Turn nucleation perturbs amyloid β self-assembly and cytotoxicity

Abstract

The accumulation of senile plaques composed of amyloid β (Aβ) fibrils is a hallmark of Alzheimer's disease, although prefibrillar oligomeric species are believed to be the primary neurotoxic congeners in the pathogenesis of Alzheimer's disease. Uncertainty regarding the mechanistic relationship between Aβ oligomer and fibril formation and the cytotoxicity of these aggregate species persists. β-Turn formation has been proposed to be a potential rate-limiting step during Aβ fibrillogenesis. The effect of turn nucleation on Aβ self-assembly was probed by systematically replacing amino acid pairs in the putative turn region of Aβ (residues 24-27) with d-ProGly ((D)PG), an effective turn-nucleating motif. The kinetic, thermodynamic, and cytotoxic effects of these mutations were characterized. It was found that turn formation dramatically accelerated Aβ fibril self-assembly dependent on the site of turn nucleation. The cytotoxicity of the three (D)PG-containing Aβ variants was significantly lower than that of wild-type Aβ40, presumably due to decreased oligomer populations as a function of a more rapid progression to mature fibrils; oligomer populations were not eliminated, however, suggesting that turn formation is also a feature of oligomer structures. These results indicate that turn nucleation is a critical step in Aβ40 fibril formation.

Figure 1

A. Schematic of major events and potential intermediates in amyloid-β self-assembly. These intermediate conformations feature a turn between residues 23 and 28. B. Cross-strand registry in Aβ40 (based on published fibril structures derived by solid-state NMR) and proposed strand alignment based on ^DPG nucleation at various positions throughout the proposed turn region spanning residues 24–27. C. NMR-derived structures of the turn region in Aβ hairpin stabilized by the Z_Ab affibody (top) and Aβ40 fibril structure (bottom).^,

Figure 2

Normalized thioflavin T (ThT) fluorescence analysis of Aβ40 and ^DPG Aβ variant peptide fibril formation. Peptide stock solutions were dissolved in phosphate-buffered saline (25 µM ThT, pH 7.4) and incubated at room temperature.

Figure 3

HPLC sedimentation analysis of fibril formation of Aβ40 and Aβ40 ^DPG variant peptides. Disaggregated peptide stock solutions were dissolved in phosphate-buffered saline (pH 7.4) and incubated at room temperature prior to sedimentation analysis. Note that the x-axis is displayed as units of days, not hours.

Figure 4

Transmission electron microscopy (TEM) images of fibrils formed by A. Aβ40; B. ^DPG-24,25; C. ^DPG-25,26; D. ^DPG-26,27.

Figure 5

X-ray diffraction d-spacings obtained from fibrils derived from Aβ40 and Aβ40 ^DPG variants.

Figure 6

FT-IR spectra of Aβ40 and Aβ40 ^DPG variant fibrils.

Figure 7

Circular dichroism (CD) spectra of Aβ40 and Aβ40 ^DPG variant fibril solutions.

Figure 8

Cytotoxicity profiles of C17.2 neural progenitor cells after exposure to: 1) freshly disaggregated Aβ40 and Aβ40 ^DPG variants (monomer); 2) Aβ40 and Aβ40 ^DPG variants grown under conditions that favor the formation of oligomers (oligomer); 3) Aged Aβ40 and Aβ40 ^DPG variants grown under conditions that favor the formation of fibrils (fibril).