Abstract
Purpose :
There are no current effective treatments for retinal pigment epithelial (RPE) cell loss in atrophic AMD (aAMD). We have recently shown that loss of Prominin-1 (Prom1) impairs autophagy and induces epithelial-mesenchymal transition (EMT) in mouse retinal pigment epithelial (mRPE) cells, recapitulating features of RPE degeneration in aAMD. Although Prom1 has diverse physiological functions beyond its structural role in photoreceptors, the molecular mechanisms contributing to mRPE degeneration due to the loss of Prom1 function are unclear.
Methods :
To detect Prom1 expression in mRPE in situ, we used both chromogenic and fluorescent RNAscope assays in mouse retinal sections. 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. The FASTQ sequencing reads were processed and filtered to remove adapters and low-quality reads. The sample read counts were normalized, and analysis of differentially expressed genes (DEGs) was performed using the DESeq2 statistical tool. To summarize a broad range of biological processes and pathways under the control of Prom1, we used gene set enrichment analysis (GSEA) to rank gene lists and identify enriched pathways. A positive normalized gene enrichment score (NES) indicates that the gene set is upregulated, and a negative suggests the gene is downregulated in the experimental group.
Results :
Our data show a list of 15 DEGs with high statistical significance in Prom1-KO vs. WT mRPE cells. The upregulated genes of relevance include Gremlin-1, Solute carrier family 7 member 11, Serpine2, Interleukin-1 receptor-like-1, and Retinoic acid-induced 14. The downregulated genes include Actin-binding LIM protein family member 1, Bone morphogenetic protein 3, and Osteoglycin. 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.
Conclusions :
These results demonstrate the signaling pathways and genes that are potentially Prom1-dependent and provide novel insights into the mechanisms by which Prom1 preserves RPE homeostasis.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.