Functional Characterization of FTDP-17 tau Gene Mutations through Their Effects on Xenopus Oocyte Maturation

Artigo Acesso aberto Revisado por pares

Functional Characterization of FTDP-17 tau Gene Mutations through Their Effects on Xenopus Oocyte Maturation

2002; Elsevier BV; Volume: 277; Issue: 11 Linguagem: Inglês

10.1074/jbc.m107716200

ISSN

1083-351X

Autores

Patrice Delobel, Stéphane Flament, Malika Hamdane, Ross Jakes, Arlette Rousseau, André Delacourte, Jean‐Pierre Vilain, Michel Goedert, Luc Buée,

Tópico(s)

Prion Diseases and Protein Misfolding

Resumo

tau gene mutations cause frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). Here we have used Xenopus oocyte maturation as an indicator of microtubule function. We show that wild-type four-repeat Tau protein inhibits maturation in a concentration-dependent manner, whereas three-repeat Tau has no effect. Of the seven four-repeat Tau proteins with FTDP-17 mutations tested, five (G272V, ΔK280, P301L, P301S, and V337M) failed to interfere significantly with oocyte maturation, demonstrating a greatly reduced ability to interact with microtubules. One mutant protein (R406W) almost behaved like wild-type Tau, and one (S305N) inhibited maturation more strongly than wild-type Tau. With the exception of R406W, wild-type Tau and all the mutants studied were similarly phosphorylated during the Xenopus oocyte maturation, and this was independent of their effects on this process. Data obtained with R406W and S305N may be related to charge changes (phosphorylation and basic amino acids). Our results demonstrate variable effects of FTDP-17 mutations on microtubules in an intact cell situation. Those findings establish Xenopus oocyte maturation as a system allowing the study of the functional effects oftau gene mutations in a quantitative manner. tau gene mutations cause frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). Here we have used Xenopus oocyte maturation as an indicator of microtubule function. We show that wild-type four-repeat Tau protein inhibits maturation in a concentration-dependent manner, whereas three-repeat Tau has no effect. Of the seven four-repeat Tau proteins with FTDP-17 mutations tested, five (G272V, ΔK280, P301L, P301S, and V337M) failed to interfere significantly with oocyte maturation, demonstrating a greatly reduced ability to interact with microtubules. One mutant protein (R406W) almost behaved like wild-type Tau, and one (S305N) inhibited maturation more strongly than wild-type Tau. With the exception of R406W, wild-type Tau and all the mutants studied were similarly phosphorylated during the Xenopus oocyte maturation, and this was independent of their effects on this process. Data obtained with R406W and S305N may be related to charge changes (phosphorylation and basic amino acids). Our results demonstrate variable effects of FTDP-17 mutations on microtubules in an intact cell situation. Those findings establish Xenopus oocyte maturation as a system allowing the study of the functional effects oftau gene mutations in a quantitative manner. three-repeat four-repeat frontotemporal dementia and parkinsonism linked to chromosome 17 germinal vesicle breakdown 4-morpholinepropanesulfonic acid wild-type A variety of sporadic and familial neurodegenerative disorders, characterized clinically by dementia and/or motor dysfunction, demonstrate intracellular accumulations of filamentous material composed of the microtubule-associated protein Tau (1Buée L. Bussière T. Buée-Scherrer V. Delacourte A. Hof P.R. Brain Res. Rev. 2000; 33: 95-130Crossref PubMed Scopus (1522) Google Scholar, 2Lee V.M.-Y. Goedert M. Trojanowski J.Q. Annu. Rev. Neurosci. 