Okadaic-Acid-Induced Inhibition of Protein Phosphatase 2A Produces Activation of Mitogen-Activated Protein Kinases ERK1/2, MEK1/2, and p70 S6, Similar to That in Alzheimer's Disease
2003; Elsevier BV; Volume: 163; Issue: 3 Linguagem: Inglês
10.1016/s0002-9440(10)63445-1
ISSN1525-2191
AutoresJin-Jing Pei, Cheng‐Xin Gong, Wenlin An, Bengt Winblad, Richard F. Cowburn, Inge Grundke‐Iqbal, Khalid Iqbal,
Tópico(s)Alzheimer's disease research and treatments
ResumoIn Alzheimer's disease (AD) brain the activity of protein phosphatase (PP)-2A is compromised and that of the extracellular signal-regulated protein kinase (ERK1/2) of the mitogen-activated protein kinase (MAPK) family, which can phosphorylate tau, is up-regulated. We investigated whether a decrease in PP-2A activity could underlie the activation of these kinases and the abnormal hyperphosphorylation of tau. Rat brain slices, 400-μm-thick, kept under metabolically active conditions in oxygenated (95% O2, 5% CO2) artificial CSF were treated with 1.0 μmol/L okadaic acid (OA) for 1 hour at 33°C. Under this condition, PP-2A activity was decreased to ∼35% of the vehicle-treated control slices, and activities of PP-1 and PP-2B were not affected. In the OA-treated slices, we observed a dramatic increase in the phosphorylation/activation of ERK1/2, MEK1/2, and p70 S6 kinase both immunohistochemically and by Western blots using phosphorylation-dependent antibodies against these kinases. Treatment of 6-μm sections of the OA-treated slices with purified PP-2A reversed the phosphorylation/activation of these kinases. Hyperphosphorylation of tau at several abnormal hyperphosphorylation sites was also observed, as seen in AD brain. These results suggest 1) that PP-2A down-regulates ERK1/2, MEK1/2, and p70 S6 kinase activities through dephosphorylation at the serine/threonine residues of these kinases, and 2) that in AD brain the decrease in PP-2A activity could have caused the activation of ERK1/2, MEK1/2, and p70 S6 kinase, and the abnormal hyperphosphorylation of tau both via an increase in its phosphorylation and a decrease in its dephosphorylation. In Alzheimer's disease (AD) brain the activity of protein phosphatase (PP)-2A is compromised and that of the extracellular signal-regulated protein kinase (ERK1/2) of the mitogen-activated protein kinase (MAPK) family, which can phosphorylate tau, is up-regulated. We investigated whether a decrease in PP-2A activity could underlie the activation of these kinases and the abnormal hyperphosphorylation of tau. Rat brain slices, 400-μm-thick, kept under metabolically active conditions in oxygenated (95% O2, 5% CO2) artificial CSF were treated with 1.0 μmol/L okadaic acid (OA) for 1 hour at 33°C. Under this condition, PP-2A activity was decreased to ∼35% of the vehicle-treated control slices, and activities of PP-1 and PP-2B were not affected. In the OA-treated slices, we observed a dramatic increase in the phosphorylation/activation of ERK1/2, MEK1/2, and p70 S6 kinase both immunohistochemically and by Western blots using phosphorylation-dependent antibodies against these kinases. Treatment of 6-μm sections of the OA-treated slices with purified PP-2A reversed the phosphorylation/activation of these kinases. Hyperphosphorylation of tau at several abnormal hyperphosphorylation sites was also observed, as seen in AD brain. These results suggest 1) that PP-2A down-regulates ERK1/2, MEK1/2, and p70 S6 kinase activities through dephosphorylation at the serine/threonine residues of these kinases, and 2) that in AD brain the decrease in PP-2A activity could have caused the activation of ERK1/2, MEK1/2, and p70 S6 kinase, and the abnormal hyperphosphorylation of tau both via an increase in its phosphorylation and a decrease in its dephosphorylation. 