Neuroprotective effects of 5-S-GAD against oxidative stress-induced apoptosis in RGC-5 cells.

N-beta-Alanyl-5-S-glutathionyl-3,4-dihydroxyphenylalanine (5-S-GAD), an antibacterial substance isolated from the flesh fly, has been described as having multipotential biological activities toward various tissues. In a previous paper, we reported a novel neuroprotective action of 5-S-GAD on rat retinal ganglion cell apoptosis induced by optic nerve injury and intraocular N-methyl-D-aspartate treatment in vivo. In the present study, we further investigated the protective mechanism of this small peptide against other types of apoptosis in cultured cells of the established rat retinal ganglion cell line RGC-5. Hydrogen peroxide and serum deprivation treatments induced intracellular reactive oxygen species levels and lipid peroxidation, revealed by 4-hydroxy-2-nonenal production, in RGC-5 cells within 9-12h. The treatments also induced cell death accompanied by nuclear condensation, DNA laddering and increases in apoptotic Bax and caspase-3 proteins in RGC-5 cells within 12-24h. 5-S-GAD at 25-50 microM clearly suppressed the cell death and apoptotic features induced by these treatments. 5-S-GAD restored the nuclear condensation, DNA laddering and increases in apoptotic proteins. Furthermore, 5-S-GAD directly activated anti-apoptotic phospho-Akt and Bcl-2 proteins in RGC-5 cells. 5-S-GAD also quenched the reactive oxygen species production and inhibited the lipid peroxidation induced by oxidative stress. Therefore, 5-S-GAD may complementarily protect RGC-5 cells against apoptosis through dual actions as a radical scavenger and an inducer of anti-apoptotic phospho-Akt and Bcl-2. Taken together, 5-S-GAD is a high-potential tool for rescuing the retinal ganglion cell apoptosis induced by a variety of glaucomatous conditions.

A novel neuroprotective role of a small peptide from flesh fly, 5-S-GAD in the rat retina in vivo.

N-beta-Alanyl-5-S-glutathionyl-3,4-dihydroxyphenylalanine (5-S-GAD), an antibacterial substance isolated from flesh fly, has been described as having multipotential biological activities toward various tissues. However, there has been no report testing its action on neural cells. In the present study, we investigate whether 5-S-GAD is neurotoxic or neuroprotective to the rat retina. 5-S-GAD at high doses (more than 200 pmol) induced apoptosis of retinal neurons 7 days after intraocular injection. 5-S-GAD at low doses (2-20 pmol) significantly attenuated the loss of retinal ganglion cells (RGCs) and the thinning of inner retina induced by NMDA in a dose-dependent manner. To understand the protective mechanism of 5-S-GAD, we investigated the influence of 5-S-GAD on the cell survival molecules, phospho-Akt and Bcl-2. 5-S-GAD (2-20 pmol) rapidly increased phospho-Akt expression 1-7 days and Bcl-2 expression 3-7 days after injection. The cellular localization of this increase was both in bipolar cells and RGCs. This neurosurvival effect of 5-S-GAD was further tested using another model of optic nerve injury. 5-S-GAD significantly blocked the apoptosis of RGCs 7 days after optic nerve crush. These results show that 5-S-GAD (2-20 pmol) protects against the NMDA- and optic nerve crush-induced apoptosis of RGCs. The neuroprotective action of 5-S-GAD in the retina might be mediated by the cell survival phospho-Akt/Bcl-2 system and offers a therapeutic option to rescue RGCs from various types of excitotoxic disease, such as glaucoma.