Proteins Associated with Phagocytosis Alteration in Retinal Pigment Epithelial Cells Derived from Age-Related Macular Degeneration Patients

Abstract

Age-related macular degeneration (AMD) is partially characterized by retinal pigment epithelial (RPE) cell dysfunction. This study focused on phagocytosis activity and its involvement in AMD. Phagocytic activity was analyzed by flow cytometry using porcine photoreceptor outer segment (POS) and fluorescent beads in basal and under oxidative stress condition induced by Fe-NTA in fifteen hiPSC-RPE cell lines (six controls, six atrophic AMD and three exudative AMD). Oxidative stress exposure inhibited phagocytosis in the same manner for control, atrophic AMD (AMDa) and exudative AMD (AMDe) cell lines. However, altered phagocytosis in basal condition in hiPSC-RPE AMDa/e was observed compared to control cell lines. Gene expression after 3 or 24 h of POS incubation was analyzed by RNA-Seq based transcriptomic profiling. Differential gene expression was observed by RNA seq after 3 and 24 h POS exposure. We have focused on the genes involved in mTOR/PI3K-AKT/MEK-ERK pathway. We investigated differences in gene expression by analyzing the expression levels and activity of the corresponding proteins by Western blot. We showed the involvement of three proteins essential for phagocytosis activity: fak, tuberin and rictor. These findings demonstrate that hiPSC-RPE AMDa/e cells have a typical disease phenotype characterized by alteration of the main function of RPE cells, phagocytosis activity.

Keywords

• age-related macular degeneration
• atrophic AMD
• exudative AMD
• iPSC-RPE phagocytosis
• RNA-seq