Mitochondrial proteomics in experimental autoimmune uveitis oxidative stress.
OBJECTIVE
Photoreceptor mitochondrial oxidative stress is the initial pathologic event in experimental autoimmune uveitis. In this study, the authors determined alterations in retinal mitochondrial protein levels in response to oxidative stress during the early phase of experimental autoimmune uveitis (EAU).


METHODS
Retinal mitochondrial fractions during early EAU were prepared and subjected to two-dimensional difference in gel electrophoresis (2D-DIGE). Protein spots showing differential expression were excised and subjected to matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) for peptide identification. Levels of these proteins were also confirmed by Western blot analysis. mRNA expression of these proteins was confirmed by real-time PCR. TUNEL staining was performed to detect apoptosis.


RESULTS
2D-DIGE analysis revealed differential expression of 13 proteins. Ten proteins were overexpressed, including manganese-SOD, alphaA crystallin, beta crystallin, and four proteins were downregulated, including adenosine triphosphate (ATP) synthase, malate dehydrogenase, and calretinin. Increased levels of alphaA crystallin, betaB2 crystallin, MnSOD, and aconitase and decreased levels of ATP synthase were confirmed by Western blot analysis. qPCR also confirmed the increased expression of alphaA crystallin, betaB2 crystallin, MnSOD, and Hsp70. Apoptosis was absent during this phase.


CONCLUSIONS
The presence of mitochondrial-specific oxidative stress-related proteins in the early EAU retina along with the downregulation of ATP synthase provides early evidence of stress-related retinal damage. The presence of high levels of alphaA and betaB2 crystallin in the mitochondria may prevent cell death during early EAU.
Saraswathy
Sindhu
S
Doheny Eye Institute, Department of Ophthalmology, Keck School of Medicine of the University of Southern California, Los Angeles, California 90033, USA.
Rao
Narsing A
NA
D000818:Animals; D017209:Apoptosis; D001327:Autoimmune Diseases / Q000378:metabolism*; D015153:Blotting, Western; D004195:Disease Models, Animal; D015180:Electrophoresis, Gel, Two-Dimensional; D005456:Fluorescent Dyes; D015870:Gene Expression; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D020287:In Situ Nick-End Labeling; D007525:Isoelectric Focusing; D051379:Mice; D008928:Mitochondria / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000378:metabolism*; D018384:Oxidative Stress; D040901:Proteomics; D012333:RNA, Messenger / Q000378:metabolism; D012160:Retina / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013482:Superoxide Dismutase / Q000378:metabolism; D014605:Uveitis / Q000378:metabolism*; D038202:alpha-Crystallin A Chain / Q000378:metabolism; D038206:beta-Crystallin B Chain / Q000378:metabolism
D005456:Fluorescent Dyes; D018840:HSP70 Heat-Shock Proteins; D024101:Mitochondrial Proteins; D012333:RNA, Messenger; D038202:alpha-Crystallin A Chain; D038206:beta-Crystallin B Chain; C113634:beta-crystallin B2; D013482:Superoxide Dismutase