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The neuronal S100B protein is a calcium-tuned suppressor of amyloid-β aggregation

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Science Advances  29 Jun 2018:
Vol. 4, no. 6, eaaq1702
DOI: 10.1126/sciadv.aaq1702
  • Fig. 1 S100B binds to Aβ42.

    (A) Isothermal titration calorimetry analysis of S100B binding to Aβ42 in the presence of calcium. The upper panel shows the raw heats of binding, and the lower panel shows the integrated data obtained after subtracting the heat of dilution from the buffer. Data fitting was performed with the nonlinear regression analyses of one binding site model obtaining the thermodynamic parameters N = 2.37 ± 0.07, Kd = 0.59 ± 0.23 μM, ΔH = −947.1 ± 29.48 cal mol−1, and ΔS = 25.3 cal mol−1 deg−1. (B) Far UV-CD spectra of 4 μM Ca2+-S100B alone (black) and with a molar ratio Aβ42/S100B of 0.25 (green), 0.5 (blue), 1 (orange), and 1.9 (red) after incubation overnight at 4°C. (C) Ratio of ellipticity at 222 nm of S100B alone and S100B + Aβ42 in the presence (blue) and absence (red) of 1.1 mM CaCl2. (D) Heteronuclear single-quantum coherence (HSQC) spectra of 100 μM 15N-labeled Aβ42 in the absence (blue) and in the presence (orange) of 20 μM S100B. (E) HSQC spectra of 100 μM 15N-Aβ42 in the absence (blue) and in the presence (orange) of 20 μM S100B with 10 mM CaCl2. ppm, parts per million.

  • Fig. 2 Mapping the interaction of Aβ42 with S100B.

    (A) HSQC spectra of 100 μM 15N-S100B in the absence (blue) and in the presence (orange) of 50 μM Aβ42. (B) HSQC spectra of 100 μM 15N-labeled S100B in the absence (blue) and in the presence (orange) of 50 μM Aβ42 with 10 mM CaCl2. (C) Structure of Ca2+-S100B [Protein Data Bank (PDB) code: 2H61], color-coded by the degree of difference in peak intensities in the absence or presence of Aβ42. Unassigned residues are colored gray, while residues that correspond to peaks that undergo large intensity changes are colored red, and residues that correspond to peaks that change little or not at all are colored blue, with a gradient for values in between. Left: Ribbon representation. Dashed line indicates the interfacial cleft. Right: Surface representation at the same orientation. Dashed circle highlights the putative Aβ42 binding region. (D) SAXS-based structural model of S100B in the absence (left) and in the presence (right) of Aβ42, both in the presence of calcium. The surface represents the best SAXS models based on the fit between the experimental data and the back-calculated SAXS data and have been overlapped with the experimental NMR structure (PDB code: 2K7O).

  • Fig. 3 S100B modulates Aβ42 aggregation.

    (A) Fibril formation of 5 μM Aβ42 (black) in 50 mM Hepes (pH 7.4) at 37°C under quiescent conditions in the presence of 10 μM (purple), 15 μM (dark blue), 20 μM (blue), 25 μM (light blue), 50 μM (dark green), 75 μM (light green), 100 μM (yellow), 150 μM (orange), 175 μM (red), and 200 μM (dark red) S100B. S100B (200 μM) alone, as control, is represented in gray. Plots represent averaged normalized intensity curves obtained from three independent replicates for each of the tested conditions. (B) Fibril formation of 10 μM Aβ42 (black) with 1.1 mM CaCl2 in 50 mM Hepes (pH 7.4) at 37°C under quiescent conditions in the presence of 10 μM (purple), 15 μM (dark blue), 20 μM (blue), 25 μM (light blue), 50 μM (dark green), 75 μM (light green), 100 μM (yellow), 150 μM (orange), 175 μM (red), and 200 μM (dark red) S100B. S100B (200 μM) alone, as control, is represented in gray. Plots represent averaged normalized intensity curves obtained from three independent replicates for each of the tested conditions. (C) Representative scheme of microscopic events occurring during Aβ42 aggregation: primary nucleation (kn) starting from monomers, elongation (k+) by monomer addiction to existing aggregates, and secondary nucleation (k2) from addition of monomers to fibril surface. (D) Log-log plot of the half-time of the Aβ42 aggregation reaction as a function of the initial Aβ42 monomer concentration in the absence (black) or in the presence (blue) of 15-fold excess of S100B. (E) Log-log plot of the half-time of the Aβ42 aggregation reaction as a function of the initial Aβ42 monomer concentration with 1.1 mM CaCl2 in the absence (black) or presence (blue) of 15-fold excess of S100B. (F) Half-times as function of increasing concentrations of S100B in the presence of Aβ42 with (blue) and without (black) 1.1 mM CaCl2. Dots represent values obtained from three independent replicates for each of the tested conditions. (G) Plot of ThT intensity of the end point of the aggregation kinetics in the presence of Aβ42 with increasing concentrations of S100B in the absence (black) and in the presence (blue) of 1.1 mM CaCl2. Dots represent averaged values obtained from three independent replicates for each of the tested conditions. (H) Plot of S100B-dependent change in microscopic rates of Aβ42 aggregation. The top panel represents the ratio of combination of kinetic rates describing primary nucleation (k+kn) with and without S100B in the absence (black) and in the presence (blue) of 1.1 mM CaCl2. The bottom panel represents the ratio of combination of kinetic rates describing secondary nucleation (k+k2) with and without S100B in the absence (black) and in the presence (blue) of 1.1 mM CaCl2. Microscopic rates were determined by individual fitting with the secondary nucleation model and with combined rate constants k+kn and k+k2 as free fitting parameters. Plots represent averaged values obtained from three independent replicates for each of the tested conditions.

