Preparation and Characterization of Zn(II)-Stabilized Aβ42 Oligomers

Abstract

Aβ oligomers are being investigated as cytotoxic agents in Alzheimer's disease (AD). Because of their transient nature and conformational heterogeneity, the relationship between the structure and activity of these oligomers is still poorly understood. Hence, methods for stabilizing Aβ oligomeric species relevant to AD are needed to uncover the structural determinants of their cytotoxicity. Here, we build on the observation that metal ions and metabolites have been shown to interact with Aβ, influencing its aggregation and stabilizing its oligomeric species. We thus developed a method that uses zinc ions, Zn(II), to stabilize oligomers produced by the 42-residue form of Aβ (Aβ₄₂), which is dysregulated in AD. These Aβ₄₂-Zn(II) oligomers are small in size, spanning the 10-30 nm range, stable at physiological temperature, and with a broad toxic profile in human neuroblastoma cells. These oligomers offer a tool to study the mechanisms of toxicity of Aβ oligomers in cellular and animal AD models.

Keywords: Alzheimer′s disease; Aβ42; neurodegeneration; oligomers; zinc.

Figure 1

Using Zn(II) for the optimization of a protocol to stabilize Aβ₄₂ oligomers: (A) ThT fluorescence profiles of the aggregation of freshly purified monomeric Aβ₄₂ at 5 μM in the absence or presence of increasing molar equivalents of Zn(II). (B) Scheme illustrating the main steps of a previously reported stabilization protocol. We propose an optimization workflow to apply this protocol for the stabilization of aggregation-prone peptides, such as Aβ, by fine-tuning key experimental variables related with (i) the preparation of the stabilization reaction; (ii) the environmental conditions for the stabilization reaction; and (iii) the isolation and manipulation of the generated oligomeric species. (C) Experimental modifications of the Aβ₄₀-Zn (II) protocol to generate stable Aβ₄₂-Zn(II) oligomeric species.

Figure 2

Biophysical features of the reaction of stabilization of Aβ₄₂ oligomers by Zn(II). (A) SDS-PAGE representing protein species distribution in supernatant (S) and pellet (P) fractions of stabilization reactions run for 10 min, 4, 8 and 20 h at 20 °C in the absence (−) or presence (+) of 1:10 molar excess of Zn(II). (B,C) Fold change of ANS-derived maximum fluorescence intensity calculated over free ANS dye and maximum ANS emission wavelength (λ_max) of stabilization reaction products run in the absence (control Aβ₄₂) or presence of Zn(II) with or without further Zn(II) enrichment of the pellet fraction (Aβ₄₂-Zn(II)). (D) F/F₀ ratio between the ThT fluorescence intensity at 485 nm in the presence (F) and absence (Fo) of protein of the supernatant fraction of the control sample and the pellet fractions of enriched and nonenriched Aβ₄₂-Zn(II) samples after 10 min, 4, 8, and 20 h of stabilization reaction. Error bars represent standard deviation. Statistical analysis was performed by a one-way ANOVA within each time point, applying Bonferroni correction for multiple comparisons. *p-value < 0.1, **p-value < 0.01, and ***p-value < 0.001.

Figure 3

Stability of Aβ₄₂ species generated over the stabilization reaction time course. Raw ThT fluorescence data representing the progression of cross-β formation over a total of 20 h of aggregation at 37 °C for the control Aβ₄₂ and enriched and nonenriched Aβ₄₂-Zn(II) samples after (A) 10 min, (B) 4 h, (C) 8 h, and (D) 20 h of stabilization reaction at 20 °C. Fold change of average cross-β formation of control Aβ₄₂, nonenriched (−), and enriched (+) Aβ₄₂-Zn(II) samples incubated for 20 h at 37 °C after (E) 10 min, (F) 4 h, (G) 8 h, and (H) 20 h of stabilization reaction.

