Abstract
Purpose :
Axonal degeneration in glaucoma causes blindness, necessitating therapies targeted at preventing axon degeneration. The canonical intrinsic apoptotic pathway, whose execution requires the protein BAX, has been implicated in axonal degeneration. We hypothesized that preventing BAX activation during glaucoma would delay both somal and axonal degeneration. To test this hypothesis, we used adeno-associated virus, serotype 2 (AAV2) to express mCherry-BCLX, a BAX antagonist, in retinal ganglion cells (RGCs) in the DBA/2J mouse glaucoma model.
Methods :
AAV2-Pgk-mCherry-BclX was cloned. The ability of the construct to prevent BAX activation was measured by quantifying GFP-BAX translocation to the mitochondria in vitro after treating ARPE-19 cells with staurosporine (STS). The effect of mCherry-BCLX transduction was measured in vivo by assessing the rate of GFP-BAX recruitment and RGC loss following optic nerve crush (ONC). Cell loss was assessed via DAPI staining and RBPMS immunofluorescence.
DBA/2J mice were transduced with virus before 6 months of age or at 7 months of age and collected at 10.5 months of age. Intraocular pressures (IOPs) were measured every 2 weeks for a separate cohort of mice to assess whether viral transduction and expression of mCherry-BCLX altered IOP. RGC pathology was assessed with DAPI staining and RBPMS immunofluorescence. Axon degeneration was quantified using paraphenyldiamine staining on optic nerve sections.
Results :
In ARPE-19 cells, mCherry-BCLX was localized to mitochondria, and prevented the recruitment of GFP-BAX after STS treatment. Similarly, RGCs transduced with mCherry-BCLX (and GFP-BAX) had less mitochondrial localization of GFP-BAX after ONC (P=0.009) and attenuation of cell loss up to 12 weeks after injury (P = 0.0004). Transduction of RGCs in DBA/2J mice showed continuous expression of mCherry-BCLX in RGC somas and axons up to 10.5 months of age. Preliminary data indicates that viral injection had no impact on the development of elevated IOP in DBA/2J mice as they age. Mice injected before 6 months of age had reduced degeneration of their optic nerves (P=0.002) and less RGC soma loss (P = 0.00003). Transduction of RGCs at 7 months, however, failed to yield a protective effect.
Conclusions :
Counteracting BAX activation using BclX gene therapy delayed somal and axonal degeneration in a glaucoma model.
This is a 2020 ARVO Annual Meeting abstract.