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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. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
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.
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.
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.
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.
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