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Sayantan Datta, Huan Mills, Marisol Cano, James T Handa; Pink1 mediated mitophagy protects ARPE19 cells from mitochondrial damage. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5779.
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© ARVO (1962-2015); The Authors (2016-present)
Autophagy and mitophagy are protective mechanisms in cells, particularly Retinal Pigment epithelium (RPE), that are constantly under stress. Autophagic dysregulation have been suggested in a variety of neurodegenerative diseases including age-related macular degeneration (AMD), but mitophagy has not been studied in depth. Induction of mitophagy/autophagy may be a treatment strategy to prevent RPE degeneration during stress. This study focuses on importance of mitophagy in ARPE19 cells under acute stress
Serum-starvation can induce generalized autophagy whereas CCCP specifically induces mitophagy. Mitophagy is initiated by mitochondrial translocation of Pink1 with Parkin recruitment, followed by the general autophagy pathway including LC3b recruitment and lysosomal degradation. ARPE19 cells with intact or knocked down (KD) Pink1 were cultured in either complete medium (CM) or serum starved (SS) conditions followed by CCCP (1uM or 10uM) treatment for 4h or 24 h. Mitophagy was assessed by Western blot and confocal imaging of LC3b and Parkin. Mitochondrial function was determined by RT- PCR assay for mtDNA/genomic DNA ratio and ATP production
CCCP induced mitophagy in cells grown in CM with significant Parkin induction (P<0.01) while SS treated cells exhibited no Parkin expression, but twice more LC3b conversion than CM treated cells (P< 0.05). Parkin co-localized with mitochondrial marker Tom20 after CCCP treatment, especially when cells were grown in CM. LC3b, on the other hand, was induced and did not colocalize with mitochondria in SS conditions. Pink1 siRNA caused 85% transcriptional and protein level knockdown, and initially protected the mitochondria from CCCP induced damage in the presence of CM with nearly undetectable LC3b conversion, and without a decrease in ATP generation or mitochondrial DNA quantity. However, after 24h, these cells performed significantly poorer on all these parameters (2 times more LC3b, 60% decrease in ATP and 50% decrease in mtDNA) than cells with intact Pink1
Cells treated with a stressor like CCCP either undergo generalized autophagy or specialized mitophagy depending on the presence of other contributing factor like serum starvation. Pink1 is necessary for early removal of damaged mitochondria and maintains mitochondrial homeostasis. Mitophagy is a protective mechanism and its failure eventually leads to more severe mitochondrial damage and dysfunction
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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