Inhibition of Protein Phosphatase 2A Overrides Tau Protein Kinase I/Glycogen Synthase Kinase 3β and Cyclin-dependent Kinase 5 Inhibition and Results in Tau Hyperphosphorylation in the Hippocampus of Starved Mouse
2001; Elsevier BV; Volume: 276; Issue: 36 Linguagem: Inglês
10.1074/jbc.m102780200
ISSN1083-351X
AutoresEmmanuel Planel, Kaori Yasutake, Shinobu C. Fujita, Koichi Ishiguro,
Tópico(s)Pancreatic function and diabetes
ResumoHyperphosphorylated tau is the major component of paired helical filaments in neurofibrillary tangles found in Alzheimer's disease (AD) brain. Starvation of adult mice induces tau hyperphosphorylation at many paired helical filaments sites and with a similar regional selectivity as those in AD, suggesting that a common mechanism may be mobilized. Here we investigated the mechanism of starvation-induced tau hyperphosphorylation in terms of tau kinases and Ser/Thr protein phosphatases (PP), and the results were compared with those reported in AD brain. During starvation, tau hyperphosphorylation at specific epitopes was accompanied by decreases in tau protein kinase I/glycogen synthase kinase 3β (TPKI/GSK3β), cyclin-dependent kinase 5 (cdk5), and PP2A activities toward tau. These results demonstrate that the activation of TPKI/GSK3β and cdk5 is not necessary to obtain hyperphosphorylated tau in vivo, and indicate that inhibition of PP2A is likely the dominant factor in inducing tau hyperphosphorylation in the starved mouse, overriding the inhibition of key tau kinases such as TPKI/GSK3β and cdk5. Furthermore, these data give strong support to the hypothesis that PP2A is important for the regulation of tau phosphorylation in the adult brain, and provide in vivo evidence in support of a central role of PP2A in tau hyperphosphorylation in AD. Hyperphosphorylated tau is the major component of paired helical filaments in neurofibrillary tangles found in Alzheimer's disease (AD) brain. Starvation of adult mice induces tau hyperphosphorylation at many paired helical filaments sites and with a similar regional selectivity as those in AD, suggesting that a common mechanism may be mobilized. Here we investigated the mechanism of starvation-induced tau hyperphosphorylation in terms of tau kinases and Ser/Thr protein phosphatases (PP), and the results were compared with those reported in AD brain. During starvation, tau hyperphosphorylation at specific epitopes was accompanied by decreases in tau protein kinase I/glycogen synthase kinase 3β (TPKI/GSK3β), cyclin-dependent kinase 5 (cdk5), and PP2A activities toward tau. These results demonstrate that the activation of TPKI/GSK3β and cdk5 is not necessary to obtain hyperphosphorylated tau in vivo, and indicate that inhibition of PP2A is likely the dominant factor in inducing tau hyperphosphorylation in the starved mouse, overriding the inhibition of key tau kinases such as TPKI/GSK3β and cdk5. Furthermore, these data give strong support to the hypothesis that PP2A is important for the regulation of tau phosphorylation in the adult brain, and provide in vivo evidence in support of a central role of PP2A in tau hyperphosphorylation in AD. Alzheimer's disease amyloid β calcium/calmodulin-dependent protein kinase II cyclin-dependent kinase 5 glycogen synthase kinase 3 c-Jun N-terminal kinase kinase activity buffer mitogen-activated protein kinase/extracellular signal-regulated kinase okadaic acid phospho- polyacrylamide gel electrophoresis paired helical filaments cAMP-dependent protein kinase A