2001; 24: 1121-1159Crossref PubMed Scopus (2098) Google Scholar). Six Tau isoforms are produced in the adult human brain by alternative mRNA splicing from a single gene (3Goedert M. Spillantini M.G. Jakes R. Rutherford D. Crowther R.A. Neuron. 1989; 3: 519-526Abstract Full Text PDF PubMed Scopus (1816) Google Scholar). They differ from each other by the presence or absence of 29- or 58-amino acid inserts located in the amino-terminal half and an additional 31-amino acid repeat located in the carboxyl-terminal half. Inclusion of the latter, which is encoded by exon 10, gives rise to the three isoforms with four repeats each; the other three isoforms have three repeats each. Similar levels of three-repeat (3R)1 and four-repeat (4R) Tau isoforms are found in normal cerebral cortex (4Goedert M. Jakes R. EMBO J. 1990; 9: 4225-4230Crossref PubMed Scopus (674) Google Scholar). These repeats constitute the microtubule-binding domains of Tau. They are made of imperfect 18-amino acids repeats separated by 13 or 14 amino acid inter-repeat (IR) regions (5Gustke N. Trinczek B. Biernat J. Mandelkow E.M. Mandelkow E. Biochemistry. 1994; 33: 9511-9522Crossref PubMed Scopus (513) Google Scholar, 6Panda D. Goode B.L. Feinstein S.C. Wilson L. Biochemistry. 1995; 34: 11117-11127Crossref PubMed Scopus (144) Google Scholar). Interestingly peptides corresponding to the first repeat (R1) and the first inter-repeat region (R1-R2 IR) alone are sufficient to suppress microtubule dynamics in a manner that is qualitatively similar to full-length Tau (6Panda D. Goode B.L. Feinstein S.C. Wilson L. Biochemistry. 1995; 34: 11117-11127Crossref PubMed Scopus (144) Google Scholar). Microtubule assembly also depends partially upon the phosphorylation state since phosphorylated Tau proteins are less effective than nonphosphorylated Tau proteins at promoting microtubule polymerization (for reviews, see Refs. 1Buée L. Bussière T. Buée-Scherrer V. Delacourte A. Hof P.R. Brain Res. Rev. 2000; 33: 95-130Crossref PubMed Scopus (1522) Google Scholar and 2Lee V.M.-Y. Goedert M. Trojanowski J.Q. Annu. Rev. Neurosci. 2001; 24: 1121-1159Crossref PubMed Scopus (2098) Google Scholar). In Alzheimer's disease, Pick's disease, progressive supranuclear palsy, and corticobasal degeneration, abnormally phosphorylated Tau proteins aggregate into filaments. The discovery of tau gene mutations in familial frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) has shown that dysfunction of Tau protein can cause neurodegeneration and dementia (7Poorkaj P. Bird T.D. Wijsman E. Nemens E. Garruto M. Schellenberg G.D. Ann. Neurol. 1998; 43: 815-825Crossref PubMed Scopus (1214) Google Scholar, 8Hutton M. Lendon C.L. Rizzu P. Baker M. Froelich S. Houlden H. Pickering-Brown S. Chakraverty S. Isaacs A. Grover A. Hackett J. Adamson J. Lincoln S. Dickson D. Davies P. Petersen R.C. Stevens M. Graaff E.D. Wauters E. Van Baren J. Hillebrand M. Joosse M. Kwon J.M. Nowotny P. Che L.K. Norton J. Morris J.C. Reed L.A. Trojanowski J.Q. Basun H. Lannfelt L. Neystat M. Fahn S. Dark F. Tannenberg T. Dodd P.R. Hayward N. Kwok D. Neary D. Owen F. Oostra B.A. Hardy J. Goate A. Van Swieten J. Mann D. Lynch T. Heutink P. Nature. 1998; 393: 702-705Crossref PubMed Scopus (2879) Google Scholar, 9Spillantini M.G. Murrell J.R. Goedert M. Farlow M.R. Klug A. Ghetti B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7737-7741Crossref PubMed Scopus (1298) Google Scholar); this has renewed interest in the mechanisms underlying the Tau pathology in Alzheimer's disease and other tauopathies. Knowntau mutations are either intronic mutations located close to the splice-donor site of the intron following exon 10 or missense, deletion, or silent mutations in the coding region (1Buée L. Bussière T. Buée-Scherrer V. Delacourte A. Hof P.R. Brain Res. Rev. 2000; 33: 95-130Crossref PubMed Scopus (1522) Google Scholar, 2Lee V.M.