Fibrillogenic Nuclei Composed of P301L Mutant Tau Induce Elongation of P301L Tau but Not Wild-type Tau

Frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), an autosomal, dominantly inherited neurodegenerative disorder caused by tau gene mutations, is neuropathologically characterized by intraneuronal filamentous inclusions of hyperphosphorylated tau protein. Biochemical and immunocytochemical analyses have shown that only mutant tau is deposited in patients harboring P301L missense mutation, whereas both wild-type and mutant tau are deposited in patients harboring R406W mutation (Miyasaka, T., Morishima-Kawashima, M., Ravid, R., Kamphorst, W., Nagashima, K., and Ihara, Y. (2001) J. Neuropathol. Exp. Neurol. 60, 872– 884 and Miyasaka, T., Morishima-Kawashima, M., Ravid, R., Heutink, P., van Swieten, J. C., Nagashima, K., and Ihara, Y. (2001) Am. J. Pathol. 158, 373–379). Here we have tested the nucleation ability of monomeric tau and the seeding ability of fibrillogenic nuclei obtained from bacterially expressed human tau. P301L mutant tau showed a higher nucleation ability than wild-type tau, whereas R406W mutant tau shows similar ability to wild-type tau. Surprisingly, fibrillogenic nuclei composed of P301L mutant tau enhanced the assembly of P301L mutant tau into filaments but did not promote filament formation from wild-type tau. In contrast, nuclei composed of R406W mutant tau supported filament formation from both wild-type tau and R406W mutant tau, as did nuclei composed of wild-type tau. Proteolytic analyses indicated that the substructure of nuclei composed of P301L mutant tau was different from that of nuclei composed of wild-type or R406W mutant tau. Thus, the interaction between fibrillogenic nuclei and monomeric protein appears to play an important role in the mechanism of tau filament assembly. Frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), an autosomal, dominantly inherited neurodegenerative disorder caused by tau gene mutations, is neuropathologically characterized by intraneuronal filamentous inclusions of hyperphosphorylated tau protein. Biochemical and immunocytochemical analyses have shown that only mutant tau is deposited in patients harboring P301L missense mutation, whereas both wild-type and mutant tau are deposited in patients harboring R406W mutation (Miyasaka, T., Morishima-Kawashima, M., Ravid, R., Kamphorst, W., Nagashima, K., and Ihara, Y. (2001) J. Neuropathol. Exp. Neurol. 60, 872– 884 and Miyasaka, T., Morishima-Kawashima, M., Ravid, R., Heutink, P., van Swieten, J. C., Nagashima, K., and Ihara, Y. (2001) Am. J. Pathol. 158, 373–379). Here we have tested the nucleation ability of monomeric tau and the seeding ability of fibrillogenic nuclei obtained from bacterially expressed human tau. P301L mutant tau showed a higher nucleation ability than wild-type tau, whereas R406W mutant tau shows similar ability to wild-type tau. Surprisingly, fibrillogenic nuclei composed of P301L mutant tau enhanced the assembly of P301L mutant tau into filaments but did not promote filament formation from wild-type tau. In contrast, nuclei composed of R406W mutant tau supported filament formation from both wild-type tau and R406W mutant tau, as did nuclei composed of wild-type tau. Proteolytic analyses indicated that the substructure of nuclei composed of P301L mutant tau was different from that of nuclei composed of wild-type or R406W mutant tau. Thus, the interaction between fibrillogenic nuclei and monomeric protein appears to play an important role in the mechanism of tau filament assembly. Neurofibrillary tangles, i.e. bundles of paired helical filaments and straight filaments, are one of the major neuropathological hallmarks of Alzheimer disease. They are composed of the microtubule-associated protein tau in a hyperphosphorylated state (1Lee V.M. Balin B.J. Otvos Jr., L. Trojanowski J.Q. Science. 1991; 251: 675-678Crossref PubMed Scopus (1252) Google Scholar, 2Hasegawa 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, 3Biernat 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, 4Goedert M. Jakes R. Crowther R.A. Six J. Lubke U. Vandermeeren M. Cras P. Trojanowski J.Q. Lee V.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5066-5070Crossref PubMed Scopus (412) Google Scholar, 5Goedert M. Jakes R. Crowther R.A. Cohen P. Vanmechelen E. Vandermeeren M. Cras P. Biochem. J. 1994; 301: 871-877Crossref PubMed Scopus (348) Google Scholar, 6Morishima-Kawashima M. Hasegawa M. Takio K. Suzuki M. 