Figure 6.

iPSC-derived neurons carrying normal PERK alleles are susceptible to ER stress and tau protein pathology when treated with PERK inhibitor. (A) NDC iPSC-derived neuronal cultures were treated with vehicle, tunicamycin (2 μg/ml) and GSK2656157 (PERKi) (200 nM) for 5 h. Representative blots for p-eIF2α, total eIF2α and HSP90 (loading control) are shown and quantified by densitometry (n = 3). One-way ANOVA with Tukey test was performed for statistical analysis. (B)Chop mRNA levels were quantified by qRT-PCR from NDC iPSC-derived neuronal cultures treated (5 h) with the indicated agents and are graphed relative to vehicle treated control. (n =3 ), ^*P-value<0.05, ^**P-value<0.005. One-way ANOVA with Tukey test was performed for statistical analysis. (C) Cleaved PARP, phosphorylated pathologic tau (AT8, PHF1), total tau (K9JA) and tubulin (loading control) were immunoblotted from NDC iPSC-derived neuronal cell lysates treated with DMF, TM or TM+PERKi for 12 h. Representative blots are shown, and protein levels were quantified by densitometry and shown relative to DMF-treated samples (n = 9). One-way ANOVA with Tukey test was performed for statistical analysis. (D)MAPT mRNA levels were quantified by qRT-PCR from iPSC-derived human neuronal cultures treated with DMF, TM or TM+PERKi for 5 h and are shown relative to DMF-treated cultures (n = 3). One-way ANOVA with Tukey test was performed for statistical analysis.