Genetic and pharmacological inhibition of ER stress-induced ATF4/CHOP prodeath pathway prevents myocilin misfolding and rescues mouse models of glaucoma

Abstract

Purpose : We recently demonstrated that ER stress-induced pro-death markers including ATF4 and CHOP are significantly increased in human post-mortem glaucomatous trabecular meshwork (TM) tissues. The purpose of this study was to further explore the role of ATF4/CHOP in myocilin misfolding in TM and IOP elevation.

Methods : ATF4 and CHOP levels were analyzed in TM of Tg-MYOC^Y437H and dexamethasone (Dex)-treated mice by Western blotting (n=3). Conscious IOP was monitored by rebound tonometer in C57 mice injected intravitreally with Ad5 Null or ATF4 virus. IOP was measured in Chop^-/-Tg-MYOC^Y437H mice and compared to Chop^+/+Tg-MYOC^Y437H and WT mice. The effects of CRISPR-Cas9 mediated knockdown of ATF4/CHOP on myocilin accumulation was studied TM-5 cells expressing mutant myocilin. Myocilin accumulation and ER stress was examined in TM cells treated with pharmacological inhibitor of ATF4/CHOP pathway, ISRIB (100 and 200nM) for 48 hours. Furthermore, ocular hypertensive Tg-MYOC^Y437H mice were treated with an intravitreal injection of ISRIB (2ul, 2mM) and IOP was measured 4 days post-injections.

Results : Western blot analysis demonstrated increased ATF4 and CHOP levels in the iridocorneal angle tissues of Tg-MYOC^Y437H and Dex-treated mice. Forced expression of Ad5.ATF4 lead to significant IOP elevation in WT mice (16mmHg in null vs 23mmHg in Ad5.ATF4-injected mice; p<0.0001, n=8). Chop^+/+Tg-MYOC^Y437H mice (22mmHg, n=16) significantly elevated IOP compared to WT Chop^+/+ littermates (16.5mmHg, n=4) while Chop^-/-Tg-MYOC^Y437H mice (19mmHg, n=8) did not elevate IOP compared to Chop^-/- littermates (18mmHg, n=6) indicating that deletion of Chop rescued ocular hypertension in Tg-MYOC^Y437H mice. In addition, Cas9 targeting ATF4 or CHOP dramatically reduced ATF-4 and CHOP level and also reduced myocilin accumulation and ER stress in TM-5 cells. Treatment of TM cells with ISRIB (100nM) dramatically reduced myocilin accumulation and also prevented ER stress. Pharmacological inhibition of ATF4/CHOP pathway via ISRIB (2mM) significantly reduced elevated IOP in Tg-MYOC^Y437H mice (14.5mmHg in vehicle vs 10mmHg in ISRIB treated Tg-MYOC^Y437H mice, p<0.0008, n=5).

Conclusions : These studies indicate that genetic or pharmacological inhibition of ATF4/CHOP pathway may provide novel treatment for glaucoma.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.