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. 2015 Apr 23;11(5):689–696. doi: 10.1016/j.celrep.2015.03.068

Figure 1.

Figure 1

Altered APP Processing and Aβ Peptide Production in Stem Cell Models of Genetic Forms of Alzheimer’s Disease

(A–C) Representative immunohistochemistry of neurons (β3-tubulin positive, blue) generated from familial Alzheimer’s disease (fAD) (PSEN1 intron 4, Y115C, and APP V717I) iPSCs, expressing transcription factors restricted to layer 6 (Tbr1, red) or layer 5 (CTIP2, green) cortical projection neurons.

(D) 3D nature of stem-cell-derived cortical cultures. Cultures cleared by passive CLARITY and immunostained for neurons (tau, red) and nuclei (DAPI, blue). Single-plane XY, XZ, and YZ (right, top, and left panels, respectively) projections of control neurons 100 days post-induction. The scale bar represents 100 μm.

(E) Generation of Aβ peptides. APP is first cleaved by β-secretase to generate membrane-bound APP-C99. This is followed by the initial γ-secretase cleavage of APP-C99, termed ε-cleavage, to generate Aβ peptides of either 48 or 49 amino acids. Aβ peptides are then subject to sequential γ-secretase carboxypeptidase cleavages, leading to extracellular release of Aβ42, 40 and 38.

(F) Neurons with three different PSEN1 mutations generate equivalent amounts of total extracellular Aβ peptides over 40 days in culture as three different healthy control lines. APP V717I neurons also do not significantly alter the production of total Aβ peptides compared to controls. This is in contrast with APP (dup) neurons, which significantly increase Aβ production. Error bars, SD; n = 3 cultures for each genotype; ∗∗p < 0.01.

(G) PSEN1 and APP V717I mutant neurons have significantly reduced Aβ40:Aβ42 ratios compared with both control and APP (dup) neurons at all time points studied, reflecting a relative increase in the generation of Aβ42. Error bars, SEM; ∗∗p < 0.01.

(H) PSEN1 and APP V717I mutant neurons exhibit a relative increase in Aβ42 compared to Aβ40 at day 80, whereas Ts21 and APP (dup) do not. All data produced from three independent cultures. Error bars, SD; ∗∗p < 0.01.

(I) Comparing ratios of Aβ40 to the sum of Aβ38 and Aβ42 at day 80, as an indicator of ε-cleavage and processing pathway choice, reveals that neither PSEN1 mutants nor increased APP dosage affects APP-C99 cleavage. By contrast, APP V717I mutants significantly bias the ε-cleavage APP-C99 to Aβ48, which is processed to both Aβ42 and Aβ38. Error bars, SD.

(J) PSEN1 mutant neurons have reduced Aβ38:Aβ42 ratios, in contrast with all other genotypes analyzed, suggesting reduced γ-secretase processivity. Error bars, SD; ∗∗p < 0.01.

(K) Proposed alternative processing pathway of APP in the presence of γ-secretase inhibitors. APP can be sequentially cleaved by β- and then γ-secretase epsilon cleavage followed by α-secretase to generate Aβ14/15/16 and AICD.

(L) Percentage of total Aβ peptides that are generated from the proposed alternative pathway (K; Aβ14/15/16) as an indicator of processivity, detected and quantified by IP-MALDI. PSEN1 mutant neurons significantly increase the percentage of Aβ peptides made up of the sum of Aβ1-14, 1-15, and 1-16, compared with control and APP V717I neurons. Error bars, SD; ∗∗p < 0.01.

(M) Percentage of total Aβ peptides that is Aβ40 as an indicator of processivity. PSEN1 mutant neurons significantly decrease the percentage of Aβ40 peptides, compared with control and APP V717I neurons. Error bars, SD; ∗∗p < 0.01.