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 using an antibody specific to the C-terminus (Fig. 6B). By dot blot assay, we estimated that the stably transfected cells expressed approximately 20-fold higher sAPP in conditioned medium than wild type neurons (data not shown). After adjusting for the level of sAPP (20x dilution with neurobasal medium), conditioned medium from sAPPα- or sAPPβ-transfected B103 cells was applied to hippocampal neurons derived from either APP wild type or APP−/− mice at DIV 2. We found the spine numbers were significantly but only partially restored by sAPPα-B103 CM (Fig. 6C). Similar to dendritic spine numbers, the number of dendritic branch points were also significantly but only partially restored by sAPPα-B103 CM (Fig. S3). On the other hand, the sAPPβ-CM did not have any effects on spine number in APP−/− neurons (Fig. 6C), confirming the notion that a major domain of the sAPP trophic property reside within the 17 amino acid region at the C-terminus of sAPPα.
