Flow cytometry-based method to analyze the change in Tau phosphorylation in a hGSK-3β and hTau over-expressing EcR-293 cell line

Neurofibrillary tangles are composed of insoluble aggregates of microtubule-associated protein Tau. In the pathology of Alzheimer's disease (AD), accumulation of hyperphosphorylated Tau results in formation of paired helical filaments. One of the main candidate to hyperphosphorylate Tau in AD is glycogen synthase kinase 3β (GSK-3β). Here we introduce a non-neuronal cell line, stably co-expressing human Tau and GSK-3β proteins, where the effect of potential kinase inhibitors on Tau phosphorylation can be monitored. The aim of our study was to establish a new flow-cytometry-based method to quantitatively analyze the changing of Tau phosphorylation, which is a suitable alternative to the well-accepted but non-quantitative Western blot technique. Our results demonstrate that the flow cytometry-based method is a convenient tool to analyze the effect of GSK-3β inhibitors on Tau phosphorylation. This new approach provides appropriate throughput for screening purposes in preclinical research for characterization of GSK-3β inhibitors, as potential drug candidate to cure Alzheimer's disease.