Effect Of Proteasome Inhibition On Microtubule-associated Protein Tau Metabolism And The Underlying Mechanism In Mouse Organotypic Hippocampal Slice Cultures

Protein degradation nearly controls all the vital movement of organisms. Many researches have shown that the aggregation of tau protein was significantly increased in the case of the inhibition of proteasome activity and dysfunction of proteasome is closely related to the metabolism of tau protein, but the mechanism still unclear. Mice organotypic hippocampal slices were used in this study to investigate the effects of proteasome inhibition on the metabolism of microtubule associated protein tau, and targeting the Akt/GSK-3β signaling pathway to explore the underlying mechanisms. Methods:14-day-old Kunming mice were dislocated executed, acute isolation of the bilateral hippocampi and400μm organotypic hippocampal slices were prepared. After the slices were cultured for different times (0,6,12,24,36,48and72hr), detecting the content of lactate dehydrogenase (LDH) in the medium to assay the activity of the slices and to derermine the culture and drug treating time; after48hr cultured, hippocampal slices were administrated with35μM protein synthesis inhibitor cycloheximide (CHX) and2.5and5μM proteasome reversible inhibitor MG132for6hr, then the expression and phosphorylation of microtubule associated protein tau and the function of microtubule-binding of tau were detected using immunoblotting, the alteration of glycogen synthase kinase-3(3(GSK-3β) activity and protein kinase B (PKB/Akt) activity and the level of heat shock protein90(Hsp90) were also detected in this study. In addition, the interaction between Akt and Hsp90, Akt and protein phosphatase-2A (PP-2A) in the organotypic hippocampal slices were investigated by co-immunoprecipitation. In this study, we found:(1) Organotypic hippocampal slices LDH value reached the peak at6hr, then decreased gradually. Between48hr to72hr, the LDH value remained stably at a relatively low level. So in the next experiments, we cultured the slices for48hr then undertook the neuropharmacological treatment.(2) Compared with controls, the level of the total tau and the phosphorylated tau at Thr231, Thr205and Ser396sites were increased in a dose-dependent manner while the level of phosphorylated tau at Ser214site was decreased after treatment of the slices with2.5and5μM MG132for6hr.(3) MG132treatent impaired the biological activity of tau in binding to the microtubule.(4) MG132treatent reduced the phosphorylation of Akt-Ser473and Thr308as well as GSK-3p-Ser9, while the level of the total Akt and GSK-3β were not changed.(5) The co-immunoprecipitation reaction between Akt and Hsp90as well as PP-2A are positive in hippocampal slices, and5μM MG132treatment decreased the immunoreactivity of interaction of Akt with Hsp90, while the immunoreactivity of interaction between PP-2A and Akt was enhanced.Our results indicated that the microtubule associated protein tau is the substrate of proteasome, inhibition of proteasome activity can lead to the obstacles of tau and increase the total amount of tau protein. In addition, inhibit proteasome activity through down-regulated Akt/GSK-3β signaling pathway activity caused the changing of multiple sites of phosphorylation of tau protein, which led to the tau protein microtubule-binding function decline. Further studies showed that decrease the activity of the Akt/GSK-3β signaling pathway were related to decline the binding capacity of Akt and Hsp90and increase dephosphorylation of Akt by PP-2A. This study provides some experimental evidence of the role of proteasome in tau pathology, and provides a solid scientific basis for the establishment of neurodegenerative diseases organotypic brain slices model.