Anti-Aβ drug screening platform using human iPS cell-derived neurons for the treatment of Alzheimer's disease

Abstract

Background: Alzheimer's disease (AD) is a neurodegenerative disorder that causes progressive memory and cognitive decline during middle to late adult life. The AD brain is characterized by deposition of amyloid β peptide (Aβ), which is produced from amyloid precursor protein by β- and γ-secretase (presenilin complex)-mediated sequential cleavage. Induced pluripotent stem (iPS) cells potentially provide an opportunity to generate a human cell-based model of AD that would be crucial for drug discovery as well as for investigating mechanisms of the disease.

Methodology/principal findings: We differentiated human iPS (hiPS) cells into neuronal cells expressing the forebrain marker, Foxg1, and the neocortical markers, Cux1, Satb2, Ctip2, and Tbr1. The iPS cell-derived neuronal cells also expressed amyloid precursor protein, β-secretase, and γ-secretase components, and were capable of secreting Aβ into the conditioned media. Aβ production was inhibited by β-secretase inhibitor, γ-secretase inhibitor (GSI), and an NSAID; however, there were different susceptibilities to all three drugs between early and late differentiation stages. At the early differentiation stage, GSI treatment caused a fast increase at lower dose (Aβ surge) and drastic decline of Aβ production.

Conclusions/significance: These results indicate that the hiPS cell-derived neuronal cells express functional β- and γ-secretases involved in Aβ production; however, anti-Aβ drug screening using these hiPS cell-derived neuronal cells requires sufficient neuronal differentiation.

Figure 1. Differentiation of forebrain neurons from hiPS cells.

(A) Experimental scheme of neural differentiation from hiPS cells, 253G4. Nestin-positive neuroepithelial cells (B) and Foxg1-positive cells (C) were observed at days 17 and 24, respectively. Scale bar, 50 µm. Expression levels of Foxg1 (D) and the neocortical markers Tbr1, Ctip2, Cux1, and Satb2 (E) at days 0, 24, and 52. Expression levels were measured by qPCR and normalized by that of GAPDH. “Fold expression” is shown as a ratio of day 24/day 0 or day52/day 0. Each column represents the mean ± SD of 3 assays. ^* p<0.05, ^** p<0.01, ^*** p<0.001, significantly different from day 0 by Dunnett's test. (F) ICC staining of Tbr1-, Ctip2-, Cux1- and Satb2-positive cells at day 52. Scale bar, 50 µm.

Figure 2. Characterization of neuronal and glial cells differentiated from hiPS cells.

(A) Time-dependent morphological changes of cells reseeded in a 24-well plate. Neuronal and glial cells were stained by anti-Tuj1 (left; red), anti-synapsin I (left; green), anti-MAP2 (right; red), and anti-GFAP (right; green) antibodies and DAPI (right; blue) at 38, 45, and 52 days. Scale bar, left; 20 µm, right; 50 µm. Expression levels of Tuj1 (B), synapsin I (C), MAP2 (D), and GFAP (E) at days 0, 24, 38, 45, and 52 were measured by qPCR and normalized by that of GAPDH. “Fold expression” is the ratio of expression at each day compared to day 0. Each point represents mean ± SD of 3 assays. ^* p<0.05, ^** p<0.01, ^*** p<0.001, significantly different from day 0 by Dunnett's test. (F–H) Neurotransmitter phenotypes at day 52. PAG (red)- and GAD (green)-positive (F), vGlut1 (green)- and Tuj1 (red)-positive (G), and GABA (green)- and Tuj1 (red)-positive cells (H). Blue, DAPI. Scale bar, 50 µm.

Figure 3. APP was expressed in hiPS cell-derived neuronal cells.

HiPS cell-derived neuronal cells express full-length APP, sAPPα, sAPPβ, APP-CTFα, APP-CTFβ and AICD at 38, 45, and 52 days. (A) Representative western blots of APP and its fragments. (B) Each column represents mean ± SD of 8 samples measured by quantitative western blot analysis and normalized by that of β-actin. “Fold expression” represents the ratio of expression on the given day compared to day 38. ^* p<0.05, ^** p<0.01, ^*** p<0.001, Tukey's test.

Figure 4. β-Secretase and γ-secretase components were expressed in hiPS cell-derived neuronal cells.

The hiPS cell-derived neuronal cells express BACE1 protein and mRNA (B), γ-secretase components; presenilin 1(PS1), nicastrin, Pen-2 (C), and Aph-1A, and Aph-1B (D) at days 38, 45, and 52. Expression levels were quantified by western blot analysis (n = 8) (B, C) or qPCR (n = 3) (D) and normalized by that of β-actin. “Fold expression” represents the ratio of expression on the given day compared to day 38. (E) The ratio Aph-1B/Aph-1A. Data represent mean ± SD. (A) Representative western blots of BACE1 and γ-secretase components at 38, 45, and 52 days. ^* p<0.05, ^** p<0.01, ^*** p<0.001, Tukey's test.

Figure 5. Aβ was produced in hiPS cell-derived neuronal cells.

(A) Aβ40 or Aβ42 secreted into the conditioned media and FL-APP were measured by sandwich ELISA and western blot analysis, respectively. Expression level of Aβ was normalized by that of FL-APP. (B) Aβ42/Aβ40 ratios. Data represent the mean ± SD of 8 assays. ^{*, #} p<0.05, ^{**, ##} p<0.01, ^{***, ###} p<0.001, Tukey's test.

Figure 6. Aβ production was modulated by β- and γ-secretase inhibitors and an NSAID.

β-Secretase inhibitor (BSI) (A, B), γ-secretase inhibitor (GSI) (C, D), and NSAID (E, F) were added into hiPS cell-derived neuronal cell cultures at day 36 (dotted line) and 50 (bold line), and two days later amounts of Aβ40 and Aβ42 secreted into the conditioned media were measured. The ratios Aβ40/FL-APP and Aβ42/FL-APP are expressed as percentages of the vehicle-treated group at day 52 and represent mean ± SD of 3 assays. A, B: There were significant main effects of day (F(1, 16) = 72.5 and 162.4, p<0.001 in Aβ40 and Aβ42, respectively) and dose (F(3, 16)  = 23.1 and 45.7, p<0.001 in Aβ40 and Aβ42, respectively), and significant interaction between day and dose (F(3, 16)  = 13.0 and 11.7, p<0.001 in Aβ40 and Aβ42, respectively) by 2-way ANOVA. C, D: There were significant main effects of day (F(1, 28)  = 240.5 and 59.1, p<0.001 in Aβ40 and Aβ42, respectively) and dose (F(6, 28)  = 70.8 and 37.8, p<0.001 in Aβ40 and Aβ42, respectively), and significant interaction between day and dose (F(6, 28)  = 23.5 and 15.1, p<0.001 in Aβ40 and Aβ42, respectively) by 2-way ANOVA. E, F: There were significant main effects of day (F(1, 20)  = 196.9 and 418.0, p<0.001 in Aβ40 and Aβ42, respectively) and dose (F(4, 20)  = 4.16, p = 0.013 and F(4, 20) = 91.9, p<0.001 in Aβ40 and Aβ42, respectively), and significant interaction between day and dose (F(4, 20) = 25.4, p<0.001 in Aβ42) by 2-way ANOVA. ^{*, #} p<0.05, ^{**, ##} p<0.01, ^{***, ###} p<0.001, significantly different from respective vehicle-treated groups by Dunnett's test.