Identification of an ERK Inhibitor as a Therapeutic Drug Against Tau Aggregation in a New Cell-Based Assay

Abstract

Formation of Tau aggregates is a common pathological feature of tauopathies and their accumulation directly correlates with cytotoxicity and neuronal degeneration. Great efforts have been made to understand Tau aggregation and to find therapeutics halting or reversing the process, however, progress has been slowed due to the lack of a suitable method for monitoring Tau aggregation. We developed a cell-based assay allowing to detect and quantify Tau aggregation in living cells. The system is based on the FRET biosensor CST able to monitor the molecular dynamic of Tau aggregation in different cellular conditions. We probed candidate compounds that could block Tau hyperphosphorylation. In particular, to foster the drug discovery process, we tested kinase inhibitors approved for the treatment of other diseases. We identified the ERK inhibitor PD-901 as a promising therapeutic molecule since it reduces and prevents Tau aggregation. This evidence establishes the CST cell-based aggregation assay as a reliable tool for drug discovery and suggests that PD-901 might be a promising compound to be tested for further preclinical studies on AD.

Keywords: CST; ERK; FRET; Tau; Tau biosensor; aggregation.

FIGURE 1

CST aggregates are detected and quantified in live cells. (A) FRET measured by sensitized emission in CST-reporter HeLa cells. Donor (cyan), acceptor (yellow) and Normalized FRET (NFRET) images (false color). White scale bar = 10 μm. (B) Box plot of NFRET values calculated on MTs network (N = 9) and on aggregates (N = 12). Box spans the standard error of the mean while whiskers indicates the standard deviation (^∗∗∗p < 0.001 t-test). (C) Magnification of cells in A and corresponding NFRET profile along the white line crossing MTs (upper panel) or aggregates (lower panel). (D) Box plot of iNFRET values calculated on MTs network (N = 9) and on aggregates (N = 19). Box spans the standard error of the mean while whiskers indicates the standard deviation (^∗∗∗p < 0.001 t-test).

FIGURE 2

CST aggregates are amyloidogenic proteopathic aggregates. (A) CST-reporter HeLa cells were stained with K114. YFP (yellow), K114 (magenta), white scale bar = 10 μm. (B) Colocalization between CST and K114 signals has been quantified by Pearson Correlation Coefficient (PCC). Box spans the standard error of the mean while whiskers indicates the standard deviation (^∗p < 0.01 t-test). (C–D) Detergent fractionation of CST-reporter and unlabeled Tau-reporter cells. Western blot of the Triton-X100 insoluble fraction (T-IF) and of the Triton-X100 soluble fraction (T-SF) developed with the Tau5 antibody.

FIGURE 3

CST aggregates are altered by phosphorylation in SH-SY5Y cells. (A) FRET measured by sensitized emission in CST reporter cells treated with seeds (CTR) or treated with seeds and STS or OA. CFP (cyan), YFP (yellow) and Normalized FRET (NFRET) images (false color). White scale bar = 10 μm. (B) Box plot of iNFRET values calculated in CTR cells (N = 37), STS treated cells (N = 21) and OA treated cells (N = 15). Box spans the standard error of the mean while whiskers indicates the standard deviation (^∗∗∗p < 0.001 ANOVA one-way test). (C) WB and corresponding quantification of phosphorylation at indicated epitopes in CST reporter cells treated with STS or OA (N = 4).

FIGURE 4

CST aggregates are reduced by PD-901 and D-JNKI-1 treatment in SH-SY5Y cells. (A) WB and corresponding quantification of phosphorylated epitopes S202/T205, S231, S356 in CST reporter cells treated with PD-901 or D-JNKI-1 (N = 4). (B) FRET measured by sensitized emission in reporter cells treated with PD-901 or D-JNKI-1. CFP (cyan), YFP (yellow) and Normalized FRET (NFRET) images (false color). White scale bar = 10 μm. (C) Box plot of iNFRET values calculated in CST reporter cells treated with seeds (CTR) (N = 37), seeds and PD-901 treated cells (N = 27), seeds and D-JNKI-1 treated cells (N = 20). Box spans the standard error of the mean while whiskers indicates the standard deviation (^∗∗∗p < 0.001 ANOVA one-way test).

FIGURE 5

CST aggregates are reduced by PD-901 in mouse primary hippocampal neurons. (A) FRET measured by sensitized emission in primary neurons treated with PD-901. CFP (cyan), YFP (yellow) and Normalized FRET (NFRET) images (false color). White scale bar = 10 μm. (B) Box plot of iNFRET values calculated in cells expressing CST (N = 58), cells treated with seeds (N = 24), cells treated with seeds and PD-901 (N = 39). Box spans the standard error of the mean while whiskers indicates the standard deviation (^****p < 0.0001; seed p < 0.0000001; PD-901 p < 0.00001 ANOVA one-way test).