Evaluation of Potential Treatment Strategies for Leber Hereditary Optic Neuropathy

Abstract

Abstract: : Purpose:To develop a treatment strategy for Leber Hereditary Optic Neuropathy (LHON) by rescue with complex II substrates or increasing cellular defenses against oxygen toxicity. Methods:To determine if complex II substrates that bypass the mutated complex I can be used to rescue LHON, cybrids cells were placed in glucose-free medium containing either the complex II substrate succinate or the complex I substrate pyruvate in addition to galactose as the main carbon source for glycolysis. This medium forces the cells to rely predominantly on oxidative phosphorylation to produce ATP. Cells harboring complex I mutations have a severe growth defect and die off in this galactose medium. In addition, to determine the role of oxygen toxicity in cell death we measured mitochondrial superoxide production using the florescent probe dihydroethidium. We evaluated the protective effect of increasing mitochondrial defenses against superoxide using adenoassociated virus (AAV) as a vector to deliver the human gene for mitochondrial superoxide dismutase (SOD2) to LHON cybrids. Results:After three days in the galactose medium containing the complex II substrate succinate, LHON cybrid cell populations dwindled 67%. This level of reduction was not significantly different than cells grown in the complex I substrate pyruvate that showed a 73% reduction. Relative to transmitochondrial LHON cybrids grown in glucose media, superoxide levels doubled in LHON cybrids placed in galactose media. Infection with AAV-expressing SOD2 increased cell survival in the galactose media, by 25%, relative to mock infection with AAV expressing GFP (p<0.008). Conclusions:We have shown here that administration of complex II substrates does not significantly improve LHON cybrid cell survival, thus suggesting that this approach may not be useful in the treatment of LHON. Moreover, despite the contribution of increased levels of superoxide to cell death and possibly optic neuropathy, the levels of antioxidant gene protection did not result in the threefold increases in cell survival that we previously reported using allotopic ND4 rescue, thus suggesting that other reactive oxygen species or reduced ATP synthesis may also play a role in cellular injury and optic neuropathy.

Keywords: mitochondria • gene transfer/gene therapy • neuro-ophthalmology: optic nerve