Prevention Of Aβ Induced Toxicity In Human Induced Pluripotent Stem Cell-derived Neurons Via Cdk2 Inhibitors

Objective To establish an in vitro Alzheimer’s disease(AD) model, screen small-molecule compounds and study the therapeutic mechanisms of relevant drug targets. Method We established β-amyloid1-42(Aβ1-42) toxicity assay to model AD using neurons derived from human induced pluoripotent stem cells(hiPSCs), based on which drug screening was performed. Methods of cell viability assay, cell immunofluorescence, western blot, and real-time PCR were used to study the mechanisms of Aβ1-42 toxicity and the protective effects from related drugs. Results1) In this study, commercially available neurons derived from one hiPSC line(iCell Neurons) were highly purified neurons with 98.4% TuJ1(+)/Nestin(-) cells; iCell Neurons were predominantly GABAergic neurons and glutamatergic neurons, with forebrain identity; iCell Neurons gradually formed neuronal network and had electrophysiological activity after post-thawing. 2) Aβ1-42 were toxic to iCell Neurons such as decreasing cell viability, inhibiting neurite outgrowth, inducing apoptosis and neuronal loss in iCell Neurons. 3) We used this kind of cellular toxicity model to screen small-molecule compounds and 19 hits were found to prevent Aβ1-42 toxicity from 354 compounds, including one Cdk2 inhibitor. We further screened a series of commercial Cdk inhibitors with different specificities to Cdk subtypes, and confirmed that Cdk2 inhibitors could efficiently protect neurons against Aβ1-42 toxicity. 4) We found that Aβ1-42 toxicity were associated with occurrence of cell cycle events(CCEs) in iCell neurons. Aβ1-42 induced iCell neurons re-entering into cell cycle S phase(DNA synthesis phase) followed by neuronal death. Moreover,Aβ1-42 induced increased expression of cell cycle proteins including Cyclin D1,phos-Rb, E2F1, Cyclin A and Cdk2. 5) Cdk2 small-molecule inhibitors could inhibit occurrence of CCEs. For example, Cdk2 inhibitor GW8510 could prevent the increased expression of Cyclin D1 and phos-Rb caused by Aβ1-42. 7) We successfully differentiated hiPS-C4(another hiPSC line) into forebrain neurons, which were also susceptible to Aβ1-42. 8) Similar with that in iCell Neurons, Aβ1-42 also induced neurons derived from hiPS-C4 into cell cycle S phase followed by neuronal death.Moreover, Aβ1-42 caused subcellular changes of cell cycle proteins in neurons derived from hiPS-C4, including Cdk4, Cyclin D1, Cdk2, and phos-Rb. 9) Cdk2 inhibitors protected neurons derived from hiPS-C4 against Aβ1-42 via prevention of decreased cell viability, neuronal apoptosis and neuronal loss. 9) We confirmed neuronal toxicity of Aβ1-42 and protective effects of Cdk2 inhibitors in neurons derived from human embryonic stem cells(H9 ES). Conclusion We demonstrated that Aβ1-42 were toxic to neurons derived from hiPSCs for the first time and drug screening was performed based on this cellular model. We found that Aβ1-42 toxic effects on neurons derived from hiPSCs were associated with occurrence of CCEs and Cdk2 inhibitors protected neurons against Aβ1-42 toxicity. This study provided an excellent example on how to use hiPSCs to establish in vitro cellular disease models and high-throughput compound screening platform for neurodegenerative diseases.