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Sujoy Bhattacharya, Jinggang Yin, Weihong Huo, Edward Chaum; Prom1-dependent Autophagy Prevents Mitochondrial Damage and Apoptosis in Human Retinal Pigment Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2017;58(8):618. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Our previous studies have demonstrated that Prom1 (CD133) modulates autophagy in the RPE through inhibition of mTORC1/2 activities and through its association with the selective autophagy receptor, sequestosome (SQSTM1)/p62. Although post-mitotic RPE cells rely on autophagy to maintain homeostasis, it is unknown whether Prom1-dependent autophagy regulates RPE apoptosis in response to stress and mitochondrial complex I inhibition.
Prom1 lentiviral constructs were used to overexpress wildtype (WT) and STGD4 Mutant (Mut-Prom1) Prom1 in ARPE19 cells. Cells were treated with p53 activator (Nutlin-3), NADPH oxidase inhibitor (Diphenyleneiodonium, DPI) in the presence and absence of autophagy inhibitor (3-Methyladenine, 3-MA). Western blotting was used to quantify Atg proteins, p62/SQSTM1, and LC3-II processing. Apoptosis was measured by activation of caspase-3 and DNA damage was measured by gamma-H2AX phosphorylation.
Nutlin-3 triggered apoptosis in confluent monolayers of ARPE-19 cells, and concomitant inhibition of autophagy blocked Nutlin-3’s ability to induce apoptosis. Overexpression of WT and STGD4 Mut-Prom1 in ARPE-19 cells conferred protection from Nutlin-3-induced apoptosis and growth arrest through activation of autophagy and inhibition of p53 signaling. Likewise, cells expressing both WT and Mut- Prom1 were protected from NADPH-oxidase inhibitor--induced mitochondrial DNA damage. However, sustained inhibition of autophagy sensitized both WT and Mut-Prom1 expressing cells to Nutlin-3-induced RPE degeneration.
We demonstrate that Prom1-dependent autophagy flux contributes to the cytoprotection of RPE cells against irreparable mitochondrial DNA damage and apoptosis.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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