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K. G. Shadrach, N. Neric, X. Gu, J. S. Crabb, S. K. Bhattacharya, M. E. Rayborn, J. G. Hollyfield, V. L. Bonilha; Retinal Pigment Epithelium (RPE) Protein Changes During Aging: A Proteomic Approach. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2160.
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Age-related changes in the retina are often precursors of visual impairment among the elderly. Functional alterations in the RPE can lead to photoreceptor degeneration. Very little is known about the mechanisms underlying age-related changes in the RPE. Toward a better molecular understanding of the aging RPE, we pursued proteomic analyses of RPE microvilli and RPE cells without microvilli from young and old rats.
RPE apical microvilli were isolated using WGA-agarose beads and the RPE cells that remained following the loss of their microvilli were also isolated from rat eyecups. RPE fractions were obtained from young adults (3-4 month-old) and aged (24-25 month-old) F1 hybrid between Fisher 344 (F344) and Brown Norway rats. These enriched fractions were further fractionated by extraction with a triton X100-containing buffer and subjected to SDS-PAGE. Proteins were excised, digested in situ with trypsin, peptides were analyzed by LC MS/MS and proteins were identified with the Mascot search engine and Swiss-Protein sequence database. In addition, Bioinformatic analysis was performed with Ingenuity Pathway Analysis 5.0.
Proteomic analyses yielded the identity of 259 RPE microvilli proteins from young rats and 289 microvilli proteins from aged rats. Following removal of the microvilli, 478 proteins were identified from the younger RPE and 226 proteins from the aged RPE cells. Immunohistochemical and Western blot analyses are underway to verify the localization of select proteins.
The RPE fractionation method utilized in this study has facilitated the detection of proteins not previously identified in these cells. Moreover, the aged RPE protein database will be important to direct future studies in age-related diseases affecting the RPE cells.
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