Interplay between autophagy and mitochondrial metabolism in retinal pigment epithelium cells obtained by differentiation of induced pluripotent stem cells generated from wet AMD patients

Abstract

AbstractPurpose: Age‐related macular degeneration (AMD) presents a limited potential of experimental studies on the target tissue from AMD patients. Therefore, it is important to have a reliable experimental model to study AMD pathogenesis. We differentiated induced pluripotent stem cells (PSCs) derived from individuals suffering from wet AMD into retinal pigment epithelium (RPE) cells (iPSCs‐RPE). Impaired autophagy and mitochondrial metabolism are recognized factors of AMD pathogenesis, but the underlined mechanism is not fully known. The aim of this study was to assess mitochondrial metabolism in dependence on autophagy in iPSCs‐RPE derived from wet AMD patients and non‐AMD controls.Methods: Bafilomycin a1 (Baf1) and MG‐132 were used as autophagy inhibitor and inducer, respectively. Expression of autophagy markers were evaluated by RT‐PCR and western blotting. The Seahorse FX test was used to evaluate mitochondrial metabolism with carbonyl cyanide‐p trifluoromethoxyphenylhydrazone (FCCP) to uncouple oxidative respiration.Results: We observed differences between the expression of the autophagy‐related genes MAP1LC3B (Microtubule Associated Protein 1 Light Chain 3 Beta) and SQSTM1 (Sequestosome 1) in RPE cells derived from wet AMD and non‐AMD controls. In addition, differences in the expression of the HSP701A (Heat Shock Protein Family A (Hsp70) Member 1A, chaperone) gene were also observed. Autophagy differentially affected mitochondrial metabolism as evaluated by basal and maximal respiration, ATP production, spare capacity and proton link.Conclusions: Autophagy differentially influences mitochondrial metabolism in iPSCs‐RPE derived from wet AMD and non‐AMD eyes.Acknowledgement: This work was supported by National Science Centre, Poland grant number 2017/27/B/NZ3/00872.