Despite recent success in treatment approaches, diabetic retinopathy (DR) remains a leading cause of progressive vision loss and blindness. Conceptual and technical breakthroughs to identify novel targets and strategies to cure this complication are paramount. We believe that such a breakthrough is offered by recent evidence of mitochondrial dysfunction in diabetic retinopathy in combination with the data from large clinical trials demonstrating a strong association between lipid abnormalities and DR progression, and the discovery that ceramide affects mitochondrial function. Mitochondria play cornerstone role in cellular metabolism and even slight modification of mitochondrial function can lead to pathology. Indeed, mitochondrial damage precedes histopathological abnormalities in DR. Recent studies demonstrate that there is an intricate connection between ceramide and mitochondrial function. Mitochondria have been shown to contain many sphingolipids including sphingomyelin and ceramide, as well as enzymes of sphingolipid pathway. Ceramide-induced restriction of respiratory chain function at the level of complex III, as well as succinate accumulation has been shown to be a causative factor in ischemia/reperfusion and stroke- induced tissue damage. In addition to effects on respiratory enzymes, ceramides were shown to contribute to mitochondrial outer membrane permeability either through S1P and hexadecenal production and activation of BAX/BAK, or directly through the formation of protein-permeable ceramide channels in mitochondrial outer membranes. These channels are shown to play a key role in the induction of apoptosis through the release of cytochrome c into the cytoplasm. We have previously demonstrated that activation of acid sphingomyelinase is an important early event in the pathogenesis of diabetic retinopathy. In this proposal we will test the overall hypothesis that increased levels of mitochondrial ceramide upon ASM activation leads to a) cytochrome c release and apoptosis and b) restriction on mitochondrial respiratory chain function in REC and RPE cells in diabetes. As traditional polarographic or fluorescence quenching (Seahorse) methods are not conducive for studies on limited amounts of available retinal tissue and cells, we are developing a novel microfluidic method for functional mitochondrial studies. We will utilize this novel methodology to assess the role of ASM activation and ceramide production in mitochondrial damage in diabetic retina.
The role of mitochondrial dysfunction in the pathogenesis of diabetic retinopathy is well documented; however, the mechanistic details of these effects are not well understood due to the lack of sensitive and specific methods capable of fully addressing the complexity of mitochondrial physiology. In this application we will develop a novel sensitive electrochemical and oxygen-sensing approach to directly measure the complex- specific currents in intact mitochondria and their impact on respiration. This methodology will be used to address the role of increased mitochondrial ceramide upon ASM activation on mitochondrial dysfunction and pathogenesis of diabetic retinopathy.
