Prominin-1 Knockdown Causes RPE Degeneration in a Mouse Model

There are currently no effective treatments for retinal pigment epithelial (RPE) cell loss in atrophic AMD (aAMD). However, our research on <i>Prominin-1</i> (<i>Prom1</i>), a known structural protein in photoreceptors (PRs), has revealed its distinct role in RPE and offers promising insights. While pathogenic <i>Prom1</i> mutations have been linked to macular diseases with RPE atrophy, the broader physiological impact of dysfunctional <i>Prom1</i> in RPE loss is unclear. We have shown that <i>Prom1</i> plays a <i>crucial</i> role in regulating autophagy and cellular homeostasis in <i>human</i> and <i>mouse</i> RPE (mRPE) cells in vitro. Nevertheless, a comprehensive understanding of its in vivo expression and function in mRPE remains to be elucidated. To characterize <i>Prom1</i> expression in RPE in situ, we used RNAscope assays and immunogold electron microscopy (EM). Our use of chromogenic and fluorescent RNAscope assays in albino and C57BL/6J <i>mouse</i> retinal sections has revealed <i>Prom1</i> mRNA expression in perinuclear regions in mRPE in situ. Immunogold EM imaging showed <i>Prom1</i> expression in RPE cytoplasm and mitochondria. To confirm <i>Prom1</i> expression in RPE, we interrogated <i>human</i> RPE single-cell RNA-sequencing datasets using an online resource, Spectacle. Our analysis showed <i>Prom1</i> expression in <i>human</i> RPE. To investigate <i>Prom1</i>'s function in RPE homeostasis, we performed RPE-specific <i>Prom1</i> knockdown (KD) using subretinal injections of AAV2/1.CMV.saCas9.U6.<i>Prom1</i>gRNA in male and female <i>mice</i>. Our data show that RPE-specific <i>Prom1</i>-KD in vivo resulted in abnormal RPE morphology, subretinal fluid accumulation, and secondary PR loss. These changes were associated with patchy RPE cell death and reduced a-wave amplitude, indicating retinal degeneration. Our findings underscore the central role of <i>Prom1</i> in cell-autonomous mRPE homeostasis. The implications of <i>Prom1</i>-KD causing aAMD-like RPE defects and retinal degeneration in a <i>mouse</i> model are significant and could lead to novel treatments for aAMD.