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Alzheimer-like Changes in Microtubule-associated Protein Tau Induced by Sulfated Glycosaminoglycans

1997; Elsevier BV; Volume: 272; Issue: 52 Linguagem: Inglês

10.1074/jbc.272.52.33118

ISSN

1083-351X

Autores

Masato Hasegawa, R. Anthony Crowther, Ross Jakes, Michel Goedert,

Tópico(s)

Endoplasmic Reticulum Stress and Disease

Resumo

Hyperphosphorylated microtubule-associated protein tau is the major proteinaceous component of the paired helical and straight filaments which constitute a defining neuropathological characteristic of Alzheimer's disease and a number of other neurodegenerative disorders. We have recently shown that full-length recombinant tau assembles into Alzheimer-like filaments upon incubation with heparin. Heparin also promotes phosphorylation of tau by a number of protein kinases, prevents tau from binding to taxol-stabilized microtubules, and produces rapid disassembly of microtubules assembled from tau and tubulin. Here, we have used the above parameters to study the interactions between tau protein and a number of naturally occurring and synthetic glycosaminoglycans. We show that the magnitude of the glycosaminoglycan effects is proportional to their degree of sulfation. Thus, the strongly sulfated glycosaminoglycans dextran sulfate, pentosan polysulfate, and heparin were the most potent, whereas the non-sulfated dextran and hyaluronic acid were without effect. The moderately sulfated glycosaminoglycans heparan sulfate, chondroitin sulfate, and dermatan sulfate had intermediate effects, whereas keratan sulfate had little or no effect. These in vitro interactions between tau protein and sulfated glycosaminoglycans reproduced the known characteristics of paired helical filament-tau from Alzheimer's disease brain. Sulfated glycosaminoglycans are present in nerve cells in Alzheimer's disease brain in the early stages of neurofibrillary degeneration, suggesting that their interactions with tau may constitute a central event in the development of the neuronal pathology of Alzheimer's disease. Hyperphosphorylated microtubule-associated protein tau is the major proteinaceous component of the paired helical and straight filaments which constitute a defining neuropathological characteristic of Alzheimer's disease and a number of other neurodegenerative disorders. We have recently shown that full-length recombinant tau assembles into Alzheimer-like filaments upon incubation with heparin. Heparin also promotes phosphorylation of tau by a number of protein kinases, prevents tau from binding to taxol-stabilized microtubules, and produces rapid disassembly of microtubules assembled from tau and tubulin. Here, we have used the above parameters to study the interactions between tau protein and a number of naturally occurring and synthetic glycosaminoglycans. We show that the magnitude of the glycosaminoglycan effects is proportional to their degree of sulfation. Thus, the strongly sulfated glycosaminoglycans dextran sulfate, pentosan polysulfate, and heparin were the most potent, whereas the non-sulfated dextran and hyaluronic acid were without effect. The moderately sulfated glycosaminoglycans heparan sulfate, chondroitin sulfate, and dermatan sulfate had intermediate effects, whereas keratan sulfate had little or no effect. These in vitro interactions between tau protein and sulfated glycosaminoglycans reproduced the known characteristics of paired helical filament-tau from Alzheimer's disease brain. Sulfated glycosaminoglycans are present in nerve cells in Alzheimer's disease brain in the early