protein kinase B phenylmethylsulfonyl fluoride serine/threonine protein phosphatases 2A, 2B, 2C), protein phosphatase (1, 2A, 2B, 2C) 2Ac, 2Bc), protein phosphatase (1, 2A, 2B) catalytic subunit phosphoserine phosphothreonine radioimmune precipitation assay tau protein kinase I tau protein kinase II 4-morpholinoethanesulfonic acid Alzheimer's disease (AD)1 is a neurodegenerative disorder characterized by the presence of two histopathological hallmarks called senile plaques and neurofibrillary tangles. The former are deposits of the β-amyloid peptide (Aβ) (1Selkoe D.J. Neuron. 1991; 6: 487-498Abstract Full Text PDF PubMed Scopus (2177) Google Scholar), whereas neurofibrillary tangles consist of hyperphosphorylated tau protein assembled in paired helical filaments (PHF) (2Grundke-Iqbal I. Iqbal K. Tung Y.C. Quinlan M. Wisniewski H.M. Binder L.I. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 4913-4917Crossref PubMed Scopus (2835) Google Scholar). Hyperphosphorylation refers to the state that tau is phosphorylated at more sites than tau from adult brain and that, for a given site, a higher than normal percentage of tau molecules is phosphorylated (3Goedert M. Ann. N. Y. Acad. Sci. 1996; 777: 121-131Crossref PubMed Scopus (103) Google Scholar). Tau is a microtubule-associated protein, and its normal physiological function is to bind and stabilize microtubules. In vitro studies have shown that PHF-tau fails to promote microtubule assembly (4Bramblett G.T. Goedert M. Jakes R. Merrick S.E. Trojanowski J.Q. Lee V.M. 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Phosphorylation of tau can be regulated by many protein kinases and phosphatases in vitro (11Imahori K. Uchida T. J. Biochem. (Tokyo). 1997; 121: 179-188PubMed Google Scholar, 12Lovestone S. Reynolds C.H. Neuroscience. 1997; 78: 309-324Crossref PubMed Scopus (263) Google Scholar, 13Billingsley M.L. Kincaid R.L. Biochem. J. 1997; 323: 577-591Crossref PubMed Scopus (367) Google Scholar). These findings, and the changes in kinases and phosphatases observed in AD, suggest that tau hyperphosphorylation in AD brain is likely to be due to an imbalance of the protein phosphorylation and dephosphorylation systems. But to date the mechanism of conversion from normal adult tau to hyperphosphorylated tau, the significance of tau hyperphosphorylation in PHF formation, and its relationship to Aβ deposition remain largely elusive. Tau hyperphosphorylation is a physiological reversible response of the brain to stressful conditions like cold water stress 2Y. Okawa, K. Ishiguro, and S. C. Fujita, manuscript in preparation.2Y. Okawa, K. Ishiguro, and S. C. Fujita, manuscript in preparation. (14Korneyev A. Binder L. Bernardis J. Neurosci. Lett. 1995; 191: 19-22Crossref PubMed Scopus (51) Google Scholar), heat-shock (15Papasozomenos S.C. J. Neurochem. 1996; 66: 1140-1149Crossref PubMed Scopus (36) Google Scholar), or starvation (16Yanagisawa M. Planel E. Ishiguro K. Fujita S.C. FEBS Lett. 1999; 461: 329-333Crossref PubMed Scopus (109) Google Scholar). Starvation induces decreases in circulating glucose, insulin, and leptin, and increases in corticosterone (17Ahima R.S. Prabakaran D. Mantzoros C. Qu D. Lowell B. Maratos-Flier E. Flier J.S. Nature. 1996; 382: 250-252Crossref PubMed Scopus (2668) Google Scholar), and results in a large decrease of glucose in many parts of the brain (18Garriga J. Cusso R. Brain Res. 1992; 591: 277-282Crossref PubMed Scopus (24) Google Scholar). 