  • Fig. 4 S100B inhibits surface-catalyzed secondary nucleation of Aβ42 oligomers.

    (A) Aggregation kinetics of 5 μM Aβ42 (black) seeded by the addition of 0.1 μM preformed Aβ42 fibrils (gray). (B) Aggregation kinetics of 5 μM Aβ42 (black) seeded by the addition of 0.1 μM preformed Aβ42 fibrils in the presence of 50 μM S100B (dark blue) and 150 μM S100B (light blue) in solution. (C) Aggregation kinetics of 5 μM Aβ42 (black) seeded by the addition of 0.1 μM preformed Aβ42 fibrils grown with S100B (gray). (D) Aggregation kinetics of 5 μM Aβ42 (black) seeded by the addition of 0.1 μM preformed Aβ42 fibrils grown with S100B in the presence of 50 μM S100B (dark blue) and 150 μM S100B (light blue) in solution. (E) TEM images with a nanogold-conjugated secondary antibody against S100B (15 nm) and Aβ42 (10 nm), showing binding of S100B to fibrils and oligomers. (F) Aggregation kinetics of 10 μM Aβ42 with and without the addition of 0.1 μM preformed Aβ42 fibrils in the presence of 50 μM Ca2+-S100B (dark blue) in solution. (G) TEM images of end points of ThT aggregation kinetics of 150 μM S100B, 10 μM Aβ42, and 10 μM Aβ42 + 150 μM S100B, with 1.1 mM CaCl2.

  • Fig. 5 S100B protects SH-SY5Y cells against Aβ42 toxicity and apoptosis.

    (A) Cell viability as measured by PrestoBlue reagent after 72 hours in differentiated SH-SY5Y cells for medium; buffer: 50 mM Hepes (pH 7.4) and 1.1 mM CaCl2, 7 μM Aβ42 with 1.1 mM CaCl2, and 7 μM Aβ42 + 84 μM S100B with 1.1 mM CaCl2. (B) Cell apoptosis as measured by caspase-3/7 activity after 72 hours in differentiated SH-SY5Y cells for medium; buffer: 50 mM Hepes (pH 7.4) and 1.1 mM CaCl2, 7 μM Aβ42 with 1.1 mM CaCl2, and 7 μM Aβ42 + 84 μM S100B with 1.1 mM CaCl2. Results represent mean and SD of two independent experiments (n = 8 and 5 wells per replicates per condition and plate). Statistically significant differences at the 95.0% confidence level using one-way analysis of variance (ANOVA) followed by Welch’s t test. ns, not significant; **P < 0.01, ***P < 0.001, ****P < 0.0001.

  • Fig. 6 S100B delays Aβ42 aggregation by suppressing primary and secondary nucleation.

    (A) In the AD brain, protein aggregation and exacerbated inflammation are well-established disease features, with elevated levels of Aβ42 and S100B. In AD animal models such as the 5XFAD mice, amyloid plaques (dotted contour) have intense staining for extracellular S100B (arrows) (see also fig. S7). (B) Extracellular S100B and Aβ42 engage in regulatory interactions, which are depicted in the scheme which summarizes the finding that S100B inhibits Aβ42 fibril formation mainly by affecting the lag phase and secondary nucleation, through interactions with monomeric and fibrillar Aβ42, suggesting a potential role of S100B as novel extracellular chaperone suppressing proteotoxicity in AD.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/6/eaaq1702/DC1

    Supplementary Methods

    fig. S1. Isothermal titration calorimetry analysis of the Aβ42/S100B interaction.

    fig. S2. BLI analysis of the Aβ42/S100B interaction.

    fig. S3. CD analysis of the Aβ42/S100B interaction in the absence of CaCl2.

    fig. S4. SAXS analysis of the Aβ42/S100B complex.

    fig. S5. Effect of apo-S100B over Aβ42 aggregation in a fragmentation-dominated regime.

    fig. S6. Effect of Ca2+-S100B over Aβ42 aggregation in a fragmentation-dominated regime.

    fig. S7. S100B inhibits Aβ25–35 aggregation.

    fig. S8. Dot blot analysis of Aβ42 aggregates formed in the presence of S100B.

    fig. S9. TEM images of Aβ aggregates formed in the presence and absence of S100B.

    fig. S10. S100B accumulates at high levels around plaques in AD mice brains.

  • Supplementary Materials

    This PDF file includes:

    • Supplementary Methods
    • fig. S1. Isothermal titration calorimetry analysis of the Aβ42/S100B interaction.
    • fig. S2. BLI analysis of the Aβ42/S100B interaction.
    • fig. S3. CD analysis of the Aβ42/S100B interaction in the absence of CaCl2.
    • fig. S4. SAXS analysis of the Aβ42/S100B complex.
    • fig. S5. Effect of apo-S100B over Aβ42 aggregation in a fragmentation-dominated regime.
    • fig. S6. Effect of Ca2+-S100B over Aβ42 aggregation in a fragmentation-dominated regime.
    • fig. S7. S100B inhibits Aβ25–35 aggregation.
    • fig. S8. Dot blot analysis of Aβ42 aggregates formed in the presence of S100B.
    • fig. S9. TEM images of Aβ aggregates formed in the presence and absence of S100B.
    • fig. S10. S100B accumulates at high levels around plaques in AD mice brains.

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