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Intracellular filamentous inclusions composed of tau are also characteristic of a number of other neurodegenerative diseases, including Pick disease, progressive supranuclear palsy, corticobasal degeneration, and frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) 3The abbreviations used are: FTDP-17, frontotemporal dementia with parkinsonism linked to chromosome PIPES, piperazine-1,4-bis(2-ethanesulfonic acid); DTT, dithiothreitol; GSK3β, glycogen synthase kinase 3β; Cdk5, cyclin-dependent kinase 5; HPLC, high performance liquid chromatography; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight; MS, mass spectrometry; CBB, Coomassie Brilliant Blue; WT, wild type. (10Lee V.M. Goedert M. Trojanowski J.Q. Annu. Rev. Neurosci. 2001; 24: 1121-1159Crossref PubMed Scopus (2145) Google Scholar). FTDP-17 is an autosomal, dominantly inherited neurodegenerative disorder caused by tau gene mutations. Currently, more than 30 exonic and intronic pathogenic mutations in the tau gene have been identified in more than 100 families with FTDP-17. Most of the exonic mutations reduce the physiological activity of tau to interact with microtubules and to promote their assembly (11Hasegawa M. Smith M.J. Goedert M. FEBS Lett. 1998; 437: 207-210Crossref PubMed Scopus (419) Google Scholar) and also increase the propensity of tau to aggregate into abnormal filaments (12Nacharaju P. Lewis J. Easson C. Yen S. Hackett J. Hutton M. Yen S.H. FEBS Lett. 1999; 447: 195-199Crossref PubMed Scopus (237) Google Scholar, 13Goedert M. Jakes R. Crowther R.A. FEBS Lett. 1999; 450: 306-311Crossref PubMed Scopus (221) Google Scholar, 14Barghorn S. Zheng-Fischhofer Q. Ackmann M. Biernat J. von Bergen M. Mandelkow E.M. Mandelkow E. Biochemistry. 2000; 39: 11714-11721Crossref PubMed Scopus (293) Google Scholar, 15von Bergen M. Barghorn S. Li L. Marx A. Biernat J. Mandelkow E.M. Mandelkow E. J. Biol. Chem. 2001; 276: 48165-48174Abstract Full Text Full Text PDF PubMed Scopus (438) Google Scholar). The other mutations, including intronic ones, affect the alternative splicing of exon 10 on tau mRNA, which contains a second repeat of the microtubule binding domain, resulting in disturbance of the normal ratio of tau isoforms (16Hasegawa M. Smith M.J. Iijima M. Tabira T. Goedert M. FEBS Lett. 1999; 443: 93-96Crossref PubMed Scopus (174) Google Scholar, 17D'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 (421) Google Scholar). However, it remains unclear that how these tau mutations lead to assembly of tau protein and why different tau mutations cause different tau pathologies. Among the tau gene mutations, the proline-to-leucine mutation at codon 301 (numbered according to the longest isoform) (P301L) is thought to be the most frequent mutation in FTDP-17 (18Spillantini M.G. Crowther R.A. Kamphorst W. Heutink P. van Am. J. Pathol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, T.D. Nochlin D. Poorkaj P. M. J. H. E. E. Schellenberg G.D. 1999; PubMed Scopus Google Scholar, M. P. M. I. Kamphorst W. R. M.G. Heutink P. Ann. Neurol. 1999; PubMed Scopus Google Scholar). wild-type tau and P301L mutant tau, it been shown that P301L mutant tau is deposited in the of patients with P301L mutation, was in between wild-type and P301L mutant tau T. Morishima-Kawashima M. R. Kamphorst W. K. Ihara Y. J. Neuropathol. Exp. Neurol. 2001; PubMed Scopus Google Scholar, P. M. R. A. Kamphorst W. van R. Heutink P. 2000; PubMed Scopus Google Scholar). In contrast, in the of the mutation at codon which been in an M. P. M. S. H. S. S. A. A. Hackett J. J. S. D. P. Petersen R.C. M. E. E. van J. M. M. P. J. Trojanowski J.Q. H. L. M. S. T. A. J. D. D. J. A. van J. D. T. Heutink P. 1998; PubMed Scopus Google a M. P. M. I. Kamphorst W. R. M.G. Heutink P. Ann. Neurol. 1999; PubMed Scopus Google and a Y. A. R. S. E. T. Suzuki K. S. PubMed Scopus Google of wild-type and R406W mutant tau in the of the T. Morishima-Kawashima M. R. Heutink P. van K. Ihara Y. Am. J. Pathol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). of the cause of these is important to not only the mechanism of tau assembly by tau mutations in FTDP-17 but also the tau in in the of other tau been as a that is characterized by the a P. von Bergen M. Mandelkow E.M. P. Mandelkow E. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar). the of monomeric tau to filament in a expressed tau is to into filaments in the of including as M. Jakes R. M.G. Hasegawa M. Smith M.J. Crowther R.A. 1996; PubMed Scopus Google as P. A. Mandelkow E.M. Mandelkow E. Biochemistry. 1998; PubMed Scopus Google as T. P. Biernat J. Mandelkow E.M. Mandelkow E. FEBS Lett. 1996; PubMed Scopus Google or as Am. J. Pathol. 1997; Google Scholar) and M. