Glycogen Synthase Kinase-3β Phosphorylates Protein Tau and Rescues the Axonopathy in the Central Nervous System of Human Four-repeat Tau Transgenic Mice
2000; Elsevier BV; Volume: 275; Issue: 52 Linguagem: Inglês
10.1074/jbc.m006219200
ISSN1083-351X
AutoresKurt Spittaels, Chris Van den Haute, Jo Van Dorpe, Hugo Geerts, Marc Mercken, Koen Bruynseels, Reena Lasrado, Kris Vandezande, Isabelle Laenen, Tim Boon, Johan Van Lint, Jacky Vandenheede, Dieder Moechars, Ruth J. F. Loos, Fred Van Leuven,
Tópico(s)Neurological diseases and metabolism
ResumoProtein tau filaments in brain of patients suffering from Alzheimer's disease, frontotemporal dementia, and other tauopathies consist of protein tau that is hyperphosphorylated. The responsible kinases operating in vivo in neurons still need to be identified. Here we demonstrate that glycogen synthase kinase-3β (GSK-3β) is an effective kinase for protein tau in cerebral neurons in vivo in adult GSK-3β and GSK-3β × human tau40 transgenic mice. Phosphorylated protein tau migrates slower during electrophoretic separation and is revealed by phosphorylation-dependent anti-tau antibodies in Western blot analysis. In addition, its capacity to bind to re-assembled paclitaxel (Taxol®)-stabilized microtubules is reduced, compared with protein tau isolated from mice not overexpressing GSK-3β. Co-expression of GSK-3β reduces the number of axonal dilations and alleviates the motoric impairment that was typical for single htau40 transgenic animals (Spittaels, K., Van den Haute, C., Van Dorpe, J., Bruynseels, K., Vandezande, K., Laenen, I., Geerts, H., Mercken, M., Sciot, R., Van Lommel, A., Loos, R., and Van Leuven, F. (1999) Am. J. Pathol. 155, 2153–2165). Although more hyperphosphorylated protein tau is available, neither an increase in insoluble protein tau aggregates nor the presence of paired helical filaments or tangles was observed. These findings could have therapeutic implications in the field of neurodegeneration, as discussed. Protein tau filaments in brain of patients suffering from Alzheimer's disease, frontotemporal dementia, and other tauopathies consist of protein tau that is hyperphosphorylated. The responsible kinases operating in vivo in neurons still need to be identified. Here we demonstrate that glycogen synthase kinase-3β (GSK-3β) is an effective kinase for protein tau in cerebral neurons in vivo in adult GSK-3β and GSK-3β × human tau40 transgenic mice. Phosphorylated protein tau migrates slower during electrophoretic separation and is revealed by phosphorylation-dependent anti-tau antibodies in Western blot analysis. In addition, its capacity to bind to re-assembled paclitaxel (Taxol®)-stabilized microtubules is reduced, compared with protein tau isolated from mice not overexpressing GSK-3β. Co-expression of GSK-3β reduces the number of axonal dilations and alleviates the motoric impairment that was typical for single htau40 transgenic animals (Spittaels, K., Van den Haute, C., Van Dorpe, J., Bruynseels, K., Vandezande, K., Laenen, I., Geerts, H., Mercken, M., Sciot, R., Van Lommel, A., Loos, R., and Van Leuven, F. (1999) Am. J. Pathol. 155, 2153–2165). Although more hyperphosphorylated protein tau is available, neither an increase in insoluble protein tau aggregates nor the presence of paired helical filaments or tangles was observed. These findings could have therapeutic implications in the field of neurodegeneration, as discussed. microtubule glycogen synthase kinase-3β wild type Alzheimer's disease paired helical filament reassembly buffer radioimmunoprecipitation assay buffer formic acid frontotemporal dementia and parkinsonism linked to chromosome 17 glial fibrillary acidic protein -2, and -5, transgenic mice of strain 1,2, and 5, respectively, overexpressing human tau homozygous mouse of strain htau40-1 tau protein kinase I Protein tau represents a family of neuronal phosphoproteins, which were originally identified as proteins that co-purify with microtubules (MT)1 and that assemble tubulin dimers into microtubules (1Weingarten M.D. Lockwood A.H. Hwo S.Y. Kirschner M.W. Proc. Natl. Acad. Sci. U. S. 