KNOCKOUT OF CASPASE-7 PROTECTS AGAINST OPTIC NERVE CRUSH-INDUCED RETINAL GANGLION CELL DEATH

Glaucoma, a leading cause of blindness worldwide is characterized by injury to the nerve of the eye leading to the death of certain eye cells called retinal ganglion cells (RGCs) and vision loss. Currently available glaucoma therapies only attempt to reduce the eye pressure without addressing the associated RGC death problem. As a result, they do not always sufficiently slow the disease progression in all glaucoma patients. Thus, there is an urgent need to develop strategies for preventing glaucoma associated RGC death. Our preliminary studies have identified a novel protein, caspase-7 as a major player in RGC death pathways. We are studying the role of caspase-7 in RGC death in a mouse glaucoma model and whether it can be targeted for better therapeutic outcome. This project is significant because it will identify a new and potentially critical component of RGC death. This will aid in the design of better therapeutic treatment for glaucoma and other degenerative diseases. Purpose (a): Optic nerve (ON) injury is involved in various ocular diseases, such as glaucoma, which leads to apoptotic death of retinal ganglion cells (RGC) and loss of vision. Caspases have been implicated previously in glaucoma and RGC death. However, the role of caspase-7, a functionally unique caspase, in ON injury and glaucomatous damage has not been studied. Therefore, the purpose of this study is to evaluate the role of caspase-7 in ON injury-induced RGC apoptosis. Methods (b): C57BL/6 (Wt) and caspase-7knockout (casp7KO) mice were used for this study. Optic nerve crush (ONC) was performed on left eyes; right eyes served as control. Western blots of the isolated retinas of Wt mice were used to assess the activation of caspase-7 at 3h, 6h, 12h, 1d, 3d, and 7d after ONC. Immunohistochemistry was performed to detect the localization of caspase-7 in RGC. RGC survival was determined by counting the RBPMS (RGC marker) labeled cells in flat-mounted retinas of Wt and casp7KO mice at 7d, 14d and 28d post injury. Both Wt and casp7KO mice were subjected to spectral-domain optical coherence tomography (SD-OCT) and scotopic threshold response of electroretinography (STR-ERG) to evaluate the retinal structural and RGC functional changes at 7d, 14d, and 28d after ONC. Results (c): Western blot data demonstrated that caspase-7 was activated in Wt retina at 12h, 1d, 3d, and 7d after ONC compared to the uninjured control retinas. The number of surviving RGCs was significantly more (3173±59 cells/mm2, mean±SEM, n=6, p<0.001) in casp7KO retinas compared to Wt retinas (1693±84 cells/mm2) at 28d post ONC. SD-OCT analysis revealed that the thickness of the inner retinal layer (ganglion cell layer, nerve fiber layer, and inner plexiform layer) in casp7KO mice was greater (54±1.1 μm, p<0.05) compared to Wt mice (42.3±1.5 μm). Most importantly, analysis of the STR-ERG response demonstrated a decline in amplitude in Wt ONC eyes (10.5±1.9 μv), whereas the response was significantly higher (20.7±2.3 μv, p<0.05) in casp7KO mice even at 28d post injury. Conclusions (d): The current study indicates that injury to the ON activates caspase-7 and knockout of caspase-7 protects inner retinal layer morphology and RGC function after ONC. Thus, caspase-7 appears to play a critical role in ONC-induced RGC death and inhibition of caspase-7 activity may be a novel therapeutic target for glaucoma and other neurodegenerative diseases of the retina.