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We found that the inhibition of PP-2A by okadaic acid (OA) induced a dramatic increase in the phosphorylation/activation of ERK1/2, MEK1/2, and p70 S6 kinase as well as the phosphorylation of tau at several of the sites seen in PHF-tau. The topography of the activation of these kinases differed markedly from one another. The selective inhibition of PP-2B by cyclosporin A (CsA) in the brain slices did not significantly change the phosphorylation/activation of any of the three kinases studied.Materials and MethodsMaterialsThe catalytic subunit of PP-2A was isolated from bovine brain according to Cohen et al.34Cohen P Alemany S Hemmings BA Resink TJ Strålfors P Tung HYL Protein phosphatase-1 and protein phosphatase-2A from rabbit skeletal muscle.Meth Enzymol. 1988; 159: 390-408Crossref PubMed Scopus (386) Google Scholar Phosphorylase kinase was purified from the skeletal muscle of White New Zealand rabbits by the method of Cohen.35Cohen P The subunit structure of rabbit-skeletal-muscle phosphorylase kinase, and the molecular basis of its activation reactions.Eur J Biochem. 1973; 34: 1-14Crossref PubMed Scopus (508) Google Scholar Inhibitor-1 was also isolated from the rabbit skeletal muscle and phosphorylated by the catalytic subunit of cAMP-dependent protein kinase (Sigma, St. Louis, MO) according to the method of Cohen et al.36Cohen P Foulkes JG Holmes CFB Nimmo GA Tonks NK Protein phosphatase inhibitor-1 and inhibitor-2 from rabbit skeletal muscle.Methods Enzymol. 1988; 159: 427-437Crossref PubMed Scopus (82) Google Scholar Antibodies to different enzymes and tau are listed in Table 1. OA (ammonium salt) was bought from Calbiochem (San Diego, CA), and CsA from Alexis Corp. (San Diego, CA).Table 1Antibodies Employed in This StudyAntibodySpecificity*P, phosphorylated epitope; NP, nonphosphorylated epitope;Phosphorylation siteDilutionSupplier [reference]Phospho-p44/42 MAP kinaseP, active ERK1/2Thr202/Tyr2041:100Cell Signaling†Beverly, MA.p44/42 MAP kinasetotal ERK1/21:100Cell SignalingPhospho-MEK1/2P, active MEK1/2Ser217/2211:100Cell SignalingMEK1/2total MEK1/21:100Cell SignalingPhospho-p70 S6 kinaseP, active p70 S6Thr421/Ser4241:100Cell Signalingp70 S6 kinasetotal p70 S61:100Cell SignalingAnti-GSK-3α/βP, active GSK-3α/βTyr279/2161:800Biosource‡Camarillo, CA.Phospho-JNK MAP kinaseP, active JNKThr183/Tyr1851:100Cell SignalingPhospho-p38 MAP kinaseP, active p38Thr180/Tyr1821:100Cell Signaling92eTotal tau1:5000[68Grundke-Iqbal I Vorbrodt AW Iqbal K Tung YC Wang GP Wisniewski HM Microtubule-associated polypeptides tau are altered in Alzheimer-paired helical filaments.Brain Res. 1988; 464: 43-52Crossref PubMed Scopus (119) Google Scholar]Tau-1NP tauSer198/199/2021:50,000[69Liu WK Moore WT Williams RT Hall FL Yen SH Application of synthetic phospho- and unphospho-peptides to identify phosphorylation sites in a subregion of the tau molecule, which is modified in Alzheimer's disease.J Neurosci Res. 1993; 34: 371-376Crossref PubMed Scopus (61) Google Scholar]12E8P tauSer262/3561:500[70Seubert P Mawal-Dewan M Barbour R Jakes R Goedert M Johnson GV Litersky JM Schenk D Lieberburg I Trojanowski JQ Lee VM-Y Detection of phosphorylated: Ser262 in fetal tau, adult tau, and paired helical filament tau.J Biol Chem. 1995; 270: 1817-1822Crossref PubMed Scopus (29) Google Scholar]PHF-1P tauSer396/4041:500[71Greenberg SG Davies P Schein JD Binder LI Hydrofluoric acid-treated tau PHF proteins display the same biochemical properties as normal tau.J Biol Chem. 1992; 267: 564-569Abstract Full Text PDF PubMed Google Scholar, 72Otvos Jr, L Feiner L Lang E Szendrei GI Goedert M Lee VM Monoclonal antibody PHF-1 recognizes tau protein phosphorylated at serine residues 396 and 404.J Neurosci Res. 1994; 39: 669-673Crossref PubMed Scopus (405) Google Scholar]R145P tauSer4221:3000[67Tanaka T Zhong J Iqbal K Trenkner E Grundke-Iqbal I The regulation of phosphorylation of τ in SY5Y neuroblastoma cells: the role of protein phosphatases.FEBS Lett. 1998; 426: 248-254Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar]* P, phosphorylated epitope; NP, nonphosphorylated epitope;† Beverly, MA.