Direct observations of amyloid β self-assembly in live cells provide insights into differences in the kinetics of Aβ(1-40) and Aβ(1-42) aggregation

Abstract

Insight into how amyloid β (Aβ) aggregation occurs in vivo is vital for understanding the molecular pathways that underlie Alzheimer's disease and requires new techniques that provide detailed kinetic and mechanistic information. Using noninvasive fluorescence lifetime recordings, we imaged the formation of Aβ(1-40) and Aβ(1-42) aggregates in live cells. For both peptides, the cellular uptake via endocytosis is rapid and spontaneous. They are then retained in lysosomes, where their accumulation leads to aggregation. The kinetics of Aβ(1-42) aggregation are considerably faster than those of Aβ(1-40) and, unlike those of the latter peptide, show no detectable lag phase. We used superresolution fluorescence imaging to examine the resulting aggregates and could observe compact amyloid structures, likely because of spatial confinement within cellular compartments. Taken together, these findings provide clues as to how Aβ aggregation may occur within neurons.

Graphical abstract

Figure 1

Amyloid Fibril Formation and Fluorescence Lifetimes of the HF488-Labeled Aβ Peptides In Vitro (A) Amyloid formation by ^HF488Aβ(1–42) (5 μM in 50 mM sodium phosphate buffer [pH 7.4]) as a function of time. The quantity of amyloid fibrils was monitored by immunochemistry using the conformation-specific antibody LOC (red circles and dashed line) to detect fibrillar species. The normalized fibril quantity (relative to the maximum value) is given on the right axis in the graph. The amyloid formation was also monitored by the decrease in fluorescence emission intensity of the HF488 dye (blue). a.u., arbitrary units. See also Figure S1A. (B) Far UV circular dichroism spectra of monomeric and fibrillar Aβ(1–42). (C) Fluorescence lifetime images of solutions of monomeric and fibrillar ^HF488Aβ(1–42). The lifetime color coding is shown in the bar to the right of the images. The frequency histograms show the per-pixel distribution of HF488 fluorescence lifetimes in each of the images. (D) Mean fluorescence lifetime (± SD) of the unconjugated HF488 dye at different pH values and for monomeric, fibrillar, and trypsin-treated monomeric ^HF488Aβ(1–40).

Figure 2

Kinetics of ^HF488Aβ(1–40) Fibril Formation Monitored by Fluorescence Lifetime Imaging Fluorescence lifetime images and corresponding frequency histograms showing the evolution of the HF488 fluorescence lifetime in a defined volume of a droplet sample of ^HF488Aβ(1–40) deposited onto a glass-bottomed culture dish and mounted on the FLIM microscope. The color coding of images and histograms relate to the color bar on the right. The ^HF488Aβ(1–40) concentration was 5 μM, and the peptide was diluted into 50 mM sodium phosphate buffer (pH 7.4). See also Figure S2.

Figure 3

Confocal Fluorescence Microscopy Images Showing the Uptake and Intracellular Localization of ^HF488Aβ(1–40) and ^HF488Aβ(1–42) in SH-SY5Y Cells (A and C) Uptake of (A) ^HF488Aβ(1–40) and (C) ^HF488Aβ(1–42) (green) and the endocytosis marker FM 4–64 (red) after 30 min of incubation. The white arrows in the overlay panels indicate colocalization in intracellular vesicles. Note that the peptides do not accumulate at the plasma membrane. (B and D) Colocalization of (B) ^HF488Aβ(1–40) and (D) ^HF488Aβ(1–42) (green) with LysoTracker red (red) after 24 hr of incubation with 500 nM peptide followed by 1 hr of incubation with 100 nM LysoTracker red. (E) Uptake of ^HF488Aβ(1–40) and ^HF488Aβ(1–42) upon incubation at 37°C (allowing endocytotic internalization) and 4°C (inhibiting endocytotic internalization). The images show the overlay of HF488 fluorescence (green) and the transmitted image. All scale bars represent 10 μm. See also Figure S3.

Figure 4

Fluorescence Lifetime Imaging of the Uptake and Accumulation of ^HF488Aβ(1–40) and ^HF488Aβ(1–42) in Live SH-SY5Y Cells (A and B) Cells treated with (A) 500 nM ^HF488Aβ(1–40) or (B) 500 nM ^HF488Aβ(1–42) for 0–6 hr. The upper panels show fluorescence intensity, the center panels show HF488 fluorescence lifetimes, and the lower panels show the normalized per-pixel HF488 fluorescence lifetime frequency (Norm. Freq.) in the corresponding image above. The histograms at the 0 hr time point show the HF488 fluorescence lifetime distribution of the extracellular peptides (E.C.). Subsequent histograms (1, 3, and 6 h) show the HF488 fluorescence lifetime distributions of the intracellular peptides only. See the color bar in (C) for the color coding of FLIM images and frequency histograms. (C) Cells treated with 500 nM ^HF488Aβ(1–40) or 500 nM ^HF488Aβ(1–42) for 24 or 48 hr. The cells were washed once prior to imaging. The images show an overlay of the color-coded FLIM image and the transmitted image. The HF488 fluorescence lifetime frequency histograms show the distribution over several analyzed images (n = 6–9). Trans., transmission. (D) Mean HF488 fluorescence lifetime (± SEM) of extracellular and intracellular ^HF488Aβ(1–40) and ^HF488Aβ(1–42). At 0 hr, the bars represent the mean HF488 fluorescence lifetime (± SEM) of the extracellular peptide (E.C.). The subsequent bars show the mean HF488 fluorescence lifetime (± SEM) of the intracellular peptides at the indicated time points after addition of 500 nM ^HF488Aβ(1–40) or 500 nM ^HF488Aβ(1–42) to the culture medium. Asterisks (^∗) over bars indicate a significant difference compared to 0 h (E.C.), and lines over bars indicate a significant difference between the two peptides at the specified time points according to Fisher’s least significant difference test (p < 0.05). ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001. Individual p values are given in Tables S1 and S2.

Figure 5

Super-Resolution Fluorescence Microscopy Imaging of Intracellular Aβ(1–40) and Aβ(1–42) (A) dSTORM images of intracellular ^HF647Aβ(1–40) and ^HF647Aβ(1–42) in fixed SH-SY5Y cells. The cells were imaged after 24 and 48 hr of incubation. Scale bars represent 1 μm. (B) Mean size of the intracellular aggregates (± SEM) estimated by pixel counting in dSTORM images. The lines above the bars indicate statistically significant mean differences (paired Student’s t test; ^∗p < 0.05; n = 18–27). Only aggregates with dimensions exceeding the resolution limit were included in the analysis. (C) Mean fluorescence lifetime (± SEM) of intracellular ^HF488Aβ(1–40) and ^HF488Aβ(1–42) at the time points corresponding to the dSTORM images. The data were extracted from Figure 4D.