Early pathogenic event of Alzheimer's disease documented in iPSCs from patients with PSEN1 mutations.

Neural differentiation and characterization of iPSCsa. Schematic diagram of our neural differentiation protocol. b. Immunofluorescence staining and quantification of neural progenitor cells (NPCs) derived from neurospheres with antibodies against Nestin (green) and SOX2 (red). The inset shows a higher magnification view. Scale bar, 50 μm. The error bars indicate SEM. n=3. c and d. Immunofluorescence staining (c) and quantification (d) of apoptosis of NPCs by TUNEL staining. Scale bar, 50 μm. The error bars indicate SEM. n=3. e to g. Immunofluorescence staining (e) and quantification (f) of proliferation abilities of NPCs as well as the percentages of BrdU+ NPCs at different passages (g). The error bars indicate SEM. n=3. h. Representative immunofluorescence images of glia (GFAP+, red) and neurons (MAP2+, green) derived from NPCs as well as other markers of neurons including TuJ1, (green), NeuN (red) and SYP (green). Scale bars, 50 μm. i. Bright field images showing whole-cell recording on a cultured iPSC under DIC microscope. Scale bars, 10 μm. j. The classification of firing capability: No AP, no action potential was observed in all voltage traces; Single AP, only one AP evoked at the beginning of suprathreshold current injection; Multiple APs, multiple APs evoked in the early time of the injection of step currents; Serial APs, serial APs evoked along whole period of step current injection. k. Statistic analysis of percentages of spiking cells recorded from neurons derived from each cell line. D31: N-NPC-1, n=12; A15-NPC-3, -4, n=8. D39: N-NPC-1, n=10; A15-NPC-3, n=10; A15-NPC-4, n=9. l. Neuronal differentiation efficiencies of each cell line by RA-induced protocol were determined by the percentage of MAP2+ cells on different days of differentiation and compared by Two-way ANOVA. The error bars indicate SEM. ns, not significant;*, p< 0.05; **, p<0.01; ***, p<0.001; n> or = 5. m. The percentages of MAP2+ cells or Nestin+ cells on day 28 of monolayer neuronal differentiation. The error bars indicate SEM.**, p<0.01; ***, p<0.001; n=3.

Premature neuronal differentiation occurs in differentiating AD-NPCsa. Representative images of neural cells at day 28 of differentiation. Scale bar, 50 μm. b. Immunofluorescence staining images of each cell line at day 28 of differentiation with MAP2 and Nestin antibodies. Scale bar, 50 μm. The inset shows a higher magnification view. Scale bars, 20 μm. c. Statistic analyses of percentages of Nestin+ and MAP2+ cells derived from each cell line after differentiation for 28 days. The percentage of MAP2+ cells of each cell line was compared with that of N-NPC-1 line by the Student's t-test (n > or = 5; ns, not significant, **p<0.01, ***p<0.001). d. The numbers of NPCs per cm2 of each cell line at different days of differentiation were compared by two-way ANOVA (n> or = 5; mean ± SEM; ns, not significant, *p<0.05, **p<0.01, ***p<0.001). e. Cell numbers of various cell line duringdifferentiation in each well of 6-well plates were counted at different days of differentiation (n=3; mean ± SEM; ns, not significant, *p<0.05, **p<0.01, ***p<0.001). f and g. Immunofluorescence staining (f) and quantification (g) of the proliferation ability of neural cells by BrdU incorporation assays on day 28 of differentiation. Scale bar, 50 μm (n = 4; mean ± SEM; ns, not significant, **p<0.01). h and i. Immunofluorescence staining (h) and quantification (i) of apoptotic neural cells by TUNEL staining assays on day 28 of differentiation. Scale bar, 50 μm (n = or > 3; mean ± SEM; ns, not significant, **p<0.01, ***p<0.001).

Typical AD pathological changes and degenerating neurons are observed in neuronal differentiation of AD-NPCsa. ELISA of Aβ40 and Aβ42 during neuronal differentiation (n = 3; mean ± SEM; ns, not significant, *p<0.05, **p<0.01). b and c. Western blotting (b) and quantification (c) of p-tau protein levels in differentiating NPCs at day 28 (n = 4; mean ± SEM;*p< 0.05). d. Neurite fragmentation in neurons from AD-NPCs, scale bars, 50 μm. Lower panels are higher magnification views of neurons. Scale bar of upper panels, 200 μm; scale bar of lower panels, 100 μm.