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the nucleation and the i.e. the monomeric protein to aggregate in a The of nuclei obtained by of tau filaments also tau into filaments P. von Bergen M. Mandelkow E.M. P. Mandelkow E. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar). is to the by as fibrillogenic tau assembly. In the we the of the accumulation of P301L mutant tau and the accumulation of wild-type and R406W mutant tau in FTDP-17 by an in of tau in the of and fibrillogenic nuclei to the nucleation and of tau filament that the interaction between fibrillogenic nuclei and monomeric protein an important role of the of tau protein wild-type and P301L mutant tau and from Ihara T. Morishima-Kawashima M. R. Kamphorst W. K. Ihara Y. J. Neuropathol. Exp. Neurol. 2001; PubMed Scopus Google Scholar). wild-type and R406W mutant tau and also from Ihara T. Morishima-Kawashima M. R. Heutink P. van K. Ihara Y. Am. J. Pathol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). was obtained from and from and of and human tau was from a human and into and The was with and and into that been with the was used to to and to in the of human tau. The of by and of and of human tau was expressed from in E. as (11Hasegawa M. Smith M.J. Goedert M. FEBS Lett. 1998; 437: 207-210Crossref PubMed Scopus (419) Google Scholar). in PIPES, DTT, by a on an at The at and the was a in The was in by was with of was by the of to and 30 at The was in and and the was The of a at was 30 protein was by protein the at In wild-type and mutant tau expressed and in by tau and at in 30 M. Jakes R. M.G. Hasegawa M. Smith M.J. Crowther R.A. 1996; PubMed Scopus Google Scholar). tau was on the of or as of of wild-type or mutant tau protein at at in 30 of assembly in 10 of 30 and at The was in 30 and with an of the on the of monomeric protein by tau at in 30 and was with as nuclei the of nuclei in the of both wild-type and mutant tau, to a of tau 30 and tau was on the of in or as of assembly and to of 30 and the was 30 The was at The was and the was in of and to of the with Coomassie Brilliant the of the tau by a S. Suzuki M. S. T. Goedert M. Hasegawa M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). was with the was used of filament of assembly to 10 with 30 of these on and with at a of on a by and and mutant tau with and in 30 10 and at M. Crowther R.A. Jakes R. Goedert M. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). by 10 a at the by and used filament Proteolytic of and tau or with at and The by the of by 10 of of of or of fibrillogenic nuclei in of 30 and at The was in 30 by with an and in The on an 30 by high performance liquid H. H. T. Hasegawa M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) and by a T. T. Hasegawa M. Biochemistry. PubMed Scopus Google Scholar) and matrix-assisted laser desorption ionization-time of a mass H. Hasegawa M. A. E. J. Takio K. T. Biol. PubMed Scopus Google Scholar). of and R406W the nucleation of and R406W tau we the of in the or of tau assembly by the tau as S. Suzuki M. S. T. Goedert M. Hasegawa M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). tau was with or R406W mutant tau in the of not of tau with in an increase of tau in with the that in P301L mutant tau wild-type and R406W mutant tau did not aggregate to P301L mutant tau to from in with (12Nacharaju P. Lewis J. Easson C. Yen S. Hackett J. Hutton M. Yen S.H. FEBS Lett. 1999; 447: 195-199Crossref PubMed Scopus (237) Google Scholar, 13Goedert M. Jakes R. Crowther R.A. FEBS Lett. 1999; 450: 306-311Crossref PubMed Scopus (221) Google Scholar). The of or R406W mutant tau in the of straight was in their that P301L mutant tau into nuclei more than wild-type or R406W mutant of tau filament formation in the of or by and P301L and or R406W and mutant tau was with 100 and or and tau filaments of by by of or P301L of tau filaments assembly of tau by formation or of nuclei composed of wild-type or P301L mutant tau as by tau filaments and with wild-type or P301L mutant tau. increase of tau was in the of and composed of wild-type tau assembly of both wild-type and P301L mutant tau and In contrast, composed of P301L mutant tau to a of P301L mutant tau, wild-type tau only and The filament formation was by by nuclei showed straight which was similar to that in the of and was in the of filaments between of and monomeric tau. that wild-type nuclei have the ability to both wild-type and P301L mutant P301L mutant tau promote the assembly only of P301L mutations. by of or R406W tested the on wild-type and R406W mutant tau. In the of increase of tau was in the of wild-type or R406W tau to and wild-type a increase of tau was in both wild-type and R406W mutant