A Novel Mechanism Causes I₂^PP2A/SET Cytoplasm Accumulation In Alzheimer’s Disease

Background: The activity of protein phosphatase-2A (PP2A) is compromised and isbelieved to be a main cause of the abnormal hyperphosphorylation of tau inAlzheimer’s disease (AD) brain. The intracellular activity of PP2A is regulated by twoendogenous proteins called I1PP2A and I2PP2A/SET. I2PP2A/SET is mainly a nuclearprotein. In AD brain, the transcription and expression of SET is increased and SET istranslocated from its primary nuclear location to the cytoplasm in the neurons whichinduced it co-localizes both with PP2A and abnormally hyperphosphorylated tau.However, until now, little is known about the detailed regulatory mechanism by whichSET was detained in cytosol and the consequent events in mammalian cells.Phosphorylation is the best-understood mechanism of nuclear transport regulation. Byradiosequencing and phosphorylated amino acid analysis, it was identified in vivoSer9was the important phosphorylation site of SET. Intriguingly, SET sequence(6-11aa) AKVSKK almost coincided with the consensus nuclear localization signal(NLS) sequence KKXXKX or XKXXKK.Objective: It was aimed to investigate the detailed regulatory mechanism by whichSET was detained in cytosol in Alzheimer’s disease brains.Methods: The longest human tau (tau441) cDNA was stably transfected into HEK293cells (HEK293/tau). The cells were cultured in Dulbecco’s modified Eagle’smedium (DMEM) containing10%fetal bovine serum, and in a humidified incubatoraerated with95%air and5%CO2at37C. The anti-SET (phospho S9) antibody rabbitpolyclonal antibody was prepared and purified with protein A-sepharose affinitychromatography from Abmart. The AD and the age-matched control brain cortexslices were generous gifts of the Dr. Iqbal. The immunoreaction was detected usinghorseradish peroxidase-labeled antibodies for1h at37C and visualized with DABtetrachloride system. Site-directed mutagenesis was carried out to mutate Ser-9ofHis-SET to either alanine (S9A) or glutamic acid (S9E), which mimicked thenon-phosphorylated (npSET) or phosphorylated (pSET) condition of SET respectively,and then mutate lysine7, lysine10and lysine11to alanine acid (K7A, K10A, K11A)respectively. After24h of subculture, cells were transfected with these mutants for48h, followed by confocal laser microscopy and image analysis to determine thesubcellular distribution of transfected SET mutants. Immunoprecipitation/Westernblot was performed to detect the binding between SET mutants and importin proteins.The effects of SET mutants on tau hyperphosphorylation at Ser396, Thr231, Ser262,Thr205, Ser214, PHF-1, Ser404and Ser199, respectively were examined by Westernblot; and the Serine/Threonine Phosphatase Assay kit (Promega, Cat.#V2460) wasused to determine PP2A activity. HEK293/tau cells were treated with100μg/mlTat-NLS (Tat-AAKVSKKE) or Tat-sNLS (Tat-KAAKSKVE) to test the effect ofTat-NLS on the subcellular localization of endogenous SET in cells.Results:In this study, we found that SET was significantly phosphorylated at Ser9; moreover,phosphorylated SET was accumulated in cytosol in AD brain cortex slices.48hoursafter transfection, SET S9E (Ser9mimic-phosphorylation) mutants showed nuclearimport defect in HEK293/tau cells, which were resulted from decreased importin-αbinding. Protein kinase CKII has been suggested to be involved in the pathology ofAD. Meanwhile, Ser9is the consensus CKII phosphorylation site. Here we confirmed that phosphorylation of SET at serine9could be induced by overexpression of CKII,which was verified by a SET S9phosphor (S9ph)-specific antibody. Importantly,CKII-mediated SET serine9phosphorylation repressed its nuclear import activity andled to reservation of SET in cytoplasm. Therefore, CKII might be a novel druggabletarget of Alzheimer disease. In addition, Ser9mimic-phosphorylation of SET inhibitsPP2A activity significantly by binding to its catalytic subunit PP2Ac with higheraffinity and causes hyperphosphorylation of tau at Ser396, Thr231, Ser262, Thr205,Ser214, PHF-1, Ser404and Ser199sites in HEK293/tau cells. Meanwhile, theputative nuclear localization signal (NLS) sequence6AKVSKK11of SET plays anessential role on SET nuclear import. His-SET (K11A) mutant showed nuclear importdefect in HEK293/tau cells, which were resulted from decreased importin-α binding.Furthermore, the binding of endogenous SET and importin-α/β system could beblocked competitively by a synthesized SET nuclear localization signal peptideTat-AAKVSKKE, which decreased SET nuclear accumulation. All results indicatethat6AKVSKK11is a potential nuclear localization signal (NLS) sequence of SET anda novel mechanism causes SET cytoplasm accumulation in specificphosphorylation-dependent fashion.Conclusion:SET is significantly phosphorylated at