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Despite the wealth of in vitro data, convincing evidence for any functional repercussion of the phosphorylation of protein tau by GSK-3β in vivo is lacking and the requirement of protein tau phosphorylation for PHF formation is still a matter of debate. We generated transgenic mice that overexpress a constitutively active form of the human kinase, i.e. GSK-3β(S9A) with serine at position 9 replaced by alanine to prevent its inactivation by phosphorylation (40Stambolic V. Woodgett J.R. Biochem. J. 1994; 303: 701-704Crossref PubMed Scopus (525) Google Scholar, 41Sutherland C. Leighton I.A. Cohen P. Biochem. J. 1993; 296: 15-19Crossref PubMed Scopus (780) Google Scholar). The GSK-3β transgenic mice were crossed with transgenic mice that overexpress the longest isoform of human protein tau, i.e. containing 2 N-terminal inserts and 4 microtubule binding domains (htau40), extensively characterized previously (42Spittaels K. Van den Haute C. Van Dorpe J. Bruynseels K. Vandezande K. Laenen I. 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A 2-fold increase in GSK-3β activity, relative to the endogenous enzymatic activity, thereby rescues nearly all neuropathological symptoms of the single htau40 transgenic mice (42Spittaels K. Van den Haute C. Van Dorpe J. Bruynseels K. Vandezande K. Laenen I. Geerts H. Mercken M. Sciot R. Van Lommel A. Loos R. Van Leuven F. Am. J. Pathol. 1999; 155: 2153-2165Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar). The conclusion that hyperphosphorylation of protein tau by GSK-3β reverses such a severe phenotype could have therapeutic implications in the field of neurodegeneration, as discussed. cDNA coding for human GSK-3β(S9A) (Refs. 40Stambolic V. Woodgett J.R. Biochem. J. 1994; 303: 701-704Crossref PubMed Scopus (525) Google Scholar and 41Sutherland C. Leighton I.A. Cohen P. Biochem. J. 1993; 296: 15-19Crossref PubMed Scopus (780) Google Scholar; gift of J. Woodgett) was ligated in the adapted mouse thy1 gene (42Spittaels K. Van den Haute C. Van Dorpe J. Bruynseels K. 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Three founder strains, i.e. htau40-1, htau40-2, and htau40-5, which transmitted the transgene in a stable Mendelian fashion, were used to generate double transgenic mice by cross-breeding with GSK-3β(S9A) animals. Single and double transgenic mice at the age of 2–4 months were subjected to three classical sensorimotor tests (46Lamberty Y. Gower A.J. Phys. Behav. 1991; 51: 81-88Crossref Scopus (77) Google Scholar), i.e. the forced swimming test, the rod walking test, and the inverted wire-grid test, performed as described previously (42Spittaels K. Van den Haute C. Van Dorpe J. Bruynseels K. Vandezande K. Laenen I. Geerts H. Mercken M. Sciot R. Van Lommel A. Loos R. Van Leuven F. Am. J. Pathol. 1999; 155: 2153-2165Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar). In addition, we scored the time that the mice needed to return upward after being forced on their back. Four groups of mice were tested: 21 wild-type FVB mice (WT), 23 homozygous htau40-1 (1HH), 17 heterozygous GSK-3β(S9A) (GSK), and 7 htau40-1 × GSK-3β(S9A) double transgenic mice (1HH × GSK). The contingency χ2 test was used to evaluate the difference. To prevent dephosphorylation during post mortem delay (47Gärtner U. Janke C. Holzer M. Vanmechelen E. Arendt T. Neurobiol. Aging. 1998; 19: 535-543Crossref PubMed Scopus (56) Google Scholar, 48Matsuo E.S. Shin R.-W. Billingsley M.L. Van de Voorde A. O'Connor M. Trojanowski J.Q. Lee V.M.-Y. Neuron. 1994; 13: 989-1002Abstract Full Text PDF PubMed Scopus (566) Google Scholar) all tissues were rapidly dissected and tissue homogenates were processed on ice and immediately stored at −70 °C after freezing in liquid nitrogen. Tissue extraction and Western blotting was performed as described previously (42Spittaels K. Van den Haute C. Van Dorpe J. Bruynseels K. Vandezande K. Laenen I. Geerts H. Mercken M. Sciot R. Van Lommel A. Loos R. 