stages of neurofibrillary degeneration, suggesting that their interactions with tau may constitute a central event in the development of the neuronal pathology of Alzheimer's disease. The paired helical filament (PHF) 1The abbreviations used are: PHF, paired helical filament; SF, straight filament; MAP, mitogen-activated protein; SAP, stress-activated protein; GSK3, glycogen synthase kinase 3; NCLK, neuronal cdc2-like kinase; PIPES, 1,4-piperazinediethanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid. 1The abbreviations used are: PHF, paired helical filament; SF, straight filament; MAP, mitogen-activated protein; SAP, stress-activated protein; GSK3, glycogen synthase kinase 3; NCLK, neuronal cdc2-like kinase; PIPES, 1,4-piperazinediethanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid. and the related straight filament (SF) are the major components of the neurofibrillary deposits that form a defining neuropathological characteristic of Alzheimer's disease and a number of other neurodegenerative disorders (reviewed in Ref. 1Goedert M. Trojanowski J.Q. Lee V.M.-Y. Rosenberg R.N. Prusiner S.B. DiMauro S. Barchi R.L. The Molecular and Genetic Basis of Neurological Disease. Butterworth/Heinemann, Oxford1997: 613-627Google Scholar). They are composed of microtubule-associated protein tau, in a hyperphosphorylated state. Mass spectrometry and immunological studies have identified a large number of phosphorylation sites in PHF-tau (2Lee V.M.-Y. Balin B.J. Otvos L. Trojanowski J.Q. Science. 1991; 251: 675-678Crossref PubMed Scopus (1240) Google Scholar, 3Hasegawa M. Morishima-Kawashima M. Takio K. Suzuki M. Titani K. Ihara Y. J. Biol. Chem. 1992; 267: 17047-17054Abstract Full Text PDF PubMed Google Scholar, 4Biernat J. Mandelkow E.M. Schröter C. Lichtenberg-Kraag B. Steiner B. Berling B. Meyer H. Mercken M. Vandermeeren M. Goedert M. Mandelkow E. EMBO J. 1992; 11: 1593-1597Crossref PubMed Scopus (422) Google Scholar, 5Goedert M. Jakes R. Crowther R.A. Six J. Lübke U. Vandermeeren M. Cras P. Trojanowski J.Q. Lee V.M.-Y. Proc. Natl. Acad. Sci. U. S. 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Goedert M. FEBS Lett. 1996; 384: 25-30Crossref PubMed Scopus (146) Google Scholar). Some of these sites are not phosphorylated in tau from normal brain, whereas others are phosphorylated to a greater extent in PHF-tau than in tau from normal brain. Many phosphorylated sites are serine/threonine-prolines. Consequently, tau protein can be phosphorylated in vitro at many of these sites by proline-directed protein kinases, such as mitogen-activated protein (MAP) kinase (12Drewes G. Lichtenberg-Kraag B. Döring F. Mandelkow E.M. Biernat J. Dorée M. Mandelkow E. EMBO J. 1992; 11: 2131-2138Crossref PubMed Scopus (491) Google Scholar, 13Ledesma M.D. Correas I. Avila J. Diaz-Nido J. FEBS Lett. 1992; 308: 218-224Crossref PubMed Scopus (185) Google Scholar, 14Goedert M. Cohen E.S. Jakes R. Cohen P. FEBS Lett. 1992; 312: 95-99Crossref PubMed Scopus (258) Google Scholar), stress-activated protein (SAP) kinases (15Goedert M. Hasegawa M. Jakes R. Lawler S. Cuenda A. Cohen P. FEBS Lett. 1997; 409: 57-62Crossref PubMed Scopus (254) Google Scholar, 16Reynolds C.H. Utton M.A. Gibb G.M. Yates A. Anderton B.H. J. Neurochem. 