-Y. Goedert M. Trojanowski J.Q. Annu. Rev. Neurosci. 2001; 24: 1121-1159Crossref PubMed Scopus (2098) Google Scholar, 7Poorkaj P. Bird T.D. Wijsman E. Nemens E. Garruto M. Schellenberg G.D. Ann. Neurol. 1998; 43: 815-825Crossref PubMed Scopus (1214) Google Scholar, 8Hutton M. Lendon C.L. Rizzu P. Baker M. Froelich S. Houlden H. Pickering-Brown S. Chakraverty S. Isaacs A. Grover A. Hackett J. Adamson J. Lincoln S. Dickson D. Davies P. Petersen R.C. Stevens M. Graaff E.D. Wauters E. Van Baren J. Hillebrand M. Joosse M. Kwon J.M. Nowotny P. Che L.K. Norton J. Morris J.C. Reed L.A. Trojanowski J.Q. Basun H. Lannfelt L. Neystat M. Fahn S. Dark F. Tannenberg T. Dodd P.R. Hayward N. Kwok D. Neary D. Owen F. Oostra B.A. Hardy J. Goate A. Van Swieten J. Mann D. Lynch T. Heutink P. Nature. 1998; 393: 702-705Crossref PubMed Scopus (2879) Google Scholar, 9Spillantini M.G. Murrell J.R. Goedert M. Farlow M.R. Klug A. Ghetti B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7737-7741Crossref PubMed Scopus (1298) Google Scholar).At present, over 20 different coding region and intronic taumutations are known in FTDP-17. Depending on their functional effects, these mutations can be divided into two largely nonoverlapping groups, namely those that have their primary effect at the RNA level and those that have their primary effect at the protein level (1Buée L. Bussière T. Buée-Scherrer V. Delacourte A. Hof P.R. Brain Res. Rev. 2000; 33: 95-130Crossref PubMed Scopus (1522) Google Scholar, 2Lee V.M.-Y. Goedert M. Trojanowski J.Q. Annu. Rev. Neurosci. 2001; 24: 1121-1159Crossref PubMed Scopus (2098) Google Scholar, 7Poorkaj P. Bird T.D. Wijsman E. Nemens E. Garruto M. Schellenberg G.D. Ann. Neurol. 1998; 43: 815-825Crossref PubMed Scopus (1214) Google Scholar, 8Hutton M. Lendon C.L. Rizzu P. Baker M. Froelich S. Houlden H. Pickering-Brown S. Chakraverty S. Isaacs A. Grover A. Hackett J. Adamson J. Lincoln S. Dickson D. Davies P. Petersen R.C. Stevens M. Graaff E.D. Wauters E. Van Baren J. Hillebrand M. Joosse M. Kwon J.M. Nowotny P. Che L.K. Norton J. Morris J.C. Reed L.A. Trojanowski J.Q. Basun H. Lannfelt L. Neystat M. Fahn S. Dark F. Tannenberg T. Dodd P.R. Hayward N. Kwok D. Neary D. Owen F. Oostra B.A. Hardy J. Goate A. Van Swieten J. Mann D. Lynch T. Heutink P. Nature. 1998; 393: 702-705Crossref PubMed Scopus (2879) Google Scholar, 9Spillantini M.G. Murrell J.R. Goedert M. Farlow M.R. Klug A. Ghetti B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7737-7741Crossref PubMed Scopus (1298) Google Scholar). Mutations that act at the RNA level affect the alternative mRNA splicing of exon 10 of the tau gene, leading to a change in the ratio of 3R to 4R Tau isoforms and resulting in an overproduction of 4R Tau. This is the case of the intronic mutations located close to the 5′-splice site of the intron following exon 10 and of some mutations in exon 10 itself that influence exon 10 splicing enhancer and silencer sequences (1Buée L. Bussière T. Buée-Scherrer V. Delacourte A. Hof P.R. Brain Res. Rev. 2000; 33: 95-130Crossref PubMed Scopus (1522) Google Scholar, 2Lee V.M.-Y. Goedert M. Trojanowski J.Q. Annu. Rev. Neurosci. 2001; 24: 1121-1159Crossref PubMed Scopus (2098) Google Scholar, 8Hutton M. Lendon C.L. Rizzu P. Baker M. Froelich S. Houlden H. Pickering-Brown S. Chakraverty S. Isaacs A. Grover A. Hackett J. Adamson J. Lincoln S. Dickson D. Davies P. Petersen R.C. Stevens M. Graaff E.D. Wauters E. Van Baren J. Hillebrand M. Joosse M. Kwon J.M. Nowotny P. Che L.K. Norton J. Morris J.C. Reed L.A. Trojanowski J.Q. Basun H. Lannfelt L. Neystat M. Fahn S. Dark F. Tannenberg T. Dodd P.R. Hayward N. Kwok D. Neary D. Owen F. Oostra B.A. Hardy J. Goate A. Van Swieten J. Mann D. Lynch T. Heutink P. Nature. 