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Mutations in the Prominin-1 (Prom1) gene disrupt photoreceptor disk morphogenesis, leading to macular dystrophies. We have shown that human retinal pigment epithelial (RPE) homeostasis is under the control of Prom1-dependent autophagy, demonstrating that Prom1 plays different roles in the photoreceptors and RPE. It is unclear if retinal and macular degeneration caused by the loss of Prom1 function is a cell-autonomous feature of the RPE or a generalized disease of photoreceptor degeneration. In this study, we investigated whether Prom1 is required for mouse RPE (mRPE) autophagy and phagocytosis, which are cellular processes essential for photoreceptor survival. We found that Prom1-KO decreases autophagy flux, activates mTORC1, and concomitantly decreases transcription factor EB (TFEB) and Cathepsin-D activities in mRPE cells. In addition, Prom1-KO reduces the clearance of bovine photoreceptor outer segments in mRPE cells due to increased mTORC1 and reduced TFEB activities. Dysfunction of Prom1-dependent autophagy correlates with both a decrease in ZO-1 and E-cadherin and a concomitant increase in Vimentin, SNAI1, and ZEB1 levels, consistent with induction of epithelial-mesenchymal transition (EMT) in Prom1-KO mRPE cells. Our results demonstrate that Prom1-mTORC1-TFEB signaling is a central driver of cell-autonomous mRPE homeostasis. We show that Prom1-KO in mRPE leads to RPE defects similar to that seen in atrophic age-related macular degeneration and opens new avenues of investigation targeting Prom1 in retinal degenerative diseases., (© 2023 The Authors. Journal of Cellular Physiology published by Wiley Periodicals LLC.)

Background: Mitochondrial dysfunction and mitochondrial DNA (mtDNA) damage in the retinal pigment epithelium (RPE) have been implicated in the pathogenesis of age-related macular degeneration (AMD). However, a deeper understanding is required to determine the contribution of mitochondrial dysfunction and impaired mitochondrial autophagy (mitophagy) to RPE damage and AMD pathobiology. In this study, we model the impact of a prototypical systemic mitochondrial defect, mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), in RPE health and homeostasis as an in vitro model for impaired mitochondrial bioenergetics., Methods: We used induced pluripotent stem cells (iPSCs) derived from skin biopsies of MELAS patients (m.3243A > G tRNA leu mutation) with different levels of mtDNA heteroplasmy and differentiated them into RPE cells. Mitochondrial depletion of ARPE-19 cells (p 0 cells) was also performed using 50 ng/mL ethidium bromide (EtBr) and 50 mg/ml uridine. Cell fusion of the human platelets with the p 0 cells performed using polyethylene glycol (PEG)/suspension essential medium (SMEM) mixture to generate platelet/RPE "cybrids." Confocal microscopy, FLowSight Imaging cytometry, and Seahorse XF Mito Stress test were used to analyze mitochondrial function. Western Blotting was used to analyze expression of autophagy and mitophagy proteins., Results: We found that MELAS iPSC-derived RPE cells exhibited key characteristics of native RPE. We observed heteroplasmy-dependent impairment of mitochondrial bioenergetics and reliance on glycolysis for generating energy in the MELAS iPSC-derived RPE. The degree of heteroplasmy was directly associated with increased activation of signal transducer and activator of transcription 3 (STAT3), reduced adenosine monophosphate-activated protein kinase α (AMPKα) activation, and decreased autophagic activity. In addition, impaired autophagy was associated with aberrant lysosomal function, and failure of mitochondrial recycling. The mitochondria-depleted p 0 cells replicated the effects on autophagy impairment and aberrant STAT3/AMPKα signaling and showed reduced mitochondrial respiration, demonstrating phenotypic similarities between p 0 and MELAS iPSC-derived RPE cells., Conclusions: Our studies demonstrate that the MELAS iPSC-derived disease models are powerful tools for dissecting the molecular mechanisms by which mitochondrial DNA alterations influence RPE function in aging and macular degeneration, and for testing novel therapeutics in patients harboring the MELAS genotype., (© 2022. The Author(s).)