Biophysical characterization of Abeta42 C-terminal fragments: inhibitors of Abeta42 neurotoxicity

Abstract

A key event in Alzheimer's disease (AD) is age-dependent, brain accumulation of amyloid beta-protein (Abeta) leading to Abeta self-association into neurotoxic oligomers. Previously, we showed that Abeta oligomerization and neurotoxicity could be inhibited by C-terminal fragments (CTFs) of Abeta42. Because these CTFs are highly hydrophobic, we asked if they themselves aggregated and, if so, what parameters regulated their aggregation. To answer these questions, we investigated the dependence of CTF aqueous solubility, aggregation kinetics, and morphology on peptide length and sequence and the correlation between these characteristics and inhibition of Abeta42-induced toxicity. We found that CTFs up to 8 residues long were soluble at concentrations >100 microM and had a low propensity to aggregate. Longer CTFs were soluble at approximately 1-80 microM, and most, but not all, readily formed beta-sheet-rich fibrils. Comparison to Abeta40-derived CTFs showed that the C-terminal dipeptide I41-A42 strongly promoted aggregation. Aggregation propensity correlated with the previously reported tendency to form beta-hairpin conformation but not with inhibition of Abeta42-induced neurotoxicity. The data enhance our understanding of the physical characteristics that affect CTF activity and advance our ability to design, synthesize, and test future generations of inhibitors.

FIGURE 1

Particle growth rate. A) Time course of average R_H was calculated from whole particle size distributions in solutions of Aβ(29–42), Aβ(30–42), Aβ(31–42), Aβ(33–42), or Aβ(30–40) at the concentrations indicated. Each data point represents mean±SEM calculated from the average R_H of eight consecutive DLS measurements during 45–60 min. Aggregation of Aβ(29–42) and Aβ(30–42) was followed until the upper limit of detection was reached. B) Average aggregation rates of Aβ(29–42), Aβ(30–42), Aβ(31–42), Aβ(33–42), and Aβ(30–40). The data represent mean±SEM of 3 independent experiments.

FIGURE 2

Time-dependent conformational change. A) Representative CD spectra of 156 µM Aβ(38–42) recorded in time intervals of 24 h. The spectra showing a minimum at 197 nm are characteristic of a statistical coil and remain unchanged for 4 days. B) Representative CD spectra of 62 µM Aβ(31–42) recorded in time intervals of 24 h. The initial spectrum showing a minimum at 197 nm is characteristic of statistical coil. The development of a maximum at 198 nm and a minimum at 218 nm indicate conformational change to β-sheet-rich structures. C) Representative time course of β-sheet formation calculated as described in Materials and Methods is shown for Aβ(29–42), Aβ(30–42), Aβ(31–42), Aβ(33–42), Aβ(34–42) and Aβ(30–40) at the concentrations indicated.

FIGURE 3

Time-dependent peptide morphology. Peptide solutions used were of the following concentrations: Aβ(28–42), 1±0.7 µM; Aβ(29–42), 14±1 µM; Aβ(30–42), 9±0.5 µM; Aβ(31–42), 22±0.6 µM; Aβ(32–42), 16±0.6 µM; Aβ(33–42), 80.0±0.1 µM; Aβ(34–42), 99±4 µM; Aβ(35–42), 122±1 µM; Aβ(30–40), 191±10 µM. Electron micrographs were recorded immediately after sample preparation (day 1) and one week later (day 7).

FIGURE 4

Relationships among biophysical and biological properties. A) Linear regression analysis correlating inhibition of Aβ42-induced toxicity with CTF solubility (r² = 0.04, p = 0.52). B) Linear regression analysis correlating aggregation rates of Aβ(29–42), Aβ(30–42), Aβ(31–42), and Aβ(33–42) with T₅₀ values of β-sheet formation (r² = 0.86, p = 0.07). C) Linear regression analysis correlating solubility of Aβ(29–42), Aβ(30–42), Aβ(31–42), and Aβ(30–40) with propensity for β-hairpin conformation (r² = 0.95, p = 0.03). D) Linear regression analysis correlating aggregation rates of Aβ(29–42), Aβ(30–42), Aβ(31–42), and Aβ(30–40) with propensity for β-hairpin conformation (r² = 0.78, p = 0.11). E) Linear regression analysis correlating T₅₀ values of β-sheet formation of Aβ(29–42), Aβ(30–42), and Aβ(31–42) with propensity for β-hairpin conformation (r² = 0.99, p = 0.01). F) Linear regression analysis correlating inhibition of Aβ42-induced toxicity of Aβ(29–42), Aβ(30–42), Aβ(31–42), and Aβ(30–40) with propensity for coil-turn conformation (r² = 0.85, p = 0.08). The symbols used in panels B-F are Aβ(29–42) – Δ, Aβ(30–42) – ✻, Aβ(31–42) –♦, Aβ(33–42) – ▢, and Aβ(30–40) – ∇.