The nanometer-scale structure of amyloid-Β visualized by atomic force microscopy

Abstract

Amyloid-beta (A beta) is the major protein component of neuritic plaques found in Alzheimer's disease. Evidence suggests that the physical aggregation state of A beta directly influences neurotoxicity and specific cellular biochemical events. Atomic force microscopy (AFM) is used to investigate the three-dimensional structure of aggregated A beta and characterize aggregate/fibril size, structure, and distribution. Aggregates are characterized by fibril length and packing densities. The packing densities correspond to the differential thickness of fiber aggregates along a zeta axis (fiber height above the x-y imaging surface). Densely packed aggregates ( > or = 100 nm thick) were observed. At the edges of these densely packed regions and in dispersed regions, three types of A beta fibrils were observed. These were classified by fibril thickness into three size ranges: 2-3 nm thick, 4-6 nm thick, and 8-12 nm thick. Some of the two thicker classes of fibrils exhibited pronounced axial periodicity. Substructural features observed included fibril branching or annealing and a height periodicity which varied with fibril thickness. When identical samples were visualized with AFM and electron microscopy (EM) the thicker fibrils (4-6 nm and 8-12 nm thick) had similar morphology. In comparison, the densely packed regions of approximately > or = 100 nm thickness observed by AFM were difficult to resolve by EM. The small, 2- to 3-nm-thick, fibrils were not observed by EM even though they were routinely imaged by AFM. These studies demonstrate that AFM imaging of A beta fibrils can, for the first time, resolve nanometer-scale, zeta-axis, surface-height (thickness) fibril features. Concurrent x-y surface scans of fibrils reveal the surface submicrometer structure and organization of aggregated A beta. Thus, when AFM imaging of A beta is combined with, and correlated to, careful studies of cellular A beta toxicity it may be possible to relate certain A beta structural features to cellular neurotoxicity.

Structural and Dynamic Features of Alzheimer's Aβ Peptide in Amyloid Fibrils Studied by Site-directed Spin Labeling

“…18 of these derivatives were based on the A␤40 peptide, and one derivative, 42R1, was based on the longer A␤42 peptide. All fibrils grown from the R1-labeled A␤ peptides display morphologies similar to those previously reported for wild type A␤ (27)(28)(29)(30). According to atomic force microscopy, electron microscopy of negatively stained samples, and thioflavin T fluorescence measurements, the introduction of R1 at each of the 19 positions did not significantly affect the ability of these modified peptides to form fibrils (data not shown).…”

“…It has previously been reported that A␤ fibrils are polymorphic and display different diameters and morphologies, including smooth, twisted, and branched fibers and fiber bundles (27)(28)(29)(30). These polymorphisms are dependent upon the time of incubation and conditions used.…”

Preparation and characterization of toxic Aβ aggregates for structural and functional studies in Alzheimer's disease research

“…In the presence of monomeric Aβ, all these species convert to mature amyloid fibrils by monomer addition. Mature amyloid fibrils have been described as insoluble, β-sheet-rich structures that are more than 1 µm long, 8-12 nm in diameter and composed of 2-6 protofilament subunits 24,25 . In vitro, synthetic and recombinant forms of Aβ peptides have been shown to form aggregates and amyloid fibrils that are indistinguishable from the ones isolated from AD brain [26][27][28] .…”

Characterization of the Transthyretin Acid Denaturation Pathways by Analytical Ultracentrifugation: Implications for Wild-Type, V30M, and L55P Amyloid Fibril Formation

“…However, several laboratories including our own have provided evidence supporting the existence of numerous preamyloid quaternary structural intermediates (7,16,17). These intermediates seem to play a major role in the amyloid fibril formation process and possibly in the pathological mechanism of amyloid disease (2,17,18).…”