PROJECT SUMMARY Dry eye disease (DED) is one of the most common visual conditions that increases with age and disproportionally affects women. DED disrupts vision, is often painful, and negatively impacts the quality of life for those affected. Current treatments have limited efficacy and there is no cure. An increase in tear osmolarity is a central feature of DED. Hyperosmolarity triggers mitochondrial dysfunction that ultimately results in caspase release and subsequent apoptosis. Work in our laboratory has found that the insulin-like growth factor binding protein-3 (IGFBP-3) is secreted from corneal epithelial cells (CEC). Secretion of IGFBP-3 is downregulated in response to hyperosmolarity. Of high importance to this proposal, the addition of recombinant IGFBP-3 to CECs cultured in hyperosmolar conditions blocks the hyperosmolar-induced decrease in mitochondrial respiration. We further provide novel data that suggests that IGFBP-3 may mediate autophagic flux in CECs during stress. The purpose of this proposal is to investigate a potential role for IGFBP-3 in mediating mitochondrial metabolism, mitophagy, and macroautophagy (autophagy) in DED. Based on these collective findings, we propose the central hypothesis that IGFBP-3 mediates cellular homeostasis in CECs during hyperosmolar stress through control of mitochondrial metabolism and autophagic mechanism(s). This hypothesis will be tested using a combination of in vitro cell cultures and in vivo animal studies to establish the relationship between IGFBP-3, mitochondrial homeostasis and metabolism. In Aim 1, we will focus on the metabolic effects of IGFBP-3 in CECs exposed to varying levels of hyperosmolar stress in vitro and characterize expression of IGFBP-3 in a desiccating stress mouse model in vivo. In Aim 2, we will focus on the role of IGFBP-3 in mitophagy and autophagy in CECs exposed to hyperosmolar culture in vitro and in vivo using CEC lines and mice that stably express the autophagosome marker, GFP-LC3, and the pH sensitive fluorophore, mt-Keima. The proposed studies will be the first to explore a pathophysiological role for IGFBP-3 in mitochondrial respiration, homeostasis, mitophagy and autophagy in CECs exposed to hyperosmolar stress. Elucidating the role of IGFBP-3 in DED may lead to the development of novel therapies to treat and mitigate disease. The outstanding research environment at UT Southwestern combined with the collective expertise from my multidisciplinary mentorship team will provide exceptional training and a solid platform to build upon as a future physician-scientist in Ophthalmology and Vision Science.