title: Complex I subunit gene therapy with NDUFA6 ameliorates neurodegeneration in EAE.
abstract: OBJECTIVE
  To address the permanent disability induced by mitochondrial dysfunction in experimental autoimmune encephalomyelitis (EAE).
  
  
  METHODS
  Mice sensitized for EAE were rescued by intravitreal injection of adeno-associated viral vector serotype 2 with the complex I subunit gene scAAV-NDUFA6Flag. Controls were injected with a mitochondrially targeted red fluorescent protein (scAAV-COX8-cherry). Another group received scAAV-COX8-cherry, but was not sensitized for EAE. Serial pattern electroretinograms (PERGs) and optical coherent tomography (OCT) evaluated visual function and structure of the retina at 1, 3, and 6 months post injection (MPI). Treated mice were killed 6 MPI for histopathology. Immunodetection of cleaved caspase 3 gauged apoptosis. Complex I activity was assessed spectrophotometrically. Expression of NDUFA6Flag in the retina and optic nerve were evaluated between 1 week to 1 month post injection by RT-PCR, immunofluorescence and immunoblotting.
  
  
  RESULTS
  Reverse transcription-PCR and immunoblotting confirmed NDUFA6Flag overexpression with immunoprecipitation and blue native PAGE showing integration into murine complex I. Overexpression of NDUFA6Flag in the visual system of EAE mice rescued retinal complex I activity completely, axonal loss by 73%, and retinal ganglion cell (RGC) loss by 88%, RGC apoptosis by 66%, and restored the 33% loss of complex I activity in EAE to normal levels; thereby, preventing loss of vision indicated by the 43% reduction in the PERG amplitudes of EAE mice.
  
  
  CONCLUSIONS
  NDUFA6 gene therapy provided long-term suppression of neurodegeneration in the EAE animal model suggesting that it may also ameliorate the mitochondrial dysfunction associated with permanent disability in optic neuritis and MS patients.
authors.affiliation: Bascom Palmer Eye Institute, University of Miami, Miller School of Medicine, Miami, Florida, United States.
authors.forename: Venu
authors.initials: V
authors.lastname: Talla
authors.affiliation: Bascom Palmer Eye Institute, University of Miami, Miller School of Medicine, Miami, Florida, United States.
authors.forename: Rajeshwari
authors.initials: R
authors.lastname: Koilkonda
authors.affiliation: Bascom Palmer Eye Institute, University of Miami, Miller School of Medicine, Miami, Florida, United States.
authors.forename: Vittorio
authors.initials: V
authors.lastname: Porciatti
authors.affiliation: Departments of Ophthalmology, University of Florida, College of Medicine, Gainesville, Florida, United States.
authors.forename: Vince
authors.initials: V
authors.lastname: Chiodo
authors.affiliation: Departments of Ophthalmology, University of Florida, College of Medicine, Gainesville, Florida, United States.
authors.forename: Sanford L
authors.initials: SL
authors.lastname: Boye
authors.affiliation: Departments of Ophthalmology, University of Florida, College of Medicine, Gainesville, Florida, United States.
authors.forename: William W
authors.initials: WW
authors.lastname: Hauswirth
authors.affiliation: Bascom Palmer Eye Institute, University of Miami, Miller School of Medicine, Miami, Florida, United States.
authors.forename: John
authors.initials: J
authors.lastname: Guy
mesh_terms: D000818:Animals; D017209:Apoptosis; D004247:DNA / Q000235:genetics; D004596:Electroretinography; D004681:Encephalomyelitis, Autoimmune, Experimental / Q000175:diagnosis / Q000235:genetics / Q000628:therapy*; D005260:Female; D005786:Gene Expression Regulation; D015316:Genetic Therapy / Q000379:methods*; D015151:Immunoblotting; D058449:Intravitreal Injections; D051379:Mice; D008811:Mice, Inbred DBA; D046529:Microscopy, Electron, Transmission; D024101:Mitochondrial Proteins / Q000008:administration & dosage* / Q000096:biosynthesis / Q000235:genetics; D012165:Retinal Ganglion Cells / Q000378:metabolism / Q000648:ultrastructure; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013053:Spectrophotometry; D041623:Tomography, Optical Coherence; D016896:Treatment Outcome
chemical_list: D024101:Mitochondrial Proteins; C530233:NDUFAF6 protein, human; D004247:DNA
keywords: mitochondria; multiple sclerosis; optic neuritis