Hyperphosphorylation and Aggregation of Tau in Laforin-deficient Mice, an Animal Model for Lafora Disease
2009; Elsevier BV; Volume: 284; Issue: 34 Linguagem: Inglês
10.1074/jbc.m109.009688
ISSN1083-351X
AutoresRajat Puri, Toshimitsu Suzuki, Kazuhiro Yamakawa, Subramaniam Ganesh,
Tópico(s)Protein Tyrosine Phosphatases
ResumoLafora progressive myoclonous epilepsy (Lafora disease; LD) is caused by mutations in the EPM2A gene encoding a dual specificity protein phosphatase named laforin. Our analyses on the Epm2a gene knock-out mice, which developed most of the symptoms of LD, reveal the presence of hyperphosphorylated Tau protein (Ser396 and Ser202) as neurofibrillary tangles (NFTs) in the brain. Intriguingly, NFTs were also observed in the skeletal muscle tissues of the knock-out mice. The hyperphosphorylation of Tau was associated with increased levels of the active form of GSK3β. The observations on Tau protein were replicated in cell lines using laforin overexpression and knockdown approaches. We also show here that laforin and Tau proteins physically interact and that the interaction was limited to the phosphatase domain of laforin. Finally, our in vitro and in vivo assays demonstrate that laforin dephosphorylates Tau, and therefore laforin is a novel Tau phosphatase. Taken together, our study suggests that laforin is one of the critical regulators of Tau protein, that the NFTs could underlie some of the symptoms seen in LD, and that laforin can contribute to the NFT formation in Alzheimer disease and other tauopathies. Lafora progressive myoclonous epilepsy (Lafora disease; LD) is caused by mutations in the EPM2A gene encoding a dual specificity protein phosphatase named laforin. Our analyses on the Epm2a gene knock-out mice, which developed most of the symptoms of LD, reveal the presence of hyperphosphorylated Tau protein (Ser396 and Ser202) as neurofibrillary tangles (NFTs) in the brain. Intriguingly, NFTs were also observed in the skeletal muscle tissues of the knock-out mice. The hyperphosphorylation of Tau was associated with increased levels of the active form of GSK3β. The observations on Tau protein were replicated in cell lines using laforin overexpression and knockdown approaches. We also show here that laforin and Tau proteins physically interact and that the interaction was limited to the phosphatase domain of laforin. Finally, our in vitro and in vivo assays demonstrate that laforin dephosphorylates Tau, and therefore laforin is a novel Tau phosphatase. Taken together, our study suggests that laforin is one of the critical regulators of Tau protein, that the NFTs could underlie some of the symptoms seen in LD, and that laforin can contribute to the NFT formation in Alzheimer disease and other tauopathies. Lafora disease (LD) 2The abbreviations used are: LDLafora diseaseDSPDdual specificity phosphatase domainCBDcarbohydrate binding domainPTGprotein targeting to glycogenNFTneurofibrillary tangleE3ubiquitin-protein isopeptide ligasePKAprotein kinase AAKTprotein kinase BPP2Aprotein phosphatase 2ACDK5cyclin-dependent kinase 5GFPgreen fluorescent protein. is an autosomal recessive and a fatal form of progressive myoclonus epilepsy characterized by the presence of Lafora polyglucosan bodies in the affected tissues (1Ganesh S. Puri R. Singh S. Mittal S. Dubey D. J. Hum. Genet. 2006; 51: 1-8Crossref PubMed Scopus (114) Google Scholar). The symptoms of LD include stimulus-sensitive epilepsy, dementia, ataxia, and rapid neurologic deterioration (1Ganesh S. Puri R. Singh S. Mittal S. Dubey D. J. Hum. Genet. 2006; 51: 1-8Crossref PubMed Scopus (114) Google Scholar, 2Van Heycop D.E. Jager H. Epilepsia. 