Overexpression of ATF-4 in trabecular meshwork causes elevation of intra ocular pressure and reduction of outflow facility in a CHOP dependent manner

Purpose: Primary open-angle glaucoma (POAG) has been primarily associated with reduced aqueous humor outflow facility through trabecular meshwork (TM) and elevated intraocular pressure (IOP). Studies based on both human as well as mice models revealed that chronic endoplasmic reticulum (ER) stress in TM is one of the causative factors responsible for TM dysfunction and ocular hypertension. The purpose of this study is to examine whether forced expression of UPR downstream pro apoptotic molecules ATF4 and CHOP leads to reduced outflow facility and IOP elevation in normal C57 mice. Methods: Ad5.ATF-4/Ad5.CHOP/Ad5.empty virus (pfu=2x107) were injected intravitreally into C57BL/6J or CHOP KO (CHOP-/-) mice. Conscious IOP of both the eyes was monitored once in a week until 7 weeks using rebound tonometer. Outflow facility was measured by constant-flow infusion technique. Also, we examined the expression levels of ATF-4 in the TM of age-matched normal and POAG donors by immunohistochemistry. Results: Forced expression of ATF-4 but not CHOP caused significant IOP elevation (23.97 mmHg in Ad5.ATF4 v/s 14.6 mmHg in Ad5.null mice) and reduced outflow facility (0.022µL/min/mmHg in Ad5.ATF4 v/s 0.04 in Ad5.null mice) in C57BL/6J mice. Elevation of IOP in C57BL/6J was prominent from 3 weeks post injection and sustained until 7 weeks. Interestingly Ad5.ATF-4 did not elevate IOP (17.7 mmHg) in CHOP-/- mice, indicating that ATF-4 interaction with CHOP is the prerequisite for ATF-4-induced IOP elevation. Also, ER stress-induced pro death marker, ATF-4 was significantly increased in human post-mortem glaucomatous TM tissues compared to normal TM tissues. Expression of ATF4 in primary TM cells induced oxidative and ER stress and also upregulated pro-apoptotic markers. Conclusions: This data indicates that chronic ER marker ATF4 is increased in the glaucomatous TM tissues, which may be associated with TM dysfunction, reduction of outflow facility and IOP elevation via induction of ER and oxidative stress. Furthermore, interaction of ATF-4 and CHOP is essential to carry out downstream signal transduction pathways.