1998; 393: 702-705Crossref PubMed Scopus (2879) Google Scholar, 9Spillantini M.G. Murrell J.R. Goedert M. Farlow M.R. Klug A. Ghetti B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7737-7741Crossref PubMed Scopus (1298) Google Scholar, 10Hasegawa M. Smith M.J. Iijima M. Tabira T. Goedert M. FEBS Lett. 1999; 443: 93-96Crossref PubMed Scopus (172) Google Scholar).Mutations that act at the protein level comprise missense and deletion mutations that are located in the microtubule-binding repeat region or close to it. Under in vitro conditions, these mutations have been shown to result in a reduced ability of Tau protein to interact with microtubules (10Hasegawa M. Smith M.J. Iijima M. Tabira T. Goedert M. FEBS Lett. 1999; 443: 93-96Crossref PubMed Scopus (172) Google Scholar, 11Hasegawa M. Smith M.J. Goedert M. FEBS Lett. 1998; 437: 207-210Crossref PubMed Scopus (414) Google Scholar, 12Hong M. Zhukareva V. Vogelsberg-Ragaglia V. Wszolek Z. Reed L. Miller B.I. Geschwind D.H. Bird T.D. McKeel D. Goate A. Morris J.C. Wilhelmsen K.C. Schellenberg G.D. Trojanowski J.Q. Lee V.M.-Y. Science. 1998; 282: 1914-1917Crossref PubMed Scopus (809) Google Scholar, 13Rizzu P. Van Swieten J.C. Joosse M. Hasegawa M. Stevens M. Tibben A. Niermeijer M.F. Hillebrand M. Ravid R. 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Moreover, overexpression of mutant Tau in transfected cells has given inconsistent results as far as effects on microtubule binding and stability are concerned (17Dayanandan R. Van Slegtenhorst M. Mack T.G.A. Ko L. Yen S.H. Leroy K. Brion J.P. Anderton B.H. Hutton M. Lovestone S. FEBS Lett. 1999; 446: 228-232Crossref PubMed Scopus (179) Google Scholar, 18Matsumura N. Yamazaki T. Ihara Y. Am. J. Pathol. 1999; 154: 1649-1656Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 19Vogelsberg-Ragaglia V. Bruce J. Richter-Landsberg C. Zhang B. Hong M. Trojanowski J.Q. Lee V.M.-Y. Mol. Biol. Cell. 2000; 11: 4093-4104Crossref PubMed Scopus (108) Google Scholar). Such studies are confounded by the problem that high expression of mutant Tau may override any effects that are present at more physiological levels.In view of these uncertainties, we have used a cell system, maturation of the Xenopus oocyte induced by progesterone, to study the effects of seven tau gene mutations (G272V, ΔK280, P301L, P301S, S305N, V337M, and R406W) on microtubule function. During maturation, there is a dramatic reorganization of the microtubule cytoskeleton that can be assessed indirectly by the presence of a white maturation spot at the animal pole and directly by the anchoring of the meiotic spindle in the plasma membrane. Besides being an indicator of microtubule function, this system also enables quantitative studies through microinjection of recombinant Tau proteins of known concentration.DISCUSSIONIt is important to understand the in vivo role of Tau protein to dissect mechanisms leading to neurodegeneration in Alzheimer's disease and other neurodegenerative disorders. Mutations in the tau gene have shown that dysfunction of Tau protein can cause neurodegeneration and dementia. In vitro studies demonstrated that missense and deletion mutations result in reduced function of Tau. However, in vivo effects have been more controversial. To avoid the variables inherent to transfection studies,X. laevis oocyte maturation was used to study the effects of Tau mutations on microtubule dynamics. Upon exposure to progesterone, oocytes undergo maturation, a process that involves a dramatic reorganization of microtubules, allowing