1963; 4: 95-119Crossref PubMed Scopus (49) Google Scholar). LD is caused by mutations in the EPM2A gene encoding laforin, a dual specificity protein phosphatase, or in the NHLRC1 gene encoding malin, an E3 ubiquitin ligase (3Minassian B.A. Lee J.R. Herbrick J.A. Huizenga J. Soder S. Mungall A.J. Dunham I. Gardner R. Fong C.Y. Carpenter S. Jardim L. Satishchandra P. Andermann E. Snead 3rd, O.C. Lopes-Cendes I. Tsui L.C. Delgado-Escueta A.V. Rouleau G.A. Scherer S.W. Nat. Genet. 1998; 20: 171-174Crossref PubMed Scopus (409) Google Scholar, 4Ganesh S. Agarwala K.L. Ueda K. Akagi T. Shoda K. Usui T. Hashikawa T. Osada H. Delgado-Escueta A.V. Yamakawa K. Hum. Mol. Genet. 2000; 9: 2251-2261Crossref PubMed Scopus (130) Google Scholar, 5Chan E.M. Young E.J. Ianzano L. Munteanu I. Zhao X. Christopoulos C.C. Avanzini G. Elia M. Ackerley C.A. Jovic N.J. Bohlega S. Andermann E. Rouleau G.A. Delgado-Escueta A.V. Minassian B.A. Scherer S.W. Nat. Genet. 2003; 35: 125-127Crossref PubMed Scopus (259) Google Scholar, 6Gentry M.S. Worby C.A. Dixon J.E. Proc. 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Delgado-Escueta A.V. Yamakawa K. Hum. Mol. Genet. 2000; 9: 2251-2261Crossref PubMed Scopus (130) Google Scholar, 7Mittal S. Dubey D. Yamakawa K. Ganesh S. Hum. Mol. Genet. 2007; 16: 753-762Crossref PubMed Scopus (75) Google Scholar), form aggresome upon proteasomal blockade (7Mittal S. Dubey D. Yamakawa K. Ganesh S. Hum. Mol. Genet. 2007; 16: 753-762Crossref PubMed Scopus (75) Google Scholar), and clear misfolded protein through ubiquitin-proteasome (8Garyali P. Siwach P. Singh P.K. Puri R. Mittal S. Sengupta S. Parihar R. Ganesh S. Hum. Mol. Genet. 2009; 18: 688-700Crossref PubMed Scopus (96) Google Scholar). Laforin has two functional domains: a phosphatase domain (dual specificity phosphatase domain; DSPD) and a carbohydrate binding domain (CBD) (9Ganesh S. Tsurutani N. Suzuki T. Hoshii Y. Ishihara T. Delgado-Escueta A.V. Yamakawa K. Biochem. Biophys. Res. Commun. 2004; 313: 1101-1109Crossref PubMed Scopus (64) Google Scholar). The CBD helps laforin to target to the glycogen particle and to the Lafora bodies (9Ganesh S. Tsurutani N. Suzuki T. Hoshii Y. Ishihara T. Delgado-Escueta A.V. Yamakawa K. Biochem. Biophys. Res. Commun. 2004; 313: 1101-1109Crossref PubMed Scopus (64) Google Scholar, 10Chan E.M. Ackerley C.A. Lohi H. Ianzano L. Cortez M.A. Shannon P. Scherer S.W. Minassian B.A. Hum. Mol. Genet. 2004; 13: 1117-1129Crossref PubMed Scopus (87) Google Scholar), and the DSPD of laforin dephosphorylates carbohydrate moieties (11Worby C.A. Gentry M.S. Dixon J.E. J. Biol. Chem. 2006; 281: 30412-30418Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). Recent studies have further shown that laforin and malin together regulate the cellular levels of PTG, the adaptor protein targeting to glycogen, and that the loss of either malin or laforin results in increased levels of PTG that eventually lead to excessive glycogen deposition (12Vilchez D. Ros S. Cifuentes D. Pujadas L. Vallès J. García-Fojeda B. Criado-García O. Fernández-Sánchez E. Medraño-Fernández I. Domínguez J. García-Rocha M. Soriano E. Rodríguez de Córdoba S. Guinovart J.J. Nat. Neurosci. 2007; 10: 1407-1413Crossref PubMed Scopus (263) Google Scholar, 13Solaz-Fuster M.C. Gimeno-Alcañiz J.V. Ros S. Fernandez-Sanchez M.E. Garcia-Fojeda B. Criado Garcia O. Vilchez D. Dominguez J. Garcia-Rocha M. Sanchez-Piris M. Aguado C. Knecht E. Serratosa J. Guinovart J.J. Sanz P. Rodriguez de Córdoba S. Hum. Mol. Genet. 