Figure 4

Progression of the mean hydrophobicity and turbidity of stabilized Aβ₄₂-Zn(II) species at physiological temperature. (A) Progression of the wavelength of maximum ANS-derived fluorescence emission of both enriched and nonenriched M-Aβ₄₂-Zn(II) samples subjected to a stabilization reaction of 20 h at 20 °C and further incubated for 8 h at 37 °C. Error bars represent standard deviation from three technical replicates. (B) Progression of the average size of enriched and nonenriched M-Aβ₄₂-Zn(II) samples under the same conditions. No significant differences were observed for both readouts after performing a one-way ANOVA with Bonferroni correction for multiple comparisons.

Figure 5

Secondary structures of Aβ₄₂-Zn(II) oligomeric species. (A) ATR-FTIR and (B) second derivative of the ATR-FTIR spectrum of Aβ₄₂-derived fibrils and enriched and nonenriched Aβ₄₂-Zn(II) samples after a stabilization reaction of 20 h at 20 °C. Oligomer-like species displayed significant antiparallel (1695 nm) and parallel (1630–1625 nm) β-sheet structure. (C) Representative dot-blot showing the antibody-specific reactivity of enriched and nonenriched Aβ₄₂-Zn(II) samples after 20 h of stabilization reaction at 20 °C, together with Aβ₄₂ fibrils. The sequence-specific 6E10 antibody was used as the peptide loading control. A11 and OC were used as conformation-specific antibodies for prefibrillar (A11) or fibrillar (OC) oligomeric or aggregate structures. (D) Total OC-derived signal per sample divided by the total 6E10-derived signal, represented as the normalized amyloid content per sample, showcasing intermediate levels of OC staining for Aβ₄₂-Zn(II) samples as compared to the control and fibrils. (E) F/F0 ratio of the ThT fluorescence intensity at 485 nm in the presence (F) and absence (F0) from the four samples tested for their antibody reactivity (Aβ42 control, non-enriched or enriched Aβ42-Zn(II) species and Aβ42 fibrils).

Figure 6

TEM images of Aβ₄₂-derived fibrils, control Aβ₄₂, and Aβ₄₂-Zn(II) samples. (A) Representative images of Aβ₄₂ fibrils (left panel), control Aβ₄₂ (middle panel), and enriched Aβ₄₂-Zn(II) (right panel) products after 20 h of stabilization reaction at 20 °C. (B–D) Control sample is composed of high heterogeneous protein populations (B, C), while enriched Zn(II) samples show greater homogeneity in terms of size and morphology (B,D). Statistical analysis was performed by a one-way ANOVA, applying Bonferroni correction for multiple comparisons. **p-value < 0.01 and ***p-value < 0.001.

Figure 7

Cytotoxicity assessment of control and Aβ₄₂-Zn(II) species. (A) Representative pictures of ROS signal (CellRox) in SH-SY5Y cells treated for 1 h with 500 nM, 1 μM, 2 μM, and 4 μM control Aβ₄₂ and enriched Aβ₄₂-Zn(II) species generated after a 20 h stabilization reaction at 20 °C. Scale bar = 100 μm. (B) Quantification of ROS production by treated cells. The CellRox-derived fluorescence (read in the RFP channel) was normalized by the total amount of cells (bright field area). Error bars represent standard deviation from three technical replicates. Statistical analysis was performed by a two-way ANOVA, applying Bonferroni correction for multiple comparisons. Ionomycin was excluded from statistical analysis. *p-value < 0.1, **p-value < 0.01, and ***p-value < 0.001. (C) Representative pictures of intracellular calcium levels (Fluo4) of SH-SY5Y cells treated for 1 h with 500 nM, 1 μM, 2 μM, and 4 μM control Aβ₄₂ and enriched Aβ₄₂-Zn(II) species generated after a 20 h stabilization reaction at 20 °C. Scale bar = 100 μm. (D) Quantification of intracellular calcium levels of treated cells. The Fluo4-derived fluorescence (read in the GFP channel) was normalized by the total amount of cells (bright field area). Error bars represent standard deviation from three technical replicates. Statistical analysis was performed by a two-way ANOVA, applying Bonferroni correction for multiple comparisons. Ionomycin was excluded from statistical analysis. *p-value < 0.1, **p-value < 0.01, ***p-value < 0.001. Vehicle corresponds to buffer enriched with Zn(II).