Wild Type Human ND4 Does Not Suppress Optic Neuropathy Induced by a Mutant Human ND4

Abstract

Purpose:: - Last year we showed that a mutant ND4 subunit made compatible with the universal genetic code and containing an arginine to histidine substitution at residue 340 induced optic disc edema followed by RGC loss and atrophy of the optic nerve when injected into the eyes of mice. Here we attempted to rescue the optic neuropathy in this murine model of Leber Hereditary Optic Neuropathy (LHON).

Methods:: - To induce LHON we injected AAV containing the allotopic ND4 gene with a His substitution at amino acid 340 into both eyes of 10 mice. Two days later we attempted rescue by injecting the right eyes with AAV containing the wild-type human ND4. As a control, we injected the left eyes with AAV-GFP at the same sitting.Animals were humanely euthanized 6 months later and the effects of the therapeutic gene transfer was assessed by light and transmission electron microscopy. In addition we injected both eyes of 20 mice with the mutant ND4 and another 20 mice with the wild-type ND4. Both had the FLAG epitope linked to the carboxyl terminus. A month later the animals were euthanized. Using a complex I immunocapture kit (MitoSciences) followed by FLAG immunobloting we assessed whether the human ND4s were assembled into the murine complex I.

Results:: - Immunocapture and immunobloting of the pooled optic nerves revealed a 52 kDa band consistent with expression and integration of the mutant and wild-type human ND4 subunits into the murine complex I. Mean RGC counts for the right eyes treated with the wild-type ND4 were 105 + 11 per 8 x10 ⁵ um². Control left eyes injected with AAV-GFP had a mean RGC count of 117 + 5 per 8 x 10 ⁵ um². These differences were statistically not significant. Comparisons to the normal uninoculated mouse eye with a mean RGC count of 149 + 3.2 per 8 x 10 ⁵ um² were statistically significant ( p<0.05) relative to the values for both eyes with nutant ND4 then normal ND4 or GFP inoculation.

Conclusions:: - Integration of the mutant human R340H ND4 subunit into the murine complex I recapitulated the hallmarks of human mitochondrial disease in the mouse. However, integration of the wild-type human ND4 subunit into the murine complex I offered no protection against RGC loss induced by the mutant ND4. The lack of rescue is likely due to the dominant effect of the mutant human ND4 in the mouse. This effect is unlike LHON in which the presence of any wild-type ND4 confers resistance to optic neuropathy. Therefore it is possible that allotopic expression of wild-type ND4 may still have a protective effect on optic neuropathy in LHON patients who have 100% mutated mitochondrial DNA.

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