ABSTRACT
BACKGROUND: An accumulation of hyperphosphorylated tau in the brain is a hallmark of Alzheimer's disease (AD). Deficits in protein phosphatase 2A (PP2A) are associated with tau hyperphosphorylation in AD. OBJECTIVE: To investigate the effects of morroniside (MOR), isolated from Cornus officinalis, on tau hyperphosphorylation and its underlying mechanisms related to PP2A. METHODS: SK-N-SH cells were pretreated with 50-200 µM MOR for 24âh followed by 20 nM okadaic acid (OA) for 6âh. PP2Ac siRNA was transfected into HEK293 cells to determine the direct interaction of MOR with PP2A. Western blotting was used to measure the expression of proteins and enzymes. PP2A activity was measured by molybdenum blue spectrophotometry. RESULTS: Pretreatment with MOR improved the cellular morphological damage and inhibited tau hyperphosphorylation in SK-N-SH cells induced by OA, a PP2A inhibitor. Moreover, MOR increased PP2A activity, concurrent with a decrease in the expression of demethylated PP2A at Leu309 and phosphorylated PP2A at Tyr307. MOR decreased protein phosphatase methylesterase 1 (PME-1) expression and the ratio of PME-1/leucine carboxyl methyltransferase 1 (LCMT-1). Furthermore, MOR treatment decreased the phosphorylation of Src at Tyr416, which regulates the phosphorylation of PP2A. MOR had no effect on PP2Ac expression and tau hyperphosphorylation in PP2Ac siRNA-transfected cells. CONCLUSION: MOR attenuated OA-induced tau hyperphosphorylation via PP2A activation, and its mechanism might be related to the regulation of PP2Ac post-translational modification and upstream enzymes such as Src and PME-1.
Subject(s)
Glycosides/pharmacology , Protein Phosphatase 2/metabolism , tau Proteins/metabolism , Analysis of Variance , Cell Line , Enzyme Inhibitors/pharmacology , Gene Expression Regulation/drug effects , Gene Expression Regulation/genetics , Glycogen Synthase Kinase 3 beta/metabolism , HEK293 Cells , Humans , Neuroblastoma/pathology , Okadaic Acid/pharmacology , Phosphorylation/drug effects , Phosphorylation/genetics , Protein Phosphatase 2/genetics , RNA, Small Interfering/genetics , Transfection , Tyrosine/metabolismABSTRACT
Aim. 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 preincubated with CIG (50, 100, and 200 µg/ml, resp.) for 24 h and then exposed to 10 µM WT and 10 µM GFX for 3 h after washing out CIG. Immunohistochemistry 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 3 ß (GSK-3 ß ), and protein phosphatase 2A (PP2A). The activity of PP2A was detected by a biochemical assay. Results. Preincubation of CIG significantly attenuated the WT/GFX-induced tau hyperphosphorylation at the sites of Thr205, Thr212, Ser214, Thr217, Ser396, 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 p-GSK-3 ß -ser9 induced by WT/GFX. However, CIG significantly elevated the activity of PP2A by reducing the demethylation of PP2A catalytic subunit (PP2Ac) at Leu309 and the ratio of PME-1/LCMT in the WT/GFX-treated cells. The results suggest that CIG may be beneficial to the treatment of AD.
