Conformational Diversity of Wild-type Tau Fibrils Specified by Templated Conformation Change

Tauopathies are sporadic and genetic neurodegenerative diseases characterized by aggregation of the microtubule-associated protein Tau. Tau pathology occurs in over 20 phenotypically distinct neurodegenerative diseases, including Alzheimer disease and frontotemporal dementia. The molecular basis of this diversity among sporadic tauopathies is unknown, but distinct fibrillar wild-type (WT) Tau conformations could play a role. Using Fourier transform infrared spectroscopy, circular dichroism, and electron microscopy, we show that WT Tau fibrils and P301L/V337M Tau fibrils have distinct secondary structures, fragilities, and morphologies. Furthermore, P301L/V337M fibrillar seeds induce WT Tau monomer to form a novel fibrillar conformation, termed WT*, that is maintained over multiple seeding reactions. WT* has secondary structure, fragility, and morphology that are similar to P301L/V337M fibrils and distinct from WT fibrils. WT Tau is thus capable of conformational diversity that arises via templated conformation change, as has been described for amyloid β, β2-microglobulin, and prion proteins. Tauopathies are sporadic and genetic neurodegenerative diseases characterized by aggregation of the microtubule-associated protein Tau. Tau pathology occurs in over 20 phenotypically distinct neurodegenerative diseases, including Alzheimer disease and frontotemporal dementia. The molecular basis of this diversity among sporadic tauopathies is unknown, but distinct fibrillar wild-type (WT) Tau conformations could play a role. Using Fourier transform infrared spectroscopy, circular dichroism, and electron microscopy, we show that WT Tau fibrils and P301L/V337M Tau fibrils have distinct secondary structures, fragilities, and morphologies. Furthermore, P301L/V337M fibrillar seeds induce WT Tau monomer to form a novel fibrillar conformation, termed WT*, that is maintained over multiple seeding reactions. WT* has secondary structure, fragility, and morphology that are similar to P301L/V337M fibrils and distinct from WT fibrils. WT Tau is thus capable of conformational diversity that arises via templated conformation change, as has been described for amyloid β, β2-microglobulin, and prion proteins. Tau filament deposition in Alzheimer disease, frontotemporal dementia, and other tauopathies correlates closely with cognitive dysfunction and cell death (1Braak H. Braak E. Acta Neuropathol.. 1991; 82: 239-259Google Scholar). About 10% of tauopathies are due to dominant mutations in the Tau gene. These diseases are collectively termed frontotemporal dementia with parkinsonism linked to chromosome 17, FTDP-17 (2Hutton 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. de Graaff E. 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. Basun H. Lannfelt L. Neystat M. Fahn S. Dark F. Tannenberg T. Dodd P.R. Hayward N. Kwok J.B. Schofield P.R. Andreadis A. Snowden J. Craufurd D. Neary D. Owen F. Oostra B.A. Hardy J. Goate A. van Swieten J. Mann D. Lynch T. Heutink P. Nature.. 1998; 393: 702-705Google Scholar, 3Spillantini M.G. Murrell J.R. Goedert M. Farlow M.R. Klug A. Ghetti B. Proc. Natl. Acad. Sci. U. S. A.. 1998; 95: 7737-7741Google Scholar, 4Poorkaj P. Bird T.D. Wijsman E. Nemens E. Garruto R.M. Anderson L. Andreadis A. Wiederholt W.C. Raskind M. Schellenberg G.D. Ann. Neurol.. 1998; 43: 815-825Google Scholar). Most of the mutations occur in the microtubule-binding region of the Tau protein, which is thought to be both its functional (5Lewis S.A. Wang D.H. Cowan N.J. Science.. 1988; 242: 936-939Google Scholar) and pathogenic (6Wischik C.M. Novak M. Thogersen H.C. Edwards P.C. Runswick M.J. Jakes R. Walker J.E. Milstein C. Roth M. Klug A. Proc. Natl. Acad. Sci. U. S. A.. 1988; 85: 4506-4510Google Scholar) “core.” Approximately 90% of tauopathies occur sporadically and involve only wild-type (WT) 2The abbreviations used are: WT, wild-type; FTIR, Fourier transform infrared spectroscopy; ThT, thioflavin T; EM, electron microscopy; MUT, P301L/V337M double mutation. 