Use of mitochondrial antioxidant defenses for rescue of cells with a Leber hereditary optic neuropathy-causing mutation.
OBJECTIVE
To explore a treatment paradigm for Leber hereditary optic neuropathy (LHON), we augmented mitochondrial antioxidant defenses to rescue cells with the G11778A mutation in mitochondrial DNA.


METHODS
Cells homoplasmic for the G11778A mutation in mitochondrial DNA were infected with an adeno-associated viral vector containing the human mitochondrial superoxide dismutase (SOD2) gene. Control cells were infected with an adeno-associated viral (AAV) vector expressing the green fluorescent protein (GFP). Two days later, the high-glucose culture medium was exchanged for a glucose-free medium containing galactose. After 1 or 2 days, cellular production of superoxide was assessed using the fluorescent probe dihydroethidium, and we used TUNEL (terminal deoxynucleotidyl transferase-mediated biotin-deoxyuridine triphosphate nick-end labeling) staining to detect apoptotic nuclei. The effect of SOD2 on LHON cell survival was quantitated after 2 or 3 days.


RESULTS
Comparisons of AAV-SOD2-infected LHON cells relative to control cells infected with AAV-green fluorescent protein showed increased expression of mitochondrial SOD that attenuated superoxide-induced fluorescence by 26% (P = .003) and suppressed TUNEL-induced fluorescence by 21% (P = .048) after 2 days of growth in galactose medium, when cell survival increased by 25% (P=.05). After 3 days in galactose medium, SOD2 increased LHON survival by 89% (P = .006) relative to controls.


CONCLUSIONS
Protection against mitochondrial oxidative stress may be useful for treatment of LHON.


CONCLUSIONS
Gene therapy with antioxidant genes may protect patients with LHON against visual loss.
Qi
Xiaoping
X
Department of Ophthalmology, College of Medicine, University of Florida, Gainesville, FL 32610, USA.
Sun
Liang
L
Hauswirth
William W
WW
Lewin
Alfred S
AS
Guy
John
J
D000975:Antioxidants / Q000378:metabolism*; D017209:Apoptosis; D002470:Cell Survival; D002478:Cells, Cultured; D004272:DNA, Mitochondrial / Q000235:genetics*; D000229:Dependovirus / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D015316:Genetic Therapy; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D020287:In Situ Nick-End Labeling; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism*; D029242:Optic Atrophy, Hereditary, Leber / Q000201:enzymology / Q000628:therapy*; D018384:Oxidative Stress; D010957:Plasmids; D017382:Reactive Oxygen Species / Q000378:metabolism; D013482:Superoxide Dismutase / Q000235:genetics*; D013481:Superoxides / Q000633:toxicity; D014162:Transfection; D000099156:Superoxide Dismutase 2
D000975:Antioxidants; D004272:DNA, Mitochondrial; D049452:Green Fluorescent Proteins; D017382:Reactive Oxygen Species; D013482:Superoxide Dismutase; D013481:Superoxides; D000099156:Superoxide Dismutase 2