Mitochondrial proteomics of the retinal pigment epithelium at progressive stages of age-related macular degeneration.
OBJECTIVE
Age-related macular degeneration (AMD) is the leading cause of vision loss in individuals over the age of 65. Histopathological changes become evident in the retinal pigment epithelium (RPE), a monolayer that provides metabolic support for the overlying photoreceptors, even at the earliest stages of AMD that precede vision loss. In a previous global RPE proteome analysis, changes were identified in the content of several mitochondrial proteins associated with AMD. In this study, the subproteome of mitochondria isolated from human donor RPE graded with the Minnesota Grading System (MGS) was analyzed.


METHODS
Human donor eye bank eyes were categorized into one of four progressive stages (MGS 1-4) based on the clinical features of AMD. After dissection of the RPE, mitochondrial proteins were isolated and separated by two-dimensional gel electrophoresis based on their charge and mass. Protein spot densities were compared between the four MGS stages. Peptides from spots that changed significantly with MGS stage were extracted and analyzed by using mass spectrometry to identify the protein.


RESULTS
Western blot analyses verified that mitochondria were consistently enriched between MGS stages. The densities of eight spots increased or decreased significantly as a function of MGS stage. These spots were identified as the alpha-, beta-, and delta-ATP synthase subunits, subunit VIb of the cytochrome c oxidase complex, mitofilin, mtHsp70, and the mitochondrial translation factor Tu.


CONCLUSIONS
The results are consistent with the hypothesis that mitochondrial dysfunction is associated with AMD and further suggest specific pathophysiological mechanisms involving altered mitochondrial translation, import of nuclear-encoded proteins, and ATP synthase activity.
Nordgaard
Curtis L
CL
Department of Ophthalmology, University of Minnesota, Minneapolis, Minnesota, USA.
Karunadharma
Pabalu P
PP
Feng
Xiao
X
Olsen
Timothy W
TW
Ferrington
Deborah A
DA
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000951:Antigens, Neoplasm / Q000378:metabolism; D015153:Blotting, Western; D018450:Disease Progression; D003576:Electron Transport Complex IV / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D005136:Eye Proteins / Q000378:metabolism*; D005260:Female; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D008268:Macular Degeneration / Q000378:metabolism* / Q000503:physiopathology; D008297:Male; D013058:Mass Spectrometry; D008875:Middle Aged; D008928:Mitochondria / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000378:metabolism; D025261:Mitochondrial Proton-Translocating ATPases / Q000378:metabolism; D009124:Muscle Proteins / Q000378:metabolism; D010444:Peptide Elongation Factor Tu / Q000378:metabolism; D010857:Pigment Epithelium of Eye / Q000378:metabolism*; D040901:Proteomics / Q000379:methods
D000951:Antigens, Neoplasm; D005136:Eye Proteins; D018840:HSP70 Heat-Shock Proteins; C088548:IMMT protein, human; D007527:Isoenzymes; D024101:Mitochondrial Proteins; D009124:Muscle Proteins; C091648:TUFM protein, human; D003576:Electron Transport Complex IV; D010444:Peptide Elongation Factor Tu; D025261:Mitochondrial Proton-Translocating ATPases