tau and in between the similar obtained composed of R406W mutant tau The of R406W mutant tau in an increase of wild-type tau, and the was the as that of R406W mutant tau and The filaments by nuclei showed similar to that in the of and was in the and that both wild-type and R406W mutant tau promote of wild-type tau as as that of R406W mutant tau. of and in the of is an i.e. patients both wild-type and mutant gene the disease. In patients both wild-type and mutant are expressed in the filament formation as also be in an more the physiological we a of wild-type and P301L mutant tau with or fibrillogenic The of tau was by tau with Coomassie Brilliant and the of wild-type or mutant tau in the was by of a to the of wild-type tau and a to P301L mutant tau or a to the of wild-type tau and a to R406W mutant tau P301L mutant tau to the of wild-type tau and P301L mutant tau, a of tau was with whereas only a increase of the tau was wild-type tau and which tau into filaments with the the tau was with and The tau with P301L mutant was with but only with that more than of the tau with P301L mutant tau was the P301L mutant and tau with wild-type tau was with these and wild-type and R406W mutant tau. tau the of tau as was with R406W tau and The of wild-type tau in the seeding by wild-type was similar to that of R406W mutant tau and R406W tau also to of both wild-type and R406W mutant tau, and was in between the and of of or that the be in of in the of of Alzheimer paired helical filaments with the and the of we thought it be to the in the of the by the we composed of or R406W tau with and the by tau was 10 with and tau was In contrast, the with to The of the composed of wild-type tau major of and and a similar was in the of the composed of R406W mutant tau major and one in the of P301L tau the of these than of the from wild-type tau the of the the by and to protein The of wild-type tau i.e. and of their which to and (numbered according to the longest The R406W mutant tau showed similar to of the wild-type tau whereas the P301L mutant tau and which to and in of their The analyses showed that the of tau composed of In the analyses of the tau and R406W mutant tau mass from to in of the P301L mutant tau mass from to and it was that the of wild-type and R406W mutant tau composed of and whereas the of P301L mutant tau composed of and protein that the of P301L mutant tau is from that of wild-type tau whereas R406W mutant tau have a similar to wild-type tau of of or by and tau composed of tau, wild-type and mutant tau by and The of tau and of tau by with tau to or The of P301L and R406W mutant tau similar to that of wild-type tau or R406W mutant tau the tau by and as by The of the composed of wild-type tau showed an and a similar was in the of R406W tau the other the of P301L mutant tau an The of wild-type or mutant tau by and by and protein of the of tau a in analyses that the of or R406W mutant tau a of the of mass from to from R406W mutant tau similar to from wild-type tau from to the from the of P301L mutant tau mass from to than of wild-type tau The analyses of the of wild-type or R406W mutant tau i.e. as a major and and as which to and (numbered according to the longest The of P301L mutant tau in a different of their i.e. as a major and and as which to and and it was that the of wild-type and R406W mutant tau composed of and whereas the of P301L mutant tau composed of and that in the state the of P301L mutant tau have a different from that of wild-type tau whereas the of R406W mutant tau was similar to that of wild-type tau The of tau gene mutations in FTDP-17 the that tau protein M. P. M. S. H. S. S. A. A. Hackett J. J. S. D. P. Petersen R.C. M. E. E. van J. M. M. P. J. Trojanowski J.Q. H. L. M. S. T. A. J. D. D. J. A. van J. D. T. Heutink P. 1998; PubMed Scopus Google Scholar, P. Bird T.D. E. E. L. A. M. Schellenberg G.D. Ann. Neurol. 1998; PubMed Scopus Google Scholar, M.G. Goedert M. A. B. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar). is not only to FTDP-17 but also to other with a neurofibrillary as Alzheimer disease, Pick disease, progressive supranuclear palsy, and corticobasal is in the which tau mutations lead to frontotemporal In the of FTDP-17 patients P301L mutation, only mutant tau was in the of their both wild-type and P301L mutant tau expressed at the T. Morishima-Kawashima M. R. Kamphorst W. K. Ihara Y. J. Neuropathol. Exp. Neurol. 