Contribution of Calpain and Caspases to Cell Death in Monkey RPE Cells Cultured Under Hypoxia or with A2E

Abstract

Purpose: : Age-related macular degeneration (AMD) is the leading cause of vision loss after age 65. Several causative mechanisms have been proposed. 1) Age-related failure of the choroidal vasculature leads to loss of the retinal pigment epithelium (RPE). 2) Accumulation of phagocytized but unreleased A₂E causes RPE dysfunction. A₂E is an auto fluorescent conjugation product of two molecules of all-trans-retinal and ethanolamine. 3) Zinc deficiency activates calpain and caspase proteases, leading to cell death. Thus, the purpose of the present study was to compare the activation of calpain and caspase pathways in monkey RPE cells cultured separately with either A₂E or under hypoxic conditions.

Methods: : Monkey primary RPE cells were cultured with synthetic A₂E or cultured under hypoxic conditions in a Gaspak pouch. Cell viability was assessed by the MTS assay. Immunoblotting was used to detect activation of calpain and caspase molecules, and also to identify enzyme-specific, α-spectrin breakdown products (SBDP). Calpain inhibitor, SNJ-1945, and pan-caspase inhibitor, z-VAD-fmk, were used to confirm activation of the proteases.

Results: : 1) A₂E and hypoxia each decreased viability of RPE cells in a time dependent manner. 2) Incubation with A₂E alone induced activation of calpain, mitochondria-dependent caspase-9, and executer caspase-3. Appearance of the calpain-specific SBDP at 145 kDa and the caspase-3-specific SBDP at 120 kDa were also observed. SNJ-1945 inhibited calpain activation and slightly inhibited caspase activation. z-VAD-fmk inhibited caspase activation and slightly inhibited calpain activation, suggesting inter-activation of one protease system with the other. 3) Incubation under hypoxia alone induced activation of calpain, but not caspases. Only the calpain-specific SBDP was formed. SNJ-1945 inhibited calpain activation, but z-VAD-fmk did not.

Conclusions: : A₂E activated both calpain and caspase pathways in monkey RPE cells, but hypoxia activated only the calpain pathway. Further investigation is necessary to determine how A₂E and hypoxia signal the activation of specific calpain or caspase systems and how these systems interact. This may become important in human AMD treatment because inhibitor drugs against calpain and/or caspase may be used to prevent RPE dysfunction caused by the putative effectors A₂E and hypoxia.Dr. Shearer receives consulting fees from, and Drs. Azuma and Nakajima are employees of, Senju Pharmaceutical Co. Ltd.

Keywords: age-related macular degeneration • retinal pigment epithelium • apoptosis/cell death