April 2010
Volume 51, Issue 13
ARVO Annual Meeting Abstract  |   April 2010
Changes in Mitochondrial Complex I Subunits in Early Stages of Experimental Autoimmune Encephalomyelitis
Author Affiliations & Notes
  • T. D. Arguello
    Ophthalmology, Bascom Palmer Eye Institute, Miami, Florida
  • J. Guy
    Ophthalmology, Bascom Palmer Eye Institute, Miami, Florida
  • Footnotes
    Commercial Relationships  T.D. Arguello, None; J. Guy, None.
  • Footnotes
    Support  R01 EY07982, P30-EY014801 and RPB Inc.
Investigative Ophthalmology & Visual Science April 2010, Vol.51, 1005. doi:
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      T. D. Arguello, J. Guy; Changes in Mitochondrial Complex I Subunits in Early Stages of Experimental Autoimmune Encephalomyelitis. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1005.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: : To evaluate the profile of different mitochondrial subunits of complex I in brain, spinal cord and retina of EAE mice.

Methods: : Experimental autoimmune encephalomyelitis (EAE) was induced in C57BL6 mice by subdermal sensitization with 0.2 ml of homologous spinal cord emulsion in complete freunds adjuvant. Total mitochondrial protein was isolated from brain, spinal cord and retina. Complex I was immunoprecipitated using a kit (MS101 Complex I immunocapture kit) following the manufacture’s specifications. Mitochondrial complex I isolates were separated in a 4-12% gradient polyacrilamide gel that was stained with Coomassie blue and also transfered to PDVF membranes. Membranes were incubated with 5 primary antibodies: NDUFA9, NDUFS3, GRIM19, NDUFB6 and anti-complex I-8 kDa subunit. Detection of the secondary antibody (Horseradish Peroxidase-conjugated bovine Anti-mouse IGg) was obtained with chemilumiscence western blotting detection reagent. Rabbit Polyclonal to VDAC1/Porin was used as mitochondria loading control. Relative intensity ratios of mitochondrial protein loading were normalized relative to porin. Densitometry was used to measure the intensity of the bands.

Results: : Coomassie blue gels revealed a decrease by one third in complex I subunits 80 KD and by half on 50 KD in EAE spinal cord relative to control brain and spinal cord. We also resolved subunits from total mitochondrial protein by western blotting. Subunit NDUFA9 (37 KD) was increased by approximately 50% in EAE spinal cord, but not in the brain or retina. Subunit NDUFS3 (30 KD) showed no differences between EAE and control. Subunit NADUFB6 (15 KD) was almost double in the retina and almost tripled in the spinal cord, but not substantially changed in brains of EAE animals relative to unsensitized controls.

Conclusions: : These findings suggest that reductions in mitochondrial ATP synthesis in EAE and loss of Complex I activity in multiple sclerosis patients may be due to alterations in assembly of certain complex I subunits.

Keywords: neuro-ophthalmology: optic nerve • mitochondria • oxidation/oxidative or free radical damage 

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