Mitochondrial proteomics of the retinal pigment epithelium at progressive stages of age-related macular degeneration
Curtis L Nordgaard 1*,
Pabalu P Karunadharma 2,
Xiao Feng 1,
Timothy W. Olsen 1,
and
Deborah A. Ferrington 3
1 Department of Ophthalmology, University of Minnesota, Minneapolis, Minnesota, United States
2 Deparment of Ophthalmology, University of Minnesota, Minneapolis, Minnesota, United States; Graduate Program in the Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota, United States
3 Department of Ophthalmology, University of Minnesota, Minneapolis, Minnesota, United States; Graduate Program in the Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota, United States
* To whom correspondence should be addressed. E-mail: nord0152{at}umn.edu.
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Abstract |
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Purpose: 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, we identified changes in the content of several mitochondrial proteins associated with AMD. Herein, we analyzed the sub-proteome of mitochondria isolated from human donor RPE graded with the Minnesota Grading System (MGS).
Methods: Human donor eye bank eyes were categorized into one of four progressive stages (MGS 1-4) based upon the clinical features of AMD. Following dissection of the RPE, mitochondrial proteins were isolated and separated based upon their charge and mass using twodimensional gel electrophoresis. Protein spot densities were compared between the four MGS stages. Peptides from spots that changed significantly with MGS stage were extracted and analyzed 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: Our 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.
Key Words:
age-related macular degeneration, mitochondria, retinal pigment epithelium, proteomics, Minnesota Grading System
