Mitochondrial fragmentation and upregulation of PINK1 and Parkin-mediated mitophagy in Fuchs Endothelial Corneal Dystrophy

Abstract

Purpose : Fuchs Endothelial Corneal Dystrophy (FECD) is a late-onset oxidative stress-induced disease characterized by progressive loss of corneal endothelial cells. We previously reported increased mitochondrial fragmentation as a possible phenomenon that explained the pathomechanism of FECD. The aim of this study is to further decipher the key checkpoints involved in mitochondrial quality control that may be aberrantly regulated in FECD.

Methods : Tissue lysates prepared from post-keratoplasty specimens from late-onset FECD cases were compared with age-matched normal donor specimens (n=3 for each group) for PINK1, phosphoParkin(Ser65), phospho-Drp1(Ser616), phosphoDrp1(Ser637). Whole cell lysates of human corneal endothelial cell line immortalized with hTert (HCEnC-21T) were treated with 50mM menadione (MN) and 10nM Bafilomycin A1 for 20 hours. HCEnC-21T cells were transfected with YFP-Parkin, fragmentation was induced with 50mM MN, and mitochondria were visualized with immunofluorescence staining of cytochrome c. Cells were treated with 1mM epoxomicin and mitochondria were purified to test for ubiquitination. Relative abundance of proteins was quantified with ImageJ.

Results : FECD ex vivo specimens showed an accumulation of full length PINK1 (3.5 fold, p = 0.0077) and increased phosphoParkin(Ser65) (25 fold, p=0.046). Drp1 and phosphoDrp1(Ser616) were not detectable in FECD tissues, whereas phosphoDrp1(Ser637) was reduced by 5-fold (p=0.003). Treatment of HCEnC-21T cells with MN resulted in a time dependent increase in fragmentation of mitochondria that induced the translocation of YFP-Parkin to fragmented mitochondria. MN also resulted in an increase in phosphoParkin(Ser65) and a decrease in Drp1 including Ser616 and Ser637. These changes in protein levels were rescued by Bafilomycin A1 in HCEnC-21T cells. In contrast, MN did not induce an accumulation of PINK1 in HCEnC-21T as seen in FECD specimens. Mitochondrial fraction of a normal CE and an FECD cell line (FECDi) showed increased phosphoParkin. Treatment with epoxomicin revealed increased ubiquitination of mitochondrial proteins (p=2.83X10^-8) in FECDi.

Conclusions : Mitochondrial quality control is abrogated in FECD that leads to a constitutive fragmentation and degradation of mitochondria through accumulation of PINK1 and activation of Parkin resulting in the increased ubiquitination of mitochondrial proteins.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.