Ser9and shows cytoplasm accumulation in ADbrains. Mimic-phosphorylation and CKII-mediated SET serine9phosphorylationinhibits SET nuclear import in HEK293/tau cells, which were resulted from decreasedimportin-α binding. Phosphorylated SET promotes the inhibition of PP2A activity andtau hyperphosphorylation in HEK293Cells. The sequence6AKVSKK11wasconfirmed to be a potential nuclear localization signal (NLS) of SET. Importantly,SET cytoplasm accumulation in AD brains was caused in specificphosphorylation-dependent fashion. Background: Berberine is an isoquinoline alkaloid extracted from Chinese herbs, such asCoptidis rhizome, which has versatile health effects and possesses a wide variety ofbiochemical and pharmacological activities, including anti-diarrheal, anti-arrhythmic, andanti-tumor activities. Over the past several years, pharmacodynamic studies have revealedpotential roles for berberine in the treatment of AD, including amelioration of spatialmemory impairment in a rat model of AD; alteration of the processing of amyloid-β proteinprecursor (AβPP) and reduction of Aβ secretion; attenuation of Aβ-induced apoptosis byreducing hippocampal neuronal intracellular free Ca2+; and anticholinesterase activities.According to these reports, berberine could be a potential drug for AD. In addition,berberine also regulates GSK-3β activity in HCT-116cells. The severity of dementia ispositively related to the number of NFTs in neocortex, constituted by hyperphosphorylatedmicrotubule-associated protein tau protein, which suggests hyperphosphorylation of tau plays a key role in Alzheimer’s disease pathogenesis. So far, it has been well recognizedthat an imbalanced regulation in protein kinases and protein phosphatases is the direct causefor the AD-like tau hyperphosphorylation. Among various kinases and phosphatases,glycogen synthase kinase-3(GSK-3) and protein phosphatase (PP)-2A are, respectively, themost implicated. However, until now, there has been no study on whether berberine affectstau phosphorylation. This study is to confirm whether berberine has any effect on tauhyperphosphorylation and the underlying mechanism. Here, we report the effect ofberberine on tau hyperphosphorylation induced by calyculin A, an inhibitor of PP2A andPP1, in tau-transfected HEK293cells.Methods: The longest human tau (tau441) cDNA was stably transfected into HEK293cells(HEK293/tau). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)containing10%fetal bovine serum, and in a humidified incubator aerated with95%air and5%CO2at37C. After24h of subculture, cells were treated with2.5nM calyculin Adiluted in serum-free medium for12h, followed by the treatments with0,5,10,20,50, or100μg/ml berberine for another24h. The CCK-8Cell Counting kit was used to determinecell viability and to get the optimal dose of berberine. Following that, microscope imaging,hoechst33342staining, TUNEL assay, flow cytometry and caspase-3activity assay werecarried out to determine effects of berberine on calyculin A-induced morphological changesand apoptosis of HEK293/tau cells. Western bolts were used to detect effects of berberineon calyculin A-induced tau hyperphosphorylation at Ser198/199/202(Tau-1sites), Ser396,Ser404, Thr205, and Thr231, respectively; and phosphorylation of glycogen synthasekinase-3β (GSK-3β) at Tyr216and Ser9after treatments with20μg/ml berberine. TheSerine/Threonine Phosphatase Assay kit (Promega, Cat.#V2460) was used to determinePP2A activity. Malondialdehyde level and superoxide dismutase activity were measured toevidence the effect of berberine on calyculin A-induced oxidative stress.Results:1) Berberine at20μg/ml reduced the cell death induced by2.5nM calyculin A; 2) Berberine markedly reversed calyculin A-induced morphological changes and apoptosisof cells;3) Berberine attenuated calyculin A-induced tau hyperphosphorylation at Ser198/199/202(Tau-1sites), Ser396, Ser404, Thr205, and Thr231sites significantly.4) Treatments of HEK293/tau cells with berberine after calyculin A restored significantlyPP2A activity and reduced GSK-3β activity, as determined by phosphatase activityassay and GSK-3β phosphorylation at Tyr216and Ser9, respectively;5) Berberine could act as an antioxidant by decreasing malondialdehyde level andincreasing superoxide dismutase activity in calyculin A-induced oxidative stress.Conclusion: Berberine attenuates Calyculin A-induced cytotoxicity and tau hyperphos-phorylation in HEK293Cells. Berberine also restored protein phosphatase2A activity andreversed glycogen synthase kinase-3β (GSK-3β) activation. Furthermore, berberinereversed both the increase of malondialdehyde and the decrease of superoxide dismutaseactivity induced by calyculin A, indicating its role in anti-oxidative stress. Our findingssuggest that berberine may be a potential therapeutic drug for AD.