title: Mitochondrial oxidative stress in the retinal pigment epithelium leads to localized retinal degeneration.
abstract: OBJECTIVE
  Oxidative stress in the RPE is widely accepted as a contributing factor to AMD. We have previously shown that ribozyme-mediated reduction in the antioxidant enzyme manganese superoxide dismutase (MnSOD) leads to some of the features of geographic atrophy in mice. To develop a mouse model independent of viral injection, we used a conditional knockout of the Sod2 gene in the RPE to elevate mitochondrial oxidative stress in that cell layer.
  
  
  METHODS
  Experimental mice in which exon 3 of Sod2 was flanked by loxP sites were also transgenic for PVMD2-rtTA and tetO-PhCMV cre, so that cre recombinase was expressed only in the RPE. Pups of this genotype (Sod2(flox/flox)VMD2cre) were induced to express cre recombinase by feeding doxycycline-laced chow to nursing dams. Controls included mice of this genotype not treated with doxycycline and doxycycline-treated Sod2(flox/flox) mice lacking the cre transgene. Expression of cre in the RPE was verified by immunohistochemistry, and deletion of Sod2 exon 3 in the RPE was confirmed by PCR. Mice were followed up over a period of 9 months by spectral-domain optical coherence tomography (SD-OCT), digital fundus imaging, and full-field ERG. Following euthanasia, retinas were examined by light and electron microscopy or by immunohistochemistry. Contour length of rod outer segments and thickness of the RPE layer were measured by unbiased stereology.
  
  
  RESULTS
  Following doxycycline induction of cre, Sod2(flox/flox) cre mice demonstrated increased signs of oxidative stress in the RPE and accumulation of autofluorescent material by age 2 months. They showed a gradual decline in the ERG response and thinning of the outer nuclear layer (by SD-OCT), which were statistically significant by 6 months. In addition, OCT and electron microscopy revealed increased porosity of the choroid. At the same interval, hypopigmented foci appeared in fundus micrographs, and vascular abnormalities were detected by fluorescein angiography. By 9 months, the RPE layer in Sod2(flox/flox) cre mice was thicker than in nontransgenic littermates, and the rod outer segments were significantly longer over most of the retina, although localized atrophy of photoreceptors was also obvious in some eyes.
  
  
  CONCLUSIONS
  Conditional tissue-specific reduction in MnSOD induced oxidative stress in mouse RPE, leading to RPE dysfunction, damage to the choroid, and death of photoreceptor cells. The RPE oxidative stress did not cause drusen-like deposits, but the model recapitulated certain key aspects of the pathology of dry AMD and may be useful in testing therapies.
authors.affiliation: Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida, Gainesville, Florida, United States.
authors.forename: Haoyu
authors.initials: H
authors.lastname: Mao
authors.affiliation: Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida, Gainesville, Florida, United States.
authors.forename: Soo Jung
authors.initials: SJ
authors.lastname: Seo
authors.affiliation: Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida, Gainesville, Florida, United States.
authors.forename: Manas R
authors.initials: MR
authors.lastname: Biswal
authors.affiliation: Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida, Gainesville, Florida, United States.
authors.forename: Hong
authors.initials: H
authors.lastname: Li
authors.affiliation: Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida, Gainesville, Florida, United States.
authors.forename: Mandy
authors.initials: M
authors.lastname: Conners
authors.affiliation: Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida, Gainesville, Florida, United States.
authors.forename: Arathi
authors.initials: A
authors.lastname: Nandyala
authors.affiliation: Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida, Gainesville, Florida, United States.
authors.forename: Kyle
authors.initials: K
authors.lastname: Jones
authors.affiliation: Departments of Medicine, Endocrinology, and Cell Biology and Harold Hamm Diabetes Center, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States.
authors.forename: Yun-Zheng
authors.initials: YZ
authors.lastname: Le
authors.affiliation: Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida, Gainesville, Florida, United States.
authors.forename: Alfred S
authors.initials: AS
authors.lastname: Lewin
mesh_terms: D000818:Animals; D004195:Disease Models, Animal; D004318:Doxycycline / Q000633:toxicity; D004596:Electroretinography; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005451:Fluorescein Angiography; D017353:Gene Deletion; D015870:Gene Expression / Q000187:drug effects; D057092:Geographic Atrophy / Q000209:etiology* / Q000378:metabolism / Q000473:pathology; D019426:Integrases / Q000235:genetics; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008822:Mice, Transgenic; D008928:Mitochondria / Q000378:metabolism*; D018384:Oxidative Stress / Q000502:physiology*; D016133:Polymerase Chain Reaction; D055213:Retinal Pigment Epithelium / Q000378:metabolism* / Q000648:ultrastructure; D012374:Rod Cell Outer Segment / Q000648:ultrastructure; D013482:Superoxide Dismutase / Q000235:genetics; D041623:Tomography, Optical Coherence; D000099156:Superoxide Dismutase 2
chemical_list: D004318:Doxycycline; D019426:Integrases; D013482:Superoxide Dismutase; D000099156:Superoxide Dismutase 2; C045073:Cre recombinase
keywords: manganese superoxide dismutase; mouse model; reactive oxygen species; retinal degeneration; retinal pigment epithelium