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FEBS Lett. 1999; 461: 329-333Crossref PubMed Scopus (109) Google Scholar). Here, we studied the mechanism of tau hyperphosphorylation in the starved mice in terms of protein kinases and phosphatases which, according to previous studies, are either implicated in the generation of tau pathological epitopes in vitro or exhibit altered activity in AD brain, and evaluated the relevance of our findings to AD. The most striking changes concomitant with tau hyperphosphorylation at specific sites were the inhibition of PP2A, concurrent with the inhibition of TPKI/GSK3β, and cdk5 activities toward tau. These results demonstrate that the activation of TPKI/GSK3β and cdk5 is not necessary to obtain hyperphosphorylated tau in vivo, and indicate that tau site-specific hyperphosphorylation in the starved mouse hippocampus involves a complex mechanism in which PP2A inhibition plays a dominant role, overriding the inhibition of key tau kinases such as TPKI/GSK3β and cdk5. Eight to ten-week-old C57BL/6NJcl male mice (Clea Japan, Tokyo) were singly housed in cages with grid floors to deny coprophagy. Food was removed for up to 3 days, but mice were allowed free access to water. Room temperature was 23 °C, and the light period was 08:00–20:00. Animals were handled according to the procedures approved by the Animal Care and Use Committee of the Mitsubishi Kasei Institute of Life Sciences. Mice were sacrificed by cervical dislocation, brains immediately removed, and hippocampi were dissected from brains in ice-chilled saline. The tissues were quickly weighed and homogenized in 10 times volume/weight of O+ buffer, modified from O'Farrell's buffer O (22O'Farrell P.H. J. Biol. Chem. 1975; 250: 4007-4021Abstract Full Text PDF PubMed Google Scholar) (62.5 mmTris-HCl, pH 6.8, 10% (w/v) glycerol, 5% (v/v) 2-mercaptoethanol, 2.3% (w/v) SDS, 100 μm orthovanadate, 1 μmokadaic acid (OA), 1 mm phenylmethylsulfonyl fluoride (PMSF), 1 mm EGTA, and 1 mm EDTA). The samples were then placed in boiling water for 5 min, centrifuged for 15 min at 20,000 × g at 4 °C, and the protein content of the supernatants was determined with the Bio-Rad Protein Assay (after proper dilution to ensure reagent compatibility) using bovine serum albumin in equivalent buffer as standard. Eighteen μg of protein (determined to fit in the linear range for quantification, calibration data not shown) were separated by 10% SDS-polyacrylamide gel electrophoresis, and electrotransferred to nitrocellulose membrane (Protran BA 85, 0.45 μm, Schleicher & Schuell). Purified rabbit polyclonal antibodies anti-tau PS199, PT231, PS262, PS396, and PS404 (23Ishiguro K. Sato K. Takamatsu M. Park J. Uchida T. Imahori K. Neurosci. Lett. 1995; 202: 81-84Crossref PubMed Scopus (82) Google Scholar) (specific to tau phosphorylated at residues indicated), anti-TPKI-C (24Yamaguchi H. Ishiguro K. Uchida T. Takashima A. Lemere C.A. Imahori K. Acta Neuropathol. (Berl.). 1996; 92: 232-241Crossref PubMed Scopus (234) Google Scholar), anti-cdk5 (anti-peptide 1), and anti-p23C (allows the detection of p35 and p25) (25Uchida T. Ishiguro K. Ohnuma J. Takamatsu M. Yonekura S. Imahori K. FEBS Lett. 1994; 355: 35-40Crossref PubMed Scopus (71) Google Scholar, 26Kobayashi S. Ishiguro K. Omori A. Takamatsu M. Arioka M. Imahori K. Uchida T. FEBS Lett. 1993; 335: 171-175Crossref PubMed Scopus (219) Google Scholar), anti-TPKI PY216 and anti-TPKI PS9 (27Tomidokoro Y. Ishiguro K. Harigaya Y. Matsubara E. Ikeda M. Park J. Yasutake K. Kawarabayashi