To verify the causal role of PSEN1 mutations for the phenotype of premature neuronal differentiation, we over-expressed either wild type PSEN1 (PSEN1-WT) or PSEN1 with mutation of A246E (PSEN1-A246E) as well as an empty vector (control) into N-iPSC-1 cells (Supplementary Figure 3a) and induced all three types of iPSCs to neural differentiation as described above. The differentiation culture of NPCs from PSEN1-A246E transfected N-iPSC-1 at day 28 contained more cells with long neurites and had an obviously lower cell density than differentiation culture of NPCs from PSEN1-WT and vector transfected N-iPSC-1, in a way similar to differentiation culture of AD-NPCs (Figure 4a). Moreover, significantly lower percentage of Nestin+ NPCs and higher percentage of MAP2+ neurons were detected in PSEN1-A246E-transfected cells (Figure 4b and 4c). The number of Nestin+ cells was dramatically reduced in PSEN1-A246E-transfected cell cultures (Figure 4d). Furthermore, over-expression of PSEN1-A246E in N-iPSC-1 drastically reduced percentage of BrdU+ proliferative cells, while no alteration was found in PSEN1-WT transduced cells (Figure 4e and 4f). In addition, introduction of PSEN1-A246E into N-iPSC-1 increased the percentage of TUNEL+ apoptotic cells during differentiation
