Abnormal mitochondrial dynamic correlats with the dysfunction of RPE phagocytosis in AMD

Abstract

Purpose : Dysfunction of Retinal Pigment Epithelial (RPE) cell phagocytosis contributes to age-related macular degeneration (AMD). However, the regulation of the phagocytosis in RPE cells has not been fully elucidated. Current studies have shown that mitochondrial fission plays a critical role in macrophage phagocytosis. In this study, we hypothesize that the alterations of mitochondrial dynamics in RPE cells from AMD donor eyes correlats with phagocytosis dysfunction.

Methods : ARPE-19 or RPE cells from AMD donor eyes were cultured with Fluoresbrite® YG Carboxylate Microspheres or pig photoreceptors out segment (OS). Z-stack of 50 optical sections was collected to cover the entire cell using a Leica TCS SP5 confocal microscope. Mean mitochondrial length and mass was measured with MetaMorphimage analysis. Protein levels were determined by Western blot. Phagocytic capacity was evaluated using flow cytometry.

Results : We found significant (p<0.001) reduction in the mean mitochondrial length in RPE cells during the phagocytosis of microspheres or photoreceptor OS. This correlated with an increase in the levels of mitochondrial fission regulator, Drp-1. MDIVI-1, a Drp-1 inhibitor, successfully inhibited mitochondrial fission and decreased RPE cell phagocytosis. To test if increased mitochondrial fission and fragmentations before phagocytosis will decrease RPE phagocytic activity, we pretreated RPE cells with low concentration of H₂O₂ to induce mitochondrial fission and fragmentation before incubating with microspheres or photoreceptor OS. This resulted in reduced RPE phagocytic activity. MDIVI-1 decreased mitochondrial fission and fragmentation induced by H₂O₂, which partially rescued RPE phagocytic activity. More importantly, increased mitochondrial fragmentation, decrease mitochondrial mass and dynamics observed in RPE cells from AMD donors compared with their normal aged matched counterparts (p<0.05), correlated with the lower phagocytic capacity.

Conclusions : Our study indicates that Drp-1-dependent mitochondrial fission plays a critical role in phagocytosis by the RPE. Reduction in RPE phagocytic function can be correlated with increased mitochondrial fragmentation, decreased mitochondrial mass and dynamics in AMD. This study not only contributes to understanding the molecular mechanisms of RPE phagocytosis but also provides novel targets for treatment of AMD.

This is a 2020 ARVO Annual Meeting abstract.