Aggregation and fibril morphology of the Arctic mutation of Alzheimer's Aβ peptide by CD, TEM, STEM and in situ AFM

Abstract

Morphology of aggregation intermediates, polymorphism of amyloid fibrils and aggregation kinetics of the "Arctic" mutant of the Alzheimer's amyloid β-peptide, Aβ((1-40))(E22G), in a physiologically relevant Tris buffer (pH 7.4) were thoroughly explored in comparison with the human wild type Alzheimer's amyloid peptide, wt-Aβ((1-40)), using both in situ atomic force and electron microscopy, circular dichroism and thioflavin T fluorescence assays. For arc-Aβ((1-40)) at the end of the 'lag'-period of fibrillization an abrupt appearance of ≈ 3 nm size 'spherical aggregates' with a homogeneous morphology, was identified. Then, the aggregation proceeds with a rapid growth of amyloid fibrils with a variety of morphologies, while the spherical aggregates eventually disappeared during in situ measurements. Arc-Aβ((1-40)) was also shown to form fibrils at much lower concentrations than wt-Aβ((1-40)): ≤ 2.5 μM and 12.5 μM, respectively. Moreover, at the same concentration, 50 μM, the aggregation process proceeds more rapidly for arc-Aβ((1-40)): the first amyloid fibrils were observed after c.a. 72 h from the onset of incubation as compared to approximately 7 days for wt-Aβ((1-40)). Amyloid fibrils of arc-Aβ((1-40)) exhibit a large variety of polymorphs, at least five, both coiled and non-coiled distinct fibril structures were recognized by AFM, while at least four types of arc-Aβ((1-40)) fibrils were identified by TEM and STEM and their mass-per-length statistics were collected suggesting supramolecular structures with two, four and six β-sheet laminae. Our results suggest a pathway of fibrillogenesis for full-length Alzheimer's peptides with small and structurally ordered transient spherical aggregates as on-pathway immediate precursors of amyloid fibrils.

Figure 1

Thioflavin-T fluorescence intensity at 482 nm for arc-Aβ_(1-40) (filled circles) and wt-Aβ_(1-40) (open circles) as a function of time from the onset of incubation of 50 μM peptide solutions in TRIS (pH 7.4).

Figure 2

CD spectra of: (A) wt-Aβ_(1-40) and (B) arc-Aβ_(1-40) peptides obtained at different time (days) after onset of incubation of 50 μM peptide solutions in Tris (pH 7.4); (C) CONTIN analysis of CD spectra of wt-Aβ_(1-40) using DICHROWEB interactive software (Whitmore and Wallace, 2004); (D) Ellipticity at 217 nm (a measure of a content of β-structures) as a function of the incubation time of arc-Aβ_(1-40).

Figure 3

AFM height images of wt-Aβ_(1-40) aggregates at different time points. All images are of size 4000 nm and grayscale coded with the height from 0 (dark) to 30 nm (bright), except for the inset images, which are of size 200 nm × 200 nm, with a height scale 0–20 nm, to better visualize individual aggregates. (A) 55 h; (B) 145 h; (C) 172 h and (D) 219 h after the onset of the sample incubation. Virtual additional faint images in inserts of A and C are unavoidable artefacts from a ‘double’ AFM tip, because of aggregates of the peptide adhered to the tip.

Figure 4

AFM height images of arc-Aβ_(1-40) aggregates at different time points. The image scales are identical to Fig. 3. (A) 2.5 h; (B) 24 h; (C) 72 h and (D) 74 h after the onset of the aggregation reaction.

Figure 5

Aggregate heights: (A–F) are probability histograms of the aggregate heights at different times after the onset of the sample incubation. Each histogram represents height measurements of 200–600 aggregates in images of scan size 2 μm. (A–C) Height histograms of the wt-Aβ_(1-40) aggregates at times 55, 145 and 172 h. (D–F) Height histograms of arc-Aβ_(1-40) aggregates at times 2.5, 24 and 72 h. The smaller inset histograms are extracted from images of scan size 1 μm. The axis scaling is identical for all histograms. (G–H) Profiles along the dashed lines in the images of Figs. 3 and 4.

Figure 6

AFM height images reveal differences between wt-Aβ_(1-40) and arc-Aβ_(1-40) in critical peptide concentrations of fibrillization. All image sizes are 10 μm. (A and B) Wild type Aβ_(1-40) at peptide concentrations 12.5 and 2.5 μM. (C and D) Fibrillization of Arctic Aβ_(1-40) at peptide concentrations 6.3 and 2.5 μM. More extensive fibril formation is observed for arc-Aβ_(1-40) despite lower concentrations of the peptide.

Figure 7

Morphology of Aβ fibrils visualized with AFM height images. The height scale is in every image grayscale coded from 0 (dark) to 18 nm (bright). (A) A typical ‘quiescent’ fibril of wt-Aβ_(1-40). (B–H) Polymorphism of arc-Aβ_(1-40) fibrils. These fibrils are classified into the different types. The Type 1 and Type 2 are non-coiled fibrils and the Type 3 – Type 5 are left-handed coiled fibrils. (I) A schematic fibril illustrating the path along the fibril axis, for which fibril cross-sections are shown in J. (J) Profiles of cross-sections of different types of both wt-Aβ_(1-40) and arc-Aβ_(1-40) fibrils. The profiles are offset in height for convenience, with minimum values shown as dashed lines.

Figure 8

(A) Selection of four major fibril types I – IV of arc-Aβ_(1-40) identified in transmission electron micrographs of negatively stained specimens. (B) Dark-field STEM image of unstained fibrils with co-deposited TMV. Fibril types I and III are present in this one field of view. White boxes show characteristic regions along fibrils, TMV and background used to calculate MPL values. (C) Major fibril types I through IV imaged in STEM. (D) Histograms of MPL measurements on the four different fibril morphologies depicted in (C). Each individual peak was fitted to a Gaussian function yielding MPL values of 19.7, 37.1, 37.0, and 54.2 kDa/nm for fibrils I through IV, respectively. The integer values above the histograms indicate for each fibril type the number of arc-Aβ_(1-40) molecules per cross-β repeat. Scale bars, 20 nm (A, C) and 100 nm (B).

Figure 9

Real time AFM height imaging of arc-Aβ_(1-40) Type 3 fibril growth on a mica surface. The panel of sequential images (A–I) shows the same 645 × 860 nm² area at different times. The reference time (0 h 00 min) is arbitrary chosen as the time for the 73 hours old sample (incubated in the test tube) in A. The time in hours and minutes is shown in each image. The vertical height scales are equal for all images, from 0 (dark) to 18 nm (bright). All images are digitally zoomed from original images of size 2 × 2 μm except for A, which was zoomed from a 6 × 6 μm image and hence it has a poorer resolution. The black arrow in all images is set as a reference point, i.e. indicating the same surface spot in each image. Apparently, a number of spherical aggregates decreases as the fibrils polymerize. A virtual additional faint image in B–D and F is an artefact from an unavoidable double AFM tip because of aggregates of the peptide adhered to the tip. The fibril partly seen in the lower right corner of each image is of Type 4.