Ritzer, L. | Search bioRxiv

bioRxiv2026

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

PurposeFuchs 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. MethodHuman corneal endothelial cell line (HCEnC-21T) and Fuchs corneal endothelial cell line (F35T) were cultured and subjected to ER stressor tunicamycin (1, 10 g/ml) 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. ResultsER-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 line. ConclusionsFuchs 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.

cell biology↗

bioRxiv2024

ATF4 regulates mitochondrial dysfunction, mitophagy, and autophagy, contributing to corneal endothelial apoptosis under chronic ER stress in Fuchs dystrophy

PURPOSEEndoplasmic reticulum (ER) stress, mitochondrial dysfunction, and mitophagy are known to contribute independently to corneal endothelial cell (CEnC) apoptosis in Fuchs endothelial corneal dystrophy (FECD). However, the role of a specific ER stress pathway (PERK-ATF4-CHOP) in regulating these events is unknown. This study aims to investigate the role of ATF4 in regulating mitochondrial dysfunction and mitophagy, which ultimately leads to CEnC apoptosis in FECD. METHODSHuman corneal endothelial cell line (21T), Fuchs corneal endothelial cell line (F35T), and primary human corneal endothelial cells were treated with ER stressor tunicamycin (Tun). ATF4 siRNA was used to knock down ATF4 in 21T cell line and primary corneal endothelial cells. Mitophagy and apoptotic proteins were analyzed using Western blotting. ATF4+/- and ATF4 +/+ mice were irradiated with UVA to assess ER stress and corneal endothelial apoptosis. RESULTSF35T cell line had significantly increased expression of the ER stress pathway as well as caspase-mediated apoptotic molecules compared to 21T at baseline, which further increased after tunicamycin treatment. F35T cells exhibited significantly decreased ATP and MMP, and increased mitochondrial fragmentation, which was further exacerbated after Tunicamycin. F35T cell line also demonstrated inhibition of mitophagy, similar to 21T, after treatment with Tunicamycin, despite the upregulation of mitophagy initiators. ATF4 knockdown attenuated ER and mitochondrial stress proteins, rescued mitochondrial membrane potential (MMP) loss, downregulated mitochondrial fragmentation, activated mitophagy, and prevented cell death under chronic ER stress. ATF4+/-mice had increased CE numbers, with improved cellular morphology and decreased ER stress CHOP expression, compared to ATF4+/+ mice post-UVA. CONCLUSIONSPro-apoptotic ATF4 induction following ER stress disrupts mitochondrial function, leading to mitophagy inhibition and CEnC apoptosis. This study highlights the importance of ATF4 in ER-mitochondrial crosstalk and its contribution to CEnC apoptosis in FECD.

cell biology↗