2008; 17: 667-678Crossref PubMed Scopus (112) Google Scholar, 14Worby C.A. Gentry M.S. Dixon J.E. J. Biol. Chem. 2008; 283: 4069-4076Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Although this model explains the genesis of Lafora bodies, the molecular etiology of LD is yet to be understood. For example, unlike this cell line study (12Vilchez D. Ros S. Cifuentes D. Pujadas L. Vallès J. García-Fojeda B. Criado-García O. Fernández-Sánchez E. Medraño-Fernández I. Domínguez J. García-Rocha M. Soriano E. Rodríguez de Córdoba S. Guinovart J.J. Nat. Neurosci. 2007; 10: 1407-1413Crossref PubMed Scopus (263) Google Scholar), the presence of Lafora bodies does not lead to neuronal cell death in the two murine models of LD (10Chan E.M. Ackerley C.A. Lohi H. Ianzano L. Cortez M.A. Shannon P. Scherer S.W. Minassian B.A. Hum. Mol. Genet. 2004; 13: 1117-1129Crossref PubMed Scopus (87) Google Scholar, 15Ganesh S. Delgado-Escueta A.V. Sakamoto T. Avila M.R. Machado-Salas J. Hoshii Y. Akagi T. Gomi H. Suzuki T. Amano K. Agarwala K.L. Hasegawa Y. Bai D.S. Ishihara T. Hashikawa T. Itohara S. Cornford E.M. Niki H. Yamakawa K. Hum. Mol. Genet. 2002; 11: 1251-1262Crossref PubMed Scopus (184) Google Scholar), and no difference in the level of PTG was seen in laforin-deficient mice (16Tagliabracci V.S. Girard J.M. Segvich D. Meyer C. Turnbull J. Zhao X. Minassian B.A. Depaoli-Roach A.A. Roach P.J. J. Biol. Chem. 2008; 283: 33816-33825Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). 3R. Puri, T. Suzuki, K. Yamakawa, and S. Ganesh, unpublished observations. However, widespread degeneration of neurons was seen in laforin-deficient mouse with the absence of Lafora bodies, suggesting that the polyglucosan bodies may not play a primary role in the epileptogenesis (15Ganesh S. Delgado-Escueta A.V. Sakamoto T. Avila M.R. Machado-Salas J. Hoshii Y. Akagi T. Gomi H. Suzuki T. Amano K. Agarwala K.L. Hasegawa Y. Bai D.S. Ishihara T. Hashikawa T. Itohara S. Cornford E.M. Niki H. Yamakawa K. Hum. Mol. Genet. 2002; 11: 1251-1262Crossref PubMed Scopus (184) Google Scholar). The laforin dominant-negative transgenic mice line also developed Lafora bodies but had no signs of neurodegeneration or epileptic seizures (10Chan E.M. Ackerley C.A. Lohi H. Ianzano L. Cortez M.A. Shannon P. Scherer S.W. Minassian B.A. Hum. Mol. Genet. 2004; 13: 1117-1129Crossref PubMed Scopus (87) Google Scholar). Thus, the neurodegenerative changes are likely to underlie the etiology of some of the LD symptoms (1Ganesh S. Puri R. Singh S. Mittal S. Dubey D. J. Hum. Genet. 2006; 51: 1-8Crossref PubMed Scopus (114) Google Scholar). The mouse model developed by the knockdown of the Epm2a gene exhibited a majority of the symptoms known in LD, including the ataxia, spontaneous myoclonic seizures, EEG epileptiform activity, and impaired behavioral responses (15Ganesh S. Delgado-Escueta A.V. Sakamoto T. Avila M.R. Machado-Salas J. Hoshii Y. Akagi T. Gomi H. Suzuki T. Amano K. Agarwala K.L. Hasegawa Y. Bai D.S. Ishihara T. Hashikawa T. Itohara S. Cornford E.M. Niki H. Yamakawa K. Hum. Mol. Genet. 2002; 11: 1251-1262Crossref PubMed Scopus (184) Google Scholar). The knock-out animals showed a number of degenerative changes that include swelling and/or loss of morphological features of mitochondria, endoplasmic reticulum, Golgi apparatus, and the neuronal processes (15Ganesh S. Delgado-Escueta A.V. Sakamoto T. Avila M.R. Machado-Salas J. Hoshii Y. Akagi T. Gomi H. Suzuki T. Amano K. Agarwala K.L. Hasegawa Y. Bai D.S. Ishihara T. Hashikawa T. Itohara S. Cornford E.M. Niki H. Yamakawa K. Hum. Mol. Genet. 2002; 11: 1251-1262Crossref PubMed Scopus (184) Google Scholar). Preliminary histochemical investigations have also suggested the possible presence of neurofibrillary tangles (NFTs) in the knock-out mice (17Machado-Salas J. Guevara P. Guevara J. Martínez I. Durón R. Bai D. Ganesh S. Yamakawa K. Suzuki T. Amano K. Cornford E. Delgado-Escueta A.V. Epilepsia. 