Abstract
Purpose :
In AMD, RPE cells show structural and functional heterogeneity, where death resistant RPE undergoing EMT exist alongside normal and dying RPE. The molecular mechanism underlying this heterogeneity is unknown. In a novel finding, we have shown that impairment of Pink1 mediated mitophagy leads to RPE EMT in an Nrf2 dependent manner. Here we try to delineate further, the mechanism by investigating the relevance of Pink1-Parkin signaling and receptor mediated mitophagy and mitochondrial retrograde signaling through ROS and TXNRD1, which respectively act up and downstream of Nrf2.
Methods :
ARPE-19 and iPS-RPE cells after 6 days of transfection with the following siRNAs--scramble, Nrf2, Pink1 and Nrf2-Pink--were tested for: EMT - using ZO1-Vimentin staining, expression of EMT TFs Snail1 and Zeb1, and cell viability using Calcein-AM. Expression of mitophagy receptors, FUNDC1, PEX13, BCL2L13, and Pink1, Parkin, P62 were quantified with RT-qPCR and after LC3B Co-IP in the mitochondrial fraction. Mito-tempo, an Mt ROS scavenger, and siRNA TXNRD1, an Nrf2 target were used to mechanistically connect them to EMT. RPE from C57BL6J, Nrf2-/- and Pink1-/- mice were examined for evidence of structural abnormality. Pink1 distribution in the RPE was assessed using IHC in AMD globes.
Results :
In AMD samples, Pink1 was reduced in dysmorphic RPE overlying the drusen, suggesting impaired mitophagy (Fig1A,B). Pink1-/- mice had morphologically abnormal RPE compared to both C57 and Nrf2-/- mice (Fig1C). In cells, Pink1 knockdown (KD) showed signs of type II EMT including cellular elongation, and Vimentin, Snail1 and Zeb1 expression (Fig2A,B). Pink1 KD cells were highly viable after oxidative stress compared to Nrf2 KD or Pink1-Nrf2 double KD cells, which showed the lowest survival (Fig2C). Pink1 KD did not affect the transcript levels of mitophagy receptors. With LC3b coIP, Pink1 KD induced an accumulation of FUNDC1 and P62 in mt fractions. Both Mito-tempo and TXNRD1 KD abrogated the Pink1 KD induced EMT phenotype.
Conclusions :
Pink1 loss caused Nrf2 dependent adaptive EMT and loss of both Pink1 and Nrf2 cells look normal but are susceptible to death. Pink1 loss induced a compensatory receptor mediated mitophagy through FUNDC1 and P62. Pink1 loss triggered a retrograde signaling cascade mediated by mtROS, Nrf2 and TXNRD1. These results thus identify novel therapeutic target for restoring RPE functionality in AMD.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.