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The solubility of protein tau in brain and spinal cord from wild type, single, and double transgenic mice was determined by sequential extraction with high salt reassembly buffer (RAB), detergents containing radioimmunoprecipitation assay buffer (RIPA) and 70% formic acid (FA), as described (55Ishihara T. Hong M. Zhang B. Nakagawa Y. Lee M.K. Trojanowski J.Q. Lee V.M.-Y. Neuron. 1999; 24: 751-762Abstract Full Text Full Text PDF PubMed Scopus (504) Google Scholar). Four htau40-1 and htau40-1 × GSK-3β transgenic mice were used, quantitative Western blot analysis was carried out using Tau-5 antibody and the amount of protein tau in the different buffers was determined, with the total transgenic human protein tau taken as 100%. The GSK-3β(S9A) protein levels in brain and spinal cord extracts were estimated by Western blotting with the monoclonal antibody TPK-I/GSK-3β (Affinity, Nottingham, United Kingdom). The GSK-3β enzymatic activity was measured in brain homogenates after immunoprecipitation and fractionation by cation exchange fast protein liquid chromatography on a Mono S column (Amersham Pharmacia Biotech, Uppsala, Sweden) as described (56Van Lint J. Khandelwal R.L. Merlevede W. Vandenheede J.R. Anal. Biochem. 1993; 208: 132-137Crossref PubMed Scopus (27) Google Scholar). Non-transgenic (n = 4), GSK-3β(S9A) (n = 4), htau40-1 (n = 8), htau40-2 (n = 8), htau40-1 × GSK (n = 4), and htau40-2 × GSK (n = 4) mice were transcardially perfused with paraformaldehyde (4% in phosphate-buffered saline). Brain and spinal cord were immersion-fixed overnight, and vibratome (40 μm) and microtome (6 μm) sections were cut and processed (42Spittaels K. Van den Haute C. Van Dorpe J. Bruynseels K. Vandezande K. Laenen I. Geerts H. Mercken M. Sciot R. Van Lommel A. Loos R. Van Leuven F. Am. J. Pathol. 1999; 155: 2153-2165Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar). Axonal dilations were detected and quantified as described (42Spittaels K. Van den Haute C. Van Dorpe J. Bruynseels K. Vandezande K. Laenen I. Geerts H. Mercken M. Sciot R. Van Lommel A. Loos R. Van Leuven F. Am. J. Pathol. 1999; 155: 2153-2165Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar). The Kruskal-Wallis test was applied to evaluate the differences. Paclitaxel-dependent isolation and re-assembly of microtubules and isolation of microtubule-associated proteins from brain and spinal cord was performed as described (57Vallee R.B. J. Cell Biol. 1982; 92: 435-442Crossref PubMed Scopus (429) Google Scholar). To increase the yield of microtubule preparations from mouse spinal cord, 3 mm CaCl2 was additionally added to the MT buffer to depolymerize cold-stable microtubules in situ(58Webb B.C. Wilson L. Biochemistry. 1980; 19: 1993-2001Crossref PubMed Scopus (84) Google Scholar). Equal amounts of proteins were loaded on 8% SDS-polyacrylamide gels after removal of mouse immunoglobulins. Densitometric quantification of Western blots of protein tau was as described (42Spittaels K. Van den Haute C. Van Dorpe J. Bruynseels K. Vandezande K. Laenen I. Geerts H. Mercken M. Sciot R. Van Lommel A. Loos R. Van Leuven F. Am. J. Pathol. 1999; 155: 2153-2165Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar), and results were normalized for tubulin and Tau-5. The blots for tubulin were developed with an antibody specific for neuronal β-III anti-N-tubulin (Promega, Madison, WI). The Wilcoxon signed-rank test was used to evaluate the differences. Mice were anesthetized and transcardially perfused with paraformaldehyde (4% in phosphate-buffered saline). Brain and spinal cord were immersion-fixed overnight and cut sagitally into two hemispheres or transversally into four tissue blocks of 9 mm, respectively. Vibratome sections (40 μm) were cut from the right hemisphere, whereas the left hemisphere was dehydrated, embedded in paraffin, and used for microtome sectioning (6 μm). Likewise, vibratome sections were cut from the thoracal part of the spinal cord, whereas the thoracolumbal part was embedded in paraffin. Primary antibodies used were monoclonal antibodies for protein tau (HT-7) and for GSK-3β (TPK-I) (Affinity), a polyclonal antiserum against synaptophysin (Dako, Glostrup, Denmark), the monoclonal antibody SMI-32 for detection of neurofilament proteins (Affinity), and an antiserum to GFAP (Dako). Other procedures were performed as described: standard hematoxylin/eosin staining for muscle and Bielschowsky's silver impregnation and thioflavine S staining for central nervous system (42Spittaels K. Van den Haute C. Van Dorpe J. Bruynseels K. Vandezande K. Laenen I. Geerts H. Mercken M. Sciot R. Van Lommel A. Loos R. Van Leuven F. Am. J. Pathol. 