2005; 46 (Suppl. 6): 75Google Scholar). In this study, we have characterized the biochemical properties of Tau protein in the animal model of LD and identified laforin as an interacting partner of Tau. Our study identifies laforin to be one of the critical regulators of Tau protein and suggests that the Tau pathology might underlie some of the symptoms seen in LD. Lafora disease dual specificity phosphatase domain carbohydrate binding domain protein targeting to glycogen neurofibrillary tangle ubiquitin-protein isopeptide ligase protein kinase A protein kinase B protein phosphatase 2A cyclin-dependent kinase 5 green fluorescent protein. The characterization of laforin-deficient mice has been described previously (15Ganesh S. Delgado-Escueta A.V. Sakamoto T. Avila M.R. Machado-Salas J. Hoshii Y. Akagi T. Gomi H. Suzuki T. Amano K. Agarwala K.L. Hasegawa Y. Bai D.S. Ishihara T. Hashikawa T. Itohara S. Cornford E.M. Niki H. Yamakawa K. Hum. Mol. Genet. 2002; 11: 1251-1262Crossref PubMed Scopus (184) Google Scholar). The animals were maintained at the RIKEN Brain Science Institute animal facilities according to the Institute guidelines for the treatment of experimental animals. Animals of 4-, 6-, or 10-month-old age groups were sacrificed by cervical dislocation, and selected tissues were dissected and fixed in appropriate fixatives or quickly frozen in liquid nitrogen and stored at −80 °C until further analysis. Brain and muscle tissues were homogenized in Tris-buffered saline containing protease and phosphatase inhibitors and used for immunoblotting analysis. The Sarkosyl-soluble and -insoluble fractions of NFTs were extracted as described (18Sahara N. Murayama M. Mizoroki T. Urushitani M. Imai Y. Takahashi R. Murata S. Tanaka K. Takashima A. J. Neurochem. 2005; 94: 1254-1263Crossref PubMed Scopus (174) Google Scholar). The following monoclonal antibodies, obtained as gifts from Dr. Peter Davies, were used for detecting the Tau protein: CP13 for phospho-Ser202 Tau, PHF1 for phospho-Ser396 Tau, and TGF5 for all forms of Tau. In addition, antibodies from Innogenetics (antibody AT8) and GenScript for the detection of phospho-Ser202 and an antibody from Epitomics (antibody E178) for the detection of phospho-Ser396 were also used. Antibodies for Gsk3β, phospho-Ser9 Gsk3β, protein kinase B (AKT), and Ser473 phospho-AKT were purchased from Cell Signaling Technology. Antibodies for protein phosphatase 2A (PP2A), Tyr307 phospho-PP2A, cyclin-dependent kinase 5 (CDK5), Ser159 phospho-CDK5, protein kinase A (PKA), Ser96 phospho-PKA, and PP1 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-GFP and anti-Myc tag antibodies were purchased from Roche Applied Science, and anti-γ-tubulin, anti-FLAG, and anti-V5 antibodies were from Sigma. Anti-ubiquitin antibody was purchased from Dako, and the secondary antibodies were obtained from Jackson ImmunoResearch. Anti-laforin antibody was raised in rabbits using a synthetic peptide corresponding to amino acid residues 85–100 of the murine laforin sequence. Immunohistochemical analysis was done on formalin-fixed, paraffin-embedded sections and was reacted with appropriate antibody, as described previously (8Garyali P. Siwach P. Singh P.K. Puri R. Mittal S. Sengupta S. Parihar R. Ganesh S. Hum. Mol. Genet. 2009; 18: 688-700Crossref PubMed Scopus (96) Google Scholar, 15Ganesh S. Delgado-Escueta A.V. Sakamoto T. Avila M.R. Machado-Salas J. Hoshii Y. Akagi T. Gomi H. Suzuki T. Amano K. Agarwala K.L. Hasegawa Y. Bai D.S. Ishihara T. Hashikawa T. Itohara S. Cornford E.M. Niki H. Yamakawa K. Hum. Mol. Genet. 