Effects Of Proteasome Inhibition On Microtubule-associated Protein Tau Phosphorylation And The Underlying Mechanism

Neurofibrillary tangles (NFTs) mainly composed of abnormal hyperphosphorylated tau proteins is one of the major hallmarks of the tauopathies, such as Alzheimer's disease (AD), Frontotemporal dementia and Parkinsonism linked to chromosome 17. The precise mechanism of tau hyperphosphorylation and NFTs formation is not clear yet. Previous studies suggested that tau ubiquitination was mediated by ubiquitin-proteasome system (UPS), as well as tau phosphorylation, co-contribute to the formation of NFTs. It was reported that phosphorylated tau coulde be ubiquitinated through E3 ubiquitin ligase and the proteasome activity was decreased in tauopathies brains, so we thought UPS might play an important role in the abnormal hyperphosphorylation of tau. Glycogen synthase kinase-3β(GSK-3β) and its upstream regulator, protein kinase B (Akt/PKB), have a direct link with tau phosphorylation, and they can phosphorylate tau at many sites. Recent study had demonstrated that the combination between heat shock protein 90 (Hsp90) and Akt could regulate Akt level and activity. In present study, we treated the HEK293 cells stably transfected with the longest human tau (tau 441) with lactacystin (a recognized irreversible 20S proteasome inhibitor) and cycloheximide-protease inhibitor (CHX) for 24 h, then the level of total or phosphorylated tau at various epitopes was detected by Western blot. The underlying mechanism involved in Hsp90/Akt/GSK-3βsignal pathway was also investigated by immunofluorescence and co-immuno-precipitation. Results are listed as following:(1) Effects of proteasome inhibition on total and phosphorylated tauProteasome inhibition could increase total tau and phosphorylated tau at Thr231, Ser396, Thr205 and Ser195/198/199/202 sites, while tau phosphorylation decreased at Ser214 epitope, which indicated proteasome can degradate normal tau and phosphorylated tau.(2) The relationship between proteasome and GSK-3βGSK-3βwas the main kinase that regulated tau phosphorylation at above-mentioned epitopes, so we detected the change of GSK-3(3 to reveal the mechinisam of tau phosphorylation after proteasome inhibition. Phosphorylation of GSK-3βat Ser9 epitope could lead to its inactivation. The levels of total and phosphorylated GSK-3βwere assayed by Western blot and immunofluorescence. Quantitative data showed that proteasome inhibition could enhance the GSK-3βactivity, which was consistent with tau hyperphosphorylation. These results suggested proteasome could indirectly regulate tau phosphorylation through upregulating GSK-3βactivity. Simultaneity, the inhibition of proteasome can enhance the total level of GSK-3β. The co-localization and co-precipitation between 20S proteasome and GSK-3βin the cells showed GSK-3βwas the client of proteasome.(3) The relationship between proteasome and AktAkt was the upstream kinase of GSK-3βand it regulated the phosphorylation of tau protein at Ser214 epitope, so we detected the effect of proteasome inhibition on Akt. We found that Akt phosphorylation at Ser473 and Thr308 epitopes (the activated form of Akt) were decreased when proteasome inhibition, which implied downregulation of Akt activity. And there was a consistency between decreased Akt activity and increased GSK-3βactivity. Decreased level of tau phosphorylation at Ser214 epitope was correlated with their co-effect. These results suggested proteasome inhibition by lactacystin activated GSK-3βthrough downregulating the Akt activity and caused tau phosphorylation in the cells. And inhibition of proteasome with different dose of lactacystin increased total Akt at the same time. We also detected the co-localization and co-precipitation between 20S proteasome and Akt in cells, suggested proteasome could also degradate Akt.(4) Effects of proteasome inhibition on heat shock proteinHsp90 could regulate Akt activity, and the result showed that proteasome inhibition induced the increase of Hsp90 and Hsp70 total level. We thought that UPS inhibition by lactacystin increased aggregation proteins, and these aggregation proteins enhanced the level of Hsp90 and Hsp70 to restore the normal function of these misfolded proteins. But this hypothesis that Hsp90 could regulate Akt activity should be conformed by further study, such as detecting the combination between Hsp90 and Akt when the proteasome was inhibited.In conclusion, the mechanism in which proteasome inhibition regulated tau phosphorylation should be as follows:①Proteasome could directly degradate tau and phosphorylated tau protein;②Proteasome could indirectly regulate tau phosphorylation through regulation of GSK-3βand Akt;③Proteasome inhibition could increase the levels of Hsp70 and Hsp90. The increase of Hsp90 by proteasome inhibition might participate in Akt activity regulation, but the precise mechnism need the further study to define.