Loss of Prom1 triggers RPE Degeneration in Atrophic AMD
The gap in our understanding of the pathobiology of atrophic AMD (aAMD) is a barrier to developing new and effective therapies for geographic atrophy (GA) of the retinal pigment epithelium (RPE). Age-related decline in phagocytic function and impaired autophagy flux in RPE cause oxidized lipid-protein accumulation, accelerating complement-mediated inflammation and leading to RPE cell damage and apoptosis. Prominin-1 (Prom1), a glycosylated membrane protein, is essential for photoreceptor (PR) disk biogenesis and outer segment (OS) structure. However, Prom1 mutations are primarily implicated in macular diseases such as Stargardt disease 4 and bull's eye macular dystrophies. The physiological impact of Prom1 mutations in the retina is not clear. We have shown earlier that Prom1 is a central regulator of autophagy in human RPE and identified Prom1 as an emerging player in RPE homeostasis. We propose to build upon these salient findings by assessing the central hypothesis that Prom1 plays a cell-autonomous role in the RPE and that defects in Prom1-dependent mTORC1-TFEB signaling impair RPE autophagy and lysosomal function, triggering mRPE degeneration and induction of epithelial-mesenchymal transition (EMT). This study investigated whether Prom1 is required for mouse RPE (mRPE) autophagy and phagocytosis, which are cellular processes essential for photoreceptor survival. 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. Transcriptomic analysis of Prom1-KO mouse RPE cells by RNA sequencing showed a list of 15 DEGs with high statistical significance in Prom1-KO vs. WT mRPE cells. GSEA and NES show significant upregulation of cell-cycle transcription factors (E2F and MYC targets, G2M checkpoint), mTORC1 signaling, unfolded protein response, reactive oxygen species, TNFA signaling via NF-kappaB, DNA repair, and oxidative phosphorylation pathways; whereas significant downregulation of apical junction and EMT pathways in Prom1-KO vs. WT 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 those seen in atrophic age-related macular degeneration and opens new avenues of investigation targeting Prom1 in retinal degenerative diseases. Overall design: In cultured mouse RPE cells, the Prom1 gene was deleted using CRISPR/Cas9-mediated gene editing. Prom1-KO was verified using genomic DNA analysis. To understand the effect of Prom1-KO in mouse RPE in situ, we used Cre-loxP-mediated gene targeting. We crossed Prom1fl/fl mice with RPE65-Cre mice to generate RPE-specific Prom1-KO in a mouse model. This mouse was gavaged with Tamoxifen to delete the Prom1 gene in the RPE but not in the photoreceptors. We used Prom1 IHC and RNAscope assays to confirm Prom1-KO in the RPE in situ. The mouse eyes were dissected, and the RPE/choroid samples from WT and RPE-specific Prom1-KO mice were used for bulk RNA sequencing. To explore the role of Prom1 in mRPE cells, we used CRISPR/Cas9-mediated gene editing to delete Prom1 in vitro. WT and Prom1-KO mRPE cells were used to perform RNAseq on the Illumina NovaSeq6000 sequencing system.