Pink1‐mitophagy and Nrf2‐stress response mediated novel mitochondrial retrograde signaling affects RPE structure and function

Abstract

PurposeDry age related macular degeneration (AMD) is the leading cause of blindness in the elderly with no effective treatment. Dysfunction and death of the retinal pigment epithelium (RPE) cells is the hallmark feature of dry AMD. In AMD, RPE cells show structural and functional heterogeneity, where death resistant RPE undergoing epithelial to mesenchymal transition (EMT) exist alongside normal and dying RPE. The molecular mechanism underlying this heterogeneity is unknown. RPE cells which form the blood retinal barrier are one of the most energy demanding and stress exposed cell types in the human body. Therefore, Pink1 mediated mitohagy and Nrf2 stress response pathways are critical for healthy RPE functioning. In a novel finding, we have shown that impairment of Pink1 mitophagy directly leads to RPE EMT in an Nrf2 dependent manner and impairment of both Pink1 and Nrf2 causes RPE death. Here we try to delineate the signaling mechanism using RNA seq and characterize the molecular and functional relevance in vivo using Pink1KO mice.MethodsARPE‐19 and iPS‐RPE cells after 6 days of transfection with the following siRNAs‐scramble, Nrf2, Pink1 and Nrf2‐Pink1 ‐ were tested for: EMT ‐ using Vimentin staining, expression of EMT TFs Snail1 and Zeb1, and cell viability using Calcein‐AM. RNA seq (HiSeq) of the 4 cell types was performed and pathways analyzed using DAVID. mtROS and AKT inhibitors were used to mechanistically connect them to EMT. RPE from C57BL6J, Nrf2KO and Pink1KO mice were examined for evidence of structural abnormality and mitophagy and EMT were biochemically investigated in Pink1KO mice using qPCR and western blots and functional tests (ERG and OKN) were done. Pink1 and Snail distribution in the RPE were assessed using IHC in AMD globes.ResultsIn human AMD samples, RPE over drusen showed higher Snail and lower Pink1 expression. Pink1KO mice had dysmorphic RPE and poor visual acuity compared to C57 mice. Western blot revealed lower pParkin and LC3b2 indicating impaired mitophagy with an increase in Vimentin in Pink1KO RPE. ARPE‐19 & iPS‐RPE cells showed cell elongation with Pink1KD. Pink1KD also had lower parkin and LC3b2 levels in mt fraction suggesting impaired mitophagy and 2‐fold increased Zeb1 and significant increase in mtROS. The double KD cells showed morphological reversal of EMT but poor cell survivability. RNA seq revealed upregulation of PI3k AKT signaling in Pink1KD cells and EMT was rescued by treatments with AKT inhibitor (A6730) and a novel dendrimer attached N‐acetyl cysteine (NAC), a mito targeted ROS quencher. The reversal of phenotype from EMT to death in Pink1KD to doubleKD cell seems to be controlled by a Notch1 switch ‐ which goes from 2 fold over‐expression to 2.3 folds downregulation in Pink1KD to double KD.ConclusionsPink1 loss caused Nrf2 dependent adaptive EMT and loss of both Pink1 and Nrf2 cells look normal but are death prone. Pink1 loss triggered a retrograde signaling cascade mediated by mtROS, Nrf2, NOTCH1 and AKT. That mitophagy and stress response pathways can control cell shape is a significant discovery and these results thus identify novel therapeutic targets for restoring RPE functionality in AMD.Support or Funding InformationSD is on a K99 grant from NEI/NIH AWARD# K99EY029010JTH is supported by Brightfocus, NIH and Robert Bond Professorship