Diagram depicting the anatomical relationships among the retinal pigmented epithelium (RPE), Bruch's membrane (BM), and drusen (Dr). Vesicles containing amyloid β that likely represent primary sites of complement activation are identified within drusen (arrows). CHOR, choroidal vasculature; CAP, capilllary.

Immunolocalization of APP and Aβ in drusen by laser scanning confocal immunofluorescence microscopy. (A–C) Anti-Aβ (6E10) binds vesicular elements within drusen (green; Cy 2 channel) of an 84-year-old male with clinical diagnosis of atrophic AMD. These “amyloid vesicles” frequently exhibit a multilamellar structure (arrows in A and B). Aβ immunoreactivity is concentrated on the outer shell of the vesicle; varying levels of Aβ are associated with the inner lamellae. Images suggesting the fusion and/or budding of vesicles are often observed (arrowheads in A and C). Autofluorescent lipofuscin particles are concentrated in the RPE cytoplasm (red; Cy3 channel). (D) A serial reconstruction of 34 0.3-μm optical sections showing a large amyloid vesicle and several smaller vesicles labeled with anti-Aβ (6E10); sectioning has partially exposed the central cavity (asterisk). Evidence of fusion or budding is apparent near the top of the vesicle (arrowhead) and on the far interior wall. (E and F) Anti-Aβ labeling of RPE cells (E, 6E10; F, 4G8). Fine, granular Aβ immunoreactivity is observed in the cytoplasm of RPE cells overlying drusen (arrows). One RPE cell contains an Aβ-positive structure that is similar in size and shape to the amyloid vesicles within drusen (arrowhead in E). (G) Secondary antibody control illustrating the absence of binding to drusen or RPE cells by the Cy2-conjugated donkey anti-mouse Ig used for the detection of anti-Aβ and anti-APP monoclonal antibodies. Autofluorescent lipofuscin particles (red; Cy3 channel) mark the RPE cells. (H) Anti-APP (22C11) localizes APP to the RPE cell cytoplasm (arrows); however, drusen are not labeled. (I and J) Double-label images illustrating labeling patterns for APP (blue; Cy5 channel) and Aβ (green; Cy2 channel). (I Left) Anti-Aβ (6E10) strongly labels a single large amyloid vesicle (arrow); (Right) In the same section, anti-APP (N-terminal peptide 44–63 polyclonal antibody) binds the same vesicle (arrow), as well as the RPE cytoplasm. (J) In merged images of different amyloid vesicles, punctate regions of both APP (blue, arrowheads) and Aβ immunoreactivity (green) are apparent. Areas of light blue fluorescence are indicative of APP and Aβ colocalization (arrows). (K) Double-label image showing the distributions of Aβ (red; Cy3) and APP (green; Cy2) in cultured human RPE cells. APP shows diffuse cytoplasmic staining, whereas punctate Aβ immunoreactivity is concentrated in the perinuclear region. (L) Anti-Aβ labeling of cultured human RPE cell cytoplasm is abrogated by preadsorption of anti-Aβ antibody (4G8) with Aβ_1→42 peptide. (M–O) Double-label confocal immunofluorescence images showing the distributions of Aβ (green; Cy2) and iC3b, an activation-specific fragment of complement C3 (red; Cy3), in amyloid vesicles. The vesicular surface is punctated by discrete, as well as overlapping, areas of Aβ and/or iC3b immunoreactivity (M). A merged projection series (N) shows that most iC3b immunoreactivity is concentrated in the interior of the vesicles, whereas Aβ predominates in the outer shell. This is illustrated most dramatically in a single optical section (O) through the same vesicle shown in N. Note that discrete areas of colocalization (yellow) are also present on the outer shell (arrows in M and N). BM, Bruch's membrane; D, drusen.

PCR analyses of APP and β-secretase expression by human RPE cells relative to human brain and primary human fibroblasts. (A) Endpoint PCR analysis of APP isoform expression. Primary RPE cell cultures (lane B), as well as an SV40 transformed RPE cell line (lane C), express transcripts for the three major APP isoforms (APP₇₇₀, APP₇₅₁, and APP₆₉₅). A similar pattern of expression is observed in primary fibroblast cultures (lane F). The APP₆₉₅ isoform predominates in both the adult (lane D) and fetal (lane E) brain, as has been reported (25). A fourth unidentified amplicon (asterisk) was detected in all samples examined. Lane A is a 100-bp size reference ladder. (B) Quantitative PCR analyses of the expression of APP₇₅₁, APP₆₉₅, and β-secretase. APP₇₅₁ transcripts are expressed at similar levels in primary RPE cell cultures (HFRPE), SV40 transformed RPE cells (SVRPE), fibroblasts, and in both adult and fetal brain. In contrast, the expression level of APP₆₉₅ in primary RPE cells is 20-fold lower than in adult brain, and more than 40-fold lower than in fetal brain. Similar expression levels are exhibited by SVRPE cells and normal human fibroblasts. The β-secretase expression level in primary RPE cells is approximately one-half of the level in the brain samples. In comparison to the primary RPE cells, SVRPE cells and fibroblasts have somewhat lower and higher β-secretase expression levels respectively. (see also Table 1). (C) Gel analysis of PCR products from the quantitative PCR analyses demonstrating the amplification of appropriately sized products for β-secretase (121 bp), APP₇₅₁ (378 bp), APP₆₉₅ (125 bp), and 18s rRNA (67 bp). (Lane 1, adult brain; Lane 2, fetal brain; lane 3, primary RPE; lane 4, SV40 RPE; lane 5, fibroblasts).