2The abbreviations used are: WT, wild-type; FTIR, Fourier transform infrared spectroscopy; ThT, thioflavin T; EM, electron microscopy; MUT, P301L/V337M double mutation. Tau. Both familial and sporadic tauopathies vary by regional involvement, disease duration, age of onset, Tau isoform expression, and fibril morphology (7Buee L. Bussiere T. Buee-Scherrer V. Delacourte A. Hof P.R. Brain Res.. 2000; 33: 95-130Google Scholar). It is unknown how the pathology of WT Tau might generate distinct disease phenotypes in sporadic tauopathies, and whether conformational diversity of the protein could potentially play a role in disease, as it does in prion disorders (8Tanaka M. Chien P. Naber N. Cooke R. Weissman J.S. Nature.. 2004; 428: 323-328Google Scholar, 9Legname G. Nguyen H.O. Peretz D. Cohen F.E. DeArmond S.J. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A.. 2006; 103: 19105-19110Google Scholar). Mutations in the Tau gene can generate conformationally distinct Tau species. Structural differences between in vitro prepared WT, G272V, N279K, P301L, V337M, and ΔK280 Tau fibrils have been observed using Fourier transform infrared spectroscopy (FTIR) (10von Bergen M. Friedhoff P. Biernat J. Heberle J. Mandelkow E.M. Mandelkow E. Proc. Natl. Acad. Sci. U. S. A.. 2000; 97: 5129-5134Google Scholar), and differential susceptibilities to protease cleavage in vitro have been described for WT and P301L Tau fibrils (11Aoyagi H. Hasegawa M. Tamaoka A. J. Biol. Chem.. 2007; 282: 20309-20318Google Scholar). Furthermore, Tau filaments extracted from diseased brain are often morphologically distinct, consisting of straight or paired helical filaments of various periodicities and widths (12Crowther R.A. Goedert M. J. Struct. Biol.. 2000; 130: 271-279Google Scholar). It is unknown whether WT Tau can assume self-propagating, structurally distinct fibrillar conformations, as has been described for amyloid β peptide (13Petkova A.T. Leapman R.D. Guo Z. Yau W.M. Mattson M.P. Tycko R. Science.. 2005; 307: 262-265Google Scholar), β2-microglobulin (14Yamaguchi K. Takahashi S. Kawai T. Naiki H. Goto Y. J. Mol. Biol.. 2005; 352: 952-960Google Scholar), and the prion protein (15Bessen R.A. Kocisko D.A. Raymond G.J. Nandan S. Lansbury P.T. Caughey B. Nature.. 1995; 375: 698-700Google Scholar). In this study, we have used biochemical and biophysical methods to test the hypothesis that WT Tau fibrils exhibit conformational diversity that is maintained by templated conformation change. Tau Expression and Purification—The microtubule-binding region (amino acids 242–364 of the 441-amino acid Tau isoform) of WT Tau and the P301L/V337M Tau double mutation (designated as MUT) used in all fibrillization reactions were subcloned into pRK172 (a gift from Dr. Virginia Lee). Recombinant Tau microtubule-binding region was prepared as described previously from Rosetta (DE3)pLacI competent cells (Novagen), exploiting the heat stability of Tau protein, followed by cation exchange chromatography (16Goedert M. Jakes R. EMBO J.. 1990; 9: 4225-4230Google Scholar). Single-use aliquots were stored at –80 °C in 10 mm HEPES and 100 mm NaCl (pH 7.4). Tau Fibrillization and Seeding Assays—4 μm Tau monomer was incubated for the indicated times at room temperature without agitation in 5 mm dithiothreitol, 10 mm HEPES (pH 7.4), 100 mm NaCl, and 150 μm arachidonic acid (Sigma-Aldrich) (17Wilson D.M. Binder L.I. J. Biol. Chem.. 1995; 270: 24306-24314Google Scholar). Tau appears in the insoluble fraction after a 15-h fibrillization reaction and 100,000 × g ultracentrifugation. Alternatively, seeded reactions were carried out using a 10% aliquot from a fibrillization reaction (i.e. 0.4 μm total Tau protein) and 4 μm monomer in three successive incubations. The primary fibrillization reaction was initiated with 150 μm arachidonic acid. After 15 h, the secondary fibrillization was initiated by exposure of 4 μm monomer to 10% of the primary reaction. After 3 h, the tertiary reaction was initiated by exposure of 4 μm monomer to 10% of the secondary reaction. After 3 h, the quaternary reaction was initiated by exposure of 4 μm monomer to 10% of the tertiary reaction, resulting in a 1,000-fold dilution of the primary seed. The quaternary reaction was allowed to proceed for 96 h before further analysis. WT* was produced by using primary MUT Tau as a seed for WT monomer in serial seeding reactions (MUT seed WT seed WT seed WT). Thioflavin T—When measuring the fibrillization rate of primary reactions, 12.5 μm thioflavin T (ThT; Sigma-Aldrich) was included in the reaction (18LeVine III, H. Protein Sci.. 1993; 2: 404-410Google Scholar). Measurements were recorded at 455 nm excitation/485 nm emission in a Safire plate reader (Tecan) every 10 min for 1 h and every 30 min for the next 14 h. When measuring the fibrillization rate of quaternary reactions, 12.5 mm ThT was mixed with aliquots of the quaternary reaction every 24 h and measured as described above. Fourier Transform Infrared Spectroscopy—WT and MUT Tau were polymerized through serially seeded reactions as described above. The polymerized Tau from quaternary reactions was pelleted by ultracentrifugation, resuspended in water, and lyophilized for 48 h. The lyophilisates were resuspended in D2O. Infrared absorbance spectra were recorded against pure D2O. Buffers and salts did not interfere with the amide I absorption band (1700-1600 cm–1). For each sample, 64 double-sided interferograms were scanned bidirectionally and averaged on a PerkinElmer System 2000 FTIR spectrometer, equipped with an MCT detector and purged with dry N2. Spectra were recorded with a 2 cm–1 instrument resolution, and 4-fold zero filling yielded one data point per 0.5 cm–1. Trace signals from water vapor were eliminated with the suppression algorithm of the software Spectrum GX V5 (PerkinElmer Life Sciences). Additionally, low frequency noise was filtered with a Fourier self-deconvolution algorithm at an 8–10 cm–1 cutoff as necessary, and the amide I band was base line-corrected. Secondary structure information was derived by amide I band decomposition as described previously (19Ollesch J. Kunnemann E. Glockshuber R. Gerwert K. Appl. Spectrosc.. 2007; 61: 1025-1031Google Scholar). Cauchy curves were numerically determined to add up to the shape of the amide I band. The Cauchy curve integrals were used to calculate the fraction of secondary structure that is assigned to the peak frequency of the curve. Curve fitting all amide I bands with identical initialization parameters for the number of bands, band position, width, and shape and the automatic determination of each intensity as well as the overall base-line values resulted in a high sensitivity for structural changes. The secondary structure fractions presented in Fig. 2G were averaged from six spectra (MUT aggregate from five) of two separate aggregation assays. Thereby, we were able to suppress the negative effects of residual water vapor and to quantify the quality of our analysis with the error bars presented. Circular Dichroism—Primary and quaternary reactions were ultracentrifuged at 100,000 × g for 1 h at room temperature, and pellets were resuspended in phosphate-buffered saline to a final concentration of ∼4 μm prior to CD. Spectra were recorded on a Jasco spectrometer and reflect four accumulations. Atomic Force Microscopy—Reactions were adsorbed onto mica chips (Ted Pella, Inc.) for 2 min, washed twice with 200 μl of water, and allowed to dry for at least 1 h prior to tapping mode atomic force microscopy (Veeco). Fiber Fragility Assays—Whole primary and quaternary fibrillization reactions of WT, MUT, and WT* were dialyzed into phosphate-buffered saline for 15 h. CD spectra were measured from 250 to 200 nm, after which the reactions were sonicated with a titanium probe on a Branson Sonifier 250 at 50% duty and 10% intensity for 1 min, and CD spectra were again measured. The changes in WT, MUT, and WT* signals were compared using Wilcoxon signed rank statistical analysis in fragility assays. The data set (n = 6) presented meets all requirements for this type of one-sided, two-tailed nonparametric analysis (α < 0.025 and *, p = 0.0156 for all fragility assays). Electron Microscopy—Quaternary fibrillization reactions were adsorbed onto glow-discharged Formvar/carbon copper grids (200 mesh; Ted Pella, Inc.). The grids were washed with ammonium acetate buffer, stained with uranyl acetate, dried, and viewed in an FEI Tecnai F20 electron microscope at 80 kV and a standard magnification of ×25,000 (20Wille H. Govaerts C. Borovinskiy A. Latawiec D. Downing K.H. Cohen F.E. Prusiner S.B. Arch. Biochem. Biophys.. 