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C. J. Scott, L. A. Levin; Effects of Superoxide Dismutase 2 Knockdown in a Differentiated Retinal Ganglion Cell Line. Invest. Ophthalmol. Vis. Sci. 2007;48(13):619.
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We have previously shown that superoxide generation is a signaling event preceding apoptosis in retinal ganglion cell-like (RGC) RGC-5 cells after axotomy (IOVS 47:1477, 2006). We compared the effect of gene silencing of superoxide dismutase 2 (SOD2) between cell viability and superoxide production when RGC-5 cells were exposed to mitochondrial electron transport chain (METC) substrates and inhibitors to show that increased superoxide production correlates to increased cell death.
RGC-5 cells were differentiated by treating with 316 nM staurosporine, then 24 hours later were transfected with SOD2 siRNA and incubated for 72 hours. Silencing efficiency of protein expression was determined by Western blotting for SOD2. Cell viability was measured by MTT assay when cells were exposed to 1 mM glutamate, 1 mM menadione, or serum free media for 24 hours. Superoxide production was measured with an HEt assay over 1 hour in cells exposed to 1 mM menadione, 100 nM rotenone, or 1 mM antimycin A.
High silencing efficiency of SOD2 was observed at the protein level, with 96% knockdown in undifferentiated RGC-5s, and 70% knockdown in differentiated cells. There was a decrease of viability in differentiated cells when exposed to SOD2 siRNA, which was not additive to the effects of glutamate and serum free media. SOD2 knockdown in undifferentiated RGC-5 cells caused an increase of superoxide production, which was greater in the presence of menadione or rotenone. In contrast, antimycin A caused a modest rise of superoxide in control cells, but a decrease in superoxide in undifferentiated RGC-5 cells in which SOD2 was knocked down cells. There was much less superoxide production in differentiated RGC-5 cells, with less effect of SOD2 knockdown.
SOD2 knockdown causes increased levels of superoxide that may induce cell death. Our results also suggest differentiation could decrease superoxide production due to a lower metabolic state, thus producing lower overall respiration and proportionally less superoxide.
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