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J. Guy, L. Sun, X. Qi, A.S. Lewin, W.W. Hauswirth; Suppression of Mitochondrial Oxidative Injury by Sod2 Gene Transfer Provides Longterm Protection Against Myelin Fiber Injury in the EAE Optic Nerve . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1197.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To investigate the role of mitochondria in the pathogenesis of optic nerve injury. Methods: Optic nerve specimens from patients with optic neuritis and animals induced with experimental autoimmune encephalomyelitis (EAE) were probed with an antibody against nitrotyrosine to identify mitochondrial proteins nitrated by peroxynitrite, formed by the reaction of superoxide and nitric oxide. Nitrated mitochondrial proteins isolated from EAE animals were identified by mass spectrometry. In vivo reactive oxygen species (ROS) were visualized in the optic nerves using DCFA, dihydroethidium, cerium chloride and immunochemistry for nitrotyrosine and nitric oxide. To suppress oxidative injury to mitochondria and to probe for a protective effect on acute optic neuritis recombinant AAV–Sod2 or rAAV–Sod1 was injected into the vitreous. Control eyes received the recombinant virus without a therapeutic gene. Mice were sensitized for EAE and followed by serial contrast enhanced MRIs. The effects of SODs on the histologic lesions of acute EAE were measured by computerized analysis of myelin fiber area at 1 month. The protective gene was then tested for a long term protective effect in chronic EAE at 3 and 12 months. Cellular RNA was extracted and probed for human Sod2 gene expression using northern blot analysis in vitro. Results: MRIs revealed contrast enhancement of the optic nerves and reductions in optic nerve diameters that occurred later at 3 months to 1 year. Western blots revealed nitrated mitochondrial proteins as early as 3 days after antigenic sensitization and prior to inflammatory cell infiltration into the optic nerve. Mass spectrometry identified nitrated proteins as mHsp70, critical to the import of nuclear encoded mitochondrial proteins from the cytosol and proteins encoded by mtDNA, cytochrome c oxidase subunit IV and NADPH–ubiquinone oxidoreductase. Optic nerve specimens revealed the presence of ROS, some within mitochondria.Sod2 increased immunogold in mitochondria, Sod1 only in the nucleus and cytosol. Sod2 levels increased 5 fold, suppressing myelin fiber injury by 31% as long, as 1 year after a single gene inoculation, but Sod1 and a ribozyme suppressing Sod2 gene expression increased myelin fiber injury. Degenerating axons, some with accumulation of mitochondria, hydropic degeneration and loss of cristae seen at 1 year were suppressed by Sod2. Conclusions: Sod2 gene delivery may be a therapeutic strategy for optic neuritis and MS.
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