2002; 11: 1251-1262Crossref PubMed Scopus (184) Google Scholar). They were visualized for light microscopy using diamino-benzidine-conjugated avidin-biotin complex kit (Vectastain ABC Elite; Vector Laboratories). For immunofluorescence staining, sections were processed with appropriate secondary antibodies that were conjugated with rhodamine or fluorescein isothiocyanate and visualized using an epifluorescence microscope, as described (8Garyali P. Siwach P. Singh P.K. Puri R. Mittal S. Sengupta S. Parihar R. Ganesh S. Hum. Mol. Genet. 2009; 18: 688-700Crossref PubMed Scopus (96) Google Scholar, 15Ganesh S. Delgado-Escueta A.V. Sakamoto T. Avila M.R. Machado-Salas J. Hoshii Y. Akagi T. Gomi H. Suzuki T. Amano K. Agarwala K.L. Hasegawa Y. Bai D.S. Ishihara T. Hashikawa T. Itohara S. Cornford E.M. Niki H. Yamakawa K. Hum. Mol. Genet. 2002; 11: 1251-1262Crossref PubMed Scopus (184) Google Scholar). Bielschowsky's silver staining was done on paraffin embedded brains sections as described previously (19Yamamoto T. Hirano A. Neuropathol. Appl. Neurobiol. 1986; 12: 3-9Crossref PubMed Scopus (306) Google Scholar). For electron microscopy studies, the Sarkosyl-insoluble materials, isolated from the laforin-deficient mice, were mildly sonicated and dispersed in phosphate-buffered saline. For negative staining, the samples were first absorbed onto glow-discharged supporting membranes on 300-mesh grids and then treated with 2% uranyl acetate, dried, and observed with a FEI Technai 20 U Twin electron microscope. For immunogold labeling, the samples were prefixed by floating the grids on drops of 4% paraformaldehyde in 0.1 m phosphate buffer for 5 min. After washing, the grids were incubated with primary antibody followed by 10-nm colloidal gold-conjugated secondary antibody and processed for negative staining with 2% sodium phosphotungstic acid and observed as described (20Zhu X. Raina A.K. Rottkamp C.A. Aliev G. Perry G. Boux H. Smith M.A. J. Neurochem. 2001; 76: 435-441Crossref PubMed Scopus (372) Google Scholar). The expression vectors containing Myc- or GFP-tagged wild-type or mutant forms of laforin were described previously (7Mittal S. Dubey D. Yamakawa K. Ganesh S. Hum. Mol. Genet. 2007; 16: 753-762Crossref PubMed Scopus (75) Google Scholar, 8Garyali P. Siwach P. Singh P.K. Puri R. Mittal S. Sengupta S. Parihar R. Ganesh S. Hum. Mol. Genet. 2009; 18: 688-700Crossref PubMed Scopus (96) Google Scholar). Expression constructs for the FLAG-tagged laforin were generated by cloning the coding regions of the EPM2A gene into the pcDNA expression vector (Invitrogen). The short hairpin RNA knockdown constructs for the Epm2a gene were purchased from Open Biosystems and validated in one of our recent studies (8Garyali P. Siwach P. Singh P.K. Puri R. Mittal S. Sengupta S. Parihar R. Ganesh S. Hum. Mol. Genet. 2009; 18: 688-700Crossref PubMed Scopus (96) Google Scholar). The expression constructs for V5-tagged Tau and its mutant form were generously provided by Dr. Michael Hutton. COS-7 or Neuro2A cells were grown in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% (v/v) fetal calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. All cells were grown at 37 °C in 5% CO2. Transfections were performed using Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer's protocol. Neuro2A cells were differentiated into neurons by culturing them in 1% fetal bovine serum as described (21Nangle L.A. Zhang W. Xie W. Yang X.L. Schimmel P. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 11239-11244Crossref PubMed Scopus (121) Google Scholar). To establish the physical interaction between laforin and Tau proteins, we used the expression construct that code for polyhistidine-tagged Tau (22Petrucelli L. Dickson D. Kehoe K. Taylor J. Snyder H. Grover A. De Lucia M. McGowan E. Lewis J. Prihar G. Kim J. Dillmann W.H. Browne S.E. Hall A. Voellmy R. Tsuboi Y. Dawson T.M. Wolozin B. Hardy J. Hutton M. Hum. Mol. Genet. 2004; 13: 703-714Crossref PubMed Scopus (590) Google Scholar). Lysates of cells that had expressed His-tagged Tau with desired protein were incubated with Ni2+-affinity resin (Sigma) for 2 h at 4 °C and processed for pull-down assays as recommended by the manufacturer. Pulled down products were detected by immunoblotting using specific antibodies. The histidine-tagged Tau protein, transiently overexpressed in COS-7 cells, was hyperphosphorylated by treating the cells with wortmannin and affinity-purified using nickel resins. Similarly, the His-tagged laforin or its mutant Q293L was expressed and purified as described (23Dubey D. Ganesh S. Hum. Mol. Genet. 2008; 17: 3010-3020Crossref PubMed Scopus (21) Google Scholar). The nickel resin-bound Tau was mixed with wild-type laforin or its mutant in the phosphatase assay buffer (50 mm HEPES, pH 6, 50 mm NaCl, 5 mm EDTA, 50 mm β-mercaptoethanol) and incubated for 2 h at 37 °C. A control reaction was performed in parallel, wherein the Tau protein was incubated with nickel resins treated with cell lysates that did not express His-tagged laforin. The reaction products were finally mixed with SDS sample buffer, boiled, and analyzed by immunoblotting. Tissue or cell lysates were preincubated with protein G-Sepharose (Bangalore Genei, India) for 2 h at 4 °C and then incubated with anti-laforin or anti-GSK3β antibody (as indicated) for 1 h at 4 °C. After incubation, protein G-Sepharose was used for precipitation. The beads were washed with lysis buffer four times and then eluted with SDS sample buffer for immunoblot analysis as described (24Ganesh S. Tsurutani N. Suzuki T. Ueda K. Agarwala K.L. Osada H. Delgado-Escueta A.V. Yamakawa K. Hum. Mol. Genet. 2003; 12: 2359-2368Crossref PubMed Scopus (40) Google Scholar). GSK3β activity was measured as described previously (25Loberg R.D. Northcott C.A. Watts S.W. Brosius 3rd, F.C. Hypertension. 2003; 41: 898-902Crossref PubMed Scopus (11) Google Scholar) after immunoprecipitation of GSK3β from 100 μg of protein. Immobilized immune complexes were washed twice with lysis buffer and twice with kinase reaction buffer and incubated with phosphoglycogen synthase peptide-2 substrate (Upstate Biotechnology) and [γ-32P]ATP for 30 min at 30 °C. After this incubation, an aliquot of samples was placed on phosphocellulose disc (Whatman 31ET CHR filter paper), air-dried, and washed three times in 0.75% phosphoric acid and once with acetone. Radioactivity in the phosphocellulose disc was counted in a β-counter (PerkinElmer Life Sciences). Protein samples were run on a 10% SDS-PAGE and transferred onto a nitrocellulose filter (MDI, India) as described previously (7Mittal S. Dubey D. Yamakawa K. Ganesh S. Hum. Mol. Genet. 2007; 16: 753-762Crossref PubMed Scopus (75) Google Scholar, 8Garyali P. Siwach P. Singh P.K. Puri R. Mittal S. Sengupta S. Parihar R. Ganesh S. Hum. Mol. Genet. 2009; 18: 688-700Crossref PubMed Scopus (96) Google Scholar). Signal intensity of the immunoblot was quantitated using NIH Image software (ImageJ; National Institutes of Health). The characterization of laforin-deficient mice was reported in one of our previous publications (15Ganesh S. Delgado-Escueta A.V. Sakamoto T. Avila M.R. Machado-Salas J. Hoshii Y. Akagi T. Gomi H. Suzuki T. Amano K. Agarwala K.L. Hasegawa Y. Bai D.S. Ishihara T. Hashikawa T. Itohara S. Cornford E.M. Niki H. Yamakawa K. Hum. Mol. Genet. 