2007; 467: 239-248Google Scholar). Electron micrographs were recorded with a Gatan UltraScan CCD camera. The magnification was calibrated using negatively stained catalase crystals and ferritin. The percentage of paired helical filaments was compared between WT, MUT, and WT* quaternary reactions using Student's t test. The data set (n = 3) presented meets all requirements for this type of two-tailed, two-sample equal variance analysis (*, p < 10–5). Fibril Seeding Strategy—We established a protocol to create pools of fibrillar protein under uniform conditions (Fig. 1, A and B). The primary WT and MUT fibrillization reactions were induced by arachidonic acid (17Wilson D.M. Binder L.I. J. Biol. Chem.. 1995; 270: 24306-24314Google Scholar). The secondary reaction was a seeded reaction in which 4 μm monomer was incubated with 10% of the primary reaction in the absence of arachidonic acid for 3 h. The secondary seeding reaction was followed by two additional seeding reactions to produce the tertiary and quaternary reactions (Fig. 1A). To induce a novel fibrillar conformation of WT Tau, termed WT*, we performed a cross-seeding reaction in which the primary MUT reaction was incubated with WT monomer. Three serial seeding reactions were then performed using WT monomer (Fig. 1B). After 96 h, quaternary reactions were ultracentrifuged, and the insoluble fractions were compared using FTIR, CD, and EM. Whole reactions were compared for fragility assays. We first determined the extent of fibrillization and the fibrillization rate for each reaction. In primary reactions, 81% of WT monomer and 89% of MUT monomer were present in the insoluble fraction after 15 h (Fig. 1, C and D), indicating that most of the protein was in an aggregated form. Primary WT and MUT reactions had similar rates of fibrillization based on ThT fluorescence (Fig. 1E) (18LeVine III, H. Protein Sci.. 1993; 2: 404-410Google Scholar). In quaternary seeded reactions, WT and WT* also had similar degrees of aggregation. 22% of WT and 21% of WT* quaternary reactions were insoluble after 96 h (Fig. 1, F and G). Monitoring ThT fluorescence of the quaternary reactions produced a t½ of 24 h for WT, 18 h for MUT, and 13 h for WT* (Fig. 1H). Despite different t½ values, all reactions reached a plateau by ∼48 h. WT and WT* Fibrils Have Secondary on Fourier Transform Infrared used FTIR to monomer and quaternary fibril The data is for structural which a of the Tau WT and MUT monomer differences in secondary structure (Fig. and G). In quaternary WT and MUT Tau fibrils distinct FTIR WT Tau had in of but also a diversity of structures, MUT Tau was (Fig. C and indicated with a of in a Student's t WT fibrils and MUT (Fig. quaternary WT* spectra were distinct from WT with and (Fig. and G). The values for the secondary structure fractions are only a structural for of Tau secondary structure analysis does not (19Ollesch J. Kunnemann E. Glockshuber R. Gerwert K. Appl. Spectrosc.. 2007; 61: 1025-1031Google Scholar). the is for differences between of a similar (19Ollesch J. Kunnemann E. Glockshuber R. Gerwert K. Appl. Spectrosc.. 2007; 61: 1025-1031Google Scholar, J. J. A. Gerwert K. D. J.. Scholar, K. J. P. H. F. de H. Gerwert K. Reed J. D. Proc. Natl. Acad. Sci. U. S. A.. 2004; Scholar). we with the structure of the FTIR differences between WT and WT* fibrils are WT and WT* Fibrils Have Secondary on Circular the FTIR we used CD spectroscopy to WT, MUT, and WT* fibrils. We prepared primary reactions as described above. After 15 h, we ultracentrifuged the reactions and resuspended the pellets in phosphate-buffered The MUT at nm nm for WT fibrils. These that MUT fibrils structure WT fibrils and that WT fibrils with data from The for MUT fibrils was also that of WT fibrils. We then compared CD spectra of quaternary WT and WT* reactions, prepared as previously again measuring only the insoluble The CD of quaternary WT* was different from that of quaternary WT (Fig. also with a These data the conformational differences by differences in WT spectra were observed between from in of arachidonic acid or Tau protein differences between WT and MUT spectra were observed each (Fig. To that the spectra observed were due to from the seeding reaction and not carried over from the primary reaction, we serially primary WT and MUT reactions. The resulting spectra from the quaternary are Fig. in the primary seeding reaction are are not to produce the signals observed in quaternary reactions. We also out the that differences in protein concentration could for the differences between the Fig. 1B). WT and WT* Fibrils Have that prion are to M. Weissman J.S. Nature.. 