Amyloid precursor protein associates with a nicastrin-dependent docking site on the presenilin 1-gamma-secretase complex in cells demonstrated by fluorescence lifetime imaging

Abstract

Gamma-secretase cleavage is the final enzymatic step generating beta-amyloid via intramembranous cleavage of the amyloid precursor protein (APP). Presenilin (PS), initially identified as a gene in which mutations account for the vast majority of early-onset autosomal dominant Alzheimer's disease, is a major component of gamma-secretase. Enzymatic activity also depends on nicastrin, Aph-1, and Pen-2. We propose a model in which gamma-secretase components assemble, interact with substrates initially at a docking site, and then cleave and release substrates. To test this model, we developed a novel morphological technique on the basis of advanced fluorescence microscopy methods, fluorescence lifetime imaging microscopy (FLIM). FLIM allows us to examine protein-protein "proximity" in intact cells. We show that, although the strongest colocalization of APP and PS1 is in the perinuclear area, the strongest interactions detected by FLIM are at or near the cell surface. We also found that APP-PS1 interactions occur even when gamma-secretase inhibitors or "dominant-negative" PS1 mutations are used to block gamma-secretase activity. Finally, using nicastrin RNA interference, we demonstrate that nicastrin is critical for APP association with PS1. We interpret these results to suggest that there is a noncatalytic docking site closely associated with PS1-gamma-secretase.

Figure 1.

A, Color-coded images of the negative (FITC only) and positive (Cy3 anti-FITC) controls. The rainbow scale shows fluorescence lifetime as color; if molecules are closer to each other, donor fluorescence (FITC) lifetime is shorter and the color will be closer to red. The graphs show lifetime distribution collected for every pixel of the images; positive control shows a shift to the left. B, The Cy3 acceptor in one-half of the cell (positive control) was destroyed by photobleaching (outlined area), leading to dequenching of the FITC fluorescence intensity and a shift to a longer lifetime.

Figure 2.

A, B, Confocal microscope images of the cells that were double immunostained with FITC-labeled PS1 (A), and Cy3-labeled APP (B) antibodies demonstrate predominantly perinuclear localization of the proteins. Scale bar, 10 μm.

Figure 3.

FLIM analysis of the proximity between APP and PS1 molecules within the cell. A, Intensity image shows standard immunostaining pattern for PS1, similar to that shown in Figure 2 A. B, Color-coded FLIM image shows lifetimes, reflecting proximity between PS1 and APP. The cell regions showing closest proximity between PS1 and APP are in the distal compartments near the cell surface. Colorimetric scale shows fluorescence lifetime in picoseconds. C, D, Enlarged boxed areas from B. The FLIM image is superimposed onto a table with calculated average lifetimes for each pixel of the image. Scale bar, 0.2 μm.

Figure 4.

Photobleach dequenching FRET between APP and PS1 demonstrates close proximity between C terminus of APP and loop region of PS1 (A). Cy3-labeled APP (emission, 568 nm) and FITC-labeled PS1 (emission, 488 nm) before and after photobleaching the acceptor (Cy3) in a selected area within the cell. DAPT does not prevent close association of APP with PS1 (B). Scale bar, 10 μm.

Figure 5.

A–D, Confocal microscope images of wtPS1 (A, B) and aspartate mutant (D257A) PS1 (C, D) double stained with biotinylatedγ-secretase inhibitor WPE-III-31C-bi (A, C) and PS1 antibody (B, D). PS1 immunostaining colocalizes with Cy3 streptavidin-labeled WPE-III-31C-bi in the cells expressing wtPS1. WPE-III-31C-bi does not bind to aspartate mutant PS1 holoprotein. Scale bar, 20 μm.

Figure 6.

Nicastrin is important for the association of APP with PS1–γ-secretase complex. A, Nct RNAi leads to a significant inhibition of nicastrin expression in the cells. B, A decrease in the FITC–PS1 lifetime in APP–PS1 double-immunostained cells is observed in mock-treated cells, indicating a close proximity between the two proteins. This association is eliminated by Nct RNAi treatment because the FITC lifetime becomes the same as in the PS1–FITC alone control.