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P Ahuja, AR Caffé, S Ahuja, I Holmqvist, T van Veen; Glutathion S-Transferase MU (GST-MU) Rescues Photoreceptors in RD/RD Mouse Retina in Culture . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1889.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Glutathione S-transferases (GSTs) are a large group of enzymes divided into two superfamilies; one soluble (e.g. alpha, mu, pi classes) and the other membrane-bound (e.g. microsomal I class). An important physiological function of GSTs is to detoxify the cell from reactive metabolites generated in the course of normal metabolism and in pathology. Current research is paying particular attention to GST-induced suppression of apoptosis. As a result the objective of our studies is to see if GSTs rescue photoreceptors in rd/rd mouse retinal explants (RE). GST-mu was investigated because the mu and alpha classes are present in rat retinal elements that are linked to photoreceptor rescue. Methods: Eyes from C3H rd/rd and +/+ mice were collected at postnatal day (PN) 2, PN7, PN14, PN21 and PN28. From each age group some eyes were fixed with 4% paraformaldehyde, 10µm-frozen sections were cut and incubated with a polyclonal antibody directed against GST-mu (1:500, Oxford Biomedical Research). The bound primary antibodies were detected using immunofluorescence. From other eye bulbs the retina was isolated, total protein extracted, separated using SDS-PAGE and proteins transferred to a nitrocellulose membrane. These blots were incubated with the GST-mu antibody (1:500) and the bound antibody detected through chromagen or chemiluminescence. PN2 and PN7 rd/rd and +/+ RE were generated as previously described and incubated with and without 10ng/ml GST-mu up to PN28. In RE sections the number of surviving photoreceptors was analyzed statistically. Results: From PN2 onwards GST-mu was present in Müller cells in both rd/rd and +/+ retina. Western blot analyses detected one protein band of around 26 kDa which further indicated that GST-mu protein levels in the rd/rd were lower than those in the +/+ mouse retina. The number of rows of photoreceptors in PN2 rd/rd RE exposed to GST-mu was 6 while PN7 RE cultured in similar conditions showed only 3 rows. In both PN2 and PN7 rd/rd RE without GST-mu, 2 to 3 rows of photoreceptor cells were present. In +/+ RE with and without GST-mu, 7 to 8 rows were observed. Conclusion: In mouse retina GST-mu is present early in development and localized to Müller cells. The lower GST-mu protein levels in the rd/rd retina might contribute to cell loss. When exogenous GST-mu is applied this treatment rescues photoreceptors only in PN2 rd/rd RE. In this model the protective potency of GST-mu is similar to that of CNTF alone and NGF+FGF2. Further study needs to establish if other members of the GST superfamily rescue rd/rd photoreceptors as well.
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