2001; PubMed Scopus Google Scholar, P. M. R. A. Kamphorst W. van R. Heutink P. 2000; PubMed Scopus Google Scholar). the other of wild-type and mutant tau in the of the by R406W mutation T. Morishima-Kawashima M. R. Heutink P. van K. Ihara Y. Am. J. Pathol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). we that the nucleation and of tau into filaments be different between the and the assembly of tau be on the of monomeric tau and fibrillogenic In we that P301L mutant tau than wild-type tau. the other the nucleation of R406W mutant tau was similar to that of wild-type tau. are with M. Jakes R. Crowther R.A. FEBS Lett. 1999; 450: 306-311Crossref PubMed Scopus (221) Google Scholar, M. M. R. J. FEBS Lett. 1999; PubMed Scopus Google Scholar). the of wild-type and mutant tau. P301L mutant tau of only P301L mutant tau, but not wild-type tau, whereas composed of wild-type tau to promote assembly of both wild-type and P301L mutant tau In contrast, R406W mutant tau of wild-type tau as as that of R406W mutant tau. on these we a that at the accumulation of tau in FTDP-17 In the by P301L mutation, mutant tau is to a number of fibrillogenic nuclei as with wild-type tau of higher propensity The is by nuclei composed of P301L mutant tau. Thus, the of P301L mutant tau on the P301L mutant tau whereas wild-type tau on the P301L mutant tau In contrast, in the of R406W mutation, both wild-type and R406W tau fibrillogenic nuclei at similar in the and the of both wild-type and R406W mutant tau on wild-type tau or mutant tau with similar we wild-type and mutant tau in the of fibrillogenic wild-type and mutant tau are to in the of composed of P301L mutant tau of P301L mutant tau more than wild-type tau wild-type tau was with R406W mutant tau, both wild-type and R406W mutant tau in in the of composed of wild-type tau, and similar obtained with composed of R406W mutant tau. with the wild-type or R406W mutant tau was in the of composed of R406W mutant tau or wild-type tau, only a increase of tau was wild-type and P301L mutant tau in the of nuclei composed of wild-type tau. is to the obtained wild-type or P301L mutant tau was with wild-type The are it is that the of wild-type and P301L mutant tau the of tau assembly into wild-type tau and P301L mutant tau interact or with one in the that nuclei composed of P301L mutant tau promote assembly of P301L mutant tau into filaments more than assembly of wild-type tau. Proteolytic is a used to the of and protein or of paired helical filaments from the of Alzheimer patients in the of paired helical filaments which with the repeat of tau M. M.J. Jakes R. J.E. C. M. A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, M. J. EMBO J. 1993; PubMed Scopus Google Scholar, M. A. Takio K. Suzuki M. T. Titani K. Ihara Y. J. 1993; PubMed Scopus Google Scholar, Bergen M. Barghorn S. M. Biernat J. Mandelkow E.M. P. U. Mandelkow E. Biochemistry. PubMed Scopus Google Scholar). the of tau mutation in the of fibrillogenic we used MS, and a protein to the of the The that the of P301L mutant tau was different from that of wild-type tau, whereas R406W mutant tau to have a similar substructure to that of wild-type tau it is that the P301L mutation the of a in and the P301L mutation is in the second repeat of tau which the the other it that R406W mutation not the of the R406W mutation is the In the of patients tau protein is in a state (1Lee V.M. Balin B.J. 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Lett. 1992; PubMed Scopus Google Scholar). of wild-type or mutant tau with and of shown in tau similar of in and R406W mutant and protein analyses of of the indicated in a P301L mutation the of fibrillogenic whereas R406W mutation The of the with tau similar to of with tau. these that a lead to a of fibrillogenic of the substructure the The of the substructure of nuclei is well other in which in the of patients with of neurodegenerative diseases, is a monomeric protein fibrillogenic it is into an a higher of M. J. M. A. D. I. Cohen Proc. Natl. Acad. Sci. U. S. A. 1993; 90: PubMed Scopus Google Scholar). is the to the substructure of tau filaments a mutation that the assembly of tau protein. the of the of fibrillogenic nuclei is we also that in the protein the nuclei are in between wild-type and P301L monomeric tau was the with nuclei the P301L that seeding are not only by the substructure of fibrillogenic nuclei but also by the of monomeric tau. The of the interaction between fibrillogenic nuclei and monomeric tau is an important in the is that the interaction between P301L mutant nuclei and P301L mutant monomeric tau is than the interaction between P301L mutant nuclei and wild-type monomeric tau, resulting in of P301L mutant tau than wild-type tau in the of P301L mutant tau In we the of the of the interaction between fibrillogenic nuclei and monomeric protein that aggregate into is with the that tau a role in with