PSEN1-A246E is responsible for the abnormal neuronal differentiation phenotypea. Representative bright field images at day 28 of differentiation form NPCs of N-iPSC-1 line transduced with the vector, PSEN1-WT and PSEN1-A246E, respectively. Scale bar, 50 μm. b. Immunofluorescence staining images of each cell line at day 28 of differentiation with antibodies against MAP2 and Nestin. Scale bar, 50 μm. c and d. Quantitative analysis of neuronal differentiation efficiencies (c) and numbers of NPCs per cm2 (d) of each cell line on day 28 (n = 5; mean ± SEM; ns, not significant, **p<0.01, ***p<0.001). e and f. Immunofluorescence staining of BrdU (e) and quantification of BrdU+ cells (f) of each cell line during differentiation on day 28. Scale bar, 50 μm (n = 5; mean ± SEM; ns, not significant, **p<0.01). g and h. TUNEL staining (g) and quantification of apoptotic cells (h) of each line at 28 days of differentiation. Scale bar, 50 μm (n = 5; mean ± SEM; ns, not significant, ***p<0.001). i. The cell morphology of A15-NPC-3 line transduced with the control and PSEN1-WT on 28 days of differentiation, respectively. Scale bar, 50 μm. j. Immunofluorescence staining of A15-NPC-3 line transduced with the control and PSEN1-WT on 28 days of differentiation, respectively, with antibodies against Nestin and MAP2. Scale bar, 50 μm. k. Quantification of percentages of MAP2+ neurons (n = 3; mean ± SEM; ns, not significant, ***p<0.001).

Knock down of PSEN1 in AD-NPCs rescues premature neuronal differentiationa. Transcript levels of PSEN1 in A15-NPC-3 transduced with a non-target sequence as negative control (shNC) and two shRNA sequences specific to PSEN1. The level of PSEN1 in A15-NPC-3 cells transduced with shNC was set as 1 (n = 3; mean ± SEM; ***p<0.001). b. Representative cell images at day 28 of differentiation in the cultures of each line. Scale bar, 50 μm. c to e. Nestin+ and MAP2+ cells of the three cell lines at day 28 of differentiation were detected by Immunofluorescence staining (c) and quantified (d and e). Scale bar, 50 μm (n = 3; mean ± SEM; *p< 0.05, ***p<0.001). f and g. Immunofluorescence staining of BrdU (f) and quantitative analysis (g) of BrdU+ cells on day 28 of neural differentiation. Scale bar, 50 μm (n = 3; mean ± SEM; **p<0.01).

Genome-wide transcriptome analyses identify genes and signaling pathways potentially associated with abnormal neuronal differentiation in differentiating AD-NPCsa. Heat map of normalized expression levels for differentially expressed genes. Fold changes of each gene are indicated by the color key. b. Top 20 enriched GO terms analyzed by DAVID based on differentially expressed genes between AD-iPSCs (A16-iPSC-1, A15-iPSC-2, A15-iPSC-3 and A15-iPSC-4) and normal pluripotent stem cells (N-iPSC-1 and N-iPSC-2) at differentiation day 32. Up-regulated genes were defined by fold changes ≥ 2 and down-regulated genes were defined by fold changes ≤ 0.5. c and d. Protein levels of DCX in AD-NPCs at day 28 of neuronal differentiation were detected by western blotting (c) and quantified (d) by image J (n = 4; mean ± SEM; *p< 0.05). e and f. Protein levels of β-catenin in AD-NPCs on day 28 of neuronal differentiation were detected by western blotting (e) and quantified (f) by image J (n = 4; mean ± SEM; *p< 0.05). g. Transcript levelsfor several down-stream genes of Wnt and Notch pathways in differentiating N-NPCs and AD-NPCs at day 28 (n = 4; mean ± SEM; *p<0.05, **p<0.01, ***p<0.001).