Anti-Aβ Drug Screening Platform Using Human iPS Cell-Derived Neurons for the Treatment of Alzheimer's Disease

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

(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.

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.

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