AD Model: Tau Phosphorylation Assay Cell Line


Categories: Cells

The microtubule associated protein Tau is the main component of the neurofribillary tangles (NFT), aberrant structures that appear in the brain of Alzheimer?s disease patients and other tauopathies, such as FTDP-17 or corticobasal degeneration. Tau protein binds to and stabilizes microtubules (MTs) but in pathological states, it aggregates and loses its important functions. These Tau aggregates are composed basically by hyperphosphorylated and truncated forms of tau. Multiple Tau gene mutations are pathogenic for hereditary FTDP-17 disease. These mutations have similar effects to hyperphosphorylation in Tau in AD and result in NFT formation.

A novel recombinant cell line has been developed for the screening of kinase modulators that affect the behavior or location of Tau protein. Innoprot offers this cell line as a stable cell line but it is also offered as vials of division-arrested cells (DA cells) in order to perform a small number of assays. Each vial of these DA cells contains 2 million cells, an enough number of cells to perform a complete experiment in a 96 well-plate.

Assay Details: U2OS stably express the human triple mutant Tau-tGFP cells were treated with log dilution series (n=3) of the LiCl during 2 hours to evaluate their effects on Tau phosphorylation and binding to microtubules. After that, the nucleus was stained with DAPI and cells Tau and MT bundles were detected by fluorescence using image analysis algorithms. % Activity was calculated relative to positive (100 mM).

IC50 value for LICl was determined by treating of U2OS TauTM-tGFP model cells with inhibitor concentrations from 10uM to 100mM during 2 hours. Followed this incubation, the intracellular bundle formation is quantified with a BD Pathway 855 High-Content Bioimager and Attovision software. Error bars represent the standard deviation among 3 replicate wells. IC50 for LiCl is 11.75 mM and z' for this experiment was 0.80 +/-0.02.

Fig 1: This assay was also performed with human Tau wild-type and single mutant Tau. Depending on the mutation, MT-bundle amount varies. These Tau mutations have similar effects to hyperphosphorylation: the triple mutant has less affinity for microtubules and consequently forms less bundles. Wt and SM shows more bundling cells than TM at rest, so their capacity to form bundles after GSK3- kinase inhibitor LiCl addition is limited.

Fig 2: U2OS stably express the human triple mutant Tau-tGFP cells were treated with log dilution series (n=3) of the LiCl during 2 hours to evaluate their effects on Tau phosphorylation and binding to microtubules.

Fig 3:IC50 value for LICl was determined by treating of U2OS TauTM-tGFP model cells with inhibitor concentrations from 10uM to 100mM during 2 hours. Followed this incubation, the intracellular bundle formation is quantified with a BD Pathway 855 High-Content Bioimager and Attovision software. Error bars represent the standard deviation among 3 replicate wells. Ic50 for LiCl is 11.75 mM and z' for this experiment was 0.80 +/-0.02.