2002; 11: 1251-1262Crossref PubMed Scopus (184) Google Scholar). The present investigation was carried out on the C57BL/6 isogenic line for the Epm2a gene knock-out, derived by back-crossing the F1 heterozygous mutants with C57BL/6 animals through 11 generations, and the animals were genotyped as described (15Ganesh S. Delgado-Escueta A.V. Sakamoto T. Avila M.R. Machado-Salas J. Hoshii Y. Akagi T. Gomi H. Suzuki T. Amano K. Agarwala K.L. Hasegawa Y. Bai D.S. Ishihara T. Hashikawa T. Itohara S. Cornford E.M. Niki H. Yamakawa K. Hum. Mol. Genet. 2002; 11: 1251-1262Crossref PubMed Scopus (184) Google Scholar). Our investigations on the neuropathological changes in the brain sections of the 10-month-old laforin-deficient mice have suggested the presence of neurofibrillary tangles (NFTs), as revealed by Bielschowsky's silver staining (Fig. 1, A–C). This was subsequently confirmed by immunohistochemical staining with antibodies E178 (26Jakes R. Novak M. Davison M. Wischik C.M. EMBO J. 1991; 10: 2725-2729Crossref PubMed Scopus (120) Google Scholar) and AT8 (27Biernat J. Mandelkow E.M. Schroter C. Lichtenberg-Kraag B. Steiner B. Berling B. Meyer H. Mercken M. Vandermeeren A. Goedert M. Mandelkow E. EMBO J. 1992; 11: 1593-1597Crossref PubMed Scopus (430) Google Scholar, 28Goedert M. Jakes R. Vanmechelen E. Neurosci. Lett. 1995; 189: 167-169Crossref PubMed Scopus (475) Google Scholar) that specifically recognize Tau protein phosphorylated at Ser396 or Ser202 residues, respectively (Fig. 1, D–I). Numerous neurons that were positive for the dyes or the phospho-Tau antibodies were seen primarily in the hippocampus, thalamus, cerebral cortex, cerebellum, and brain stem of the laforin-deficient mice but not in the corresponding regions of the wild-type littermates (Fig. 1, A–I). Identical observations were made with antibodies CP13 and PHF1 as well (supplemental Fig. S1, A–D). The NFTs in laforin-deficient mice also stained intensely with ubiquitin antibody (supplemental Fig. S1, E–G). NFTs were not observed in the 2-, 4-, or 6-month-old knock-out mice analyzed. In addition to brain, Tau protein is known to express in muscle tissues (29Askanas V. Engel W.K. Bilak M. Alvarez R.B. Selkoe D.J. Am. J. Pathol. 1994; 144: 177-187PubMed Google Scholar, 30Mirabella M. Alvarez R.B. Bilak M. Engel W.K. Askanas V. J. Neuropathol. Exp. Neurol. 1996; 55: 774-786Crossref PubMed Scopus (103) Google Scholar). We have therefore checked for the presence of hyperphosphorylation of Tau protein in the muscle tissues of the 10-month-old laforin-deficient mice. Phospho-Tau-specific antibodies identified immunoreactive cytoplasmic inclusions in the muscle sections from the knock-out mice but not the wild-type littermates (Fig. 1, J–M). Such inclusions were not seen in the muscle sections of 4- and 6-month-old knock-out mice. Consistent with the immunohistochemical observations, immunoblot analysis of Tau protein from the 10-month-old animals showed a significant increase in the phosphorylation levels at the Ser202 and Ser396 positions, both in muscle and brain tissues of the laforin-deficient mice, as compared with the wild-type littermates (Fig. 2, A and B, and supplemental Fig. S2A). This difference, however, was not obvious in the 4-month-old mice (Fig. 2A). The phosphorylation levels of Tau were nearly the same in wild-type and heterozygous animals of the 10-month age group (supplemental Fig. S2C). Because Tau is known to form insoluble aggregates upon hyperphosphorylation (31Lewis J. McGowan E. Rockwood J. Melrose H. Nacharaju P. Van Slegtenhorst M. Gwinn-Hardy K. Paul Murphy M. Baker M. Yu X. Duff K. Hardy J. Corral A. Lin W.L. Yen S.H. Dickson D.W. Davies P. Hutton M. Nat. Genet. 