1999; 155: 2153-2165Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar, 44Moechars D. Dewachter I. Lorent K. Reversé D. Baekelandt V. Naidu A. Tesseur I. Spittaels K. Van den Haute C. Checler F. Godaux E. Cordell B. Van Leuven F. J. Biol. Chem. 1999; 274: 6483-6492Abstract Full Text Full Text PDF PubMed Scopus (618) Google Scholar). For transmission electron microscopy, 4–8-month-old htau40-1HH (n = 8), htau40-1HH × GSK-3β (n = 4), and wild-type (n = 4) mice were perfused with 4% glutaraldehyde in phosphate-buffered saline or 4% paraformaldehyde and 0.1% glutaraldehyde. Areas of neocortex, hippocampus, and spinal cord were excised from 40-μm-thick vibratome sections, postfixed with OsO4, and embedded in epon. For immunoelectron microscopy areas of cerebral neocortex, hippocampus, thalamus and subiculum were excised from 40-μm-thick vibratome sections of 6-month-old htau40-1HH (n = 4) and htau40-1HH × GSK-3β (n = 2) mice and processed as described (59Van Dorpe J. Smeijers L. Dewachter I. Nuyens D. Spittaels K. Van den Haute C. Mercken M. Moechars D. Laenen I. Kuiperi C. Bruynseels K. Tesseur I. Loos R. Vanderstichele H. Checler F. Van Leuven F. Am. J. Pathol. 2000; 157: 1283-1298Abstract Full Text Full Text PDF PubMed Google Scholar). The following antibodies were used: B19 (anti-polyclonal; gift of J. P. Brion, Free University of Brussels, Belgium), anti-Tau (polyclonal; BioMakor, Rehovot, Israel), and AT8 (monoclonal; Innogenetics, Gent, Belgium) against protein tau; and anti-GFAP (polyclonal; Dako, Glostrup, Denmark). Fragments of hippocampus from an Alzheimer's disease patient, processed in the same way, were used as a positive control for tangle detection. We have generated transgenic mice that express a mutant form of human GSK-3β, denoted GSK-3β(S9A), containing alanine in position 9 instead of the wild-type serine, to prevent inactivation by phosphorylation (60Woodgett J.R. EMBO J. 1990; 9: 2431-2438Crossref PubMed Scopus (1178) Google Scholar). The cDNA was incorporated in a recombinant DNA construct based on the mousethy1 gene promoter (Fig. 1 a), and transgenic mice were generated by micro-injection, in the FVB mouse strain (42Spittaels K. Van den Haute C. Van Dorpe J. Bruynseels K. Vandezande K. Laenen I. Geerts H. Mercken M. Sciot R. Van Lommel A. Loos R. Van Leuven F. Am. J. Pathol. 1999; 155: 2153-2165Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar, 43Moechars D. Lorent K. De Strooper B. Dewachter I. Van Leuven F. EMBO J. 1996; 15: 1265-1274Crossref PubMed Scopus (178) Google Scholar, 44Moechars D. Dewachter I. Lorent K. Reversé D. Baekelandt V. Naidu A. Tesseur I. Spittaels K. Van den Haute C. Checler F. Godaux E. Cordell B. Van Leuven F. J. Biol. Chem. 1999; 274: 6483-6492Abstract Full Text Full Text PDF PubMed Scopus (618) Google Scholar). The human GSK-3β protein was demonstrated by Western blotting in brain and spinal cord (Fig. 1, b and c). The transgene was enzymatically active on a synthetic peptide substrate, resulting in a doubling of the total GSK-3β kinase activity in GSK-3β mouse brain homogenates, relative to wild-type mice (Fig. 1 d). Transgenic mice that overexpress the longest human protein tau isoform (htau40) (Fig. 1, f–h) were characterized extensively (42Spittaels K. Van den Haute C. Van Dorpe J. Bruynseels K. Vandezande K. Laenen I. Geerts H. Mercken M. Sciot R. Van Lommel A. Loos R. Van Leuven F. Am. J. Pathol. 1999; 155: 2153-2165Abstract Full Text Full Text PDF PubMed Scopus (346) Google
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