Mitochondrial Dysfunction In Retinal Ganglion Cells In Glutamate Cytotoxicity

Abstract

Purpose: : Glaucoma is an optic neuropathy characterized by gradual cupping of the optic disc and the degeneration of the optic nerve, resulting in a progressive loss of visual field, ultimately leading to blindness. In vivo studies have shown that the final pathway in glaucoma leading to blindness is retinal ganglion cell (RGC) death via apoptosis. The neural retina is highly oxygenated, and constantly irradiated tissue and thus considered to be especially prone to damage by reactive oxygen species (ROS) formed as a consequence of mitochondrial respiration and via photosensitized reactions. It also contains higher proportions of unsaturated fatty acids, which can easily form lipid peroxides as a result of oxidation by ROS. Membrane oxidation, and the ensuing loss of integrity is most readily apparent at the level of the mitochondria where loss of inner membrane impermeability is reflected by loss of membrane potential, and eventually, by cell death. We hypothesize that mitochondrial destability due to oxidative damage is central to pathogenesis of glaucoma leading to apoptosis of RGCs and damage to ONH.

Methods: : RGC–5 cells were treated with L–glutamic acid (5 mM). Glutamate cytotoxicity induced oxidative damage was assessed by measuring glutamate/cystine anti–porter levels and activity, gamma–glutamylcystein synthetase levels, and glutathione levels. The mitochondrial membrane potential, against glutamate challenge, was determined using live cell confocal microscopy with JC–1 mitochondrial dye.

Results: : Glutamate challenge to RGC–5 cells resulted in decreased gamma–glutamylcystein synthetase levels, ³⁵S–cysteine uptake, lowering of glutathione levels, and in a loss of mitochondrial membrane potential. The inclusion of N–acetyl cysteine reversed the effects of glutamate cytotoxicity.

Conclusions: : Glutamate cytotoxicity resulted in oxidative damage of RGC–5 cells via loss of mitochondrial membrane potential, and the glutathione synthesis pathway. Furthermore, the data support the hypothesis that antioxidants may be useful in the neuroprotection of retinal ganglion cells in ocular pathologies such as glaucoma.

Keywords: ganglion cells • retinal culture • apoptosis/cell death