T. Okamoto K. Shoji M. Neurosci. Lett. 2001; 299: 169-172Crossref PubMed Scopus (92) Google Scholar), were described previously. T1.7, a mouse monoclonal antibody specific to TPKI/GSK3β was also reported (28Fujita S.C. Takahashi M. Hasegawa J. Imahori K. Neurochem. Res. 1995; 20: 327Crossref Scopus (27) Google Scholar). Purified polyclonal antibodies for mitogen-activated protein kinase/extracellular signal regulated kinase (MAPK/ERK), phospho-Akt, and Akt (or protein kinase B, PKB) were purchased from New England Biolabs; antibodies to cdk5 (C-8), c-Jun N-terminal kinase (JNK) JNK1 (FL), c-AMP dependent protein kinase A (PKA) PKAα cat (C-20), PP1 (E-9), and PP2A (C-20) from Santa Cruz Biotechnology, Inc.; anti-active calcium/calmodulin-dependent protein kinase II (CaMKII), anti-active JNK, anti-active MAPK from Promega; anti-PP2Bα from Calbiochem-Novabiochem. Monoclonal antibodies AT8 (Innogenetics; specific to tau dually phosphorylated at Ser202 and Thr205 (29Goedert M. Jakes R. Vanmechelen E. Neurosci. Lett. 1995; 189: 167-169Crossref PubMed Scopus (470) Google Scholar)) and Tau-1 (Roche Molecular Biochemicals; recognizes tau dephosphorylated at Ser195, Ser198, Ser199, and Ser202 (30Szendrei G.I. Lee V.M. Otvos Jr., L. J. Neurosci. Res. 1993; 34: 243-249Crossref PubMed Scopus (193) Google Scholar)) were also used. Membrane blocking and antibody incubations were done according to New England Biolab MAPK Immunoblotting Protocol (New England Biolabs number 9910), with appropriate primary and secondary Ig dilutions. Anti-mouse, anti-rabbit, and anti-goat horseradish peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology. The bands were developed with SuperSignal West Pico Chemiluminescent Substrate (Pierce) or with 4-chloro-1-naphthol and H2O2. The images obtained with the LAS-1000plus Luminescent Image Analyzer (Fuji Film), or scanned from stained membranes, were quantified using a Macintosh version of Fuji Films Science Lab 99 Image Gauge. Statistical analysis of the results was performed by one-way analysis of variance (ANOVA). Significant ANOVA (p < 0.05) was followed by the Tukey/Kramer test of pairwise multiple comparisons. In all the figures, error bars represent the standard deviation (S.D.), while * and ** indicate significant difference with p < 0.05 and p < 0.01, respectively. LiCl experiments were conducted on 6 groups of 3 mice. At 10:00 on day 1 through day 3, mice of groups 2 and 5 received intraperitoneal injection with 400 μl of 0.3 mNaCl, and mice of groups 3 and 6 were injected with 400 μl of 0.3m LiCl. Mice of groups 4 to 6 were deprived of food at 10:00 on day 2. The mice were sacrificed in the morning of day 4, and hippocampi were taken and analyzed as described above. Mouse hippocampi were homogenized in 5 × volume/weight of ice-chilled RIPA buffer (50 mmTris-HCl, 1% Nonidet P-40, 0.25% Na deoxycholate, 150 mmNaCl, 1 mm EGTA, 1 mm NaF, 1 mmNa3VO4, 1 μg/ml each of leupeptin, aprotinin, and pepstatin, 1 mm of PMSF, 1 μm okadaic acid), centrifuged for 15 min at 20,000 × g and 4 °C, and the protein content of the supernatants was determined. Immunoprecipitation was carried out at 4 °C. Five μl of fresh brain extract were mixed with complexes of T1.7 or Cdk5 (C-8) antibodies coupled to IgG beads (Dynabeads, rat anti-mouse IgG1 M-450 or sheep anti-rabbit M-280, prepared according to the manufacturer's instructions) suspended in 50 μl of cell extraction buffer (10 mm Tris-HCl, pH 7.4, 50 mm NaCl, 50 mm NaF, 1 mm EDTA, 1 mm EGTA, 