siRNA Suppression of MnSOD in Neuroprogenitor and Retinal Cells Directly Impacts Survival

Abstract

Abstract: : Purpose: To assess the impact of siRNA suppression of mitochondrial manganese superoxide dismutase (MnSOD) on the survival of adult neuroprogenitor cells and cultured retinal ganglion cells. Although most studies have documented increased oxidative stress in apoptotic cells, sources and sites of reactions for reactive oxygen species (ROS) in inducing apoptosis are not currently well-characterized. It is known, however, that mitochondria are the major subcellular sources of ROS due to energy charge/redox state impairments and/or disruption of mitochondrial membrane integrity. MnSOD most likely plays a major part in the mechanism for halting ROS disruption of mitochondrial membrane integrity and subsequent cytochrome C release leading to apoptosis. Methods: Primary rodent hippocampal neuroprogenitor cells (AHPC) and retinal ganglion cells were cultured for 24 hours before being exposed to MnSOD siRNA for 48 hours. In parallel, cells were treated with a PKC activator, PMA, to upregulate MnSOD expression. Multiple trial real-time PCR quantitation was used to assess the amount of MnSOD gene mRNA transcripts. Comparison of cell viability was then determined by dye exclusion assay followed by coulter counter assessment. Results: MnSOD siRNA application effectively suppressed MnSOD expression. Forty-eight hours after the treatment, MnSOD siRNA cell groups displayed over 100 fold reduction in MnSOD mRNA as compared to normals. While in the cells treated with PMA, we observed more than a two fold increase of MnSOD mRNA relative to normals. Concomitantly, there was a three to four-fold reduction in cell survival after MnSOD siRNA treatment as compared to controls. In contrast, groups treated with PMA possessed a two fold (190%) increase in cell number, compared to control, suggesting growth promotion. Conclusions: Mitochondrial SOD expression, specifically, is critical to cell survival in neuroprogenitor cells and in cultured retinal ganglion cells. Neuronal dependence on MnSOD expression suggests a key mechanism determinant for cell survival.

Keywords: gene/expression • mitochondria • cell death/apoptosis