beebe), a yeast geneticist (Dr. Alan Tartakoff) and an expert in epithelium membrane transport mechanisms (Dr. Ulrich Hopfer). I believe the proposed exploratory goals are ideally suited for support by the R21 mechanism, as a successful outcome is expected to open up a vast field of investigation that may altogether have profound implications for redox homeostasis in the aging eye.
Oxidation has been considered as one of the major risk factors in age-related nuclear cataract formation, affecting over 16 million people over the age of 40 in the United States. The major antioxidant, glutathione, decreases with age and we have now found that it is playing a crucial role in age-related cataract formation. However, how the lens maintains its glutathione homeostasis, especially in aging process, is still unclear. We now have an animal model we can use for these studies. Using this model, we will study how the glutathione can be delivered into lens. We hope to find that this will help to increase the lens glutathione and thus to protect the lens from cataract development.
