ROLE OF C/EBP HOMOLOGOUS PROTEIN (CHOP) IN THE SURVIVAL OF RETINAL GANGLION CELLS AFTER RETINAL ISCHEMIA/REPERFUSION INJURY

Glaucoma is one of the leading causes of blindness and visual impairment, affecting 70 million people worldwide. A major characteristic of this disease is the irreversible death of retinal ganglion cells (RGCs), retinal neurons that transmit visual information from the eye to the brain. There is an unmet need to develop novel therapeutic strategies for glaucoma. One of the major causes of glaucoma is increased pressure inside the eye i.e., increased intraocular pressure. Increased IOP leads to obstruction of the central retinal artery. Obstruction of this artery leads to insufficient blood supply to the eye, which in turn prevents adequate supply of oxygen and nutrients to the eye, causing ischemia. All these events result in improper folding of proteins in the endoplasmic reticulum (ER). Unfolded or misfolded proteins in the ER lead to ER stress which is one of the major pathways of RGC death. C/EBP Homologous protein (CHOP) is a player in this pathway of cell death. We are studying the mechanisms of RGC death in mice. Our mouse model is called the ischemia/reperfusion (I/R) model. In this model, IOP is increased above the normal level for an hour. This prevents blood supply to the eye and leads to ischemia. After an hour, IOP is brought back to normal and blood supply is restored i.e., reperfusion, which causes considerable damage to the eye. Damage caused to the eye in this model simulates the mechanism of RGC death caused in glaucoma. The goal of our project is to investigate if the presence of CHOP in mice causes RGC death after ischemia/reperfusion injury. If it does, then we would study if the absence of CHOP in mice helps in the survival of RGCs. If absence of CHOP in mice is found to protect retinal ganglion cells, then results of the proposed study could lead to the development of a novel therapy for glaucoma. Purpose (a): Retinal ischemia/reperfusion (I/R) causes apoptotic death of retinal ganglion cells (RGC). CHOP is a pro-apoptotic protein and a unfolded protein response (UPR) marker that plays a role in ER-stress mediated apoptotic cell death. The purpose of this study was to investigate the role of CHOP in mouse RGC survival following retinal I/R injury. Methods (b): Retinal I/R was induced in adult C57BL/6J (WT) and CHOP-/- mice by cannulation of the anterior chamber of the left eye with a needle connected to a reservoir of saline. Intraocular pressure was increased to 120 mmHg for 60 min, after which the needle was withdrawn to restore retinal circulation. Uninjured right eyes served as controls. Expression of CHOP protein and other UPR markers (p-eIF2α and BiP) in WT mice post-I/R was studied using Western blot and immunohistochemistry. To compare RGC survival between WT and CHOP-/- mice, retinal flat mount staining with RGC marker, Brn3a was performed. Scotopic threshold response electroretinography (STR-ERG) was performed at 0.03 mcd.s/m2 light intensity to evaluate retinal function. Results (c): CHOP protein was up-regulated by 30 % in I/R injured eyes (1.30 ± 0.11 arbitrary units (a.u.)) compared to control eyes (1 ± 0.07 a.u.) in WT mice three days after I/R injury (p < 0.05). Protein levels of p-eIF2α and BiP also increased by 19% (I/R: 1.19 ± 0.15 a.u., Control: 1 ± 0.06 a.u.) and 11% (I/R: 1.11 ± 0.02 a.u., Control: 1 ± 0.03 a.u.) respectively (both p < 0.05). Co-localization of CHOP and Brn3a confirmed the up-regulation of CHOP specifically in the RGCs. In the uninjured control eyes, CHOP knockout did not affect baseline RGC density or STR-ERG amplitude. I/R injury decreased RGC densities and STR-ERG amplitudes in both WT and CHOP-/- mice. However, survival of RGCs in I/R-injured CHOP-/- mouse eyes (3337.1 ± 316.4 RGC/mm2) was 48% higher (p < 0.05) than that of I/R-injured WT mouse eyes (2248.7 ± 225.9 RGC/mm2) three days after I/R injury. STR-ERG amplitudes were 83 % higher in CHOP-/- I/R eyes (18.6 ± 1.1 μV) compared to WT I/R eyes (10.1 ± 0.9 μV) (p < 0.05). Conclusions (d): Absence of CHOP partially protects against the loss of RGCs and reduction in retinal function (STR-ERG) after I/R injury. These results indicate that CHOP and thus ER-stress play an important role in RGC apoptosis in retinal I/R injury.