Purchase this article with an account.
D. Garland, F. Li, K. Speicher, D. Speicher, E. Pierce; Proteomic Analysis of Bruch's Membrane/Choroid from Efemp1-R345W Knockin Mice. Invest. Ophthalmol. Vis. Sci. 2009;50(13):790.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
The EFEMP1-R345W mutation is pathogenic for inherited macular degeneration Doyne Honeycomb Retinal Dystrophy/Malattia Leventinese and the Efemp1-R345W mutation is pathogenic in knockin mice. The mutant mice develop basal deposits between Bruch’s membrane and the RPE. Since basal deposits are considered precursor to drusen, this study was initiated to elucidate the processes involved in the formation of the deposits. Proteomic technology was used to analyze the Bruch’s membrane/choroid of knockin and control mice.
After removal of neural retina and RPE, the deposits remain with the Bruch’s membrane/choroid. Bruch’s membrane with adherent choroid was peeled from the eyes of homozygous Efemp1-R345W and control mice, 8 and 14 month, ages before and after deposit formation, respectively. Samples combined from 2-3 mice were sonicated briefly in a solubilizing cocktail. The solubilized proteins were separated on 1D SDS gels. Each lane was cut into 20 slices which were subjected to in-gel digestion with trypsin. The peptide masses were determined using an LTQ-FT ICR mass spectrometer. Peptide sequences and proteins were identified and analyzed using both Bioworks and Scaffold. Protein quantities were estimated by spectral counting.
In each sample about 1000 proteins were identified by 2 or more peptides. About 20% of the proteins were determined to have altered levels as determined by both the absolute differences and the relative ratios among samples from wild type and mutant mice. The identification of cell processes that may be involved in the pathogenesis of basal deposit formation was based on changes in clusters of proteins associated with the process. Of particular significance were the changes at both ages in expression of extracellular matrix (Bruch’s membrane) components, the increased expression of proteins associated with antigen presentation and the complement pathway including complement component C4b, and the decreased expression of proteins involved in vesicle biology.
The identification of changes in the immune related pathways like antigen presentation and the complement pathway suggest the pathogenesis of basal deposit formation may involve multiple immune responses. The significant increase in complement component C4b supports a role of the classical complement pathway. The identification of altered cellular processes before basal deposits develop suggests additional avenues for investigation in elucidating mechanisms of basal deposit formation.
This PDF is available to Subscribers Only