Purpose: Prominin-1 (Prom1) is a transmembrane glycoprotein, which is expressed in stem cell lineages, and has recently been implicated in cancer stem cell survival. Mutations in the Prom1 gene have been shown to disrupt photoreceptor disk morphogenesis and cause an autosomal dominant form of Stargardt-like macular dystrophy (STGD4). Despite the apparent structural role of Prom1 in photoreceptors, its role in other cells of the retina is unknown. The purpose of this study is to investigate the role of Prom1 in the highly metabolically active cells of the retinal pigment epithelium (RPE).
Methods: Lentiviral siRNA and the genome editing CRISPR/Cas9 system were used to knockout Prom1 in primary RPE and ARPE-19 cells, respectively. Western blotting, confocal microscopy, and flow sight imaging cytometry assays were used to quantify autophagy flux. Immunoprecipitation was used to detect Prom1 interacting proteins.
Results: Our studies demonstrate that Prom1 is primarily a cytosolic protein in the RPE. Stress signals and physiological aging robustly increase autophagy with concomitant upregulation of Prom1 expression. Knockout of Prom1 increased mTORC1 and mTORC2 signaling, decreased autophagosome trafficking to the lysosome, increased p62 accumulation, and inhibited autophagic puncta induced by activators of autophagy. Conversely, ectopic overexpression of Prom1 inhibited mTORC1 and mTORC2 activities, and potentiated autophagy flux. Through interactions with p62 and HDAC6, Prom1 regulates autophagosome maturation and trafficking, suggesting a new cytoplasmic role of Prom1 in RPE function.
Conclusions: Our results demonstrate that Prom1 plays a key role in the regulation of autophagy via upstream suppression of mTOR signaling and also acting as a component of a macromolecular scaffold involving p62 and HDAC6.