Role of aggregation conditions in structure, stability, and toxicity of intermediates in the Abeta fibril formation pathway

Abstract

beta-amyloid peptide (Abeta) is one of the main protein components of senile plaques associated with Alzheimer's disease (AD). Abeta readily aggregates to forms fibrils and other aggregated species that have been shown to be toxic in a number of studies. In particular, soluble oligomeric forms are closely related to neurotoxicity. However, the relationship between neurotoxicity and the size of Abeta aggregates or oligomers is still under investigation. In this article, we show that different Abeta incubation conditions in vitro can affect the rate of Abeta fibril formation, the conformation and stability of intermediates in the aggregation pathway, and toxicity of aggregated species formed. When gently agitated, Abeta aggregates faster than Abeta prepared under quiescent conditions, forming fibrils. The morphology of fibrils formed at the end of aggregation with or without agitation, as observed in electron micrographs, is somewhat different. Interestingly, intermediates or oligomers formed during Abeta aggregation differ greatly under agitated and quiescent conditions. Unfolding studies in guanidine hydrochloride indicate that fibrils formed under quiescent conditions are more stable to unfolding in detergent than aggregation associated oligomers or Abeta fibrils formed with agitation. In addition, Abeta fibrils formed under quiescent conditions were less toxic to differentiated SH-SY5Y cells than the Abeta aggregation associated oligomers or fibrils formed with agitation. These results highlight differences between Abeta aggregation intermediates formed under different conditions and provide insight into the structure and stability of toxic Abeta oligomers.

Figure 1.

Aβ aggregation as measured by Congo red binding as a function of time after initial dissolution of the peptide. One hundred millimolar Aβ was incubated at 37°C (A) with gentle agitation and (B) under quiescent conditions.

Figure 2.

Representative size exclusion chromatograms of 100 μM Aβ at different times during aggregation. Aβ samples were incubated at 37°C for (A) 2, 4, 8, and 24 h with gentle agitation and (B) 4, 8, 24, and 72 h under quiescent conditions.

Figure 3.

Representative EM images of 100 μM Aβ at different times during aggregation at 37°C; (A) fresh Aβ, (B) 2 h of aggregation with gentle agitation, (C) 24 h of aggregation with gentle agitation, (D) 8 h of aggregation under quiescent conditions, and (E) 72 h of aggregation under quiescent conditions.

Figure 4.

Secondary structure of Aβ at different times during aggregation with gentle agitation (A) and under quiescent conditions (B). All times indicate time of incubation at 37°C after initial dissolution of 100 μM Aβ. Representative CD spectra are shown. (Light thin solid line) fresh Aβ; (dark thin solid line) 2 h; (light thick solid line) 4 h; (dark thick solid line) 8 h; (light thick broken line) 10 h; (dark thick broken line) 12 h.

Figure 5.

Effect of guanidine hydrochloride (GuHCl) on structure of different Aβ aggregates as measured by CD intensity at 220 nm. Aβ samples (100 μM) were aggregated either with gentle agitation or under quiescent conditions at 37°C for various lengths of time, after which Aβ samples were mixed with GuHCl to a final concentration of 10 μM Aβ and 0.5–7 M GuHCl. CD intensity at 220 nm of Aβ samples in GuHCl were monitored for 2 h, until no further detectable changes in structure or CD intensity occurred. CD intensities at 220 nm were used for Aβ stability tests. (Filled triangles) fresh Aβ; (open squares) Aβ aggregated with gentle agitation for 24 h; (filled circles) Aβ aggregated for 8 h under quiescent conditions.

Figure 6.

Relative cell viability of differentiated SY5Y cells treated with 100 μM Aβ that had been aggregated with gentle agitation (A) or under quiescent conditions (B) for different lengths of time prior to addition to cells. Cells were incubated with Aβ for 2 h prior to assessment of viability via staining with annexin and 7AAD followed by flow cytometry.