for GVBD, translocation of the germinal vesicle, and anchoring of meiotic spindles at the plasma membrane of the animal pole. A known amount of Tau protein (wild type or mutant) was microinjected into the oocyte, and the biological effect on microtubules was assessed.Microinjection of wild-type 4R Tau into the oocyte perturbed the maturation process in a dose-dependent manner (from 0.6 to 4.7 μm), whereas 5 μm wild-type 3R Tau was without effect. Altogether these data suggest that there is a direct interaction between 4R Tau, but not 3R Tau, and microtubules. This is consistent with previous findings showing that four-repeat Tau has a higher affinity for microtubules than 3R Tau (4Goedert M. Jakes R. EMBO J. 1990; 9: 4225-4230Crossref PubMed Scopus (674) Google Scholar, 5Gustke N. Trinczek B. Biernat J. Mandelkow E.M. Mandelkow E. Biochemistry. 1994; 33: 9511-9522Crossref PubMed Scopus (513) Google Scholar, 6Panda D. Goode B.L. Feinstein S.C. Wilson L. Biochemistry. 1995; 34: 11117-11127Crossref PubMed Scopus (144) Google Scholar, 28Butner K.A. Kirschner M.W. J. Cell Biol. 1991; 115: 717-730Crossref PubMed Scopus (439) Google Scholar, 29Goode B.L. Denis P.E. Panda D. Radeke M.J. Miller H.P. Wilson L. Feinstein S.C. Mol. Biol. Cell. 1997; 8: 353-365Crossref PubMed Scopus (234) Google Scholar).Regarding the functional effects of Tau mutants, three groups could be distinguished. The first includes mutations G272V, ΔK280, P301L, P301S, and V337M. These mutations allowed for normal oocyte maturation as visualized by the appearance of a white spot indicating that they failed to interact correctly with microtubules. Only the microinjection of high concentrations of mutant Tau led to abnormalities such as the formation of ectopic meiotic spindles. It was previously shown that these mutations cause a decreased ability of Tau to promote microtubule assembly in vitro, although there were discrepancies regarding their relative effects (11Hasegawa M. Smith M.J. Goedert M. FEBS Lett. 1998; 437: 207-210Crossref PubMed Scopus (414) Google Scholar, 12Hong M. Zhukareva V. Vogelsberg-Ragaglia V. Wszolek Z. Reed L. Miller B.I. Geschwind D.H. Bird T.D. McKeel D. Goate A. Morris J.C. Wilhelmsen K.C. Schellenberg G.D. Trojanowski J.Q. Lee V.M.-Y. Science. 1998; 282: 1914-1917Crossref PubMed Scopus (809) Google Scholar). In the present study, mutations G272V, P301L, P301S, and V337M allowed for normal maturation with only a few changes in the organization of meiotic spindles. A higher percentage of ectopic meiotic spindles was found in oocytes microinjected with the Tau ΔK280 mutant suggesting that this deletion mutation allows for better microtubule binding than the above missense mutations. Another explanation of the maturation inhibition could be related to differences in the degree of phosphorylation of wild-type and mutant Tau isoforms during oocyte maturation. However, wild-type 3R and 4R Tau were found to be phosphorylated to the same extent as the G272V, ΔK280, P301L, P301S, and V337M mutants. Thus, differential phosphorylation cannot explain the data obtained for the first group of Tau mutants. The data show that these mutations strongly reduce the ability of Tau to interact with microtubules.Two additional mutations (R406W and S305N) were studied, and they were found to perturb oocyte maturation; they make up the second and third groups, respectively. R406W mutant Tau showed less phosphorylation than the other Tau proteins studied. A loss of about 25% of AD2 and 12E8 immunoreactivities may explain the increased ability of 4R R406W Tau to inhibit maturation when compared with wild-type protein. Phosphorylation of sequences recognized by AD2 and 12E8 decreases Tau-microtubule interactions (30Biernat J. Gustke N. Drewes G. Mandelkow E.M. Mandelkow E. Neuron. 