1997; 68: 1736-1744Crossref PubMed Scopus (189) Google Scholar), glycogen synthase kinase-3 (GSK3) (17Hanger D.A. Hughes K. Woodgett J.R. Brion J.P. Anderton B.H. Neurosci. Lett. 1992; 147: 58-62Crossref PubMed Scopus (642) Google Scholar, 18Mandelkow E.M. Drewes G. Biernat J. Gustke N. Van Lint J. Vandenheede J.R. Mandelkow E. FEBS Lett. 1992; 314: 315-321Crossref PubMed Scopus (479) Google Scholar, 19Ishiguro K. Shiratsuchi A. Sato S. Omori A. Arioka M. Kobayashi S. Uchida T. Imahori K. FEBS Lett. 1993; 325: 167-172Crossref PubMed Scopus (363) Google Scholar), and neuronal cdc2-like kinase (NCLK) (20Paudel H.K. Lew J. Ali Z. Wang J.H. J. Biol. Chem. 1993; 268: 23512-23518Abstract Full Text PDF PubMed Google Scholar, 21Baumann K. Mandelkow E.M. Biernat J. Piwnica-Worms H. Mandelkow E. FEBS Lett. 1993; 336: 417-424Crossref PubMed Scopus (416) Google Scholar). Moreover, cAMP-dependent protein kinase and Ca2+/calmodulin-dependent protein kinase II phosphorylate tau at specific sites in vitro, some of which are also phosphorylated in PHF-tau (22Steiner B. Mandelkow E.M. Biernat J. Gustke N. Meyer H.E. Schmidt B. Mieskes G. Söling H.D. Drechsel D. Kirschner M.W. Goedert M. Mandelkow E. EMBO J. 1990; 9: 3539-3544Crossref PubMed Scopus (255) Google Scholar, 23Scott C.W. Spreen R.C. Herman J.L. Clow F.P. Davison M.D. Young J. Caputo C.B. J. Biol. Chem. 1993; 268: 1166-1173Abstract Full Text PDF PubMed Google Scholar, 24Litersky J.M. Johnson G.V.W. Jakes R. Goedert M. Lee M. Seubert P. Biochem. J. 1996; 316: 655-660Crossref PubMed Scopus (120) Google Scholar). Hyperphosphorylation of tau results in its inability to bind to microtubules and is believed to precede PHF assembly (25Bramblett G.T. Goedert M. Jakes R. Merrick S.E. Trojanowski J.Q. Lee V.M.-Y. Neuron. 1993; 10: 1089-1099Abstract Full Text PDF PubMed Scopus (742) Google Scholar, 26Yoshida H. Ihara Y. J. Neurochem. 1993; 61: 1183-1186Crossref PubMed Scopus (202) Google Scholar, 27Braak E. Braak H. Mandelkow E.M. Acta Neuropathol. 1994; 87: 554-567Crossref PubMed Scopus (661) Google Scholar). However, it is unclear whether hyperphosphorylation of tau is either necessary or sufficient for PHF formation. We have recently shown that a phosphorylation-independent interaction between recombinant tau and sulfated glycosaminoglycans leads to the formation of Alzheimer-like filaments under physiological conditions in vitro (28Goedert M. Jakes R. Spillantini M.G. Hasegawa M. Smith M.J. Crowther R.A. Nature. 1996; 383: 550-553Crossref PubMed Scopus (836) Google Scholar, 29Goedert M. Spillantini M.G. Hasegawa M. Jakes R. Crowther R.A. Klug A. Cold Spring Harbor Symp. Quant. Biol. 1996; 61: 565-573Crossref PubMed Google Scholar). Three repeat-containing tau isoforms gave rise to paired helical-like filaments, whereas four repeat-containing isoforms formed straight filaments, thus suggesting an explanation for the two tau assemblies present in Alzheimer's disease brain. We also showed that heparin prevents tau from binding to microtubules and promotes microtubule disassembly. Heparan sulfate and hyperphosphorylated tau have been found to co-exist in nerve cells in Alzheimer's disease brain at the earliest known stages of neurofibrillary pathology (28Goedert M. Jakes R. Spillantini M.G. Hasegawa M. Smith M.J. Crowther R.A. Nature. 1996; 383: 550-553Crossref PubMed Scopus (836) Google Scholar). Moreover, phosphorylation of tau by cdc28, cAMP-dependent protein kinase, GSK3, and several SAP kinases is markedly stimulated by heparin (11Hasegawa M. Jakes R. Crowther R.A. Lee V.M.-Y. Ihara Y. Goedert M. FEBS Lett. 1996; 384: 25-30Crossref PubMed Scopus (146) Google Scholar, 15Goedert M. Hasegawa M. Jakes R. Lawler S. Cuenda A. Cohen P. FEBS Lett. 1997; 409: 57-62Crossref PubMed Scopus (254) Google Scholar, 30Mawal-Dewan M. Sen P.C. Abdel-Ghany M. Shalloway D. Racker E. J. Biol. Chem. 1992; 267: 19705-19709Abstract Full Text PDF PubMed Google Scholar, 31Brandt R. Lee G. Teplow D.P. Shalloway D. Abdel-Ghany M. J. Biol. Chem. 1994; 269: 11776-11782Abstract Full Text PDF PubMed Google Scholar, 32Yang S.D. Yu J.S. Shiah S.G. Huang J.J. J. Neurochem. 