Stable mutated tau441 transfected SH-SY5Y cells as screening tool for Alzheimer's disease drug candidates

Abstract

The role of hyperphosphorylation of the microtubule-associated protein tau in the pathological processes of several neurodegenerative diseases is becoming better understood. Consequently, development of new compounds capable of preventing tau hyperphosphorylation is an increasingly hot topic. For this reason, dependable in vitro and in vivo models that reflect tau hyperphosphorylation in human diseases are needed. In this study, we generated and validated an in vitro model appropriate to test potential curative and preventive compound effects on tau phosphorylation. For this purpose, a stably transfected SH-SY5Y cell line was constructed over-expressing mutant human tau441 (SH-SY5Y-TMHT441). Analyses of expression levels and tau phosphorylation status in untreated cells confirmed relevance to human diseases. Subsequently, the effect of different established kinase inhibitors on tau phosphorylation (e.g., residues Thr231, Thr181, and Ser396) was examined. It was shown with several methods including immunosorbent assays and mass spectrometry that the phosphorylation pattern of tau in SH-SY5Y-TMHT441 cells can be reliably modulated by these compounds, specifically targeting JNK, GSK-3, CDK1/5, and CK1. These four protein kinases are known to be involved in in vivo tau phosphorylation and are therefore authentic indicators for the suitability of this new cell culture model for tauopathies.

Fig. 1

Characterization of TMHT expression in SH-SY5Y-TMHT441 cells and in brain tissue from TMHT mice. a Schematic presentation of the tau441 construct with two mutations (TMHT). b–d SH-SY5Y-TMHT441 cells were subjected to indirect immunofluorescence analysis using antibodies against HT7 (tau, red) and DAPI (nuclei, blue). Images show secondary antibody control (b), untransfected SH-SY5Y cells (c), and SH-SY5Y-TMHT441 cells (d). Scale bar: 100 μm. e Western blot of SH-SY5Y-TMHT441 cells compared to untransfected cells (wt) and cortical (CTX) and hippocampal (HC) brain tissue homogenates of 12-month-old TMHT transgenic mice. Antibody Tau-5 labels human and mouse tau protein. AD2, AT270, AT180, and AT8 antibodies detect Ser396/Ser404, Thr181, Thr231/Ser235, and Ser202 phosphorylation, respectively. A representative blot of two independent experiments is shown. f, g Measurement of total tau (f) and pThr231 (g) levels in untransfected and stable TMHT441 transfected cells by Mesoscale Discovery (N = 2). **P < 0.01; unpaired t test. Graphs represent mean ± SEM

Fig. 2

JNK inhibitor SP600125 strongly inhibits tau phosphorylation at residue Thr181, Thr231, and Ser199 and to a lesser extent Ser396 in SH-SY5Y-TMHT441 cells. a–c Effect of JNK inhibitor SP600125 (50 μM) on Thr181 (a), Thr231 (b), and Ser396 (c) phosphorylation as measured by Phospho-Tau SRM assay (N = 1). d–f Effect of JNK inhibitor SP600125 on Thr181 (d), Thr231 (e), and Ser396 (f) phosphorylation as measured by Mesoscale Discovery. First lane (black) shows protein of untreated cells, second lane (dark gray) protein of vehicle treated cells and gray bars show SP600125 treated cells (N = 2). *P < 0.05, **P < 0.01. Graphs represent mean ± SEM. One-way ANOVA followed by Dunnett’s post hoc test. g Western Blot of untreated (wt), vehicle (v) treated, and SP600125 (concentration ranging from 0.1–50 μM) treated SH-SY5Y-TMHT441 cells showing total tau (Tau-5), β-actin as loading control, pThr181 (AT270), pThr231/235 (AT180), and pSer396 (AD2) levels. A representative blot of two independent experiments is shown. h Effect of JNK inhibitor SP600125 on Ser199 as measured by Phospho-Tau SRM assay (N = 1). i Effect of JNK inhibitor SP600125 on Ser262 as measured by Phospho-Tau SRM assay (N = 1)

Fig. 3

GSK3-β inhibitor AR-A014418 inhibits Thr231 and Ser396 phosphorylation but not Thr181 tau phosphorylation in SH-SY5Y-TMHT441 cells. Effect of GSK3-β inhibitor AR-A014418 on Thr181 (a), Thr231 (b), and Ser396 (c) phosphorylation as measured by Mesoscale Discovery (N = 2). First lane (black) shows protein of untreated cells. Second lane (dark gray) shows protein of vehicle treated cells. Light gray bars represent AR-A014418 treated cells; dashed bars: treatment caused cell death as measured by MTT assay. *P < 0.05, **P < 0.01. Graphs represent mean ± SEM. One-way ANOVA followed by Dunnett’s post hoc test. d Western blot of untreated (wt), vehicle (v) treated, and AR-A014418 treated (concentration ranging from 0.1 to 50 μM) SH-SY5Y-TMHT441 cells showing total tau (Tau-5), β-actin as loading control, pThr181 (AT270), pThr231/235 (AT180), and pSer396 (AD2) levels. A representative blot of two independent experiments is shown

Fig. 4

Effect of CDK1/5 and CK-I inhibitor on the phosphorylation status of tau in SH-SY5Y-TMHT441 cells. Effect of CDK1/5 inhibitor RP106 on Thr181 (a), Thr231 (b), and Ser396 (c) phosphorylation as measured by Mesoscale Discovery (N =2). Effect of CK-I inhibitor IC261 on Thr181 (d), Thr231 (e), and Ser396 (f) phosphorylation as measured by Mesoscale Discovery (N=2). First lane (black) shows protein of untreated cells; second lane (dark gray) shows protein of vehicle treated cells; light gray bars represent inhibitor treated cells; dashed bars: treatment caused cell death as measured by MTT assay. Graphs represent mean ± SEM. One-way ANOVA followed by Dunnett’s post hoc test