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X. Qi, L. Sun, A. Lewin, J. Guy; Allotopic Expression of Mutant ND4 Subunit of Complex I Induces Optic Neuropathy . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1198.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To develop an animal model of Leber Hereditary Optic Neuropathy (LHON). LHON is a disorder characterized by severe bilateral visual loss in patients harboring mutated mitochondrial DNA. The most common mutation is a G to A transition at nucleotide 11778 that results in an argenine to histidine substitution at amino acid 340 in the ND4 subunit of complex I. Methods: Ten mice received intraocular injections of an AAV–ribozyme directed against the murine mt 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 (R340H ND4) in the right eyes and AAV–gfp was injected into the left eyes. A third group received both the mouse ND4 Rz followed by the allotopic mutant ND4 in the right eyes, but the human RzND4 and GFP respectively in the left eyes. The right eyes of a fourth group received the wild–type R340R ND4. Animals underwent three dimensional contrast enhanced fat suppressed MRIs of the optic nerves and brains in a 4.7 Tesla superconducting magnet and were sacrificed for histopathologic examination 1 month post injection. 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 mitochondrial expression of wild–type R340R ND4FLAG within the ganglion cells of the retina, and axons and astrocytes of the optic nerve in 40% of the animals. No histologic abnormality was found in these animals. In contrast animals injected with the AAV expressing mutant R340H ND4 showed ganglion cells with hyperchromatic cytoplasm and nuclear condensation suggestive of apoptosis. Optic nerves showed secondary demyelination with axonal loss. Transmission electron microscopy revealed swelling and vacuolization of mitochondria with dissolution of cristie. Conclusions: While allotopic expression of a normal complex I subunit (ND4) appears to be safe, allotopic expression of a mutant R340H ND4 results in histopathology similar to patients with LHON harboring the G11778A mutation in mitochondrial DNA.
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