1993; 11: 153-163Abstract Full Text PDF PubMed Scopus (642) Google Scholar, 31Utton M.A. Vandecandelaere A. Wagner U. Reynolds C.H. Gibb G.M. Miller C.C. Bayley P.M. Anderton B.H. Biochem. J. 1997; 323: 741-747Crossref PubMed Scopus (86) Google Scholar, 32Biernat J. Mandelkow E.M. Mol. Biol. Cell. 1999; 10: 727-740Crossref PubMed Scopus (110) Google Scholar, 33Schneider A. Biernat J. von Bergen M. Mandelkow E. Mandelkow E.M. Biochemistry. 1999; 38: 3549-3558Crossref PubMed Scopus (445) Google Scholar). A 50% loss of phosphorylation was observed for 3R R406W that was not sufficient to perturb oocyte maturation. This is not surprising since wild-type 3R Tau did not perturb oocyte maturation and since in vitro studies have indicated that 3R R406W Tau has a weak effect on tubulin polymerization (11Hasegawa M. Smith M.J. Goedert M. FEBS Lett. 1998; 437: 207-210Crossref PubMed Scopus (414) Google Scholar). One hypothesis can be raised to explain the effects of 4R R406W Tau. With injections of low amounts of 4R wild-type Tau, the protein is phosphorylated but does not interact sufficiently with microtubules to allow perturbation of oocyte maturation. Conversely 4R R406W mutant Tau is less phosphorylated allowing for a better binding to microtubules than wild-type Tau and thus perturbation of oocyte maturation. At the highest Tau concentration, the effect of the R406W mutation on microtubule binding may override the effect on phosphorylation. As a result, wild-type 4R Tau perturbs the maturation process more than the R406W mutant Tau. Altogether these data suggest that this mutation has a weak effect on microtubule interactions in agreement with in vitro findings (11Hasegawa M. Smith M.J. Goedert M. FEBS Lett. 1998; 437: 207-210Crossref PubMed Scopus (414) Google Scholar). They are also consistent with the fact that it causes late onset of disease when compared with other FTDP-17 mutations (for review, see Ref. 34Heutink P. Hum. Mol. Genet. 2000; 9: 979-986Crossref PubMed Scopus (99) Google Scholar). However, the R406W mutation may have other effects, including increased fibrillogenesis, increased proteolysis susceptibility, and differences in protein phosphatase 2A and glycogen synthase kinase 3 binding (35Nacharaju P. Lewis C. Easson S. Yen S. Hackett M. Hutton M. Yen S.H. FEBS Lett. 1999; 447: 195-199Crossref PubMed Scopus (235) Google Scholar, 36Yen S.H. Hutton M. DeTure M. Ko L.W. Nacharaju P. Brain Pathol. 1999; 9: 695-705Crossref PubMed Scopus (40) Google Scholar, 37Goedert M. Satumtira S. Jakes R. Smith M.J. Kamibayashi C. White C.L. Sontag E. J. Neurochem. 2000; 75: 2155-2162Crossref PubMed Scopus (87) Google Scholar, 38Connell J.W. Gibb G.M. Betts J.C. Blackstock W.P. Gallo J. Lovestone S. Hutton M. Anderton B.H. FEBS Lett. 2001; 493: 40-44Crossref PubMed Scopus (31) Google Scholar).The most pronounced inhibitory effect on oocyte maturation was observed with S305N Tau, even following microinjection of small amounts of Tau protein. This effect was independent of phosphorylation of the five epitopes studied here. The mutation changes the last amino acid of exon 10 and is one of the exonic mutations that modify the ratio of 3R and 4R Tau isoforms, resulting in the overexpression of 4R Tau (for reviews, see Refs. 1Buée L. Bussière T. Buée-Scherrer V. Delacourte A. Hof P.R. Brain Res. Rev. 2000; 33: 95-130Crossref PubMed Scopus (1522) Google Scholar and 2Lee V.M.-Y. Goedert M. Trojanowski J.Q. Annu. Rev. Neurosci. 