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Jianwen Liu, Venu Talla, Sacide S. Ozdemir, Tsung-Han Chou, Vittorio Porciatti Porciatti, John Guy; Overexpression of the NDUFA6 Subunit of Complex I Ameliorates Neurodegeneration in Experimental Optic Neuritis. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1908.
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We have previously shown that peroxynitrite mediates nitration of nuclear-encoded complex I subunit NDUFA6 in the EAE animal model of MS and that genetic knockdown of another supernumerary complex I subunit (NDUFA1) mediates neurodegeneration that contributes to the permanent disability in optic neuritis and MS. Here we overexpressed NDUFA6 to rescue visual loss and optic neuropathy.
EAE was induced in female DBA/1J mice (n=20). Ten mice were rescued by intravitreal injection of scAAV-NDUFA6 to which a FLAG epitope was attached to the C terminus. Ten controls were injected with cherry to which a mitochondrial targeting sequence (COX8) was appended to the N terminus (scAAV-COX8-mcherry). Another group of 10 mice were injected with scAAV-COX8-mcherry but were not sensitized for EAE. Serial PERG and OCT evaluated visual function and structure of the inner retina at 1, 3 and 6 months post injection (MPI). All mice were sacrificed 6MPI for histopathology. Expression of NDUFA6-FLAG in the retina and ONs were evaluated at 15 days post injection by RT-PCR, immunofluorescence (IF) and western blotting (WB).
Expression: Immunofluorescence revealed a typical punctate and perinuclear expression of NDUFA6FLAG that co-localized with mitochondrial porin and RGC thy1.2. RT-PCR and WB confirmed NDUFA6FLAG overexpression in the retina and ONs. Rescue: PERG analysis at 3M and 6MPI showed a 42% and 45% reduction in amplitude of EAE-mCherry compared to control mCherry (p<0.005,). NDUFA6 injection rescued this amplitude by 100% and 77.6% respectively (p<0.05). PERG latency was delayed by 15% and 21% in EAE-mCherry compared to mCherry control (p<0.05), whereas the NDUFA6 injected mice rescued the delay by 100% and 76% respectively at 3M and 6MPI. OCT images showed a significant thinning in EAE-mCherry retina compared to unsensitized mCherry animals at 3M (16%) and 6MPI (15%) p<0.05, whereas NDUFA6 rescued this thinning by 100% (p<0.05). The ultrastructural analysis of the EAE ONs demonstrated different levels of degeneration in axon, myelin and connective tissues. Degenerated axons showed a varied mitochondrial number, shape and morphology. NDUFA6 ONs showed a relatively continuous myelin with organized microtubules and normal looking mitochondria in the axons.
NDUFA6 gene therapy suppressed RGC degeneration and optic neuropathy in experimental optic neuritis suggesting that it may also ameliorate neurodegeneration in optic neuritis and MS patients.
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