1994; 63: 1416-1425Crossref PubMed Scopus (56) Google Scholar). Taken together, these findings suggest that an interaction between tau protein and sulfated glycosaminoglycans may be a central event in the development of the neurofibrillary pathology of Alzheimer's disease. In the present study we show that the degree of sulfation of glycosaminoglycans is of crucial importance for their ability to induce tau filament formation, to prevent tau from binding to microtubules, and to promote microtubule disassembly. Of those tested, dextran sulfate, pentosan polysulfate, and heparin are the most sulfated, followed by dermatan sulfate, heparan sulfate, chondroitin sulfate, and keratan sulfate. Hyaluronic acid and dextran are not sulfated (see Table I for degrees of sulfation). We also show that the phosphorylation of tau by MAP kinase, NCLK, and GSK3β is markedly stimulated in the presence of heparin, at heparin concentrations lower than those required for tau filament formation. Phosphorylation of tau by MAP kinase, NCLK, and GSK3β is also stimulated by heparan sulfate, dextran sulfate, and pentosan polysulfate, but not by dextran and hyaluronic acid, with the magnitude of stimulation of tau phosphorylation being proportional to the degree of glycosaminoglycan sulfation. Tau phosphorylation by MAP kinase, NCLK, and GSK3 is also stimulated in the presence of nucleic acids and tubulin. Nucleic acids had little effect on the binding of tau to microtubules. However, incubation of tau with tRNA led to the formation of filaments, in confirmation of a recent report (33Kampers T. Friedhoff P. Biernat J. Mandelkow E.M. Mandelkow E. FEBS Lett. 1996; 399: 344-349Crossref PubMed Scopus (402) Google Scholar).Table IDegrees of sulfation of glycosaminoglycansSulfates/disaccharide unitDextran sulfate4–6Pentosan polysulfate4–6Heparin1.6–3Dermatan sulfate1–3Heparan sulfate0.4–2Keratan sulfate0.9–1.8Chondroitin sulfate0.1–1.3Hyaluronic acid0Dextran0 Open table in a new tab These results raise the possibility that an interaction between tau protein and negatively charged polymers with a sugar backbone, as found in sulfated glycosaminoglycans and nucleic acids, results in a conformational change in tau that induces polymerization of tau molecules via the microtubule-binding repeats of individual tau molecules, resulting in the formation of filaments like those present in Alzheimer's disease and other neurodegenerative disorders. The 3-repeat 381-amino acid (htau37) and the 4-repeat 441-amino acid (htau40) isoforms of human tau (34Goedert M. Spillantini M.G. Jakes R. Rutherford D. Crowther R.A. Neuron. 1989; 3: 519-526Abstract Full Text PDF PubMed Scopus (1779) Google Scholar) were expressed in Escherichia coli and purified as described (28Goedert M. Jakes R. Spillantini M.G. Hasegawa M. Smith M.J. Crowther R.A. Nature. 1996; 383: 550-553Crossref PubMed Scopus (836) Google Scholar). The tau mutants htau24S262A, htau24S356A, and htau24S262AS356A have been described previously (9Seubert P. Mawal-Dewan M. Barbour R. Jakes R. Goedert M. Johnson G.V.W. Litersky J.M. Schenk D. Lieberburg I. Trojanowski J.Q. Lee V.M.