2001; 24: 1121-1159Crossref PubMed Scopus (2098) Google Scholar). The S305N mutation improves the binding of U1 small nuclear RNA and destabilizes a stem loop structure located at the boundary between exon 10 and the following intron (39Varani L. Hasegawa M. Spillantini M.G. Smith M.J. Murrell J.R. Ghetti B. Klug A. Goedert M. Varani L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8229-8234Crossref PubMed Scopus (197) Google Scholar). This mutation leads to exon 10 being preferentially spliced and no longer subject to regulation (10Hasegawa M. Smith M.J. Iijima M. Tabira T. Goedert M. FEBS Lett. 1999; 443: 93-96Crossref PubMed Scopus (172) Google Scholar, 39Varani L. Hasegawa M. Spillantini M.G. Smith M.J. Murrell J.R. Ghetti B. Klug A. Goedert M. Varani L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8229-8234Crossref PubMed Scopus (197) Google Scholar, 40D'Souza I. Poorkaj P. Hong M. Nochlin D. Lee V.M. Bird T.D. Schellenberg G.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5598-5603Crossref PubMed Scopus (415) Google Scholar, 41Hartmann A.M. Rujesku D. Giannakouros T. Nikolakaki E. Goedert M. Mandelkow E.M. Gao Q.S. Andreadis A. Stamm S. Mol. Cell. Neurosci. 2001; 18: 80-90Crossref PubMed Scopus (87) Google Scholar). It was previously shown that S305N Tau has a slightly increased ability to promote microtubule assembly in vitro when compared with wild-type protein (10Hasegawa M. Smith M.J. Iijima M. Tabira T. Goedert M. FEBS Lett. 1999; 443: 93-96Crossref PubMed Scopus (172) Google Scholar). The present findings are consistent with these observations. However, the magnitude of the effect was greater in the Xenopusoocyte. This may be explained by phosphorylation of wild-type Tau at Ser-305 (42Drewes G. Trinczek B. Illenberger S. Biernat J. Schmitt-Ulms G. Meyer H.E. Mandelkow E.M. Mandelkow E. J. Biol. Chem. 1995; 270: 7679-7688Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar, 43Paudel H.K. J. Biol. Chem. 1997; 272: 1777-1785Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 44Reynolds C.H. Betts J.H. Blackstock W.P. Nebreda A.R. Anderton B.H. J. Neurochem. 2000; 74: 1587-1595Crossref PubMed Scopus (309) Google Scholar). The S305N mutation abolishes this phosphorylation, and it may increase the ability of the mutant to bind microtubules. In fact, at the level of microtubules, the S305N mutation behaves in an opposite manner to other missense mutations. It remains to be seen whether this contributes to the clinical and neuropathological picture.The present study indicates that in vivo Tau missense mutations either strongly reduce interactions with microtubules or increase these interactions. It also demonstrates that theXenopus oocyte is an interesting model system for following perturbations in microtubule function. A variety of sporadic and familial neurodegenerative disorders, characterized clinically by dementia and/or motor dysfunction, demonstrate intracellular accumulations of filamentous material composed of the microtubule-associated protein Tau (1Buée L. Bussière T. Buée-Scherrer V. Delacourte A. Hof P.R. Brain Res. Rev. 2000; 33: 95-130Crossref PubMed Scopus (1522) Google Scholar, 2Lee V.M.-Y. Goedert M. Trojanowski J.Q. Annu. Rev. Neurosci. 2001; 24: 1121-1159Crossref PubMed Scopus (2098) Google Scholar). Six Tau isoforms are produced in the adult human brain by alternative mRNA splicing from a single gene (3Goedert M. Spillantini M.G. Jakes R. Rutherford D. Crowther R.A. Neuron. 