Spontaneous Aggregation and Cytotoxicity of the β-Amyloid Aβ1–40: A Kinetic Model

Abstract

The time dependency of the spontaneous aggregation of the fibrillogenic beta-amyloid peptide, Abeta1-40, was measured by turbidity, circular dichroism, HPLC, and fluorescence polarization. The results by all methods were comparable and they were most consistent with a kinetic model where the peptide first slowly forms an activated monomeric derivative (AM), which is the only species able to initiate, by tetramerization, the formation of linear aggregates. The anti-Abeta antibody 6E10, raised against residues 1-17, at concentrations of 200-300 nM delayed significantly the aggregation of 50 microM amyloid peptide. The anti-Abeta antibody 4G8, raised against residues 17-24, was much less active in that respect, while the antibody A162, raised against the C-terminal residues 39-43 of the full-length Abeta was totally inactive at those concentrations. Concomitant with the aggregation experiments, we also measured the time dependency of the Abeta1-40-induced toxicity toward SH-EPI cells and hippocampal neurons, evaluated by SYTOX Green fluorescence, lactate dehydrogenase release, and activation of caspases. The extent of cell damage measured by all methods reached a maximum at the same time and this maximum coincided with that of the concentration of AM. According to the kinetic scheme, the latter is the only transient peptide species whose concentration passes through a maximum. Thus, it appears that the toxic species of Abeta1-40 is most likely the same transient activated monomer that is responsible for the nucleation of fibril formation. These conclusions should provide a structural basis for understanding the toxicity of Abeta1-40 in vitro and possibly in vivo.

Tissue damage in the amyloidoses: Transthyretin monomers and nonnative oligomers are the major cytotoxic species in tissue culture

“…The authors suggest that there may also be a shared prefibrillar conformation, not detectable in mature fibrils, which is responsible for cytotoxicity in tissue culture, and perhaps in vivo. This hypothesis is supported by other reports where solutions containing early aggregates (as well as their precursors) of TTR amyloidogenic mutants (20,21), and of amyloid-␤ (22), have shown cytotoxic effects on neurons and other cell lines. Our data suggest that the size of such cytotoxic intermediates can range from that of the monomer to something approaching a hexamer (Ͻ100 kDa).…”

The Interaction of Amyloid-β with ApoE

“…The integrity of the ApoE reconsituted lipoprotein particles (made the same day of the experiments) was checked by ultracentrifligation prior to use in the experiments and the AP(l-40) aggregation reactions were carried out in a standardized format which gave highly reproducible results . The AP(l-40) aggregation reaction proceeded in a three step process consistent with that reported by Taylor et al (2003); a lag phase of 40 minutes, a phase in which intermediate sized species were formed and a third phase in which the formation of fibrils occurred. Based on kinetic anaylsis the intermediate species of Ap reached its highest concentration at 60 minutes which coincided with the time that the lipidated ApoE demonstrated an isoform specific binding to the Ap(l-40) aggregate.…”

Temperature dependence of the nucleation constant rate in β amyloid fibrillogenesis

“…One of the best ways to achieve purity and homogeneity of the starting material is by pretreatment with acid or solvents, such as HFIP [22]. In addition to rigorous sample purification, the aggregation mixture needs to be stirred at a constant speed in order to obtain rates consistent from day to day [23]. the typical S-shape is observed.…”

“…Several authors [23,[26][27][28] have indicated that, prior to the nucleation step, the unactivated monomer must be activated. This process of activation is a slow conformational transition, but has to take place in the peptide for the transformation of soluble peptide to amyloid peptide.…”