Oxidative Injury Induced by Complex I Deficiency is Protected With AAV Mediated Gene Transfer of Human SOD2

Abstract

Abstract: : Purpose: To determine the role of oxygen toxicity in cellular injury of complex I deficiency that is believed to associate with the human disease, Leber Hereditary Optic Neuropathy (LHON). Methods: we have shown that ribozymes designed to reduce expression of the nuclear encoded NDUFA1 subunit of complex I of the respiratory chain induced massive cell death in vitro and Leber-like optic neuropathy in vivo. To determine if cellular injury associated with reduced expression of the NDUFA1 subunit gene of complex I is mediated by reactive oxygen species, we measured superoxide and hydrogen peroxide generation in murine cell lines infected with Adeno associated virus (AAV) expressing ribozyme RzM338 or RzM217, both targeting NDUFA1 mRNA at different sites. Fluorescent probes were used to measure generation of reactive oxygen species: dihydroethidium for mitochondrial superoxide production and 2-7 dichlorofluorescein diacetate (DCFDA) for hydrogen peroxide. Fluorescence was quantitated using a fluorescence activated cell sorter (FACS). To determine if increasing cellular defenses against reactive oxygen species protects against cellular injury and cell death we introduced AAV expressing the human SOD2 gene then tested for any rescue effect by measurement of cell survival. After AAV infections, cells were trypsinized then a aliquot counted using an automated Coulter Z-100 particle cell counter. Results: Mean cell counts were 945,900 cells/ml with ribozyme RzM338, but were much less (561,500 cells/ml) with the more potent ribozyme RzM217. Relative to cells mock-infected with AAV expressing GFP, superoxide levels did not significantly change with cellular infection of either ribozyme. In contrast, hydrogen peroxide levels increased 2.8 fold with RzM338 and 1.3 fold with RzM217. Infection with AAV-expressing human SOD2 had a protective effect. Relative to mock infection with AAV expressing GFP, SOD2 infection increased cell survival by 17% in cells co-infected with ribozyme RzM338 and by 54% in cells co-infected with RzM217. Conclusions: We have shown here that reactive oxygen species appear to contribute to the death of cell lines with experimentally induced complex I deficiency. The rescue of these cells with a gene that detoxifies reactive oxygen species (SOD2) suggests that this approach may be useful to treat patients with complex I deficiencies such as LHON.

Keywords: gene transfer/gene therapy • antioxidants • animal model