OBJECTIVE The aim of the present study was to investigate the effect of cornel iridoid glycoside(CIG)on tau hyperphosphorylation induced by wortmannin(WT)and GF-109203X(GFX)and the underlying mechanisms.METHODS Human neuroblastoma SK-N-SH cells were pre-incubated with CIG(50,100,and 200μg·mL-1,respectively)for 24 h,and then exposed to WT 10μmol·L-1 and GFX 10μmol·L-1for 3hafter washing out CIG.Immuno-fluorescence was used to observe the microtubular cytoskeleton of the cultured cells.Western blotting was used to measure the phosphorylation level of tau protein,glycogen synthase kinase 3β(GSK-3β)and protein phosphatase 2A(PP2A).The activity of PP2 Awas detected by a biochemical assay.RESULTS Pre-incubation of CIG significantly attenuated the WT/GFX-induced tau hyperphosphorylation at the sites of Thr205,Thr212,Ser214,Thr217Ser396 and PHF-1,and improved the damage of morphology and microtubular cytoskeleton of the cells.CIG did not prevent the decrease in p-AKT-ser473 and pGSK3β-ser9 induced by WT/GFX.However,CIG significantly elevated the activity of PP2 Aby reducing the demethylation of PP2AC at Leu309 and the ratio of PME-1/LCMT in the WT/GFX-treated cells.CONCLUSION CIG obviously attenuated WT/GFX-induced tau hyper-phosphorylation at multiple AD-related sites by increasing the activity of PP2A.The mechanism may be involved in the reduced demethylation of PP2 Avia down regulating the ratio of PME/LCMT.
OBJECTIVE Protein phosphatase 2A(PP2A),a major protein phosphatase,have been reported to be involved in the microtubule-associated protein tau hyperphosphorylation and aggregation in Alzheime disease(AD).Morroniside(MOR)is the isolated component from Cornus officinalis Sieb.et Zucc.The present study is to investigate the inhibitory effect of MOR on tau hyperphosphorylation and the underlying mechanisms.METHODS SK-N-SH cells were pretreated with MOR 50-200μmol·L-1 for 24 hand then treated with okadaic acid(OA)(20nmol·L-1)for 6h to induce tau hyperphosphorylation by inhibiting PP2A activity.To determine whether the inhibitory effect of MOR on tau hyperphosphorylation was dependent on PP2A directly,we transfected PP2Ac siRNA into HEK293 cells.Cell morphology was visualized under contrast microscope.Western blotting was used to measure the expressions of phosphorylated tau,total tau,Protein phosphatase-2A(PP2A),phosphorylated PP2 Aat Tyr307(P-PP2A),demethylated PP2 Aat Leu309(DM-PP2A),protein phosphatase methylesterase 1(PME-1),Leucine carboxyl methyltransferase 1(LCMT-1),phosphorylated Src at Tyr416 and Tyr529,total Src,glycogen synthase kinase-3β(GSK-3β)and phospho-GSK3β(Ser9).The activity of PP2 A was measured by aprotein phosphatases activity assay kit.RESULTS Compared with the control,the OA-treated cells became retracted and rounded up and their tau phosphorylation levels at pSer199/202,pT205,pT212,pS214,pT217 markedly increased.Pretreatment with MOR improved the cellular morphology and reduced OA-induced tau hyperphosphorylation.In addition,MOR treatment increased PP2 Aactivity accompanied by a decrease of DM-PP2 Aand P-PP2 Aexpression.MOR decreased PME-1expression and the ratio of PME/LCMT-1.Furthermore,MOR treatment altered the level of Src phosphorylated at Tyr416,which can regulate phosphorylation of PP2 A.PP2Ac siRNA could inhibit PP2Ac expression and induce tau hyperphosphorylation.MOR had no effect on PP2Ac expression,correspondingly,didn′t affect tau hyperphosphorylation in PP2Ac siRNA transfec
