Fig. 1: Increased levels of S100B sequester zinc ions.

a Treatment of hippocampal cultures with 30 μM S100B or S100Bmut for 24 h at DIV10. The mean intracellular signal intensity of Zinpyr1 fluorescence is shown. Treatment of hippocampal cultures with S100B significantly reduces the free intracellular Zn²⁺ concentration assessed by the analysis of 35 cells per condition. No significant decrease in intracellular Zinpyr1 fluorescence can be observed in cultures treated with S100Bmut (one-way ANOVA, F_2,77 = 8.86; p < 0.0001; Post-hoc analysis: control vs. S100B, p = 0.0037; control vs. S100Bmut, p = 0.2469; S100B vs. S100Bmut, p < 0.0001). b Exemplary images showing signal intensities of Zinpyr1 in a color-coded manner. c–g Increased levels of S100B lead to a decrease in zinc-dependent SHANK proteins at the synapse. c Treatment of hippocampal cultures with 30 μM S100B for 24 h. The signal intensity of SHANK2 is significantly reduced 24 h after exposure to S100B. Similarly, the signal intensity of SHANK3 puncta shows a trend toward a reduction. No significant reduction of SHANK2 and SHANK3 was observed 24 h after applying S100Bmut. For all analyses, 8–10 cells per condition were used (one-way ANOVA: SHANK2: F_2,27 = 502.009, p = 0.001; Post-hoc analysis: Control vs. S100B p = 0.0013, S100B vs. S100Bmut p = 0.0029; Shank3: F_2,27 = 278.257, p = 0.005; Post-hoc analysis: Control vs. S100B p = 0.497, S100B vs. S100Bmut p = 0.0037) (n = 10 cells per condition). d Exemplary images showing anti-SHANK2 and SHANK3 staining of hippocampal neurons 24 h after treatment with S100B and S100Bmut. e The mean number of immunoreactive signals per dendrite length was evaluated. No significant alterations were detected. f Using Western blot experiments (from three different preparations), a significant decrease of SHANK2 protein levels in the crude membrane fraction can be seen (t-test, p = 0.0265). g Cells were again treated with S100B for 24 h at DIV8. A significant reduction of SHANK2 and SHANK3 proteins is visible. This reduction was abolished in case cells were treated with 30 μM S100B that was saturated with 60 μM ZnCl₂ for 1 h on ice before application (one-way ANOVA: SHANK2: F_3,36 = 12.2454, p < 0.001; Post-hoc analysis: Control vs. S100B p = 0.001, S100B vs. saturated S100B p = 0.001; SHANK3: F_3,36 = 15.9105, p = 0.005; Post-hoc analysis: Control vs. S100B p = 0.001, S100B vs. saturated S100Bmut p = 0.001, p = 0.0037) (n = 10 cells per condition).