SOD2 knockdown mouse model of early AMD.
OBJECTIVE
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.
Justilien
Verline
V
Department of Molecular Genetics, University of Florida, Gainesville, Florida, USA.
Pang
Ji-Jing
JJ
Renganathan
Kutralanathan
K
Zhan
Xianquan
X
Crabb
John W
JW
Kim
So Ra
SR
Sparrow
Janet R
JR
Hauswirth
William W
WW
Lewin
Alfred S
AS
D000818:Animals; D017209:Apoptosis; D000229:Dependovirus / Q000235:genetics; D004195:Disease Models, Animal; D004596:Electroretinography; D020868:Gene Silencing; D005822:Genetic Vectors; D008268:Macular Degeneration / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D051379:Mice; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D018345:Mice, Knockout; D018384:Oxidative Stress; D010775:Photic Stimulation; D010857:Pigment Epithelium of Eye / Q000187:drug effects / Q000201:enzymology; D011726:Pyridinium Compounds / Q000378:metabolism; D016337:RNA, Catalytic / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D012160:Retina / Q000378:metabolism / Q000648:ultrastructure; D012176:Retinoids / Q000378:metabolism; D013482:Superoxide Dismutase / Q000235:genetics* / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D000099156:Superoxide Dismutase 2
D011726:Pyridinium Compounds; D016337:RNA, Catalytic; D012333:RNA, Messenger; D012176:Retinoids; D013482:Superoxide Dismutase; D000099156:Superoxide Dismutase 2; C112040:A2-E (N-retinylidene-N-retinylethanolamine)