GSK-3β 억제제를 확인하기 위해 타우가 안정적으로 발현하는 인간배아 신장 세포를 이용하여 세포기반 실험 개발

Abstract

Glycogen synthase kinase-3β (GSK-3β), a serine/threonine kinase is also called tau phosphorylating kinase I, which has been implicated in pathogenic conditions of Alzheimer’s disease. Many investigators have focused on searching GSK-3 inhibitor as a therapeutic drug. Here, we established a cell-based assay, which named in situ-ELISA, to assess an imbalance of tau phosphorylation. For this, four-repeat (4R) tau cDNAs were stably expressed in HEK293 cells (HEK293-Tau). Followed by the treatment of the increased concentrations of GSK-3β inhibitor, endogenous GSK-3β and stable-expressed tau were not changed their protein levels in HEK293-Tau cells. Proliferation potential in HEK293-Tau was exhibited with no significant difference from that in HEK293 cells, measured by BrdU-ELISA. The phosphorylation state of taus396 was examined by western blot and by in situ-ELISA with treatment of GSK-3β inhibitors, LiCl, Chir98023 and SB415286, obtained consistent data. We also compared cell-based methods between western blot and in situ-ELISA, ELISA method was more sensitive in screening GSK-3β inhibitor. These data demonstrate that the in situ-ELISA method in HEK293-Tau cells can be applied to the cell-based assay system for screening of GSK-3β inhibitor.