Phenotypic Screening Identifies Modulators of Amyloid Precursor Protein Processing in Human Stem Cell Models of Alzheimer's Disease

Abstract

Human stem cell models have the potential to provide platforms for phenotypic screens to identify candidate treatments and cellular pathways involved in the pathogenesis of neurodegenerative disorders. Amyloid precursor protein (APP) processing and the accumulation of APP-derived amyloid β (Aβ) peptides are key processes in Alzheimer's disease (AD). We designed a phenotypic small-molecule screen to identify modulators of APP processing in trisomy 21/Down syndrome neurons, a complex genetic model of AD. We identified the avermectins, commonly used as anthelmintics, as compounds that increase the relative production of short Aβ peptides at the expense of longer, potentially more toxic peptides. Further studies demonstrated that this effect is not due to an interaction with the core γ-secretase responsible for Aβ production. This study demonstrates the feasibility of phenotypic drug screening in human stem cell models of Alzheimer-type dementia, and points to possibilities for indirectly modulating APP processing, independently of γ-secretase modulation.

Keywords: Alzheimer's disease; Down syndrome; amyloid beta; dementia; human neurons; iPSCs; ivermectin; neural stem cells; phenotypic screening; selamectin.

Figure 1

Phenotypic Screen of the Prestwick Chemical Library for Modifiers of Aβ Production in TS21 Cortical Neurons (A) Fifty days after the initiation of neural induction, cortical neurons differentiated from TS21 iPSCs express microtubule-associated protein 2 (MAP2), and the cerebral cortex neuronal markers CTIP2 and TBR1. Scale bar represents 50 μm. (B) Timeline of the primary screen of the Prestwick Chemical library in TS21 iPSC-derived cortical neurons. d, day. (C and E) Coefficients of variation (CV) for vehicle (DMSO)-treated cultures for (C) Aβ38/Aβ42 and (E) Aβ40/Aβ42 ratios indicate a stable and sensitive platform for identifying hit compounds (dashed line represents recommended upper limit of 15% [Inglese et al., 2007]). (D and F) A primary screen of the Prestwick Chemical library (black symbols) identified a number of primary hits (blue symbols) altering the Aβ38/Aβ42 (D) and Aβ40/Aβ42 (F) ratios (dashed line represents B-score >3). Primary hits were validated in a secondary screen (green symbols) and then confirmed with a dose response (red symbols).

Figure 2

Avermectins Increase the Aβ38/Aβ42 Ratio in Human TS21 Neurons, in a Manner Phenotypically Similar to Previously Identified γ-Secretase Modulators The γ-secretase modulators (R)-flurbiprofen (A) and E2012 (B) dose-dependently increase the ratio of Aβ38 to Aβ42 in human TS21 cortical neurons. The avermectins abamectin (C) and ivermectin (D), identified in the primary screen, as well as emamectin benzoate (E) and selamectin (F), also dose-dependently increase the Aβ38/Aβ42 ratio, while the structurally related milbemycin moxidectin (G) has no consistent effect. n = 3–6 cultures/concentration. Error bars represent SD. Note that abamectin, ivermectin, and emamectin benzoate are all mixtures of B1a (R = CH₂CH₃) and B1b (R = CH₃) components.

Figure 3

The Avermectin-Induced Increase in the Aβ38/Aβ42 Ratio Is Driven by an Increase in Aβ38 and/or a Decrease in Aβ42 (A and C) When the contribution of each Aβ species is considered as a percentage of total as detected by immunoassay, avermectins cause a dose-dependent increase in Aβ38 (A) and/or a decrease in Aβ42 (C). (B) With the exception of emamectin benzoate, the avermectins also cause a dose-dependent decrease in Aβ40 levels (B). n = 3–6 cultures/concentration. Error bars represent SD.

Figure 4

Selamectin Has Complex Effects on Aβ Peptide Production as Measured with IP-MALDI (A and B) Representative Aβ IP-MALDI traces after treatment with DMSO vehicle (A) and selamectin (B). Peptides selected for quantitative analysis are highlighted in red. (C) Analysis of peak areas of selected peptides as a percentage of total peak areas indicates an increase in Aβ1-37 and a decrease in Aβ1-40 following selamectin treatment. n = 4 cultures per treatment. Error bars represent SD.

Figure 5

Avermectins Do Not Influence Aβ Processing via Their Known Pharmacological Targets (A and B) The effects of ivermectin and selamectin are not blocked by the GABA_A receptor antagonist picrotoxin (A) and are not phenocopied by the GABA_A receptor agonist muscimol (B). (C) Likewise, the glycine receptor antagonist strychnine exhibited no dose-dependent antagonism of avermectin effects of Aβ processing (see also Figure S1). n = 3–6 cultures/treatment. Error bars represent SD.

Figure 6

Avermectins Do Not Cause Accumulation of C-terminal Fragments of γ-Secretase Substrates, and Their Effects on Aβ Are Not Due to Direct Action on the Core γ-Secretase Complex (A) In human cortical neurons, treatment with the conventional GSI DAPT causes significant accumulation of C-terminal fragments of the γ-secretase substrates APP and N-cadherin, whereas treatment with the milbemycin moxidectin or the avermectins ivermectin or selamectin does not (see also Figure S2). (B) In a cell-free γ-secretase assay, the conventional GSI L-685,458 inhibits cleavage of APP C-terminal fragment by γ-secretase, and reduces the production of AICD. (C) Moxidectin and the avermectins have no consistent effects, although 1 μM ivermectin causes a significant reduction of AICD production (C). (D–F) In the same assay, milbemycins and avermectins induce no dose-dependent changes in the production of Aβ38 (D), Aβ40 (E), or Aβ42 (F) over DMSO control. Western blots in (A) and (B) are representative of two to four cultures per treatment (A) and three independent experiments (B), respectively, and data shown in (C) to (F) are from three independent experiments. Error bars represent SD. ^∗p < 0.05, Holm-Šídák adjusted one-sample t test versus 100% control value.

Figure 7

The Effect of Selamectin on Aβ Production in Cortical Neurons Is Reversible and Not Specific to the TS21 Genotype (A) After 10 days of compound treatment, withdrawal of selamectin leads to a reduction in effect compared with continued treatment over 4 days (A). (B) The effect of selamectin on Aβ processing is reproduced in neurons differentiated from patients carrying APP or PSEN1 mutations, indicating that the effect is not specific to the TS21 genotype (B). n = 3–6 cultures/treatment. Error bars represent SD.