2000; 25: 402-405Crossref PubMed Scopus (1141) Google Scholar, 32Bandyopadhyay B. Li G. Yin H. Kuret J. J. Biol. Chem. 2007; 282: 16454-16464Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar), we further assessed the amount of Tau in the Sarkosyl-insoluble fractions derived from the brain and muscle tissues of the 10-month-old animals. A large amount of insoluble and phosphorylated forms of Tau was recovered from the brain and muscle tissue lysates of the laforin-deficient mice as compared with lysates of wild-type littermates (Fig. 2C). The Sarkosyl-insoluble material recovered from the laforin-deficient mice was further investigated with transmission electron microscopy. The NFTs observed in the Sarkosyl-insoluble fraction appeared to be straight filaments of about 10–20 nm in diameter (Fig. 2D). Labeling with antibodies against phosphorylated Tau (Ser396) revealed reasonably abundant Tau-containing filaments in the preparation (Fig. 2, E and F). Such filaments were not seen in the preparations obtained from the age-matched wild-type littermates (data not shown), and the gold particles were not seen when the primary antibody was omitted for the immunodetection for the samples from the knock-out mice (data not shown). Taken together, the biochemical and ultrastructural analyses strongly suggest the presence of NFT-like Tau aggregates in the brain and muscle tissue of the laforin-deficient mice. Having established the difference in the phosphorylation levels of Tau protein in laforin-deficient mice, we next explored whether loss of laforin leads to changes in the phospho forms of key kinases and phosphatases that are known to regulate Tau. For this analysis, we have selected six kinases and two phosphatases (see Fig. 3). Activation of GSK3β is known to phosphorylate Tau protein (33Sperber B.R. Leight S. Goedert M. Lee V.M. Neurosci. Lett. 1995; 197: 149-153Crossref PubMed Scopus (198) Google Scholar, 34Imahori K. Uchida T. J. Biochem. 1997; 121: 179-188PubMed Google Scholar). The active and inactive forms of GSK3β were quantitated by looking at the phosphorylation status of Tyr216 and Ser9 residues by using antibodies that are specific to these two phospho forms (35Bhat R.V. Shanley J. Correll M.P. Fieles W.E. Keith R.A. Scott C.W. Lee C.M. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 11074-11079Crossref PubMed Scopus (354) Google Scholar, 36Cross D.A. Alessi D.R. Cohen P. Andjelkovich M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4376) Google Scholar) and also by enzymatic assays using a substrate (Fig. 3, A and B). Although the total level of GSK3β was comparable among the wild-type and laforin-deficient animals, the levels of the inactive form of GSK3β (phospho-Ser9) were found to be significantly lower in the muscle and brain tissues of the 10-month-old knock-out mice (Fig. 3, A and D, and supplemental Fig. S2B). We therefore measured the GSK3β activity by 32P labeling in an in vitro assay and found that the GSK3β from laforin-deficient mice indeed show increased activity as compared with that from age-matched wild-type mice (Fig. 3B). No difference in the phosphorylation levels of Tyr216 residue was observed in the analyzed tissues of the two age groups (Fig. 3A). Using a similar approach, we next examined how a few other regulators (kinases/phosphatases) might contribute to hyperphosphorylation of Tau protein in laforin-deficient mice. As shown in Fig.
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