Project Summary/Abstract Diabetic retinopathy (DR) remains the first cause of blindness in the working age population and the increasing number of diabetic cases only promises further increase in the healthcare burden associated. Anti-VEGF is a powerful new treatment option for proliferative diabetic retinopathy and macular edema, late stages of DR; however there are still no therapies available to prevent early alterations of the neuroretina. aA- and aB-crystallins are chaperone proteins promoting cell survival in acute stress conditions, which have been proposed as promising therapeutic targets for neurodegenerative conditions. The molecular chaperone aA-crystallin is highly upregulated in the retina during diabetes and while we showed that its phosphorylation on S148 (rodent) or T148 (human) residue is critical for its protective role, there is a fundamental gap in our knowledge regarding the molecular mechanisms by which it promotes the survival of retinal cells and how those mechanisms are regulated by this phosphorylation. The long-term goal of this research is to determine how the chaperone functions of ?-crystallins can be manipulated to preserve vision in patients with diabetes. The objective of this project is to identify the molecular mechanisms by which the function of ?A-crystallin protein is modulated in retinal neurons and glia in diabetes. Emerging from our preliminary data and previously published work, the central hypothesis guiding this project is that aA-crystallin promotes neuronal survival via both cell autonomous and non-autonomous mechanisms regulated by the phosphorylation of the S148/T148 residue. The rationale for the proposed research is that determining the cellular function of aA-crystallin in the neuro-glial unit will lead to strategies to enhance retinal cell viability in chronic retinal diseases such as diabetic retinopathy. This hypothesis will be tested by pursuing 3 specific aims: i) Determine the molecular mechanisms by which pS148/pT148 controls the protective function of aA-crystallin in retinal neurons; ii) Determine the function of aA-crystallin in Mller glia; and iii) Determine the kinase and phosphatases regulating phosphorylation of aA-crystallin on S148/T148. Under the first 2 aims, we will establish how aA-crystallin and its phosphorylation regulate the balance of apoptotic and survival signaling pathways in retinal neurons in vitro, while human tissues will be used to confirm their relevance, and AAV-based constructs in animal models will allow to test their potential in vivo. While aim 1 will focus on the cell autonomous mechanisms in retinal neurons themselves, aim 2 will focus on the non-cell autonomous mechanisms involving Mller glia. Finally aim 3 will use in vitro screening followed by in vivo validation to identify the kinase and how it is affected by diabetes. This research project will reveal the multifaceted and critical regulatory functions played by aA-crystallin in the retina, potentially leading to the identification of approaches for utilizing this intrinsic protective pathway to promote retinal cell survival in chronic neurodegenerative conditions and, thereby, preserve visual function.