Abstract
Purpose :
Beyond its role in photoreceptor disk morphogenesis, our previous studies have demonstrated that Prom1 (CD133) is a novel regulator of autophagy in the RPE. However, the cellular and molecular mechanisms governing Prom1-mediated RPE autophagy have not previously been determined. We show that Prom1 modulates autophagy in the RPE through inhibition of Akt/mTOR activities and through its association with the selective autophagy receptor, sequestosome (SQSTM1)/p62.
Methods :
The CRISPR/Cas 9 nuclease system was used to knockout Prom1 in the RPE and Prom1 lentiviral constructs were also used to overexpress Prom1 in ARPE19 cells. Western blotting and flow cytometry assays were used to quantify LC3-II processing and punctae formation in the presence and absence of lysosomal inhibitors as cellular readouts for autophagic flux. Immunoprecipitation was used to detect Prom1 interacting proteins and Western blotting was performed to analyze the mTOR and Akt pathway-associated proteins: p-ULK1 (Ser555), p-AMPKα (Thr172), p-mTOR (Ser2448), pAkt (Ser473), p4EBP1(Thr37/46), and LC3-II/LC3-I after stimulation of autophagy.
Results :
Overexpression of Prom1, significantly reduced phosphorylation of S6 ribosomal protein at Ser235/236 and Akt at Ser473, surrogate markers for mTORC1 and mTORC2 activities, respectively, and decreased p62 levels. Prom1 overexpression increased Atg5, and Atg7 expression, LC3I/LC3-II turnover, EBSS-induced autophagy flux, and decreased Bafilomycin-induced p62 accumulation. Immunoprecipitation experiments revealed that Prom1 interacted with p62 and HDAC6, both critical mediators of autophagy flux and autophagosome maturation. Conversely, the Prom1 KO increased mTOR activity, delayed autophagosome trafficking to the lysosome, increased p62 accumulation, and impaired stress-induced autophagy.
Conclusions :
We demonstrate a novel regulation of Prom1-dependent autophagy via upstream inhibition of the mTOR/Akt pathway, and through the protein acting as a molecular scaffold for p62/HDAC6 association in the autophagosomal-lysosomal fusion pathway.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.