‡ Camarillo, CA. Open table in a new tab For immunohistochemical studies of AD brain, formalin-fixed frozen 100-μm sections of hippocampi from AD cases at Braak stage V37Braak H Braak E Neuropathological staging of Alzheimer-related changes.Acta Neuropathol. 1991; 82: 239-259Crossref PubMed Scopus (11332) Google Scholar were obtained from Dr. Heiko Braak of J.W. Goethe University, Frankfurt, Germany.Preparation of Rat Brain Slices and Treatment with Protein Phosphatase InhibitorsCD rats (Caesarean derived from a Wistar rat in Charles River Lab, Wilmington, MA), male, 2 to 3 months old, were injected intraperitoneally with 75 mg/kg Nembutal. The animals were decapitated when deeply anesthetized. The brains were immediately removed and cooled down in ice-cold (4°C) oxygenated artificial cerebrospinal fluid (CSF) consisting of 126 mmol/L NaCl, 3.5 mmol/L KCl, 1.2 mmol/L NaH2PO4, 1.3 mmol/L MgCl2, 2.0 mmol/L CaCl2, 11 mmol/L D(+)glucose, 25 mmol/L NaHCO3 (pH 7.4) for 7 to 8 minutes. Each brain was then divided sagittally and 400-μm-thick coronal slices were made with a Camden Vibraslicer (WP Inc., Sarasota, FL). The slices were transferred into a chamber containing the oxygenated artificial CSF and incubated at room temperature for 1 hour, followed by incubation at 33°C for 1 to 3 hours. The PP inhibitors were included in the artificial CSF during incubation at 33°C. Typically eight slices were prepared from one brain within 10 minutes. Half of them were treated with PP inhibitors and the other half with artificial CSF alone as controls. The oxygenation of the artificial CSF was carried out by bubbling the solution with a mixture of 95% O2 and 5% CO2 during the entire procedure.At the end of incubation, the brain slices were either homogenized at 4°C for biochemical analyses or fixed for immunocytochemical studies (see below). For biochemical analyses, the brain slices were rinsed briefly with homogenizing buffer [50 mmol/L Tris-HCl (pH 7.0), 10 mmol/L β-mercaptoethanol, 1.0 mmol/L ethylenediaminetetraacetate (EDTA), 0.1 mmol/L phenylmethyl sulfonyl fluoride, 2.0 mmol/L benzamidine and 2.0 μg/ml each of aprotinin, leupeptin, and pepstatin A] and homogenized at a ratio of 9.0 ml buffer/1.0-g tissue slices. The homogenates were divided into two parts. One was centrifuged at 16,000 × g for 10 minutes and the resulting supernatant was used for PP activity assays. Into the other half, an equal volume of phosphatase-inhibitor cocktail (20 mmol/L β-glycerophosphate, 2.0 mmol/L Na3VO4 and 100 mmol/L NaF, pH 7.0) was added immediately and the samples were stored at −80°C for Western blotting. Protein concentrations of all samples were quantitated by the method of Bradford38Bradford MM A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal Biochem. 