Activation of PINK1-Parkin-Mediated Mitophagy Degrades Mitochondrial Quality Control Proteins in Fuchs Endothelial Corneal Dystrophy.

Corneal endothelium (CE) is a monolayer of mitochondria-rich cells, critical for maintaining corneal transparency compatible with clear vision. Fuchs endothelial corneal dystrophy (FECD) is a heterogeneous, genetically complex disorder, where oxidative stress plays a key role in the rosette formation during the degenerative loss of CE. Increased mitochondrial fragmentation along with excessive mitophagy activation has been detected in FECD; however, the mechanism of aberrant mitochondrial dynamics in CE cell loss is poorly understood. Here, the role of oxidative stress in mitophagy activation in FECD is investigated. Immunoblotting of FECD ex vivo specimens revealed an accumulation of PINK1 and phospho-Parkin (Ser65) along with loss of total Parkin and total Drp1. Similarly, modeling of rosette formation with menadione (MN), led to phospho-Parkin accumulation in fragmented mitochondria resulting in mitophagy-induced mitochondrial clearance, albeit possibly in a PINK1-independent manner. Loss of PINK1, phospho-Drp1, and total Drp1 was prominent after MN-induced oxidative stress, but not after mitochondrial depolarization by carbonyl cyanide m-chlorophenyl hydrazone. Moreover, MN-induced mitophagy led to degradation of Parkin along with sequestration of Drp1 and PINK1 that was rescued by mitophagy inhibition. This study shows that in FECD, intracellular oxidative stress induces Parkin-mediated mitochondrial fragmentation where endogenous Drp1 and PINK1 are sequestered and degraded by mitophagy during degenerative loss of post-mitotic cells of ocular tissue.

A systematic search for endoplasmic reticulum (ER) membrane-associated RING finger proteins identifies Nixin/ZNRF4 as a regulator of calnexin stability and ER homeostasis.

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 membrane-anchored 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.

Induction of mitophagy in the HEI-OC1 auditory cell line and activation of the Atg12/LC3 pathway in the organ of Corti.

Autophagy is a highly evolutionary conserved quality control defense mechanism within cells, which has also been implicated in cell death processes. In the mammalian inner ear, autophagy has been shown to play a role during early morphogenesis as well as in adult cochlear hair cells exposed to ototoxic insults. Mitophagy, a selective autophagic cell process targeting mitochondria, hasn't been studied in the inner ear so far. On this work, we searched for molecular indicators of mitophagy within House Ear Institute-Organ of Corti-1 (HEI-OC1) cells as well as in the organ of Corti (OC). We first tested for the expression of Pink1/Park2 mRNA in 5-day-old C57BL/6 mice's cochleae using RT-PCR. We focused on the induction of mitophagy in HEI-OC1 cells as well as in the OC and investigated a possible mitophagic potential of the aminoglycoside agent gentamicin. The induction of mitophagy in HEI-OC1 cells was detected by objectivizing the translocation of fluorescence-tagged LC3 to mitochondria using confocal microscopy after a 6-h incubation with a well-described mitochondrial uncoupler and mitophagy-inducing agent: carbonyl cyanide m-chlorophenyl hydrazone (CCCP). Incubation with gentamicin generated no mitochondrial translocation of LC3. Protein levels of COXIV, Atg5/12 and LC3 were evaluated by an immunoblot analysis after a 24-h CCCP treatment as well as gentamicin. We demonstrated mitophagy after CCCP exposure in HEI-OC1 cells by showing a downregulation of COXIV. A downregulation of COXIV could also be visualized in the OC after CCCP. A significant oxygen consumption rate (OCR) changed in cells treated with CCCP as well as significant morphological changes of mitochondria by electron microscopy (EM) strengthen this assumption. Gentamicin exposure generated no impact on OCR or mitochondrial morphological changes by EM. Finally, we demonstrated changes in the expression of Atg12 and LC3 proteins in both the OC and HEI-OC1 cells after CCCP exposure but not after gentamicin. Our data indicate that gentamicin had no impact in the activation of mitophagy-neither in the HEI-OC1 cell line nor in the OC. Therefore, we speculate that mitophagic-independent mechanisms may underly aminoglycoside ototoxicity.

Activation of mitophagy leads to decline in Mfn2 and loss of mitochondrial mass in Fuchs endothelial corneal dystrophy.

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.

Menadione-Induced DNA Damage Leads to Mitochondrial Dysfunction and Fragmentation During Rosette Formation in Fuchs Endothelial Corneal Dystrophy.

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 ΔΨm 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. Antioxid. Redox Signal. 24, 1072-1083.

Proteasome-mediated quality control of S-nitrosylated mitochondrial proteins.

Accumulating low level mitochondrial insults are thought to be key to aging processes and neurodegeneration. Among other stressors, protein damage due to nitrosative stress negatively impacts mitochondrial function and is linked to neurodegeneration. Using biotin switch technique, we show that mitochondrial proteins are S-nitrosylated not only in the presence but also in the absence of exogenous nitrosative stress. In addition, we revealed a role for the ubiquitin-proteasome system and the outer mitochondrial membrane associated degradation (OMMAD)-component p97 in the quality control of S-nitrosylated mitochondrial. Taken together, constant proteasome-mediated clearance of nitrosatively-damaged proteins from mitochondria is likely important for maintaining organelle function.