2006; Scholar), and β2-microglobulin amyloid fibrils exhibit CD at nm fibrils R. K. H. K. Naiki H. Goto Y. J. Biol. Chem.. 2007; 282: Scholar), that CD changes before and after can be used to fibril as an additional of structural differences between the fibril we a to the sensitivity of WT, MUT, and WT* fibrils to we that quaternary WT Tau but not MUT or WT* based on atomic force microscopy (Fig. To quantify fragility, we compared the CD spectra at nm of the primary reactions before and after at 10% β2-microglobulin of Tau fibrils resulted in fibrils that to a at After six the of MUT CD was of the WT (Fig. and Fig. A and B). MUT fibrils to an extent similar to WT fibrils after at intensity not WT* fibrils were also WT fibrils and by WT* fibrils 21% of the of that of quaternary WT fibrils (Fig. and Fig. C and the differences between WT and WT* observed by FTIR and CD spectroscopy are by distinct fibril WT and WT* Fibrils Have on Electron a final of the distinct structures, we used negative to the of the three fibril WT fibrils of paired helical filaments (Fig. A and MUT Tau fibrils were similar to straight were the paired helical and a of (Fig. C and WT* fibrils were distinct from WT fibrils in of paired helical structure and had similar and to MUT Tau (Fig. and When three separate were and we differences in the of paired helical filaments in WT fibril MUT and in WT of WT fibrils as paired helical only of MUT fibrils and of WT* fibrils were helical in and as fibrils (Fig. with the data our that WT Tau fibrils can a distinct, fibrillar structure that is via templated conformation change. In this study, we the hypothesis that WT Tau can assume distinct fibrillar conformations that are serially by templated conformation change. We have an protein, MUT Tau, two mutations as a with which to WT Tau into a distinct fibrillar conformation, termed We have compared WT, MUT, and WT* fibril conformations using four FTIR, CD spectroscopy, sensitivity to and analysis of fibril of that WT* a novel conformation of WT fibril that is maintained over through templated conformation change. data are with the that sporadic tauopathies, which involve only WT Tau, might diversity from conformational differences in WT Tau fibrils. In to MUT Tau we have also observed that ΔK280 Tau fibrils can seed a conformation of WT Tau Fig. that the to induce WT Tau to conformations by templated conformation is not to a seed. It to be determined to extent a Tau protein in can of the wild-type and whether other might play a role. the that induce WT Tau to form fibrils of a conformation in sporadic could be a seed amyloid a with which to diversity in the sporadic WT Tau is a conformational the it this distinct conformation via templated conformation change. from of prion disease a between the conformation of a fibrillar protein and the (8Tanaka M. Chien P. Naber N. Cooke R. Weissman J.S. Nature.. 2004; 428: 323-328Google Scholar, 9Legname G. Nguyen H.O. Peretz D. Cohen F.E. DeArmond S.J. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A.. 2006; 103: 19105-19110Google Scholar). to this a protein a fibrillar conformation, that conformation is over through seeded fibrillization reactions. The of a fibril type as fragility, and play an role in M. Weissman J.S. Nature.. 2006; Scholar). data by that multiple have the to form distinct based on templated conformation (13Petkova A.T. Leapman R.D. Guo Z. Yau W.M. Mattson M.P. Tycko R. Science.. 2005; 307: 262-265Google Scholar, K. Takahashi S. Kawai T. Naiki H. Goto Y. J. Mol. Biol.. 2005; 352: 952-960Google Scholar). the seeding that has been between P301L Tau and WT Tau is of prion (11Aoyagi H. Hasegawa M. Tamaoka A. J. Biol. Chem.. 2007; 282: 20309-20318Google Scholar, T. M. R. K. Y. J. Neurol.. Scholar) and is with our which for cross-seeding to between Tau other amyloid as amyloid β, β2-microglobulin, and prion protein, WT Tau can multiple distinct fibrillar conformations that are maintained over via templated conformation change. In it be to whether diversity in sporadic tauopathies can be linked to WT Tau fibril We Weissman for of the atomic force microscope and and for of Jasco with