-Y. J. Biol. Chem. 1995; 270: 18917-18922Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar, 24Litersky J.M. Johnson G.V.W. Jakes R. Goedert M. Lee M. Seubert P. Biochem. J. 1996; 316: 655-660Crossref PubMed Scopus (120) Google Scholar). Activated recombinant p42 MAP kinase was prepared as described (14Goedert M. Cohen E.S. Jakes R. Cohen P. FEBS Lett. 1992; 312: 95-99Crossref PubMed Scopus (258) Google Scholar). GSK3β was purified from rabbit skeletal muscle, as described (35Sutherland C. Leighton I.A. Cohen P. Biochem. J. 1994; 296: 15-19Crossref Scopus (740) Google Scholar). Active NCLK was reconstituted from recombinant cyclin-dependent protein kinase-5 and a recombinant fragment of the brain-specific activator p35 (36Qi Z. Huang Q.-Q. Lee K.-Y. Lew J. Wang J.H. J. Biol. Chem. 1995; 270: 10847-10854Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Antiserum 134 recognizes the carboxyl terminus of tau in a phosphorylation-independent manner (34Goedert M. Spillantini M.G. Jakes R. Rutherford D. Crowther R.A. Neuron. 1989; 3: 519-526Abstract Full Text PDF PubMed Scopus (1779) Google Scholar). AT8 is a phosphorylation-dependent monoclonal antibody which recognizes tau protein phosphorylated at Ser-202 and Thr-205 (8Goedert M. Jakes R. Vanmechelen E. Neurosci. Lett. 1995; 192: 209-212Crossref PubMed Scopus (46) Google Scholar). The monoclonal antibody 12E8 recognizes tau protein phosphorylated at Ser-262 and/or Ser-356 (9Seubert P. Mawal-Dewan M. Barbour R. Jakes R. Goedert M. Johnson G.V.W. Litersky J.M. Schenk D. Lieberburg I. Trojanowski J.Q. Lee V.M.-Y. J. Biol. Chem. 1995; 270: 18917-18922Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar). Heparin (from bovine intestinal mucosa), dextran, dextran sulfate, pentosan polysulfate, and poly-l-glutamic acid were purchased from Sigma. Heparan sulfate (from bovine kidney), chondroitin-4-sulfate (from bovine trachea), dermatan sulfate (from porcine skin), keratan sulfate (from bovine cornea), and hyaluronic acid (from human umbilical cord) were obtained from Fluka Chemie AG. Tubulin was purchased from Cytoskeleton Inc. Double-stranded DNA was obtained from CLONTECHand tRNA from Boehringer Mannheim. Oligonucleotides (30–60-mers) were made on an Applied Biosystems DNA synthesizer. Phosphorylation assays (0.050 ml) were carried out at 30 °C and comprised 25 mm Tris-HCl, pH 7.4, 0.1 mm EGTA, 0.1 mm sodium orthovanadate, 2.5 μm PKI (a specific inhibitor of cyclic AMP-dependent protein kinase), protease inhibitors (0.5 mm phenylmethylsulfonyl fluoride, 5 μg/ml aprotinin, 5 μg/ml leupeptin, and 0.5 μg/ml pepstatin), tau protein (4 μm), 10 mm magnesium acetate, 2 mm [γ-32P]ATP (approximately 106 cpm/nmol), and 1 unit/ml activated p42 MAP kinase, 1 unit/ml GSK3β, or 5 units/ml recombinant reconstituted NCLK. Reactions were initiated with ATP and aliquots were removed at various times ranging from 10 min to 24 h and used for SDS-polyacrylamide gel electrophoresis and immunoblotting. Immunoblots were performed as described (15Goedert M. Hasegawa M. Jakes R. Lawler S. Cuenda A. Cohen P. FEBS Lett. 1997; 409: 57-62Crossref PubMed Scopus (254) Google Scholar). Alternatively, incorporation of 32P radioactivity was measured after adsorption to Whatman P-81 paper, as described (14Goedert M. Cohen E.S. Jakes R. Cohen P. FEBS Lett. 1992; 312: 95-99Crossref PubMed Scopus (258) Google Scholar). Glycosaminoglycans and nucleic acids were included in the assays at 50 μg/ml and tubulin at 20 μm. For microtubule binding, recombinant htau40 (4 μm, 0.18 mg/ml) was incubated with different concentrations of glycosaminoglycans and nucleic acids (10, 50, 100, 500, and 1,000 μg/ml) in assembly buffer (80 mmPIPES, 1 mm MgCl2, 1 mm EGTA, 1 mm dithiothreitol, 1 mm GTP, pH 6.8) for 10 min at 37 °C, then added to 10 μm taxol-stabilized microtubules and incubated for a further 20 min. Following ultracentrifugation, aliquots of supernatants (free tau) and pellets (microtubule-bound tau) were subjected to SDS-polyacrylamide gel electrophoresis. Protein concentrations were estimated by scanning the gels with a Molecular Dynamics computing densitometer (Model 300 A), and were expressed as percentage of tau bound to microtubules in the absence of glycosaminoglycans and nucleic acids (taken as 100%). For microtubule assembly, recombinant htau40 (2 μm) was incubated with tubulin (10 μm) in assembly buffer at 37 °C. After 5 min, glycosaminoglycans and nucleic acids (100 μg/ml) were added and incubated for a further 5 min. Polymerization and depolymerization of microtubules were monitored by measuring the turbidity at 350 nm. Purified recombinant htau37 (40 μm) or htau40 (40 μm) was incubated with various concentrations of glycosaminoglycans and nucleic acids (ranging from 5 to 20 μm) in 25 μl of 30 mm MOPS, 1 mm 4-(2-aminoethyl)benzenesulfonylfluoride (Calbiochem), pH 7.4, at 37 °C for 48 h, as described (28Goedert M. Jakes R. Spillantini M.G. Hasegawa M. Smith M.J. Crowther R.A. Nature. 1996; 383: 550-553Crossref PubMed Scopus (836) Google Scholar). In some experiments various concentrations of CaCl2, MgCl2, ZnCl2, and AlCl3 were added. Control experiments consisted of using tau alone, glycosaminoglycans alone, tau plus di- and trivalent cations, and glycosaminoglycans plus di- and trivalent cations. Samples were examined by electron microscopy, as described (28Goedert M. Jakes R. Spillantini M.G. Hasegawa M. Smith M.J. Crowther R.A. Nature. 1996; 383: 550-553Crossref PubMed Scopus (836) Google Scholar). Recombinant htau40 was incubated for various times (ranging from 10 min to 24 h) with 5 units/ml recombinant reconstituted NCLK, in the presence or absence of 50 μg/ml heparin. After 24 h tau incorporated 3.8 mol of phosphate/mol of protein in the absence of heparin and 11.4 mol of phosphate/mol of protein in the presence of heparin (Fig. 1). The stimulation of tau phosphorylation by heparin was apparent throughout the incubation period and was particularly evident at early time points (Fig. 1). A dose-response curve showed that the effect was maximal at 30 μg/ml heparin. Phosphorylation of tau by NCLK in the presence of heparin produced the epitopes of phosphorylation-dependent anti-tau antibodies which recognize (S/T)P sites in tau, as shown in Fig.2 for antibody AT8 which recognizes tau phosphorylated at Ser-202 and Thr-205 (8Goedert M. Jakes R. Vanmechelen E. Neurosci. Lett. 1995; 192: 209-212Crossref PubMed Scopus (46) Google Scholar). Moreover, tau was immunoreactive for antibody 12E8 which recognizes the phosphorylated non-(S/T)P sites Ser-262 and/or Ser-356 (9Seubert P. Mawal-Dewan M. Barbour R. Jakes R. Goedert M. Johnson G.V.W. Litersky J.M. Schenk D. Lieberburg I. Trojanowski J.Q. Lee V.M.-Y. J. Biol. Chem. 1995; 270: 18917-18922Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar) (Fig. 2). Phosphorylation of htau24S262A, htau24S356A, and htau24S262AS356A by NCLK plus heparin showed that Ser-262, but not Ser-356 was phosphorylated (Fig. 2). The effects of glycosaminoglycans (50 μg/ml) other than heparin on tau phosphorylation by NCLK were investigated after 18 h of incubation (Fig. 3). Dextran sulfate produced a large effect, resulting in an approximately 3-fold stimulation of tau phosphorylation. Heparan sulfate and pentosan polysulfate also produced a large effect, with smaller effects for dermatan sulfate, chondroitin sulfate, and keratan sulfate. Hyaluronic acid, dextran, and poly-l-glutamic acid were without a significant effect. Addition of 50 μg/ml nucleic acids also stimulated tau phosphorylation by NCLK, with