June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
BAX Oligomerization Kinetics and its Role in Retinal Ganglion Cell Death
Author Affiliations & Notes
  • Margaret Maes
    Ophthalmology and Visual Sciences, University of Wisconsin - Madison, Madison, WI
  • Cassandra Schlamp
    Ophthalmology and Visual Sciences, University of Wisconsin - Madison, Madison, WI
  • Robert W Nickells
    Ophthalmology and Visual Sciences, University of Wisconsin - Madison, Madison, WI
  • Footnotes
    Commercial Relationships Margaret Maes, None; Cassandra Schlamp, None; Robert Nickells, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 4952. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to Subscribers Only
      Sign In or Create an Account ×
    • Get Citation

      Margaret Maes, Cassandra Schlamp, Robert W Nickells; BAX Oligomerization Kinetics and its Role in Retinal Ganglion Cell Death. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4952.

      Download citation file:


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

      ×
  • Supplements
Abstract

Purpose: Glaucoma is characterized by retinal ganglion cell (RGC) apoptosis. RGC death occurs through the BAX-dependent intrinsic apoptotic pathway in which BAX oligomerization marks the committed step of this process. The study of BAX oligomerization kinetics can provide an understanding of the mechanism and timing of BAX function in RGC death, which can establish a window of opportunity for therapeutic intervention to slow or prevent RGC commitment to apoptosis.

Methods: To monitor BAX oligomerization kinetics in vitro, HCT116BAX-/-/BAK-/- and differentiated 661W cells transiently expressing fluorescently labeled BAX protein were challenged with an apoptotic stimulus. Cells were imaged using spinning disc confocal microscopy and changes in BAX fluorescence at individual mitochondria were quantified using IMARIS 7.7. The curve fitting function of the SciPy library was used to fit the resulting quantitative data to determine the time of nucleation and the rate of BAX oligomerization. To monitor BAX oligomerization kinetics in vivo, optic nerve crush (ONC) was performed on mice after intraocular injection of AAV2-GFP-BAX. One, three and five days post-ONC, retinas were whole mounted and imaged.

Results: BAX mutants and cytochrome c release confirmed functional BAX oligomer formation for in vitro studies. BAX oligomerization kinetics were quantified at individual mitochondria in a living cell and BAX oligomerization follows a sigmoidal growth function. Mitochondria in individual cells exhibited nearly identical nucleation times but had varying rates of BAX oligomerization, although once initiated, this process is complete within 20 minutes regardless of cell type and apoptotic stimulus. Oligomerization of GFP-BAX was induced in RGCs by optic nerve damage, with peak levels detected at 3 days post-ONC.

Conclusions: These results demonstrate that the study of BAX oligomerization kinetics can be a powerful tool for providing insight into the mechanism of BAX function in apoptosis and suggest a therapeutic window of approximately 3 days after axonal injury may be available to inhibit the apoptotic program.

×
×

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.

×