Abstract
Purpose: :
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.
Methods: :
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).
Results: :
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.
Conclusions: :
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.
Keywords: gene transfer/gene therapy • optic nerve • mitochondria