Aβ binding of Zn2+ regulates synapse loss and synaptic levels of ProSAP2/Shank3 in hippocampal cell culture. A,B) Effect of Aβ and Zn2+ on synapse density assessed by treating cultured hippocampal neurons (DIV15) with Aβ1-40 followed by equimolar ZnCl2 supplementation or Aβ1-40 preincubated with equimolar ZnCl2. Synapse density was determined by quantifying the number of ProSAP2/Shank3 and Bassoon colocalizing puncta per unit length of MAP2 positive primary and secondary dendrites (arrow, right panel B). A) Quantification of synapse density on neurons treated for 1, 6 and 24 h with DMSO (control/vehicle), 1 μM Zn2+, 1 μM Aβ1-40 and 1 μM Aβ1-40 preincubated for 1 h on ice with 1 μM ZnCl2. B) Treatment of hippocampal neurons for 18 and 24 h with DMSO or Aβ1-40, 18 h with DMSO and 6 h with 1 μM ZnCl2 and 18 h with 1 μM or 10 μM Aβ1-40, followed by 1 μM or 10 μM ZnCl2 supplementation for 6 h. Synapse density is significantly higher in cultures supplied with Aβ saturated with Zn2+ than in those treated with 1 μM Aβ1-40 alone. C,D) Synaptic levels of ProSAP2/Shank3 in hippocampal cultures treated with Zn2+ and/or Aβ C) Quantification of ProSAP2/Shank3 signal grey values colocalizing with Bassoon puncta along MAP2 positive primary and secondary dendrites of neurons treated for 1, 6 and 24 h with DMSO (control), 1 μM Zn2+, 1 μM Aβ1-40 and 1 μM Aβ1-40 preincubated for 1 h on ice with 1 μM ZnCl2. A significantly higher ProSAP2/Shank3 level compared to treatment with 1 μM Aβ1-40 was measured after 24 h in cultures supplied with Zn2+-saturated Aβ D) Neurons treated for 18 and 24 h with DMSO or Aβ1-40, 18 h with DMSO and 6 h with 1 μM ZnCl2 and 18 h with 1 μM or 10 μM Aβ1-40, followed by 1 μM or 10 μM ZnCl2 supplementation for 6 h. Supplementation of ZnCl2 for 6 h after 18 h treatment with Aβ1-40 leads to a rescue of ProSAP2/Shank3 levels at the synapse (p < 0.05*; < 0.01**; < 0.001***). E) Western blots of P2 membrane fractions from hippocampal neurons cultured for 15 DIV and then treated for 18 or 24 h with Aβ1-40, 24 h Aβ1-40 preincubated for 1 h on ice with ZnCl2 and 18 h Aβ1-40 followed by 6 h incubation with ZnCl2. Compared to untreated cells at time-point 0 h, a decrease in the amount of ProSAP2/Shank3 could be detected after 18 and 24 h of Aβ treatment. In contrast, treatment for 24 h with Zn2+ saturated Aβ1-40 and 18 h Aβ1-40 followed by 6 h incubation with ZnCl2 leads to ProSAP2/Shank3 levels comparable to control conditions. Note PSD-95 and β-III Tubulin levels did not change under these conditions.