September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Increased Mitochondrial Fragmentation Upregulates Mitophagy in Fuchs Endothelial Corneal Dystrophy
Author Affiliations & Notes
  • Anne-Sophie Benischke
    Schepens Eye Research Institute, Boston, Massachusetts, United States
  • Takashi Miyai
    Schepens Eye Research Institute, Boston, Massachusetts, United States
  • Cailing Liu
    Schepens Eye Research Institute, Boston, Massachusetts, United States
  • Ula V Jurkunas
    Schepens Eye Research Institute, Boston, Massachusetts, United States
  • Footnotes
    Commercial Relationships   Anne-Sophie Benischke, None; Takashi Miyai, None; Cailing Liu, None; Ula Jurkunas, None
  • Footnotes
    Support  NEI/NIH R01 EY020581, Massachusetts Lions Eye Research Fund
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 5291. doi:
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      Anne-Sophie Benischke, Takashi Miyai, Cailing Liu, Ula V Jurkunas; Increased Mitochondrial Fragmentation Upregulates Mitophagy in Fuchs Endothelial Corneal Dystrophy. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5291.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Purpose:
Fuchs Endothelial Corneal Dystrophy (FECD) is a late-onset oxidative stress-induced disease, which we previously have linked to mitochondrial dysfunction. In FECD, mitochondrial dysfunction is characterized by the loss of mitochondrial membrane potential and enhanced mitochondrial fragmentation. The aim of this study is to elucidate whether disrupted fragmentation stems from impaired mitochondrial fusion in FECD.

Methods : Methods: Mitophagy was analyzed by western blotting to detect Mfn2 and LC3 in post-keratoplasty specimens from late-onset FECD cases. Whole cell lysates of immortalized normal human corneal endothelial and FECD cell lines (HCECi and FECDi, respectively) were immunoprecipitated with Mfn2 antibody and immunoblotted for Mfn2 and ubiquitin. Whole cell lysates from the HCECi and FECDi were also tested for the expression of Mfn2 and LC3 by western blotting. VDAC was used as a loading control for mitochondrial fractions. Cell lines were treated with 50μM of MG132 for 6h to inhibit proteasome-mediated degradation. Mitochondrial mass was examined by flow cytometry in HCECi and FECDi incubated with 50nM MitoTracker Green FM for 30 min at 37°C. Mitophagy was inhibited in cell lines with 10nM bafilomycin for 20h prior to flow cytometry.

Results : Results: Mfn2 protein levels in FECDi (p<0.05) as well as in FECD ex vivo specimens (n=11; p=0.001) were significantly lower indicating lack of fusion. Treatment with the proteasome inhibitor MG132 stabilized Mfn2 protein compared to untreated FECDi. In addition, immunoprecipiration of Mfn2 revealed an increased ubiquitination in FECDi. LC3-II, a critical component of the mitophagy pathway, was increased in both whole cell lysates as well as mitochondrial fractions of FECDi. LC3-II levels in ex vivo specimens corroborated the in vitro results. Mitochondrial mass was decreased in FECDi compared to HCECi but was rescued with the mito/autophagy inhibitor bafilomycin

Conclusions : Conclusion: This study shows that there is impaired mitochondrial fusion due to decreased Mfn2 protein levels in FECD. Reduced Mfn2 levels are caused by increased ubiquitination and enhanced proteasomal degradation. In addition, increased conversion of LC3-I to –II indicates activation of mitophagy which leads to loss of mitochondrial mass. This is the first study presenting that imbalance of fusion and fission activates mitochondrial degradation in FECD.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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