Abstract
Purpose: :
Mitochondrial dysfunction mediates neurodegeneration in optic neuritis and multiple sclerosis (MS) that contributes to permanent loss of function. This dreaded sequelae has no remedy. We rescued retinal ganglion cells (RGCs) and optic neuropathy in experimental optic neuritis using gene therapy with the single subunit yeast complex I (NDI1).
Methods: :
Experimental autoimmune encephalomyelitis (EAE) was induced in female DBA/1J mice (n=20). Mice got intravitreal scAAV-NDI1 (n=10) and controls received an scAAV that contained the 23 amino acid COX8 mitochondrial targeting sequence appended to the N terminus of cherry. An additional 10 mice received scAAV-Cox8-mcherry but were unsensitized. RGC's were assessed by serial PERG and OCT at 1, 3 and 6 months post injection (MPI). 6MPI mice were sacrificed, for histolopathology. An additional 6 mice that received NDI1 (OD) and GFP (OS) were sacrificed 3MPI and the retinas were assessed by TUNEL, DHE and DCFDA staining. Expression of the NDI1 in the retina and ONs were evaluated at 15d PI by RT-PCR, immunofluorescence (IF) and western blotting (WB).
Results: :
Expression: IF revealed punctate and perinuclear expression of NDI1, colocalized with GRIM19 and Thy1.2 indicating expression in mitochondria of RGCs. RT-PCR and WB confirmed the NDI1 expression in the retina and ONs. Rescue: PERG analysis at 3M and 6MPI showed a significant reduction, in amplitude of EAE-mcherry compared to control mcherry (42%, 45%) (p=0.0035, p=0.0004), whereas NDI1 rescued mice showed a 13% and 9% reduction in the amplitude (p>0.05). NDI1 significantly rescued PERG amplitude by 67% and 80% in EAE (p=0.029,p=0.0015). PERG latency was delayed by 15% and 21% in EAE-mcherry compared to mcherry control (p<0.05), whereas the NDI1 injected mice rescued this delay by 100% and 56% (p<0.05) at 3M and 6MPI. OCT images showed a significant thinning in EAE-mcherry compared to mcherry control at 3M (16%) and 6MPI (15%) p<0.05. Whereas NDI1 rescued mice showed thickening by 16% and 12% (p<0.05). There was 14.3% TUNEL + cells in RGC layer of GFP control compared to 4% in NDI1 injected eyes (p=0.02). In addition, DHE and DCFDA staining indicated more mitochondrial stress in retina and ONs of GFP control vs NDI1. Ultrastructural analysis of the EAE ONs demonstrated different levels of degeneration in axon, myelin and connective tissues whereas NDI1 ONs showed relatively healthy axons with continuous myelin organized microtubules and healthy looking mitochondria.
Conclusions: :
NDI1 gene therapy is a good candidate for suppression of mitochondrial induced neurodegeneration in optic neuritis and multiple sclerosis.
Keywords: optic nerve • mitochondria • gene transfer/gene therapy