tRNA producing a larger effect than DNA (Fig. 3). Incubation of tau with NCLK in the presence of 20 μm tubulin led to an approximately 50% stimulation of tau phosphorylation (Fig. 3).Figure 2Phosphorylation of wild-type and mutated recombinant htau24 (383-amino acid isoform of human tau) with 5 unit/ml recombinant reconstituted NCLK + 50 μg/ml heparin. Immunoblots were stained with anti-tau serum 134 and phosphorylation-dependent monoclonal anti-tau antibodies AT8 and 12E8. Lanes: 1, htau24 + heparin; 2, htau24 + heparin + NCLK; 3, htau24S262A + heparin;4, htau24S262A + heparin + NCLK; 5, htau24S356A + heparin; 6, htau24S356A + heparin + NCLK; 7, htau24S262AS356A + heparin; 8, htau24S262AS356A + heparin + NCLK.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Stimulation of tau phosphorylation by NCLK, GSK3β, and MAP kinase in the presence of glycosaminoglycans, nucleic acids, and tubulin. Recombinant htau40 (441-amino acid isoform of human tau) was incubated for 18 h with protein kinase (kinase) or protein kinase + 50 μg/ml heparin (H), heparan sulfate (HS), chondroitin 4-sulfate (CS), dermatan sulfate (DS), keratan sulfate (KS), hyaluronic acid (HA), pentosan polysulfate (PPS), dextran sulfate (Dex S), dextran (Dex), poly-l-glutamic acid (PGA), DNA, RNA, or 20 μm tubulin (T). The results are expressed as means ± S.E. (n = 2).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Recombinant htau40 was incubated for 18 h with 1 unit/ml GSK3β purified from skeletal muscle or 1 units/ml activated recombinant p42 MAP kinase, in the presence or absence of 50 μg/ml glycosaminoglycans, nucleic acids, and 20 μmtubulin. As shown before, phosphorylation of tau by GSK3β was strongly stimulated by heparin. Dextran sulfate had a similar effect, with an almost 5-fold stimulation of phosphorylation (Fig. 3). Addition of heparan sulfate, chondroitin sulfate, pentosan polysulfate, and dermatan sulfate resulted in a 2.0–3.0-fold stimulation of tau phosphorylation by GSK3β, whereas keratan sulfate, hyaluronic acid, dextran, and poly-l-glutamic acid were without a significant effect. RNA and DNA produced a 2.5–3.0-fold stimulation of tau phosphorylation by GSK3β; an effect of similar magnitude was obtained upon addition of tubulin (Fig. 3). Phosphorylation of htau40 by MAP kinase was stimulated 1.5–3.0-fold by heparin, heparan sulfate, pentosan polysulfate, dextran sulfate, and RNA (Fig. 3). Chondroitin sulfate, dermatan sulfate, keratan sulfate, hyaluronic acid, dextran, poly-l-glutamic acid, and DNA were without a significant effect. Incubation of tau with GSK3β or MAP kinase in the presence of 20 μm tubulin led to a 3-fold stimulation of tau phosphorylation (Fig. 3). Recombinant htau40 was incubated with different concentrations (50, 100, 250, 500, and 1,000 μg/ml) of glycosaminoglycans or nucleic acids and added to taxol-stabilized microtubules, followed by ultracentrifugation to separate unbound tau from microtubule-bound tau. The addition of heparin, dextran sulfate, and pentosan polysulfate resulted in a dose-dependent inability of tau to bind to microtubules, with IC50 values of approximately 100 μg/ml (Fig. 4). Heparan sulfate and dermatan sulfate produced a smaller, but significant, reduction which amounted to 40% at 1 mg/ml (Fig. 4). Only small reductions in the ability of tau to bind to microtubules were observed upon addition of RNA and DNA. Hyaluronic acid, chondroitin sulfate, keratan sulfate, and poly-l-glutamic acid had no significant effect, even at high concentrations (Fig. 4). Recombinant htau40 was incubated with tubulin and microtubule assembly monitored by