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D.M. Kumar, J.W. Simpkins, Z.Y. Cai, D. Covey, N. Agarwal; Neuroprotective Mechanisms of the Non–Feminizing Estrogen Analogue ZYC–3 Against Glutamate Induced Cytotoxicity in RGC–5 Cells . Invest. Ophthalmol. Vis. Sci. 2005;46(13):4658.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To determine the neuroprotective mechanisms of the novel estrogen analogue ZYC–3, against glutamate induced cytotoxicity of rat retinal ganglion cells (RGC–5 cells). Methods: RGC–5 cells were pretreated with ZYC–3 followed by an insult with L–glutamic acid (5 mM). Cell viability was assessed using the neutral red dye uptake assay. Glutamate/cystine anti–porter levels and activity, γ–glutamylcysteinsynthetase levels, buthionine–sulfoxamine cell viability studies, and glutathione levels were used to determine the ability of ZYC–3 to affect the glutathione synthesis pathway. ZYC–3’s ability to effect mitochondrial membrane potential, against glutamate challenge, was determined using live cell confocal microscopy with JC–1 mitochondrial dye. Modulation of pro versus anti–apoptotic signaling cascades by ZYC–3, were examined by inhibitor studies and Western blot analysis. Results: ZYC–3 treatment enhanced 35S–cysteine uptake, enhanced γ–glutamylcysteinsynthetase levels, and attenuated glutathione loss in glutamate challenged RGC–5 cells. Furthermore, cell viability was comparable to control in these studies, reiterating ZYC–3’s neuroprotective capability. ZYC–3 pretreament prevented loss of mitochondrial membrane potential observed with glutamate challenge of RGC–5 cells. ZYC–3 promoted cell survival signals: Bcl–2 levels and activation of the Akt pathway were significantly enhanced in RGC–5 cells pretreated with ZYC–3. Conclusions: The non–feminizing estrogen analogue ZYC–3 protects RGC–5 cells against glutamate induced cytotoxicity by protecting the glutathione synthesis pathway, preventing loss of mitochondrial membrane potential, and activating cell survival signaling cascades. These are the first such reports for a non–feminizing estrogen analogue. The data support the hypothesis that ZYC–3 may be useful in the neuroprotection of retinal ganglion cells in ocular pathologies such as glaucoma.
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