July 2019
Volume 60, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2019
Glaucoma-associated E50K-optineurin mutation impairs mitochondrial-derived vesicle trafficking
Author Affiliations & Notes
  • James Powers
    Ophthalmology, Duke Eye Center, Durham, North Carolina, United States
  • Kayla Trautman-Buckley
    Northeast Ohio Medical University, Ohio, United States
  • Emily Sun
    Ophthalmology, Duke Eye Center, Durham, North Carolina, United States
  • Charlaine Chen
    Ophthalmology, Duke Eye Center, Durham, North Carolina, United States
  • Denise M Inman
    Northeast Ohio Medical University, Ohio, United States
  • Henry Tseng
    Ophthalmology, Duke Eye Center, Durham, North Carolina, United States
  • Footnotes
    Commercial Relationships   James Powers, None; Kayla Trautman-Buckley, None; Emily Sun, None; Charlaine Chen, None; Denise Inman, None; Henry Tseng, None
  • Footnotes
    Support  American Glaucoma Society, International Retinal Research Foundation, NEI K08-EY021520
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 667. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      James Powers, Kayla Trautman-Buckley, Emily Sun, Charlaine Chen, Denise M Inman, Henry Tseng; Glaucoma-associated E50K-optineurin mutation impairs mitochondrial-derived vesicle trafficking. Invest. Ophthalmol. Vis. Sci. 2019;60(9):667.

      Download citation file:


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

      ×
  • Supplements
Abstract

Purpose : Although optineurin (OPTN) is known to function in the turnover of mitochondria, it is unclear how the E50K OPTN mutation associated with normal-tension glaucoma impacts this process. We tested the hypothesis that E50K OPTN disrupts mitochondrial derived vesicle (MDV) formation and trafficking.

Methods : Cos7 cells and mouse embryonic fibroblasts (MEFs) were utilized. Cos7 cells were transfected with fluorescently tagged wildtype-OPTN, E50K-OPTN or green fluorescent protein as control. MEFs were purified from wildtype mice (as control) and E50K OPTN transgenic mice generated in our laboratory that exhibit a normal-tension glaucoma phenotype. To promote MDV formation, cells were treated with 40µM Antimycin A (AA, a complex III inhibitor) for 2 hours. Mitochondrial turnover and MDVs were evaluated using immunofluorescent staining for Tom20 (outer mitochondrial membrane protein) and pyruvate dehydrogenase. Cells were stained with 10µg/mL dihydroethidium to detect reactive oxygen species (ROS). The student’s t-test was used for statistical comparison.

Results : E50K and wt OPTN MEFs had comparable numbers of mitochondria. With AA exposure, mitochondria decreased in wt MEFs but not in E50K MEFs, suggesting impaired mitochondrial turnover. We next found greater numbers of MDVs in E50K cells than wt cells (p<0.05), yet following AA exposure, wt and E50K cells showed similar number of MDVs, indicating that the E50K mutation does not disrupt MDV formation. Colocalization between E50K-OPTN and MDVs was decreased compared to wt regardless of AA exposure (p<0.01 and p<0.0001, respectively) suggesting the E50K mutation may weaken optineurin’s interaction with MDVs. E50K-OPTN colocalization with mitochondria (p<0.001) was decreased as compared to wt. With AA exposure, association of wt-OPTN with mitochondria declined (p<0.0001), but did not change for the E50K cells. Finally, we found that E50K cells had greater ROS levels compared to control (p<0.0001) and, with AA exposure, had greater ROS levels than wt cells (p<0.05).

Conclusions : Our results support our hypothesis that E50K OPTN disrupts the trafficking and therefore the degradation of oxidized proteins in the MDV-pathway. While the formation of MDVs is maintained in E50K cells, perhaps the impaired trafficking of MDVs in these cells leads to increased ROS levels, increased MDV buildup, and inefficient mitochondrial turnover.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×