Amyloid seeds formed by cellular uptake, concentration, and aggregation of the amyloid-beta peptide

Abstract

One of the neuropathological hallmarks of Alzheimer's disease (AD) is the amyloid plaque, primarily composed of aggregated amyloid-beta (Abeta) peptide. In vitro, Abeta(1-42), the major alloform of Abeta found in plaques, self-assembles into fibrils at micromolar concentrations and acidic pH. Such conditions do not exist in the extracellular fluid of the brain where the pH is neutral and Abeta concentrations are in the nanomolar range. Here, we show that extracellular soluble Abeta (sAbeta) at concentrations as low as 1 nM was taken up by murine cortical neurons and neuroblastoma (SHSY5Y) cells but not by human embryonic kidney (HEK293) cells. Following uptake, Abeta accumulated in Lysotracker-positive acidic vesicles (likely late endosomes or lysosomes) where effective concentrations (>2.5 microM) were greater than two orders of magnitude higher than that in the extracellular fluid (25 nM), as quantified by fluorescence intensity using laser scanning confocal microscopy. Furthermore, SHSY5Y cells incubated with 1 muM Abeta(1-42) for several days demonstrated a time-dependent increase in intracellular high molecular weight (HMW) (>200 kDa) aggregates, which were absent in cells grown in the presence of Abeta(1-40). Homogenates from these Abeta(1-42)-loaded cells were capable of seeding amyloid fibril growth. These results demonstrate that Abeta can be taken up by certain cells at low physiologically relevant concentrations of extracellular Abeta, and then concentrated into endosomes/lysosomes. At high concentrations, vesicular Abeta aggregates to form HMW species which are capable of seeding amyloid fibril growth. We speculate that extrusion of these aggregates may seed extracellular amyloid plaque formation during AD pathogenesis.

Fig. 1.

Cell uptake of FITC-Aβ. (A) SHSY5Y cells, (B) primary murine cortical neurons, and (C) HEK293 cells were cultured in the presence of 250 nM human FITC-Aβ_1–42 for 24 h, then imaged with confocal/phase-contrast microscopy. Vesicular uptake was observed only in the neurons and SHSY5Y cells. (D) SHSY5Y cells were incubated with 250 nM fluorescein alone, (E) FITC-scrambled Aβ_1–42, or (F) FITC-Aβ_1–40 for 24 h. Vesicular uptake was observed with FITC-Aβ_1–42 and Aβ_1–40, but not with FITC-scrambled-Aβ_1–42 or fluorescein alone.

Fig. 2.

Intracellular co-localization of Aβ_1–42 with LysoTracker. SHSY5Y cells, grown in the presence of 250 nM TMR-Aβ_1–42 for 24 h, were imaged 30 min after 50 nM LysoTracker was added to the culture medium. (A, D, and G) TMR- Aβ_1–42 was detected in vesicles that co-stained with LysoTracker (B, E, and H); merged fluorescent images with phase contrast image (C, F, and I) demonstrate co-localization of Aβ and LysoTracker-stained vesicles.

Fig. 3.

Dose- and time-dependence of vesicular uptake of TMR-Aβ_1–42. SHSY5Y cells were grown in varying concentrations of TMR-labeled Aβ_1–42 (1–250 nM as indicated (A), then imaged using confocal microscopy after 24 h. Fluorescent vesicles were quantified, and demonstrated dose-dependent uptake (B). SHSY5Y cells were grown in 250 nM TMR-Aβ_1–42,imaged at varying times (0–72 h) thereafter (Fig. S1A), and fluorescent vesicles were quantified (C). SHSY5Y cells were grown in 250 nM TMR-Aβ_1–42 for 24 h, TMR-Aβ_1–42 was washed out of the medium, imaged a various times thereafter (Fig. S1C), and fluorescent vesicles were quantified (D). After washout, the number of fluorescent vesicles decreased with time, disappearing by 48 h. Error bars, s.e.m. from three independent experiments.

Fig. 4.

SHSY5Y cells, grown in the presence of 25 nM TMR-Aβ_1–42 for 24 h, were imaged using confocal microscopy. The phase image of a group of cells is shown in the x-y plane with the fluorescence intensity plotted on the z axis and projected onto the image. The fluorescence originating from intracellular vesicles is up to two orders of magnitude greater than fluorescence in the extracellular medium (see Fig. S3 A and B), suggesting high effective concentrations of Aβ in the vesicles.

Fig. 5.

Aggregation of intracellular Aβ_1–42 into HMW forms. (A) SHSY5Y cells, grown in the presence or absence of Aβ_1–42 (1 μM) for 5 days, were homogenized and run on a Tris-Tricine gel and blotted with an anti-Aβ antibody (6E10). Culture medium incubated with Aβ_1–42 (1 μM) for 5 days show the presence of only monomers. Extracts from cells grown in Aβ_1–42 show HMW aggregates, while cell grown without Aβ do not (note the non-specific bands in the 10–40 kDa range from the cell extracts). (B–D) SHSY5Y cells were grown in the presence of Aβ_1–42 (0–1,000 nM as indicated) for 5 days (B), for varying times (0–7 days, 1 μM Aβ) (C), or in the presence of human Aβ_1–42, Aβ_1–40, or rat Aβ_1–42 for 5 days (as indicated in D). Cell homogenates were then run on an agarose gel and blotted with an anti-Aβ antibody (6E10). Intracellular Aβ was found to aggregate in a concentration- and time-dependent manner. Human Aβ_1–42 forms HMW intracellular aggregates, but human Aβ_1–40 and rat Aβ_1–42 do not. Human Aβ_1–42 incubated in culture medium alone for 5 days did not form HMW aggregates (D, right lane).

Fig. 6.

Cell extracts from Aβ_1–42-loaded cells seed the formation of amyloid fibrils. SHSY5Y cells incubated with or without 1 μM unlabeled Aβ_1–42 or scrambled Aβ_1–42 for 5 days were homogenized and then incubated with 100 nM TMR-Aβ_1–42 for 48 h. Phase contrast images of the cell extracts show the absence of cells (A–C). Extracts from control cells (grown in the absence of Aβ) did not show TMR fluorescence (D) or Thioflavin-S staining (G); Aβ-loaded cell extracts developed TMR precipitates (E) which stained for Thioflavin-S (H); while scrambled-Aβ-loaded cell extracts showed neither (F and I). These results suggest that intracellular Aβ aggregates can seed the formation of Thioflavin-positive aggregates.