Calpains Play a Role in Retinal Ganglion Cell Apoptosis Using Glaucoma Death Stimuli

Abstract

Purpose:: Calpains are calcium dependent cysteine proteases that have been shown to mediate neuronal apoptosis. The purpose of this study was to investigate whether these enzymes play a role in apoptosis of a retinal ganglion cell (RGC) line (RGC-5) and in the ganglion cell layer (GCL) of retinal explants in glaucomatous paradigms.

Methods:: RGC-5 cells were subjected to death stimuli relevant to the pathophysiolgy of glaucoma, namely calcium ionophore (excitotoxicity) and serum starvation (neurotrophin deprivation). Flow cytometry was used to confirm apoptosis. Western blotting was employed to analyse the role of latent and active calpains, of the calpain inhibitor calpastatin, and of active calpain breakdown products in such death. Subsequently, using an axotomy model, retinal explants were cultured in serum free media. Terminal dUTP Nick End Labelling (TUNEL) was employed to confirm apoptosis, and protein analysis of GCL shavings was done to examine the expression of active and latent calpains, and active calpain breakdown products.

Results:: RGC-5 cells and cells within the GCL underwent significant apoptosis in both models. There was significant upregulation of active calpains and concurrent downregulation of latent calpains in RGC-5 cells and retinal explant models. Fodrin, a breakdown product of active calpains was increased and there was decreased expression of the endogenous inhibitor of calpains, calpastatin.

Conclusions:: Calpains play a role in the apoptotic death of RGCs in serum starved and excitotoxic paradigms in RGC-5 cells and in the serum starved GCL of retinal explants. As these conditions are believed to contribute to the upstream pathogenesis of glaucoma, calpains may partake in the pathological mechanisms of this disease.

Keywords: ganglion cells • apoptosis/cell death