IOVS Applied and Environmental Microbiology
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(Investigative Ophthalmology and Visual Science. 2007;48:4407-4420.)
© 2007 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.07-0432
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SOD2 Knockdown Mouse Model of Early AMD

Verline Justilien,1 Ji-Jing Pang,2 Kutralanathan Renganathan,3 Xianquan Zhan,3 John W. Crabb,3 So Ra Kim,4 Janet R. Sparrow,4 William W. Hauswirth,2 and Alfred S. Lewin1

1From the Departments of Molecular Genetics and 2Ophthalmology University of Florida, Gainesville, Florida; 3Cole Eye Institute and Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio; and the 4Department of Ophthalmology, Columbia University, New York, New York.

PURPOSE. To test the hypothesis that oxidative injury to the retinal pigment epithelium (RPE) may lead to retinal damage similar to that associated with the early stages of age-related macular degeneration (AMD).

METHODS. A ribozyme that targets the protective enzyme manganese superoxide dismutase (MnSOD) was expressed in RPE-J cells, and adeno-associated virus (AAV) expressing the ribozyme gene was injected beneath the retinas of adult C57BL/6 mice. The RPE/choroid complex was examined for SOD2 protein levels and protein markers of oxidative damage using immunoblot analysis and LC MS/MS-identification of proteins and nitration sites. Lipids were extracted from retinal tissue and analyzed for the bis-retinoid compounds A2E and iso-A2E. The mice were analyzed by full-field electroretinography (ERG) for light response. Light and electron microscopy were used to measure cytological changes in the retinas.

RESULTS. The treatment of RPE-J cells with Rz432 resulted in decreased MnSOD mRNA and protein as well as increased levels of superoxide anion and apoptotic cell death. When delivered by AAV, Rz432 reduced MnSOD protein and increased markers of oxidative damage, including nitrated and carboxyethylpyrrole-modified proteins in the RPE-choroid of mice. Ribozyme delivery caused a progressive loss of electroretinograph response, vacuolization, degeneration of the RPE, thickening of Bruch’s membrane, and shortening and disorganization of the photoreceptor outer and inner segments. Progressive thinning of the photoreceptor outer nuclear layer resulted from apoptotic cell death. Similar to the eyes of patients with AMD, ribozyme-treated eyes exhibited increased autofluorescence and elevated levels of A2E and iso-A2E, major bis-retinoid pigments of lipofuscin.

CONCLUSIONS. These results support the hypothesis that oxidative damage to the RPE may play a role in some of the key features of AMD.






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