50 mm β-glycerophosphate, 3 mm benzamidine, 0.05% NaN3, 100 μmNa3VO4, 1 μg/ml each of leupeptin, aprotinin and pepstatin, 1 mm PMSF, 1 μm okadaic acid) and incubated for 1 h at 4 °C on rotating agitator. After washing 3 times with cell extraction buffer, and twice with kinase activity buffer (KAB; 100 mm MES-NaOH, pH 6.2, 1 mm Mg acetate, 1 mm EGTA, 10% glycerol, 0.02% Tween 20, 1 mm PMSF, 1 μm okadaic acid), the samples were resuspended in 10 μl of KAB. Kinase activity was measured by phosphorylation of human recombinant tau-441 (Panvera). Ten-μl aliquots of suspension of immunoprecipitated kinases in KAB were mixed with 10 μl of KAB containing 200 μm ATP and 100 μg/ml tau, and incubated at 30 °C for 20 min (for TPKI) or 2 h (for cdk5). The reaction was stopped by adding 20 μl of O+ buffer and boiling for 5 min. Five μl of this mixture were loaded per lane on 10% SDS-PAGE gels, followed by immunoblotting and band quantification with phospho-tau-specific antibodies as described above. To prepare phosphatase substrate, 1 μl of fresh RIPA buffer extract of hippocampus (10 × v/w) was added to 25 μl of tau phosphorylation mixture (100 μg/ml recombinant tau, 1 mm ATP, 10 mm Tris-HCl, pH 7.5, 2 mm 2-mercaptoethanol, 2 mm Mg acetate, 1 mm PMSF, 1 μm okadaic acid, 1 μg/ml aprotinin, leupeptin, and pepstatin) and incubated for 4 h at 37 °C. This procedure allows incorporation of 6–7 mol of phosphate/mol of recombinant tau, as determined by a standard radiometric assay using [γ-32P]ATP (data not shown). The reaction was terminated by boiling for 5 min followed by centrifugation for 15 min at 20,000 × g and 4 °C. After addition of trichloroacetic acid (1/9, w/v), the supernatant was incubated for 30 min on ice, and centrifuged for 10 min at 20,000 × g and 4 °C. The pellet was washed 3 times with 20% trichloroacetic acid, redissolved in the same volume of substrate solubilization buffer (50 mm Tris-HCl, pH 8.5, 0.1 mm EDTA, 5 mm 2-mercaptoethanol, 0.01% Tween 20) as used during phosphorylation, and dialyzed twice overnight at 4 °C against 1 liter of dialysis buffer (25 mm Tris-HCl, pH 7.5, 0.1 mm EDTA, 5 mm 2-mercaptoethanol, 0.01% Tween 20), to remove any traces of OA. Phosphatase activity was measured by decrease in phosphorylation of tau by the brain extracts as analyzed by Western blotting with anti-phospho-tau antibodies. Mouse hippocampi were homogenized in 5 × volume/weight of ice-chilled phosphatase sample buffer (50 mm Tris-HCl, pH 7.0, 0.25m sucrose, 10 mm 2-mercaptoethanol, 0.1 mm EDTA, 1 mm benzamidine, 1 μg/ml each of aprotinin, leupeptin, and pepstatin, 1 mm PMSF) modified from Gong et al. (31Gong C.X. Singh T.J. Grundke-Iqbal I. Iqbal K. J. Neurochem. 1993; 61: 921-927Crossref PubMed Scopus (454) Google Scholar), centrifuged for 30 min at 20,000 × g and 4 °C, and the protein contents were determined as above. One μl of phosphatase extract was added to 20 μl of phosphatase activity mixture (50 μg/ml phosphorylated tau, 37.5 mm Tris-HCl, pH 7.5, 0.1 mm EDTA, 5 mm 2-mercaptoethanol, 0.01% Tween 20, 1 mmPMSF, 1 μg/ml each of aprotinin, leupeptin, and pepstatin) and incubated at 30 °C for various periods of time. The reaction was stopped by addition of an equal volume of O+ buffer and boiling for 5 min. Five-μl aliquots were loaded on 10% SDS-PAGE gels and analyzed as above. PP1 and PP2A activities in the brain extracts toward phosphorylated recombinant tau were assessed after 10 min of incubation at 30 °C by analyzing the dephosphorylation of AT8 epitope in the presence or absence of 5.0 μm OA (inhibitory to PP2A and PP1, Calbiochem), or 1.0 μm inhibitor-2 (PP1-specific inhibitor, Sigma) (32Cohen P. Methods Enzymol. 