Chronic ER Stress Disrupts Mitochondrial-Associated ER Membrane Integrity in Corneal Endothelial Cells.

Abstract

Purpose: Fuchs endothelial corneal dystrophy (FECD) is an age-related degenerative disease of the corneal endothelium cells (CEnCs), affecting 4% of the US population over 40. While Endoplasmic reticulum (ER) and mitochondrial stress have been independently associated with FECD pathogenesis, few studies have examined ER-mitochondrial interactions/ER-mitochondrial contact sites/mitochondria-associated ER membrane (MAM), or MAM proteins, and their contribution to ER and mitochondrial stress in FECD. This study aims to characterize alterations in MAMs and identify key MAM proteins associated with ER and mitochondrial stress in FECD. Methods: Human corneal endothelial cell line (HCEnC-21T) and Fuchs corneal endothelial cell line (F35T) were cultured and subjected to ER stressor tunicamycin for 6 and 24 hours. MAM proteins were isolated by subcellular fractionation, and key ER and mitochondrial-damage-sensor proteins, such as PERK and Parkin, respectively, were identified by immunoblotting. ER-mitochondrial contact sites were quantified using the MAM plasmid and transmission electron microscopy (TEM) in normal and Fuchs cell lines, as well as in human tissues under chronic ER stress. Results: ER-mitochondrial contact distance significantly increased in Fuchs tissues compared with normal tissues, and a similar increase was observed in 21T cell line after tunicamycin treatment. There was a significant increase in the intensity of the MAM plasmid upon tunicamycin treatment at 6 hours in the 21T cell line compared to the non-treated control. However, MAM plasmid intensity significantly decreased at 24 hours compared to 6 hours post-tunicamycin treatment in 21T cell line. Analysis of MAM function by quantifying phosphatidylserine synthase 1 (PSS1 [gene PTDSS1]) expression in 21T cells showed a reduction in PTDSS1 expression after 24 hours of tunicamycin treatment. ER stress protein PERK and mitochondria damage sensor protein (Parkin) significantly increased in the MAM fraction after tunicamycin at 24 hours in 21T cell. Conclusions: Fuchs cell lines and tissues demonstrate decreased ER-mitochondrial interactions/MAMs, which are also seen in 21T cell line after chronic ER stress. Under chronic ER stress, ER and mitochondrial stress mediator proteins are translocated to MAM. This study highlights the importance of MAMs as a potential mediator of ER-mitochondria crosstalk in degenerating corneal endothelial cells for FECD.