The effect of Cu(2+) and Zn(2+) on the Aβ42 peptide aggregation and cellular toxicity

Abstract

The coordination chemistry of Cu and Zn metal ions with the amyloid β (Aβ) peptides has attracted a lot of attention in recent years due to its implications in Alzheimer's disease. A number of reports indicate that Cu and Zn have profound effects on Aβ aggregation. However, the impact of these metal ions on Aβ oligomerization and fibrillization is still not well understood, especially for the more rapidly aggregating and more neurotoxic Aβ42 peptide. Here we report the effect of Cu(2+) and Zn(2+) on Aβ42 oligomerization and aggregation using a series of methods such as Thioflavin T (ThT) fluorescence, native gel and Western blotting, transmission electron microscopy (TEM), and cellular toxicity studies. Our studies suggest that both Cu(2+) and Zn(2+) ions inhibit Aβ42 fibrillization. While presence of Cu(2+) stabilizes Aβ42 oligomers, Zn(2+) leads to formation of amorphous, non-fibrillar aggregates. The effects of temperature, buffer, and metal ion concentration and stoichiometry were also studied. Interestingly, while Cu(2+) increases the Aβ42-induced cell toxicity, Zn(2+) causes a significant decrease in Aβ42 neurotoxicity. While previous reports have indicated that Cu(2+) can disrupt β-sheets and lead to non-fibrillar Aβ aggregates, the neurotoxic consequences were not investigated in detail. The data presented herein including cellular toxicity studies strongly suggest that Cu(2+) increases the neurotoxicity of Aβ42 due to stabilization of soluble Aβ42 oligomers.

Fig. 1

ThT fluorescence for Aβ₄₂ incubated with various stoichiometric ratios of (a) Cu²⁺ and (b) Zn²⁺ for 24 h at 25 °C in PBS. Conditions: [Aβ₄₂] = 25 μM, [M²⁺] = 0–2 equiv., [ThT] = 10 μM.

Fig. 2

TEM images and native gel/Western blots of Aβ₄₀ and Aβ₄₂ fibrils in absence and presence of Cu and Zn. Top: (a) Aβ₄₀; (b) Aβ₄₀ + Cu²⁺; (c) Aβ₄₀ + Zn²⁺; (d) Aβ₄₂; (e) Aβ₄₂ + Cu²⁺; (f) Aβ₄₂ + Zn²⁺; bottom: Western analysis of same samples. Conditions: 24 hours with agitation in PBS at 37 °C, [Aβ] = [M] = 25 μM. The scale bar represents 500 nm.

Fig. 3

TEM images of samples containing (a) Aβ₄₂ (20 μM HEPES, 150 μM NaCl, pH 6.6); (b) Aβ₄₂ + Cu²⁺ (20 μM HEPES, 150 μM NaCl, pH 6.6); (c) Aβ₄₂ (20 mM HEPES, pH 6.6); (d) Aβ₄₂ + Cu²⁺ (20 mM HEPES, pH 6.6); (e) Aβ₄₂ (20 mM HEPES, pH 7.4); (f) Aβ₄₂ + Cu²⁺ (20 mM HEPES, pH 7.4); (g) Aβ₄₂ (PBS, pH 7.4); (h) Aβ₄₂ + Cu²⁺ (PBS, pH 7.4). Conditions: 24 hours with agitation at 37 °C, [Aβ] = [Cu²⁺] = 25 μM. The scale bar represents 500 nm.

Fig. 4

TEM images and native gel/Western blot of Aβ₄₂ aggregation in absence and presence of Cu²⁺. Left: TEM images for (a) Aβ₄₂ aggregation for 72 h at 25 °C; (b) Aβ₄₂ + Cu²⁺ aggregation for 72 h at 25 °C; (c) Aβ₄₂ aggregation for 24 h at 37 °C; (d) Aβ₄₂ + Cu²⁺ aggregation for 24 h at 37 °C. Right: the corresponding Western blots, (e) molecular weight ladder. Conditions: [Aβ] = [Cu²⁺] = 25 μM, PBS. The scale bar represents 500 nm.

Fig. 5

Top: TEM images and for samples containing 25 μM Aβ₄₂ with: (a) 0 equiv. Cu²⁺; (b) 0.25 equiv. Cu²⁺ (6.25 μM); (c) 0.5 equiv. Cu²⁺ (12.5 μM); (d) 1.0 equiv. Cu²⁺ (25 μM); (e) 1.5 equiv. Cu²⁺ (37.5 μM); (f) 2 equiv. Cu²⁺ (50 μM). Bottom: the corresponding native gel/Western blots, (g) molecular weight ladder. Conditions: 24 h, 25 °C, PBS. The scale bar represents 500 nm.

Fig. 6

TEM images of samples containing 25 μM Aβ₄₂ with: (a) 0 equiv. Zn²⁺; (b) 0.25 equiv. Zn²⁺ (6.25 μM); (c) 0.5 equiv. Zn²⁺ (12.5 μM); (d) 1 equiv. Zn²⁺ (25 μM); (e) 1.5 equiv. Zn²⁺ (37.5 μM); (f) 2 equiv. Zn²⁺ (50 μM). Conditions: 24 h, 25 °C, PBS. The scale bar represents 500 nm.

Fig. 7

Native gel/Western blots for time-dependent aggregation of Aβ₄₂ with and without Cu²⁺ or Zn²⁺. Panel A: lane 1, Aβ₄₂, day 0; lane 2, Aβ₄₂ + Cu²⁺, day 0; lane 3, Aβ₄₂, day 1; lane 4, Aβ₄₂ + Cu²⁺, day 1; lane 5, Aβ₄₂, day 2; lane 6, Aβ₄₂ + Cu²⁺, day 2; lane 7, Aβ₄₂, day 3; lane 8, Aβ₄₂ + Cu²⁺, day 3; lane 9, Aβ₄₂, day 4; lane 10, Aβ₄₂ + Cu²⁺, day 4. Panel B: lane 1, Aβ₄₂, day 0; lane 2, Aβ₄₂ + Zn²⁺, day 0; lane 3, Aβ₄₂, day 1; lane 4, Aβ₄₂ + Zn²⁺, day 1; lane 5, Aβ₄₂, day 2; lane 6, Aβ₄₂ + Zn²⁺, day 2; lane 7, Aβ₄₂, day 3; lane 8, Aβ₄₂ + Zn²⁺, day 3; lane 9, Aβ₄₂, day 4; lane 10, Aβ₄₂ + Zn²⁺, day 4; lanes 11, molecular weight ladder. Conditions: 24 h, 25 °C, PBS.

Fig. 8

Cell viability (% of control) upon incubation of Neuro-2A cells with (1) monomeric Aβ₄₂ (MAβ); (2) Aβ₄₂ oligomers (OAβ); (3) Aβ₄₂ fibrils (FAβ); (4) Cu²⁺; (5) MAβ₄₂ + Cu²⁺; (6) Zn²⁺; (7) MAβ₄₂ + Zn²⁺. Conditions: [Aβ₄₂] = [M²⁺] = 20 μM. The t-test analysis of experimental data reveals values of p < 0.001 for any two of the above lanes, except for lanes 2 and 5 that are not statistically different (p = 0.206).