1976; 72: 248-254Crossref PubMed Scopus (213510) Google Scholar using the Protein Assay Reagent from Bio-Rad (Hercules, CA) and bovine serum albumin as a standard.Involvement of MEK1/2-ERK1/2 Cascade in Phosphorylation of TauSome rat brain slices were prepared by using a McIlwain Tissue Chopper (Brinkmann, Westbury, CT) as described previously.24Gong C-X Lidsky T Wegiel J Zuck L Grundke-Iqbal I Iqbal K Phosphorylation of microtubule-associated protein tau is regulated by protein phosphatase 2A in mammalian brain. Implications for neurofibrillary degeneration in Alzheimer's disease.J Biol Chem. 2000; 275: 5535-5544Crossref PubMed Scopus (390) Google Scholar The tissue slices (400-μm-thick) were immediately washed twice with oxygenated artificial CSF, and the slices from each brain were evenly divided into 5 portions for parallel treatments with various compounds. After incubation of the tissue slices in oxygenated artificial CSF with or without specific inhibitors at 33°C for 2 hours, the tissue was harvested, washed twice with ice-cold homogenizing buffer (20 mmol/L β-glycerophosphate, pH 7.0, 10 mmol/L β-mercaptoethanol, 1.0 mmol/L EDTA, 0.1 mmol/L phenylmethyl sulfonyl fluoride, 2.0 mmol/L benzamidine, 2.0 mmol/L NaVO4, 100 mmol/L NaF, and 2.0 μg/ml each of aprotinin, leupeptin, and pepstatin A) and then homogenized in 9 volumes of the same homogenizing buffer. The homogenates were stored at −80°C until further analysis.Assays of Protein Phosphatases and Protein KinasesThe activities of PP-1 and PP-2A in the extracts of brain slices were assayed using [32P]phosphorylase a as a substrate as described by us previously.39Gong C-X Grundke-Iqbal I Iqbal K Dephosphorylation of Alzheimer disease abnormally phosphorylated tau by protein phosphatase-2A.Neurosci. 1994; 61: 765-772Crossref PubMed Scopus (139) Google Scholar A PP-1 specific inhibitor, Inhibitor 1,40Atiken A Bilham T Cohen P Complete primary structure of protein phosphatase inhibitor-1 from rabbit skeletal muscle.Eur J Biochem. 1982; 126: 235-246Crossref PubMed Scopus (118) Google Scholar was added in the assays for PP-2A activity. PP-1 activity was calculated by subtracting the PP-2A activity from the total phosphorylase phosphatase activity (PP-1 + PP-2A) assayed in the absence of Inhibitor-1. PP-2B activity was assayed using [32P]phosphorylase kinase as a substrate as described previously41Gong C-X Shaikh S Grundke-Iqbal I Iqbal K Inhibition of protein phosphatase-2B (calcineurin) activity towards Alzheimer abnormally phosphorylated tau by neuroleptics.Brain Res. 1996; 741: 95-102Crossref PubMed Scopus (45) Google Scholar except that 1.0 μmol/L calyculin A was added into the reaction mixture to inhibit PP-1 and PP-2A. The activation of ERK1/2, MEK1/2, and p70 S6 kinase were determined by immunostaining with antibodies specific to the activated/phosphorylated forms of these enzymes (see Table 1).Western Blot AnalysesThe levels of tau phosphorylation and the immunoreactivities of different enzymes were analyzed by Western blots using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis as described originally by Laemmli.42Laemmli UK Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature. 1970; 227: 680-685Crossref PubMed Scopus (206057) Google Scholar The protein bands were transferred to Immobilon-P membrane (Millipore, Bedford, MA) and probed with different antibodies listed in Table 1. The blots were developed with alkaline phosphatase-conjugated secondary antibodies, and 5-bromo-4-chloro-3-indoyl phosphate p-toluidine salt and p-nitro blue tetrazolium chloride as substrates.ImmunohistochemistrySome of the rat brain slices were fixed in periodate/lysine/paraformaldehyde solution for 3 to 5 hours and embedded in paraffin.24Gong C-X Lidsky T Wegiel J Zuck L Grundke-Iqbal I Iqbal K Phosphorylation of microtubule-associated protein tau is regulated by protein phosphatase 2A in mammalian brain. Implications for neurofibrillary degeneration in Alzheimer's disease.J Biol Chem. 2000; 275: 5535-5544Crossref PubMed Scopus (390) Google Scholar Paraffin-embedded sections (6 μm) were cut from the tissue slices. Before processing for immunostaining, the sections were placed in 0.01 mol/L citric acid (pH 6.0) and microwaved as described.43Kass L Varayoud J Ortega H Munoz de Toro M Luque EH Detection of bromodeoxyuridine in formalin-fixed tissue: DNA denaturation following microwave or enzymatic digestion pretreatment is required.Eur J Histochem. 