Figure 3

Application of soluble Aβ oligomers decreases Zn²⁺ loading of ProSAP2/Shank3. Cos7 cells grown in 5 μM Zn²⁺-supplemented medium were transfected with GFP-ProSAP2/Shank3. The intracellular Zn²⁺ level, visualized by Zinquin ethyl ester, and subcellular distribution were compared to GFP-ProSAP2/Shank3. A) In control cells, GFP-ProSAP2/Shank3 colocalizes with Zn²⁺ (t = 0 min) (left panel). After application of TPEN, Zn²⁺-ions were removed from ProSAP2/Shank3 clusters (t = 10 min). Supplementation with 10 μM ZnCl₂ restores and increases the initial Zn²⁺-association with GFP-ProSAP2/Shank3 clusters (left panel t = 50 min). Twenty min application of 10 μM Aβ_1-40(red fluorescence) followed by supplementation with 10 μM ZnCl₂ for 20 min only leads to a minor increase in Zn²⁺ loading of ProSAP2/Shank3 (middle panel t = 50 min). Application of 10 μM Aβ_1-40(red fluorescence) preloaded with 10 μM ZnCl₂ followed by supplementation with 10 μM ZnCl₂ leads to a significantly higher increase in ProSAP2/Shank3 Zn²⁺ loading (right panel t = 50 min) (scale bar = 50 μm). B) Magnification of Zn²⁺ signals colocalizing with ProSAP2/Shank3 cluster under the conditions described in A) (scale bar = 25 μm). C) Quantification of Zn²⁺ fluorescence, visualized with Zinquin, colocalizing with ProSAP2/Shank3 clusters. The ratio of mean grey values between control cells (t = 0 min) and treated cells is shown.