1991; 201: 389-398Crossref PubMed Scopus (204) Google Scholar, 33McKintosh C. Grahame Hardie D. Protein Phosphorylation: A Practical Approach. Oxford University Press, Oxford1993: 197-230Google Scholar, 34Gong C.X. Lidsky T. Wegiel J. Zuck L. Grundke-Iqbal I. Iqbal K. J. Biol. Chem. 2000; 275: 5535-5544Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar). Phosphorylated tau was incubated in 20 μl of phosphatase activity mixture for 1 h at 30 °C in the presence or absence of 62.5 microunits/μl of recombinant catalytic subunit of PP1 (New England Biolabs). Hippocampal neurons were prepared from 18-day-old embryonic rat brains and cultured as described previously (35Takashima A. Noguchi K. Sato K. Hoshino T. Imahori K. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7789-7793Crossref PubMed Scopus (379) Google Scholar). These cultures were largely free of non-neuronal cells. Seven days after plating, OA was added to the medium to 100 nm. After 1 h of exposure, the cells were lysed in 5 volumes of RIPA buffer, and the extracts centrifuged for 15 min at 20,000 ×g and 4 °C. The supernatants were analyzed by immunoblotting using anti-tau and anti-protein kinase antibodies. One μg of protein was used for Tau-C, 10 μg for p35 and TPKI PS9, and 2 μg for other antibodies. Immunoblots were reacted with anti-rabbit or anti-mouse secondary antibodies conjugated to alkaline phosphatase and developed with 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (Promega). Batches of three or five young adult mice were starved for up to 3 days and some were refed for up to 1 week after 2 days of starvation. Hippocampal extracts of these mice were individually analyzed by immunoblotting with antibodies to protein kinases and phosphatases (Figs. Figure 1, Figure 2, Figure 3, Figure 4). As described qualitatively (16Yanagisawa M. Planel E. Ishiguro K. Fujita S.C. FEBS Lett. 1999; 461: 329-333Crossref PubMed Scopus (109) Google Scholar), tau undergoes phosphorylation and dephosphorylation, illustrated here by phosphorylation-dependent anti-tau antibody AT8 (Fig.1, A1 and B1). The two isoforms of rat GSK3, GSK3α and TPKI/GSK3β, are encoded by different genes and share a 85% homology at the amino acid level (36Woodgett J.R. EMBO J. 1990; 9: 2431-2438Crossref PubMed Scopus (1140) Google Scholar). TPKI/GSK3β is a leading candidate protein kinase responsible for tau hyperphosphorylation in AD brains (11Imahori K. Uchida T. J. Biochem. (Tokyo). 1997; 121: 179-188PubMed Google Scholar). This enzyme phosphorylates tau at PHF sites Ser199, Thr231, Ser396, Ser404, and Ser413 (23Ishiguro K. Sato K. Takamatsu M. Park J. Uchida T. Imahori K. Neurosci. Lett. 1995; 202: 81-84Crossref PubMed Scopus (82) Google Scholar) (numbering of amino acids according to the longest human tau (37Goedert M. Spillantini M.G. Jakes R. Rutherford D. Crowther R.A. Neuron. 