an increase in turbidity. After 5 min, when assembly was maximal, glycosaminoglycans or nucleic acids (100 μg/ml) were added and microtubule disassembly monitored for a further 5 min by a decrease in turbidity. Addition of heparin, pentosan polysulfate, dextran sulfate, and DNA caused rapid and complete microtubule disassembly (Fig. 5). Heparan sulfate and dermatan sulfate had an intermediate effect (Fig. 5). RNA and poly-l-glutamic acid produced only a small effect on microtubule disassembly, whereas addition of hyaluronic acid, chondroitin sulfate, keratan sulfate, and dextran had no significant effect (Fig. 5). Incubation of the three repeat-containing tau isoform htau37 with glycosaminoglycans led to bulk assembly into twisted filaments with a morphology similar to the PHFs from Alzheimer's disease brain (Table II, Figs.6 and 7). As observed before (28Goedert M. Jakes R. Spillantini M.G. Hasegawa M. Smith M.J. Crowther R.A. Nature. 1996; 383: 550-553Crossref PubMed Scopus (836) Google Scholar, 29Goedert M. Spillantini M.G. Hasegawa M. Jakes R. Crowther R.A. Klug A. Cold Spring Harbor Symp. Quant. Biol. 1996; 61: 565-573Crossref PubMed Google Scholar), incubation of four repeat-containing tau isoforms with glycosaminoglycans gave straight filaments with a morphology similar to the SFs from Alzheimer's disease brain (data not shown). Incubation of htau37 with heparin of molecular masses ranging from 3 to 30 kDa gave large numbers of twisted filaments (Fig. 6,A and B). A second class of filament appearing as thinner, wavy structures was also observed (shown in Fig.6 C). These may correspond to half-twisted filaments, as they are sometimes seen extending from the ends of twisted filaments. This would imply that the filaments formed in vitro are two-stranded, like Alzheimer filaments (37Crowther R.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2288-2292Crossref PubMed Scopus (269) Google Scholar). The relative numbers of each type of filament were strongly dependent on the tau:heparin ratios, with a 2-fold change in the ratios being sufficient to switch from a predominance of half-filaments to a preponderance of twisted PHF-like filaments. Filaments with very similar morphologies were observed, when heparan sulfate was used instead of heparin (Fig.6 C). However, the total number of filaments was always higher with heparin. Addition of 1–10 μmZnCl2 significantly increased the number of twisted filaments formed following addition of heparan sulfate to htau37 (Fig.6 D). Addition of 10 mm MgCl2 led to a small increase in the number of twisted filaments, whereas CaCl2 and AlCl3 were without effect. Chondroitin sulfate and dermatan sulfate induced the assembly of tau into filaments with a morphology similar to those formed after addition of heparin or heparan sulfate (Fig. 7, A and B). However, the number of filaments was small relative to that observed after addition of heparin. Keratan sulfate, hyaluronic acid, dextran, and poly-l-glutamine failed to induce filament formation (Table II). This contrasted with the results obtained using the highly sulfated synthetic glycosaminoglycans dextran sulfate and pentosan polysulfate which induced the formation of very large numbers of short twisted tau filaments (Fig. 7, C and D, TableII). In contrast to the filaments obtained with naturally occurring sulfated glycosaminoglycans, these filaments were much shorter, consisting mostly of pieces less than one turn. We also examined the effects of tRNA and both single-stranded and double-stranded DNA on tau filament format

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