DIV 16-21 and spine density was assessed. In cultures with wild type neuron conditioned medium, the deficit in spine density seen in APP−/− neurons was essentially restored to normal levels (Fig. 5). In addition, the number of branch points in APP−/− neurons was also restored by adding APP WT-CM (Fig. S3, APP WT-CM: 28.4 ± 1.0; APP+/−: 29.7 ± 1.6; APP−/−: 17.1 ± 1.0; p < 0.001, one-way ANOVA with Tukey’s post-hoc test). This indicated that molecules, absent in APP−/− mice, and constitutively secreted from normal neurons, are necessary to induce or maintain a normal level of dendritic spine, and likely synapse, numbers. A possible candidate is obviously sAPP. To test this hypothesis, we generated stable cell lines in mouse B103 cells that express full-length mouse APP or only sAPPα or sAPPβ by introducing a stop codon at the α- or β-secretase site, respectively, to truncate APP at the C-terminal (Fig. 6A & B). B103 cells were chosen because they lack endogenous mouse APP (Schubert and Behl, 1993). As expected, western blot analyses of the various transfected cell lines confirmed the presence of sAPP in culture media but these constructs lack the C-terminus when immunoblotted in cell lysates, the latter
