Abstract
Purpose :
Endoplasmic reticulum (ER) stress has been linked with various acute and chronic neurodegenerative diseases. We previously found that optic nerve (ON) injury, ocular hypertension, and optic neuritis induce neuronal ER stress in retinal ganglion cells (RGCs). We further demonstrated that germ-line knockout of CHOP, a downstream pro-apoptotic molecule of ER stress, preserves the structure and function of glaucomatous RGC somata and axons. CHOP can also cause cell death by up-regulating protein synthesis and oxidative stress when it forms heterodimers with its upstream transcription factor ATF4. It is clinically important and scientifically interesting to determine the neuroprotective role of neuronal-specific ATF4/CHOP inhibition in glaucoma.
Methods :
Taking advantage of ATF4 and CHOP floxed mouse lines and our recently developed RGC-specific promoter mouse γ-synuclein (mSncg) and silicone oil-induced ocular hypertension (SOHU) mouse glaucoma models, we evaluated the effects of RGC-specific deletion of ATF4, CHOP, or both in RGC and ON survival by histology assay and optical coherence tomography (OCT) and visual function by pattern ERG (PERG) and optokinetic response (OKR). All floxed transgenic mice received intravitreal injection of 3x109 vector genomes (vg) of AAV2-Cre in 2uL. We also tested a small molecule inhibitor of ATF4/CHOP, ISRIB, and CRISPR-mediated knockdown of Gadd45α, an ATF4 downstream effector to further demonstrate the neuroprotection effect of blocking the ATF4/CHOP pathway in glaucoma. The Gadd45α was knocked down by intravitreal injection of the mix of AAV-Cas9 and AAV-gRNAs at 2:1 ratio with a total volume 3x109 vg/eye. Each treatment group contained 9-15 mice at age of 5-8 weeks old.
Results :
RGC-specific deletion of ATF4 or CHOP promotes significant RGC and ON survival and preserves visual function in the mouse ON crush model and SOHU glaucoma model. Double KO of ATF4 and CHOP have an even better effect. Consistently, topical application of ATF4/CHOP chemical inhibitor ISRIB and CRISPR-mediated knockdown of Gadd45α also deliver significant neuroprotection of RGC somata and axons and visual function in the SOHU glaucoma model.
Conclusions :
Our studies demonstrate that blocking the neuronal intrinsic ATF4/CHOP axis of ER stress is a promising neuroprotection strategy for glaucomatous neurodegeneration.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.