1989; 3: 519-526Abstract Full Text PDF PubMed Scopus (1827) Google Scholar)), but can also phosphorylate other sites in combination with other kinases (38Wang J.Z. Wu Q. Smith A. Grundke-Iqbal I. Iqbal K. FEBS Lett. 1998; 436: 28-34Crossref PubMed Scopus (173) Google Scholar). Phosphorylation of TPKI/GSK3β at Tyr216 is essential for its activity, while phosphorylation at Ser9leads to partial inhibition (39Cohen P. Philos. Trans. R. Soc. Lond. B Biol. Sci. 1999; 354: 485-495Crossref PubMed Scopus (133) Google Scholar). Phosphorylation at Tyr216did not change significantly (Fig. 1, A2 and B2), but starvation induced a dramatic elevation of phospho-Ser9level (Fig. 1, A3 and B2). PKB phosphorylates TPKI/GSK3β at Ser9 (39Cohen P. Philos. Trans. R. Soc. Lond. B Biol. Sci. 1999; 354: 485-495Crossref PubMed Scopus (133) Google Scholar), but the active-PKB epitope was increased only during the first day of refeeding (Fig. 1, A7and B6). Interestingly, the activating phosphorylation of MAPK was transiently increased after 1 day of starvation (Fig. 1,A4 and B3). Activating phosphorylation of JNK, a kinase reported to phosphorylate tau at Thr181, Ser202/Thr205, Thr231, Ser396, and Ser422 epitopes (40Reynolds C.H. Utton M.A. Gibb G.M. Yates A. Anderton B.H. J. Neurochem. 1997; 68: 1736-1744Crossref PubMed Scopus (192) Google Scholar), rose during starvation (Fig. 1, A5 and B4). Levels of phospho-CaMKII immunoreactivity during starvation were not significantly different from control values (Fig. 1, A6 andB5). These observations indicate that, during starvation, MAPK is transiently activated, JNK activated, while TPKI/GSK3β is inhibited.Figure 2Protein levels of tau and protein kinases during starvation. Panel A, changes in kinase immunoreactivities (A2-A8) and Tau-C (A1), in the hippocampi of bib6 male mice, after normal feeding (lanes 2–6); starvation for 1 (lanes 7–11), 2 (lanes 12–16), or 3 days (lanes 17–21); starvation for 2 days followed by refeeding for 1 day (lanes 22–26), or 1 week (lanes 27–31). Each lane (from 2 to31) represents an extract of individual mouse. Lanes 1 and 32 represent molecular weights markers (M). Panel B, immunoblot bands were quantified and averaged (n = 5). All the quantitative results are expressed as percentages of the fed control. Data are presented as mean ± S.D.; * and ** indicate significant difference from fed controls with p < 0.05 and p < 0.01, respectively. Repetition of these experiments using different batches of mice (n = 3 or 4) led to similar results.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Tau and TPKI/GSK3β immunoreactivities after intraperitoneal injections of solutions of LiCl or NaCl. Panel A, each lane represents a hippocampal extract from an individual mouse. Lanes 1–9, fed mice; lanes 10–18, 2-day starved mice. Mice 1–3 and 10–12 were not injected. Mice 4–6 and 13–15 received daily injections of NaCl, and mice 7–9 and 16–18 daily injections of LiCl for 3 days and were sacrificed on the fourth day. Immunoblots were developed using Tau-C and various phospho-dependent tau and TPKI/GSK3β antibodies. Panel B, immunoblot bands were quantified (n = 3). All the quantitative results are expressed as percentages of the fed, non-injected control. Data are presented as mean ± S.D.; * and ** indicate significant difference from fed controls (except where indicated bybrackets) with p < 0.05 andp < 0.01, respectively. Repetition of these experiments using different batches of mice (n = 2 or 5) led to similar results.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Protein levels of phosphatase catalytic subunits during starvation. Panel A, changes in phosphatase catalytic subunits in the hippocampi of bib6 male mice, after normal feeding (lanes 2–6); starvation for 1 (lanes 7–11), 2 (lanes 12–16), or 3 days (lanes 17–21); starvation for 2 days followed by refeeding for 1 day (lanes 22–26) or 1 week (lanes 27–31). Each lane (from 2 to 31) represents an extract of individual mouse. Lanes 1 and 32 represent molecular weights markers (M). Panel B, immunoblot bands were quantified and averaged (n = 5). All the quantitative results are expressed as percentages of the fed control. Data are presented as mean ± S.D.; ** indicates significant difference from fed controls with p < 0.01. Repetition of these experiments using different batches of mice (n = 3 or 4) led to similar results.