title: SOD2 protects against oxidation-induced apoptosis in mouse retinal pigment epithelium: implications for age-related macular degeneration.
abstract: OBJECTIVE
  Oxidative stress from reactive oxygen species (ROS) has been implicated in many diseases, including age-related macular degeneration (AMD), in which the retinal pigment epithelium (RPE) is considered a primary target. Because manganese superoxide dismutase (SOD2), localized in mitochondria, is known to be a key enzyme that protects the cells against oxidative stress, this study was undertaken to examine oxidation-induced apoptosis in cultured RPE cells with various levels of SOD2.
  
  
  METHODS
  Primary cultures of RPE cells were established from wild-type (WT), heterozygous Sod2-knockout mouse (HET) and hemizygous Sod2 mice with overexpression of the enzyme (HEMI). Purity of the RPE cell cultures was verified by immunostaining with antibody to RPE65 and quantified by flow cytometry. Oxidative stress was induced in RPE cells by exposing them to H(2)O(2) (0-500 muM) for 1 hour and reculturing them in normal medium for various times (0-24 hours). Apoptosis in the RPE was examined by TUNEL staining and quantified by cell-death-detection ELISA. Mitochondrial transmembrane potential (MTP) was measured by a cationic dye, and cytochrome c leakage from mitochondria was analyzed by Western blot analysis.
  
  
  RESULTS
  More than 95% of the cells in each culture were RPE65 positive, and the relative SOD2 levels in HET, WT, and HEMI cells were 0.6, 1.0, and 3.4, respectively. H(2)O(2)-induced apoptotic cell death was both dose and time dependent, and apoptosis in these cells was related to the cellular SOD2 level. Disruption of MTP and release of cytochrome c were observed to occur before apoptotic cell death, and they correlated with cellular SOD2.
  
  
  CONCLUSIONS
  The results demonstrate a critical role of SOD2 in protection against oxidative challenge. Cells from HET mice showed greater apoptotic cell death, whereas in those from HEMI mice, cell death induced by oxidative injury was suppressed.
authors.affiliation: Kellogg Eye Center, Department of Ophthalmology and Visual Sciences, University of Michigan, 1000 Wall Street, Ann Arbor, MI 48105, USA.
authors.forename: Emiko
authors.initials: E
authors.lastname: Kasahara
authors.forename: Li-Ren
authors.initials: LR
authors.lastname: Lin
authors.forename: Ye-Shih
authors.initials: YS
authors.lastname: Ho
authors.forename: Venkat N
authors.initials: VN
authors.lastname: Reddy
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D002352:Carrier Proteins; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D045304:Cytochromes c / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005136:Eye Proteins / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry; D006861:Hydrogen Peroxide / Q000633:toxicity; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D008268:Macular Degeneration / Q000201:enzymology* / Q000378:metabolism; D008297:Male; D008564:Membrane Potentials; D051379:Mice; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008928:Mitochondria / Q000502:physiology; D016877:Oxidants / Q000633:toxicity*; D018384:Oxidative Stress; D010857:Pigment Epithelium of Eye / Q000201:enzymology* / Q000378:metabolism / Q000473:pathology; D013482:Superoxide Dismutase / Q000502:physiology*; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase; D000099156:Superoxide Dismutase 2
chemical_list: D002352:Carrier Proteins; D045304:Cytochromes c; D005136:Eye Proteins; D006861:Hydrogen Peroxide; D016877:Oxidants; D013482:Superoxide Dismutase; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase; D000099156:Superoxide Dismutase 2