Aims: Fuchs endothelial corneal dystrophy (FECD), a leading cause of age-related corneal edema requiring transplantation, is characterized by rosette formation of corneal endothelium with ensuing apoptosis. We sought to determine whether excess of mitochondrial reactive oxygen species leads to chronic accumulation of oxidative DNA damage and mitochondrial dysfunction, instigating cell death. Results: We modeled the pathognomonic rosette formation of postmitotic corneal cells by increasing endogenous cellular oxidative stress with menadione (MN) and performed a temporal analysis of its effect in normal (HCEnC, HCECi) and FECD (FECDi) cells and ex vivo specimens. FECDi and FECD ex vivo specimens exhibited extensive mtDNA and nDNA damage as detected by quantitative PCR. Exposure to MN triggered an increase in mitochondrial superoxide levels and led to mtDNA and nDNA damage, while DNA amplification was restored with NAC pretreatment. Furthermore, MN exposure led to a decrease in DCm and adenosine triphosphate levels in normal cells, while FECDi exhibited mitochondrial dysfunction at baseline. Mitochondrial fragmentation and cytochrome c release were detected in FECD tissue and after MN treatment of HCEnCs. Furthermore, cleavage of caspase-9 and caspase-3 followed MN-induced cytochrome c release in HCEnCs. Innovation: This study provides the first line of evidence that accumulation of oxidative DNA damage leads to rosette formation, loss of functionally intact mitochondria via fragmentation, and subsequent cell death during postmitotic cell degeneration of ocular tissue. Conclusion: MN induced rosette formation, along with mtDNA and nDNA damage, mitochondrial dysfunction, and fragmentation, leading to activation of the intrinsic apoptosis via caspase cleavage and cytochrome c release.
To identify novel regulators of endoplasmic reticulum (ER)-linked protein degradation and ER function, we determined the entire inventory of membrane-spanning RING finger E3 ubiquitin ligases localized to the ER. We identified 24 ER membraneanchored ubiquitin ligases and found Nixin/ZNRF4 to be central for the regulation of calnexin turnover. Ectopic expression of wild type Nixin induced a dramatic down-regulation of the ER-localized chaperone calnexin that was prevented by inactivation of the Nixin RING domain. Importantly, Nixin physically interacts with calnexin in a glycosylation-independent manner, induces calnexin ubiquitination, and p97-dependent degradation, indicating an ER-associated degradation-like mechanism of calnexin turnover.The endoplasmic reticulum (ER) 3 is a major cellular site for production, folding, quality control, and distribution of proteins. Many regulatory mechanisms are in place to keep these processes in balance and therefore to ensure cellular fitness and survival, with ubiquitin-dependent protein degradation playing an important part (1). One major challenge the ER faces is an overload with unfolded or folding proteins. An excess of folding proteins in the ER triggers a cellular response called the unfolded protein response (UPR) (2, 3). UPR entails lowering of the protein load by the attenuation of protein translation and the up-regulation of chaperones thereby increasing the protein folding capacity of the cells. If the capacity of UPR is exceeded, the cell utilizes ER-associated degradation (ERAD), a system for the recognition of terminally misfolded proteins and their disposal (4). Misfolded proteins destined for ERAD are ubiquitinated by the RING domain containing ubiquitin ligases (5), Hrd1 (6), and Doa1 (7, 8), retrotranslocated across the ER membrane into the cytosol by the AAA-ATPase p97 (9), and then degraded by the 26 S proteasome.Upon entry into the ER, most nascent polypeptides are recognized by glycosidases and modified on specific asparagine residues (Asn-Xaa-(Thr/Ser)) with the N-glycan GlcNAc 2 Man 9 Gluc 3 (10). Core glycosylation of nascent polypeptides decreases their overall hydrophobicity. Trimming of the terminal two glucose residues by glucosidase I allows for binding of the lectins/chaperones calreticulin and calnexin thereby facilitating the proper folding of the newly synthesized protein (10,11).Although the importance of regulated degradation of ER resident proteins is firmly established, only a small number of RING finger-containing ubiquitin ligases are known to be involved in such processes to date, namely SYVN1/hHrd1 (6, 12), AMFR/gp78 (13), TEB4/MARCH6 (14), RNF5/Rma1 (15), RNF77/TRIM13 (16), and RNF13 (17). Given the importance of protein metabolism and degradation in the ER and the vast number of ubiquitin ligases encoded in the human genome, we asked whether other ubiquitin ligases are involved in the regulation of ER-related degradation processes.Based on the assumption that the ER lumen is devoid of E1 and E2 ubiquitination activity and on the...
Human corneal endothelial cells (HCEnCs) are terminally differentiated cells that have limited regenerative potential. The large numbers of mitochondria in HCEnCs are critical for pump and barrier function required for corneal hydration and transparency. Fuchs Endothelial Corneal Dystrophy (FECD) is a highly prevalent late-onset oxidative stress disorder characterized by progressive loss of HCEnCs. We previously reported increased mitochondrial fragmentation and reduced ATP and mtDNA copy number in FECD. Herein, carbonyl cyanide m-chlorophenyl hydrazone (CCCP)-induced mitochondrial depolarization decreased mitochondrial mass and Mfn2 levels, which were rescued with mitophagy blocker, bafilomycin, in FECD. Moreover, electron transport chain complex (I, V) decrease in FECD indicated deficient mitochondrial bioenergetics. Transmission electron microscopy of FECD tissues displayed an increased number of autophagic vacuoles containing degenerated and swollen mitochondria with cristolysis. An elevation of LC3-II and LAMP1 and downregulation of Mfn2 in mitochondrial fractions suggested that loss of fusion capacity targets fragmented mitochondria to the pre-autophagic pool and upregulates mitophagy. CCCP-induced mitochondrial fragmentation leads to Mfn2 and LC3 co-localization without activation of proteosome, suggesting a novel Mfn2 degradation pathway via mitophagy. These data indicate constitutive activation of mitophagy results in reduction of mitochondrial mass and abrogates cellular bioenergetics during degeneration of post-mitotic cells of ocular tissue.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.