An Ultrastructural Study of Amyloid Intermediates in Aβ_1–42 Fibrillogenesis

Abstract

Many attempts have been made to define early stages and intermediates in amyloid fibrillogenesis that may be susceptible to inhibition. We have developed an in vitro system, based on the use of A beta1-42 peptides, in which the development of prestages of protofilaments and protofilament and fibril formation could, for the first time, be followed by electron microscopy, supported by fluorescence spectrometry. The first recognizable ultrastructures after incubation of A beta1-42 peptides at 37 degrees C were globular subunits (4-5 nm in diameter) that gradually became organized into short protofilaments (30-100 nm), which in turn formed fibrils mainly by lateral association. At this stage, part of the protofilaments were seen first as collaterals protruding from the fibrils and then, as they were gradually incorporated, as buds on the fibril surface. A continuous growth of A beta1-42 fibrils was observed, seemingly originating from a nucleus, which appeared to consist of aggregates of amyloid intermediates. That protofilaments are intermediates also in the in vivo formation of amyloid was supported by the finding that AL fibrils isolated from amyloid tissues also exhibited radiating protofilaments. The demonstrated globular subunits and early formed protofilaments may be attractive targets for inhibition of fibril formation.

Structural properties of Aβ protofibrils stabilized by a small molecule

“…Thus, in an incubation of A␤(1-40) without CLC, aggregates collected at day 2 (within the ThT lag phase) are curvilinear, rough-edged protofibrils (Fig. 4a), as reported (1,2,7,(19)(20)(21). Aggregates collected from the same reaction at day 14 are typical amyloid fibrils (Fig.…”

“…CLC aggregates exhibit fine structure highly reminiscent of a particular type of protofibril often observed by EM (2,19,20) and atomic force microscopy (1,7,21). Normal A␤ protofibrils incubated in the absence of excess A␤ monomer tend to dissociate (7); whereas CLC stabilizes the A␤ aggregates, minimizing dissociation, these aggregates are incapable of elongating via monomer addition (Fig.…”

Localization of Human Serum Amyloid P Component and Heparan Sulfate Proteoglycan in In Vitro‐Formed Aβ Fibrils

“…When extracted from amyloid tissue the AA amyloid ®brils appeared to consist of, from SAP and HSPG detached, ®laments which after coiling assembled into parallel, twisting structures. Contrary to these ®ndings, we could not identify any stage that corresponded to these twisting structures when the in vitro ®brillogenesis of Ab 1±42 was monitored from the oligomeric state to the mature ®bril by EM [9]. Furthermore, when coincubated with Ab peptides the SAP molecules aligned on the surface of the mature ®brils.…”

The Chlamydia pneumoniae Adhesin Pmp21 Forms Oligomers with Adhesive Properties

“…Amyloids are insoluble protein aggregates derived from the conversion of protofibrils into amyloid fibrils, which are formed by proteins characterized by a typical ␤-sheet structure. The commercially available human amyloid ␤ 42 (A␤ 42 ) is an amyloid-like peptide that can also form amyloid fibrils in vitro.…”

“…Our data demonstrate that the monomer (D-Wt-M) forms oligomers that adopt an amyloid-like structure. The Pmp21-D oligomers (collectively referred to as D-Wt-O) are comparable in size and shape to protofibrils of A␤ 42 . Comparison of the oligomerization capacity of D-Wt with that of a previously analyzed mutant form (D-Mt) with poor adhesion properties revealed that the FXXN motif in D-Wt contributes significantly to the formation of oligomers.…”