Aβ-40 Y10F increases βfibrils formation but attenuates the neurotoxicity of amyloid-β peptide

Abstract

Alzheimer's disease (AD) is characterized by the abnormal aggregation of amyloid-β peptide (Aβ) in extracellular deposits known as senile plaques. The tyrosine residue (Tyr-10) is believed to be important in Aβ-induced neurotoxicity due to the formation of tyrosyl radicals. To reduce the likelihood of cross-linking, here we designed an Aβ-40 analogue (Aβ-40 Y10F) in which the tyrosine residue was substituted by a structurally similar residue, phenylalanine. The aggregation rate was determined by the Thioflavin T (ThT) assay, in which Aβ-40 Y10F populated an ensemble of folded conformations much quicker and stronger than the wild type Aβ. Biophysical tests subsequently confirmed the results of the ThT assay, suggesting the measured increase of β-aggregation may arise predominantly from enhancement of hydrophobicity upon substitution and thus the propensity of intrinsic β-sheet formation. Nevertheless, Aβ-40 Y10F exhibited remarkably decreased neurotoxicity compared to Aβ-40 which could be partly due to the reduced generation of hydrogen peroxide. These findings may lead to further understanding of the structural perturbation of Aβ to its fibrillation.

Keywords: Alzheimer’s disease; amyloid-β peptide; neurotoxicity; βfibrils.

Figure 1

UV region circular dichroism (CD) spectra of various Aβ peptides. Molar ellipticity of wild type Aβ-40 (A) and Aβ-40 Y10F (B) was recorded in the UV region (190–240 nm). Aβ peptides were diluted using 10 mM phosphate buffer (pH 7.4) to a final concentration of 50 μM in each sample. The average data of six run is shown.

Figure 2

Kinetic measurements of β-aggregation resulting from incubation of wild type Aβ-40 and Aβ-40 Y10F. ThT assay was performed with 10 μM Aβ, 10 μM ThT and 10 mM phosphate buffer at 37 °C. ThT fluorescence was monitored at an excitation of 450 nm and emission of 485 nm. Each point represents the average of six independent experiments.

Figure 3

Negatively stained transmission electron micrographs of Aβ-40 (A) and Aβ-40 Y10F (B). The peptide was diluted in phosphate buffer to 10 μM, and then incubated at 37 °C for 72 h before experiments. Both peptides presented fibrillar morphologies, wild type Aβ-40 showed the combined morphologies of fibrils (5–10 nm in diameter and 400 nm in length) and curvilinear protofibrils (6–8 nm in diameter and <100 nm in length), while Aβ-40 Y10F gave rise to longer mature fibrils (long and extended fibrils >2 μm in length). Scale bar: 200 nm.

Figure 4

Effect of variant Aβ peptides on ANS fluorescence. Fluorescence spectra were collected from 430 to 650 nm, with excitation at 350 nm. Solid lines symbolize unbound ANS in phosphate buffer, while ANS binding to Aβ-40 and Aβ-40 Y10F are represented by dotted and dashed lines, respectively. Each point represents the average of three independent experiments.

Figure 5

Neurotoxicity of variant Aβ peptides. Primary neuronal cells were grown for 7 days in vitro, and then neurons were exposed to various Aβ peptides at the final concentration of 5 μM for 96 h. Cell viability was determined by measuring the reduction of MTS. Data are presented as the mean ± S.E.M. of three independent experiments. Student’s t test was applied to determine the significance, denoted as ^** P < 0.01.

Figure 6

H₂O₂ production induced by wild type Aβ-40 or Aβ-40 Y10F. Primary cortical neurons were exposed to 5 μM wild type Aβ-40 or Aβ-40 Y10F for 96 h. Absorbance readings were taken and compared to vehicle (Student’s t test, ^** P < 0.01, n = 3).