September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Chronic oxidative stress depletes ATP in retinal pigment epithelial cells inducing apoptosis without drusen-related protein expression
Author Affiliations & Notes
  • Faye M Drawnel
    Pharma Research and Early Development, F. Hoffmann-La Roche Ltd, Basel, Switzerland
  • Carolin Willburger
    Pharma Research and Early Development, F. Hoffmann-La Roche Ltd, Basel, Switzerland
  • Ulrich F O Luhmann
    Pharma Research and Early Development, F. Hoffmann-La Roche Ltd, Basel, Switzerland
  • Footnotes
    Commercial Relationships   Faye Drawnel, F. Hoffman-La Roche Ltd (E); Carolin Willburger, F. Hoffman-La Roche Ltd (E); Ulrich Luhmann, F Hoffman-La Roche Ltd (E)
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 5007. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Faye M Drawnel, Carolin Willburger, Ulrich F O Luhmann; Chronic oxidative stress depletes ATP in retinal pigment epithelial cells inducing apoptosis without drusen-related protein expression. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5007.

      Download citation file:


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

      ×
  • Supplements
Abstract

Purpose : Chronic oxidative stress and altered mitochondrial function, developing as a consequence of age, are contributory factors to the development of age-related macular degeneration. Energetic failure and altered protein production in the aged, pro-oxidative retinal environment are thought to contribute to the development of extracellular protein deposits (drusen) which disrupt retinal function. Here, we test whether antioxidant withdrawal promotes phenotypes related to AMD in cultures of human foetal retinal pigment epithelial cells (RPE), by examining the effect of glutathione (GSH) depletion.

Methods : Cultured RPE have highly efficient antioxidant systems to detoxify reactive oxygen species (ROS). To deplete glutathione and mimic the consequences of age-dependent antioxidant decline, human foetal RPE were cultured for 3 weeks and treated with 300 µM buthionine sulfoximine (BSO) for 5 days. Cellular ROS, GSH and ATP were measured with luciferase assays, whilst apoptosis was quantified using a high-content image assay. The energetic properties of the culture were monitored using the Seahorse Metabolic analyzer. Mitochondrial morphology was visualized with confocal microscopy, whilst RNA expression of drusen related proteins (DRP) e.g CLU, APP, BACE1 was measured using qPCR.

Results : Treatment of RPE with BSO for 5 days resulted in depletion of intracellular GSH and tonic accumulation of ROS. Cellular ROS level was further increased by co-administration of H2O2. BSO treatment induced apoptotic cell death in 20% of the cell culture after 5 days, which was associated with a 50% decrease in the total ATP per cell. Investigation of the energetic properties of the culture showed that BSO treatment increased resting oxygen consumption rate and proton leak from the mitochondrial electron transport chain, indicative of mitochondrial dysfunction. In addition, mitochondrial morphology became fragmented. However, RNA expression of DRP genes was not increased.

Conclusions : Persistent oxidative stress induces mitochondrial dysfunction in RPE which promotes apoptotic cell death. However, oxidative stress is not sufficient to induce expression of DRP. This suggests that age-related changes in the RPE that promote formation of extracellular deposits cannot solely be explained by loss of antioxidant activity, reinforcing a multifactorial hypothesis for AMD aetiology.

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

×
×

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.

×