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

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 the TM of Tg-MYOCY437H and dexamethasone (Dex)-treated mice by Western blotting (n=3). Conscious IOP was monitored by rebound tonometer in C57BL/6 mice injected intravitreally with Ad5 Null or ATF4 virus. IOP was measured in Chop-/-Tg-MYOCY437H mice and compared to Chop+/+Tg-MYOCY437H and WT mice. The effects of CRISPR-Cas9 mediated knockdown of ATF4/CHOP on myocilin accumulation was studied in TM-5 cells expressing mutant myocilin. Myocilin accumulation and ER stress was examined in TM cells treated with pharmacological inhibition of the ATF4/CHOP pathway with 100 and 200nM of ISRIB (an inhibitor of the integrated stress response) for 48 hours. Furthermore, ocular hypertensive Tg-MYOCY437H 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-MYOCY437H and Dex-treated mice. Forced expression of Ad5.ATF4 led to significant IOP elevation in WT mice (16mmHg in null vs 23mmHg in Ad5.ATF4-injected mice; p+/+Tg-MYOCY437H mice (22mmHg, n=16) had significantly elevated IOP compared to WT Chop+/+ littermates (16.5mmHg, n=4), while Chop-/-Tg-MYOCY437H mice (19mmHg, n=8) did not elevate IOP compared to Chop-/- littermates (18mmHg, n=6) indicating that deletion of Chop rescues ocular hypertension in Tg-MYOCY437H mice. In addition, Cas9 targeting of ATF4 or CHOP dramatically reduced ATF-4 and CHOP levels 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 the ATF4/CHOP pathway with ISRIB (2mM) significantly reduced elevated IOP in Tg-MYOCY437H mice (14.5mmHg in vehicle vs 10mmHg in ISRIB treated Tg-MYOCY437Hmice, p Conclusion: These studies indicate that genetic or pharmacological inhibition of the ATF4/CHOP pathway may provide a novel approach to glaucoma treatment.