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Sayantan Datta, Marisol del Valle Cano, Kie Ito, Hiromi Sesaki, James T Handa; Pink1 deficiency induces Nrf2 dependent EMT and metabolic dysfunction in RPE cells. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1605.
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© ARVO (1962-2015); The Authors (2016-present)
Defects in anti-oxidative response and mitochondrial functioning have been implicated in the development of AMD, but the interplay of these two pathways in energy demanding RPE cells has not been examined. Here we sought to look at the effect of two important genes –Nrf2 transcription factor, master regulator of the anti-oxidant pathway and Pink1 kinase, initiator of mitophagy, on human RPE structure and function.
ARPE19 cells were treated with various siRNA (scramble, Nrf2, Pink1, or both Nrf2-Pink1) and stressors and grown for 5 days. Cell morphology was photographed and cells were harvested for RNA and protein that were used for RT-qPCR and Western analysis, respectively. Phenotypic readouts like Mitosox superoxide, TMRM mitochondrial membrane potential (ΔΨ), ATP and lactate levels were measured and Seahorse mito-stress test was performed using the XF96 analyzer.
Mitotracker-lysotracker assays showed Pink1 knockdown(KD) impaired mitophagy. Cells with Pink1KD or cells treated with EGF started elongating on day 4 whereas the cells with scramble, Nrf2 or Nrf2-Pink1 double KD did not change shape. Both Nrf2KD and treatment with Nrf2 inhibitor Trigonelline prevented cell elongation induced by Pink1KD. This change correlated with a two-fold increase in Twist1 and Zeb1 gene expression and marked Vimentin immuno-staining, suggesting that these morphologic changes are due to epithelial-mesenchymal transition EMT (Fig1). The Pink1KD cells exhibited increased mitochondrial superoxide and lactate levels, decreased (ΔΨ) and ATP production - all of which were ameliorated by Nrf2KD. Seahorse data revealed a 50% decrease in reserve respiratory capacity with Pink1KD which was partially rescued by Nrf2KD (Fig2). Among mitochondrial biogenesis associated genes, PGC1a gene expression showed strong correlation with induction and rescue of the EMT phenotype.
Pink1 deficiency impaired mitochondrial respiration necessitating a switch to glycolysis and induced EMT in RPE cells. Paradoxically, Nrf2 signaling appears necessary for EMT. RPE of AMD patients have been shown to undergo EMT changes and have mitochondrial dysfunction. This study shows direct induction of EMT by Pink1KD in an Nrf2 dependent manner and provides evidence of an interplay of oxidative stress and mitophagy pathways in controlling cell morphology and energy metabolism which can be used as novel AMD therapeutic targets.
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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