PROJECT SUMMARY/ABSTRACT: Our long-term goal is to contribute to the prevention and treatment of cataracts, which are the major global cause of visual impairment. Age-related lens optical aberrations manifest around age forty and gradually progress into a cataract. Accrual of Hydrogen peroxide (H2O2) due to UV radiation, normal cellular metabolism, etc., is a major cause of age-related cataract (ARC) formation. The scavenging activity of Glutathione (GSH) and Glutathione Peroxidase 1 (GPX1) helps to detoxify cellular H2O2. As the lens ages, GPX1 activity is reduced, causing gradual H2O2 accumulation which affects homeostasis and transparency. Homeostasis of the avascular lens significantly depends on microcirculation to carry in nutrients and eliminate metabolic waste. Aquaporin (AQP) water channels and Connexin (Cx) gap junction channels are integral to the lens microcirculation. Lens epithelial cells express AQP1, AQP5, Cx43 and Cx50, and fiber cells express AQP0, AQP5, Cx46 and Cx50. AQP0, the most abundant lens membrane protein, exists as intact and end-cleaved forms. Mutation or knockout (KO) of AQP0, Cx46 and Cx50 causes cataracts. Under stressful conditions, AQP1 or AQP5 KO lenses develop cataracts. Lens Cxs play a role in transporting GSH to the nucleus. Our preliminary intact lens studies show that lenticular AQPs are permeable to H2O2. In this grant proposal, we, for the first time, seek to explore the non-canonical role of lens AQPs as peroxiporins or H2O2 transporters, alongside the protective roles of Cxs and GPX1 in scavenging lenticular H2O2. Main Hypothesis is: Lens AQPs transport extracellular H2O2 into epithelial and peripheral fiber cells, where it is detoxified by GSH/GPX1, thus removing extracellular H2O2 from the circulation to central fiber cells, which have low GSH and no GPX1. Age-induced reductions in the function of GPX1 cause H2O2-initiated damage to lens AQPs, Cxs and Na+/K+-ATPase, thus compromising the microcirculation and contributing to ARC. To test, in Aim 1, we will quantify age-related changes in transparency in relation to alteration in H2O2 transport by AQPs using the lenses of WT and AQP-KO models. We will test if post-translationally end-cleaved AQP0s transport H2O2 and are regulated by pH, calcium and zinc, using intact lenses of WT and a transgenic mouse model. We will investigate whether the increase in H2O2 level during aging will alter the intracellular pH of the lens. In Aim 2, we will explore the protective role of GPX1 on AQPs, Cxs and Na+/K+-ATPase to maintain the lens microcirculatory system during aging, using WT and GPX1-KO lenses. We will test the spatial and temporal H2O2, and reduced (GSH) and oxidized GSH (GSSG) levels in GPX1-KO in relation to ARC. In Aim 3, we intend to enhance GPX1 activity through a diet containing GPX1-mimic Ebselen in WT and GPX1-KO mice, to lessen GPX1-reduction- induced H2O2 surge and adverse oxidative stress during aging. We believe that the emerging data as a funct...