The overall goal of this project is to test the hypothesis that high glucose (HG)-induced upregulation of mitochondrial fission genes and inhibition of mitochondrial fusion genes, concomitant with decreased autophagy and mitochondrial connexin 43 (mtCx43) downregulation, promotes mitochondrial fragmentation and dysfunction; amelioration of these events would protect mitochondrial function and thereby prevent retinal vascular cell loss in diabetic retinopathy (DR). The hypothesis is based on findings that mitochondrial fission/fusion, autophagy, and mtCx43 play critical roles in maintaining mitochondrial morphology and function. Our previous studies show HG induces mitochondrial fragmentation and breakdown of the mitochondrial networks resulting in increased mitochondrial membrane potential heterogeneity, decreased oxygen consumption, altered extracellular acidification, increased cytochrome c release, and ultimately apoptosis of retinal vascular cells. Furthermore, HG decreases autophagy and mtCx43 expression in these cells. Our preliminary data indicates that HG increases expression of fission genes, Fis1 and Drp1, and decreases expression of fusion genes, Opa1 and Mfn2, in retinal vascular cells. Additionally, HG reduces autophagy/mitophagy, a process that removes dysfunctional cellular components including mitochondrial fragments, and thereby contributes to accumulation of fragmented mitochondria. We also observed HG reduces mtCx43 expression, and that HG-induced mtCx43 channel inhibition alters mitochondrial morphology and cytochrome c release in rat retinal endothelial cells. Furthermore, mitochondrial fragmentation was noted in retinal vascular cells of diabetic rats, and new data indicate decreased number of acellular capillaries and pericyte ghosts in retinal capillaries of diabetic Drp1+/- mice and Opa1+/- mice suggesting attenuation of mitochondrial fragmentation could be beneficial. Based on the information and preliminary data, we propose three Specific Aims to determine 1) whether inhibition of HG-induced mitochondrial fragmentation prevents apoptosis in retinal endothelial cells and pericytes in vitro, as well as in retinas of diabetic rats; 2) whether HG-induced decreased autophagy promotes accumulation of mitochondrial fragments and retinal vascular cell loss; and 3) whether altered mtCx43 expression contributes to the development of retinal vascular lesions in experimental DR. The proposed project is expected to identify novel mechanism(s) underlying retinal vascular cell loss involving mitochondrial abnormalities, and thus, provide insight into potential strategies to prevent these abnormalities related to vascular cell death in DR.