2000; 44: 185-191PubMed Google Scholar To eliminate nonspecific bindings, the sections were treated with 5% H2O2/methanol for 10 minutes, then 3% normal goat serum in 50 mmol/L TBS (pH 7.4), for 30 minutes at room temperature. The sections were then incubated with primary antibodies listed in Table 1 at 4°C overnight, followed by incubation with biotinylated anti-mouse or anti-rabbit IgG at a dilution of 1:200 for 2 hours, and visualized using the avidin-biotin-peroxidase complex Elite kit (Vector, Burlingame, CA) and 3–3′-diaminobenzidine-4 HCl/H2O2 (DAB; Sigma) as a substrate.Double-Immunofluorescent Staining and Confocal MicroscopyAutopsied brain tissue blocks including the entorhinal, hippocampal, and temporal cortices and/or amygdala from two normal controls and two individuals with stage V neurofibrillary degeneration according to Braak and Braak37Braak H Braak E Neuropathological staging of Alzheimer-related changes.Acta Neuropathol. 1991; 82: 239-259Crossref PubMed Scopus (11332) Google Scholar were used in this study. The tissue was fixed by immersion in a mixture of 4% paraformaldehyde and picric acid at pH 7.0 for 48 hours.44Braak E Braak H Mandelkow EM A sequence of cytoskeleton changes related to the formation of neurofibrillary tangles and neuropil threads.Acta Neuropathol. 1994; 87: 554-567Crossref PubMed Scopus (664) Google Scholar The frozen tissue blocks were sectioned at 100 μm.Double-immunofluorescent staining of floating sections was carried out using CY3-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories Inc., West Grove, PA) to visualize bound antibodies to active forms of MEK1/2, ERK1/2, and p70 S6 kinase, and CY2-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories Inc.) to label-bound mAb AT8. Radiance PLUS, a Bio-Rad laser-scanning confocal imaging system, equipped with a Nikon Eclipse inverted microscope (TE300) was used to determine co-localization of the CY3 (red)-labeled active forms of ERK1/2, MEK1/2, and p70 S6 kinase with the CY2 (green)-labeled hyperphosphorylated tau (AT8 staining). An argon ion laser that excites at 488 nm with a dichroic beamsplitter 560DCLP and a bandpass filter HQ515/30 was used to detect CY2 (green)-labeled abnormal tau (AT8 staining). A HeNe laser that excites at 543 nm with E570LP emission filter was used to measure the active forms of ERK1/2, MEK1/2, and ERK1/2 labeled by CY3 (red). A Nikon 60×/1.4 NA oil immersion objective was used. Images scanned on the two channels (red and green) were merged to produce a single profile. In this mode, all structures exhibiting colocalization display yellow fluorescence. Fluorescence images were collected at 1× or 2.5× zoom using the Bio-Rad Lasersharp software package and processed using Adobe Photoshop 5.0.Dephosphorylation with Purified PP-2AIn some cases, tissue sections and blots were dephosphorylated with purified PP-2A before incubation with primary antibody. The dephosphorylation was carried out by overlaying the sections and blots with PP-2A (20 μg/ml) in buffer containing 60 mmol/L Tris (pH 7.0), 0.1% β-mercaptoethanol, and 1.0 mmol/L MnCl2, at room temperature overnight. For the purpose of comparison, the PP-2A-treated and untreated tissue sections and blots were immunostained in parallel.ResultsActive ERK1/2, MEK1/2, and p70 S6 Kinase Are Associated with Tangle-Bearing Neurons in AD BrainPrevious studies have revealed no significant differences in the immunoreactivity of total ERK1/223Pei JJ Sersen E Iqbal K Grundke-Iqbal
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