Abundant lipid and protein components of drusen

Abstract

Background: Drusen are extracellular lesions characteristic of aging and age-related maculopathy, a major retinal disease of the elderly. We determined the relative proportions of lipids and proteins in drusen capped with retinal pigment epithelium (RPE) and in RPE isolated from non-macular regions of 36 human retinas with grossly normal maculas obtained <6 hr after death.

Methodology/principal findings: Druse pellets were examined by light and electron microscopy. Component proteins were extracted using novel methods for preserved tissues, separated, subjected to tryptic digestion and LC-MS(MS)(2) analysis using an ion trap mass spectrometer, and identified with reference to databases. Lipid classes were separated using thin layer chromatography and quantified by densitometry. Major druse components were esterified cholesterol (EC), phosphatidylcholine (PC), and protein (37.5+/-13.7, 36.9+/-12.9, and 43.0+/-11.5 ng/druse, respectively). Lipid-containing particles (median diameter, 77 nm) occupied 37-44% of druse volume. Major proteins include vitronectin, complement component 9, apoE, and clusterin, previously seen in drusen, and ATP synthase subunit beta, scavenger receptor B2, and retinol dehydrogenase 5, previously seen in RPE. Drusen and RPE had similar protein profiles, with higher intensities and greater variability in drusen. C8, part of the complement membrane attack complex, was localized in drusen by immunofluorescence.

Conclusions/significance: At least 40% of druse content is comprised by lipids dominated by EC and PC, 2 components that are potentially accounted for by just one pathway, the secretion of lipoproteins by RPE. Manipulating genes encoding apolipoprotein pathways would be a fruitful approach to producing drusen with high EC content in laboratory animals. Therapies that directly mitigate drusen should prepare for the substantial volume of neutral lipids. The catalog of major druse proteins is nearing completion.

Figure 1. Lipid localization in isolated RPE-capped drusen.

A. Light micrograph of RPE-capped drusen, isolated from extra-macular retina, pelleted, post-fixed by the OTAP method, and sectioned (1 µm). Two drusen in the panel are both considered hard. Bar, 50 µm. B, C. Transmission electron micrographs of RPE-capped drusen that are either untreated (B-1, B-2) or extracted with chloroform-methanol to remove lipids (C-1, C-2). RPE is at the top of B-1 and C-1. B-1, B-2. Drusen have abundant electron-dense (dark) lipid droplets. L, lipofuscin granule; d, druse interior; arrowhead, basal infolding; asterisk, basal laminar deposit. Bar in B-1, 1 µm. Bar in B-2, 200 nm. C-1, C-2. Lipid droplets are removed by chloroform-methanol extraction, leaving electron-lucent profiles (C-2).

Figure 2. Lipid-containing particles in drusen.

Electron-dense profiles were measured by digital planimetry in electron micrographs of drusen like Figure 1B-1, B-2, and equivalent diameters were determined. Descriptive statistics for this population of ∼900 particles are shown. Area fraction, the proportion of druse cross-sectional area occupied by electron-dense lipid, is reported for 2 eyes. See Methods for measurement details.

Figure 3. Lipid Composition.

Mole% of major lipid classes in RPE-capped drusen, RPE, as determined by thin-layer chromatography and densitometry compared to that in BrM lipoprotein particles . Abbreviations for 6 lipid classes are given in the notes to Table 2. As described in the Methods, EC, TG, FA, and UC were separated by a petroleum ether: diethyl ether: acetic acid solvent system, and SPM and PC were separated by chloroform: methanol: ammonium hydroxide.

Figure 4. Complement factor 8 (C8) confirmed in drusen.

Each column shows the same isolated RPE-capped drusen viewed with differential interference contrast microscopy (A,D), a rhodamine filter cube for immunofluorescence (B,E), and immunofluorescence merged with autofluorescence visualized with a fluorescein filter cube, resulting in a bronze -colored RPE (C,F). A, B, C. C8 immunoreactivity D, E, F. Control experiment using goat IgG.

Figure 5. Proteins in RPE-capped Drusen and RPE.

Spectral count ion intensities from mass spectrometry for each of 20 proteins meeting quality criteria (see Methods) from RPE-capped drusen (n = 6 eyes) and RPE (5 of the same 6 eyes). The ion intensity data was log transformed (base 10), visualized using the enhanced heat map feature in Mayday version 2.9 and sorted by druse signal strength. Similar proteins were detectable in drusen and RPE, with a log unit higher intensities in drusen.