June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
Increased mitochondrial fission in Fuchs Endothelial Corneal Dystrophy
Author Affiliations & Notes
  • Takashi Miyai
    Ophthalmology, Massachusetts Eye and Ear/ Schepens Eye Research Institute, Boston, MA
    Ophthalmology, Harvard Medical School, Boston, MA
  • Anne-Sophie Benischke
    Ophthalmology, Massachusetts Eye and Ear/ Schepens Eye Research Institute, Boston, MA
    Ophthalmology, Harvard Medical School, Boston, MA
  • Ula V Jurkunas
    Ophthalmology, Massachusetts Eye and Ear/ Schepens Eye Research Institute, Boston, MA
    Ophthalmology, Harvard Medical School, Boston, MA
  • Footnotes
    Commercial Relationships Takashi Miyai, None; Anne-Sophie Benischke, None; Ula Jurkunas, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 2577. doi:
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    • Get Citation

      Takashi Miyai, Anne-Sophie Benischke, Ula V Jurkunas; Increased mitochondrial fission in Fuchs Endothelial Corneal Dystrophy. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2577.

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

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Abstract
 
Purpose
 

Fuchs Endothelial Corneal Dystrophy (FECD) is a genetic oxidative stress-related disorder. Previously, we have shown loss of mitochondrial membrane potential (MMP) and increase in mitochondrial DNA damage in FECD. In depolarized mitochondria, Parkin, an E3 ubiquitin ligase involved in mitochondrial quality control, is known to translocate to mitochondria and ubiquitinates outer mitochondrial membrane proteins, triggering mitochondrial fission and fusion pathways. The aim of this study is to investigate the role of mitochondrial fusion and fission activation in pathogenesis of FECD.

 
Methods
 

Methods: Mitochondrial morphology and protein expression were investigated in post-keratoplasty FECD specimens and immortalized normal and FECD human corneal endothelial cell lines (HCECi and FECDi, respectively) with and without menadione (MN), an oxidant specifically relevant for study of FECD. HCECi cells were exposed to MN (0, 25, 50, 100 µM for 2 hours) and mitochondrial morphology was assessed by cytochrome c staining using confocal microscopy. Mitochondrial fractions were obtained by suspending the fresh cells in mitochondrial isolation buffer and passing them 15 times through 25G needle. Samples were centrifuged at 500 rpm, 5min, at 4℃ and 10,000 rpm, 30min, at 4℃. The protein levels of Parkin of whole cell lysates and mitochondrial and cytosolic fractions were compared by immunoblotting. VDAC-1 was used as a loading control of mitochondrial fractions.

 
Results
 

Fragmented mitochondria were observed in FECD corneal endothelium compared to normal specimens. MN induced rosette formation in HCECi seen in FECD specimens. MN (50 and 100 µM) induced mitochondrial fragmentation and loss of tubular structures seen in normal untreated cells in a dose-dependent fashion. Furthermore, whole cell lysate levels of Parkin were higher in FECD specimens compared to normals. Parkin was not detected in cytosolic and mitochondrial fractions of HCECi but showed upregulation of both cytosolic and mitochondrial fractions in FECDi. Intensity percentages were 90.75% in cytosolic fraction in FECDi and 9.25% in mitochondrial fraction.

 
Conclusions
 

Loss of MMP in FECD leads to mitochondrial fragmentation and upregulation of Parkin. These findings suggest that Parkin may play an important role in abnormal mitochondrial quality control pathway in contribute to mitochondrial fission in FECD.  

 
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