Mechanism of Apoptosis in Rat Retinal Ganglion Cell Line RGC–5 Involves Calpain–Mediated Proteolysis: Calpain Inhibition Provides Functional Neuroprotection

Abstract

Abstract: : Purpose: Progressive degeneration of the retinal ganglion cells (RGCs) in the optic nerve head diminishes vision in glaucoma patients. Degeneration of RGCs may also occur in course of development of the optic neuritis (ON), leading to loss of visual acuity in patients with ON as well. The mechanism of degeneration of RGCs in these neurodegenerative diseases of the central nervous system is not well understood. We hypothesized that increase in intracellular free Ca2+ levels and activation of calpain, a Ca2+–dependent protease, could cause apoptosis of RGCs in cell culture. To test this hypothesis, in the current investigation, we examined the mechanism of apoptosis of the rat retinal ganglion cell line RGC–5 following exposure to ionomycin (a Ca2+ ionophore) and interferon–gamma (IFN–g) and also tested the functional neuroprotection with calpeptin, a cell–permeable inhibitor of calpain. Methods: We used Wright staining and ApopTag assay to detect apoptotic death, fura–2 assay to measure intracellular free Ca2+ levels, Western blottings for detection of specific protein bands, and electrophysiological recordings for determining whole–cell membrane potential. Results: Wright staining and ApopTag assay detected apoptosis morphologically and biochemically, respectively, following exposure of RGC–5 cells to 250 nM ionomycin or 300 units/ml IFN–g for 24 h. Apoptosis in RGC–5 cells was associated with a significant increase in intracellular free Ca2+ levels, as determined by fura–2 assay. A pretreatment of RGC–5 cells with 2 µM calpeptin for 1 h prevented Ca2+ influx. Western blot analyses showed an increase in Bax:Bcl–2 ratio, release of cytochrome c from mitochondria, and increase in the activities of calpain, caspase–12, caspas–9, and caspase–3, during apoptosis. Pretreatment with calpeptin attenuated all these apoptosis related molecular events and prevented apoptosis of RGC–5 cells. Most importantly, pretreatment with calpeptin maintained whole–cell membrane potential and thus provided functional neuroprotection. Conclusions: Increases in intracellular free Ca2+ result in activation of calpain, triggering apoptotic death in RGCs. Calpeptin, a cell–permeable inhibitor of calpain, provides functional neuroprotection. Our results imply that calpeptin may serve as a potential therapeutic agent for functional neuroprotection in glaucoma and ON.

Keywords: apoptosis/cell death • calcium • neuroprotection