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Tau protein, as detected with anti-Tau-C antibody, occurred in normally fed mice as a strong band at 56 kDa and a faint one at 60 kDa, with a smear between 60 and 70 kDa (Fig. 2, A1, lanes 2–6). After 2 days of starvation, the 60-kDa band became prominent, the 56-kDa band weakened while 2 additional bands appeared at 64 and 70 kDa (Fig. 2, A1, lanes 12–16). These mobility shifts reflecting tau phosphorylation (41Goedert M. Spillantini M.G. Cairns N.J. Crowther R.A. Neuron. 1992; 8: 159-168Abstract Full Text PDF PubMed Scopus (895) Google Scholar) were not accompanied by significant changes in total tau protein levels (Fig. 2,B1). Protein levels of TPKI/GSK3β, MAPK, PKB, JNK, or catalytic subunits of PKA, did not show changes that correlated with tau phosphorylation levels (Fig. 2, A2–5, 8 andB2–5, 8). Tau protein kinase II (TPKII) is a heterodimer of a cdk5 catalytic subunit and p25, a 25-kDa regulatory subunit derived proteolytically from p35 (25Uchida T. Ishiguro K. Ohnuma J. Takamatsu M. Yonekura S. Imahori K. FEBS Lett. 1994; 355: 35-40Crossref PubMed Scopus (71) Google Scholar, 42Tsai L.H. Delalle I. Caviness Jr., V.S. Chae T. Harlow E. Nature. 1994; 371: 419-423Crossref PubMed Scopus (808) Google Scholar). TPKII phosphorylates tau in vitro at PHF sites Ser202, Thr205, Ser235, and Ser404 (23Ishiguro K. Sato K. Takamatsu M. Park J. Uchida T. Imahori K. Neurosci. Lett. 1995; 202: 81-84Crossref PubMed Scopus (82) Google Scholar). Cdk5 immunoreactivity did not change significantly during starvation (Fig. 2, A7 and B7). Interestingly, p35 displayed a slight upward mobility shift upon starvation, followed by a decrease in intensity, and the strengthening of two lighter bands at 33 and 34 kDa, which are likely to be partial degradation products (Fig. 2,A6). One day of refeeding restored the upper band to its original intensity, and 1 week canceled the band shift (Fig. 2,A6 and B6). The level of p25 was undetectable. Thus, during starvation, the protein levels of tau or the kinases studied did not change, except for cdk5 activator p35 which displayed a slight mobility shift followed by a decrease. As starvation induced a sharp rise in TPKI/GSK3β phospho-Ser9, the role of this enzyme in tau hyperphosphorylation was further studied with an inhibitor of this enzyme, lithium, which reduces tau phosphorylation by inhibiting GSK3 both in vitro and in vivo (43Hong M. Chen D.C. Klein P.S. Lee V.M. J. Biol. Chem. 1997; 272: 25326-25332Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar, 44Munoz-Montano J.R. Moreno F.J. Avila J. Diaz-Nido J. FEBS Lett. 1997; 411: 183-188Crossref PubMed Scopus (308) Google Scholar). Daily injections of solutions of LiCl or control NaCl were given to fed or starved mice, and hippocampal extracts were analyzed for tau and TPKI/GSK3β phosphorylation (Fig. 3). Two days of starvation induced increases in tau phosphorylation levels by ∼50 fold at AT8 and PT231, ∼5 fold at Tau-1 and PS262, and ∼65% at PS396, but not at PS199 and PS4
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