Effect of Genetically Altered Expression of Manganese Superoxide Dismutase (SOD2) on Oxidation–Induced Apoptosis in Cultured Mouse Retinal Pigment Epithelium (RPE)

Abstract

Abstract: : Purpose: Oxidative stress from reactive oxygen species (ROS) has been implicated in many diseases including age–related macular degeneration (AMD). RPE is considered a primary target in AMD because cell death is observed in the early phase of this disease. Since deficiency of SOD2 and the resulting accumulation of ROS play a central role in apoptosis, we compared oxidation–induced apoptosis in cultured RPE derived from wild–type (WT) and mutant mice with SOD deficiency (HET) and over–expression of SOD2 (HEMI). Methods: Primary cultures of RPE were established from the three groups of mice – WT, partial knockout (HET) and HEMI with different levels of SOD2. Purity of the cell cultures was established by immunostaining with antibody to RPE–65 – a specific marker for RPE. Apoptosis in the three groups of cells was examined by TUNEL (terminal deoxynucleotidyl transferase–mediated dUTP nick–end labeling) staining and cell death detection ELISA–kit following exposure to different levels of H2O2 (0–500µM) for 1 hour and reculturing in normal media for varying time periods (0–24 hours). Results: More than 90% of the cells in each culture were positive to RPE–65 staining and the relative SOD2 levels, as determined in Western blots, in HET:WT:HEMI were 0.6:1.0:3.4 respectively. H2O2–induced apoptotic cell death was both dose and time dependant. Apoptosis was first detected at 200µM level in WT and HET but none in HEMI. Also, time dependency for apoptosis was related to cellular SOD2 level. Thus, SOD2 deficient cells (HET) were more susceptible to oxidative injury compared to cells fromWT and HEMI. Conclusions: The results demonstrate a critical role of SOD2 in protection against oxidative challenge; deficiency of the enzyme causes greater H2O2–induced apoptosis and that over–expression of the enzyme suppresses or protects against oxidation–induced cell death. It is concluded that under conditions of decreased expression of SOD2 or its loss from mitochondria during aging may lead to increased accumulation of ROS and apoptotic cell death in AMD and other degenerative diseases of the eye.

Keywords: apoptosis/cell death • retinal pigment epithelium • transgenics/knock-outs