Abstract
Abstract: :
Purpose: To develop an animal model for Leber Hereditary Optic Neuropathy (LHON) and rescue it. Methods: Ten sedated 8 week–old DBA/1J mice received intravitreal injections of 2 ul of AAV–ribozyme directed against the murine mitochondrial ND4 subunit of complex I into the right eyes. As controls, left eyes received AAV with a ribozyme directed against human ND4 that does not cleave murine ND4 mRNA. A second group of mice received an allotopic version expressing the LHON mutant ND4, encoding for the histidine substitution at amino acid 340 (G11778A ND4) OD and AAV–gfp was injected OS. A third group received both the mouse ND4 Rz followed two weeks later by the allotopic mutant ND4 OD, but the human RzND4 and GFP respectively OS. Next, we attempted rescue. Two additional groups of animals were inoculated with the mouse ribozymes into both eyes, then we attempted rescue with an allotopic version of normal ND4 (340R) or the human SOD2 gene OD. All animals underwent three dimensional contrast enhanced fat suppressed MRIs of the optic nerves and brains in a 4.7 Tesla superconducting magnet at 4 weeks post injection. Optic nerve diameters (ONDs) were measured at the retrobulbar, mid–orbital and orbital apex regions. Statistical analysis of the differences between the right and left sides were done using Students t test. Prior to animal testing, complex I activity and cell survival of infected 3T3 cells were evaluated in galactose media. Results: In vitro, murine 3T3 cells infected with the murine AAV–RzND4 dwindled by 30% in galactose relative to infection with the human AAV–RzND4 that does not cleave mouse ND4 mRNA. Complex I activity of 3T3 cells infected with the murine AAV–RzND4 and grown in normal media containing glucose revealed a 55% reduction relative to infection with the human RzND4. In vivo we found that relative to the human RzND4, the murine RzND4 decreased optic nerve diameters by 10% (p<0.001). The mutant R340H ND4 alone had no effect on OND. However, the addition of the mutant ND4 to the RzND4 reduced OND by 17% (p<0.02). Rescue of murine RzND4 inoculated eyes with the allotopic ND4 revealed a 13% increase in OND relative to the unprotected left side (p<0.001). However, no effect was seen with SOD2 rescue. Conclusions: Our results show that suppression of complex I activity with a ribozyme targeting the ND4 subunit alone and the Rz combined with a mutant LHON ND4 induces optic neuropathy. The rescue effect seen with a normal ND4, but not with an antioxidant gene (SOD2) suggests that optic nerve injury may initially be due to loss of oxidative phosphorylation rather than oxygen toxicity.
Keywords: mitochondria • gene transfer/gene therapy