1989; 3: 519-526Abstract Full Text PDF PubMed Scopus (1816) Google Scholar). They differ from each other by the presence or absence of 29- or 58-amino acid inserts located in the amino-terminal half and an additional 31-amino acid repeat located in the carboxyl-terminal half. Inclusion of the latter, which is encoded by exon 10, gives rise to the three isoforms with four repeats each; the other three isoforms have three repeats each. Similar levels of three-repeat (3R)1 and four-repeat (4R) Tau isoforms are found in normal cerebral cortex (4Goedert M. Jakes R. EMBO J. 1990; 9: 4225-4230Crossref PubMed Scopus (674) Google Scholar). These repeats constitute the microtubule-binding domains of Tau. They are made of imperfect 18-amino acids repeats separated by 13 or 14 amino acid inter-repeat (IR) regions (5Gustke N. Trinczek B. Biernat J. Mandelkow E.M. Mandelkow E. Biochemistry. 1994; 33: 9511-9522Crossref PubMed Scopus (513) Google Scholar, 6Panda D. Goode B.L. Feinstein S.C. Wilson L. Biochemistry. 1995; 34: 11117-11127Crossref PubMed Scopus (144) Google Scholar). Interestingly peptides corresponding to the first repeat (R1) and the first inter-repeat region (R1-R2 IR) alone are sufficient to suppress microtubule dynamics in a manner that is qualitatively similar to full-length Tau (6Panda D. Goode B.L. Feinstein S.C. Wilson L. Biochemistry. 1995; 34: 11117-11127Crossref PubMed Scopus (144) Google Scholar). Microtubule assembly also depends partially upon the phosphorylation state since phosphorylated Tau proteins are less effective than nonphosphorylated Tau proteins at promoting microtubule polymerization (for reviews, see Refs. 1Buée L. Bussière T. Buée-Scherrer V. Delacourte A. Hof P.R. Brain Res. Rev. 2000; 33: 95-130Crossref PubMed Scopus (1522) Google Scholar and 2Lee V.M.-Y. Goedert M. Trojanowski J.Q. Annu. Rev. Neurosci. 2001; 24: 1121-1159Crossref PubMed Scopus (2098) Google Scholar). In Alzheimer's disease, Pick's disease, progressive supranuclear palsy, and corticobasal degeneration, abnormally phosphorylated Tau proteins aggregate into filaments. The discovery of tau gene mutations in familial frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) has shown that dysfunction of Tau protein can cause neurodegeneration and dementia (7Poorkaj P. Bird T.D. Wijsman E. Nemens E. Garruto M. Schellenberg G.D. Ann. Neurol. 1998; 43: 815-825Crossref PubMed Scopus (1214) Google Scholar, 8Hutton M. Lendon C.L. Rizzu P. Baker M. Froelich S. Houlden H. Pickering-Brown S. Chakraverty S. Isaacs A. Grover A. Hackett J. Adamson J. Lincoln S. Dickson D. Davies P. Petersen R.C. Stevens M. Graaff E.D. Wauters E. Van Baren J. Hillebrand M. Joosse M. Kwon J.M. Nowotny P. Che L.K. Norton J. Morris J.C. Reed L.A. Trojanowski J.Q. Basun H. Lannfelt L. Neystat M. Fahn S. Dark F. Tannenberg T. Dodd P.R. Hayward N. Kwok D. Neary D. Owen F. Oostra B.A. Hardy J. Goate A. Van Swieten J. Mann D. Lynch T. Heutink P. Nature. 1998; 393: 702-705Crossref PubMed Scopus (2879) Google Scholar, 9Spillantini M.G. Murrell J.R. Goedert M. Farlow M.R. Klug A. Ghetti B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7737-7741Crossref PubMed Scopus (1298) Google Scholar); this has renewed interest in the mechanisms underlying the Tau pathology in Alzheimer's disease and other tauopathies. Knowntau mutations are either intronic mutations located close to the splice-donor site of the intron following exon 10 or missense, deletion, or silent mutations in the coding region (1Buée L. Bussière T. Buée-Scherrer V. Delacourte A. Hof P.R. Brain Res. Rev. 2000; 33: 95-130Crossref PubMed Scopus (1522) Google Scholar, 2Lee V.M.-Y. Goedert M. Trojanowski J.Q. Annu. Rev. Neurosci. 2001; 24: 1121-1159Crossref PubMed Scopus (2098) Google Scholar, 7Poorkaj P. Bird T.D. Wijsman E. Nemens E. Garruto M. Schellenberg G.D. Ann. Neurol. 1998; 43: 815-825Crossref PubMed Scopus (1214) Google Scholar, 8Hutton M. Lendon C.L. Rizzu P. Baker M. Froelich

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Functional Characterization of FTDP-17 tau Gene Mutations through Their Effects on Xenopus Oocyte Maturation