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Robert W Nickells, Margaret E. Maes, Ryan Donahue, Jenna R. Gustafson, Donna M Peters; Live Cell Imaging of BAX Recruitment in Retinal Ganglion Cells Reveals Characteristics of Anoikis. Invest. Ophthalmol. Vis. Sci. 2020;61(7):4334.
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Anoikis is the term for intrinsic apoptosis initiated by loss of cell-cell/cell-ECM attachment and is characterized by activation of the BAX pro-apoptotic protein. After optic nerve damage, RGCs exhibit significant structural remodeling, suggesting they undergo widespread detachment from their environment. We hypothesize that RGCs undergo anoikis after optic nerve damage.
GFP-BAX recruitment was monitored after optic nerve crush (ONC) in mice. RGCs were preloaded with GFP-BAX by AAV2/2 viral gene transfer. Some RGCs were doubly transduced with viruses carrying mCherry-BAX or cytochrome c-GFP. After ONC, retinas were isolated and mounted as explants for live cell imaging, or were fixed and mounted for static imaging. Some naïve mice preloaded with GFP-BAX were subjected to a single intravitreal injection of 10 nmoles of the focal adhesion kinase (FAK) inhibitor PF573228 to induce anoikis. For comparison, the kinetics of GFP-BAX recruitment were also measured in differentiated 661W cells with forced expression of HDAC3 and in naïve RGCs treated with staurosporine (STS). IMARIS was used for kinetic analysis of time-lapse videos.
ONC induced the recruitment of GFP-BAX in RGCs, which was significantly elevated at 3 days pONC (P<0.05) and peaked at 7 days. The rate of ONC-induced BAX recruitment (-5.1±1.5 Log2RFU/min) was slower than in 661W cells (-2.3±1.2, P<0.001), but comparable to the rate induced in RGCs by STS treatment (-6.0±1.3). Approximately 6% of imaged RGCs exhibited BAX recruitment followed by it’s retrotranslocation to the cytosol, a feature of detached cultured cells undergoing anoikis but are then replated. Similarly, cells undergoing anoikis exhibit a delay between BAX recruitment and cytochrome c release. Preliminary results showed that 41.2±11.1% of RGCs with recruited mCherry-BAX retained mitochondrial cytochrome c-GFP 4 days pONC, while this dropped to 22.5±2.2% by 7 days (P=0.012). Treatment of naïve retinas with PF573228 induced a significant increase in GFP-BAX recruitment within 24 hours after intravitreal injection (P=0.025).
This study marks the first attempt to characterize BAX activation in a complex tissue environment. RGCs exhibit differences in this process relative to tissue culture cells, although it shares characteristics with anoikis. This suggests that RGC detachment may be an important initiator of intrinsic apoptosis after ONC.
This is a 2020 ARVO Annual Meeting abstract.
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