Soluble Aβ oligomers reduce synapse density and dysregulate ProSAP/Shank family members in hippocampal cell culture. A) Changes in synapse density along the dendrites of hippocampal neurons, cultured for 15 DIV, treated with 1 μM Aβ1-40 and fixed after 0, 1, 3, 6, and 24 h. Synapses along MAP2 positive dendrites were identified with antibodies against Bassoon as presynaptic marker and ProSAP2/Shank3 (left panel) or Shank1. Synapse density was calculated measuring the number of synapses per unit dendrite length of ten cells of three independent experiments for every time-point and condition (right panel). B) Spine maturation state after 24 h Aβ1-40 treatment was assessed by quantifying spine morphology (using ProSAP2/Shank3 immunoreactivity) along MAP2 positive dendrites (left panels). Spines were classified as "filopodia like", "thin" (immature) and "mushroom and stubby" (mature). The overall fraction of filopodia like and thin synapses is higher after 24 h Aβ treatment compared to control conditions (24 h treatment with DMSO). C) Aβ treatment causes a progressive synaptic loss of ProSAP2/Shank3 and Shank1. Cultured hippocampal neurons were immunostained with antibodies against Bassoon and ProSAP2/Shank3 or Shank1 (upper left panel) and the ratio of mean grey values per mean signal area between treated and untreated neurons were measured after 1, 3, 6 or 24 h treatment with Aβ1-40 (upper right panel). Cumulative histograms illustrate that the puncta intensity values are shifted across the entire populations of ProSAP2/Shank3 and Shank1 puncta (bottom panels). Data derive from 3 independent experiments at each time-point and condition representing approx. 2,500 signals per experiment.