Asparagine residue 368 is involved in Alzheimer's disease tau strain–specific aggregation
2020; Elsevier BV; Volume: 295; Issue: 41 Linguagem: Inglês
10.1074/jbc.ra120.013271
ISSN1083-351X
AutoresShotaro Shimonaka, Shin‐ei Matsumoto, Montasir Elahi, Koichi Ishiguro, Masato Hasegawa, Nobutaka Hattori, Yumiko Motoi,
Tópico(s)Dementia and Cognitive Impairment Research
ResumoIn tauopathies, tau forms pathogenic fibrils with distinct conformations (termed “tau strains”) and acts as an aggregation “seed” templating the conversion of normal tau into isomorphic fibrils. Previous research showed that the aggregation core of tau fibril covers the C-terminal region (243–406 amino acids (aa)) and differs among the diseases. However, the mechanisms by which distinct fibrous structures are formed and inherited via templated aggregation are still unknown. Here, we sought to identify the key sequences of seed-dependent aggregation. To identify sequences for which deletion reduces tau aggregation, SH-SY5Y cells expressing a series of 10 partial deletion (Del 1–10, covering 244–400 aa) mutants of tau-CTF24 (243–441 aa) were treated with tau seeds prepared from a different tauopathy patient's brain (Alzheimer's disease, progressive supranuclear palsy, and corticobasal degeneration) or recombinant tau, and then seed-dependent tau aggregation was assessed biochemically. We found that the Del 8 mutant lacking 353–368 aa showed significantly decreased aggregation in both cellular and in vitro models. Furthermore, to identify the minimum sequence responsible for tau aggregation, we systematically repeated cellular tau aggregation assays for the delineation of shorter deletion sites and revealed that Asn-368 mutation suppressed tau aggregation triggered by an AD tau seed, but not using other tauopathy seeds. Our study suggested that 353–368 aa is a novel aggregation-responsible sequence other than PHF6 and PHF6*, and within this sequence, the Asn-368 residue plays a role in strain-specific tau aggregation in different tauopathies. In tauopathies, tau forms pathogenic fibrils with distinct conformations (termed “tau strains”) and acts as an aggregation “seed” templating the conversion of normal tau into isomorphic fibrils. Previous research showed that the aggregation core of tau fibril covers the C-terminal region (243–406 amino acids (aa)) and differs among the diseases. However, the mechanisms by which distinct fibrous structures are formed and inherited via templated aggregation are still unknown. Here, we sought to identify the key sequences of seed-dependent aggregation. To identify sequences for which deletion reduces tau aggregation, SH-SY5Y cells expressing a series of 10 partial deletion (Del 1–10, covering 244–400 aa) mutants of tau-CTF24 (243–441 aa) were treated with tau seeds prepared from a different tauopathy patient's brain (Alzheimer's disease, progressive supranuclear palsy, and corticobasal degeneration) or recombinant tau, and then seed-dependent tau aggregation was assessed biochemically. We found that the Del 8 mutant lacking 353–368 aa showed significantly decreased aggregation in both cellular and in vitro models. Furthermore, to identify the minimum sequence responsible for tau aggregation, we systematically repeated cellular tau aggregation assays for the delineation of shorter deletion sites and revealed that Asn-368 mutation suppressed tau aggregation triggered by an AD tau seed, but not using other tauopathy seeds. Our study suggested that 353–368 aa is a novel aggregation-responsible sequence other than PHF6 and PHF6*, and within this sequence, the Asn-368 residue plays a role in strain-specific tau aggregation in different tauopathies. Tau is a natively highly soluble, unfolded protein (1Schweers O. Schonbrunn-Hanebeck E. Marx A. Mandelkow E. Structural studies of tau protein and Alzheimer paired helical filaments show no evidence for β-structure.J. Biol. Chem. 1994; 269 (7929085): 24290-24297Abstract Full Text PDF PubMed Google Scholar, 2Margittai M. Langen R. Side chain-dependent stacking modulates tau filament structure.J. Biol. Chem. 2006; 281 (17023423): 37820-3782710.1074/jbc.M605336200Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), and physiologically it participates in the assembly and stabilization of microtubules (MTs) (3Weingarten M.D. Lockwood A.H. Hwo S.Y. Kirschner M.W. A protein factor essential for microtubule assembly.Proc. Natl. Acad. Sci. U. S. 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These findings suggested that the C-terminal region including the MBD plays an essential role in the aggregation of tau. This understanding was supported by the discovery of the PHF6 (306–311 aa: VQIVYK) and PHF6* (275–280 aa: VQIINK) sequences, which were found in the protease-resistant C-terminal fragment of tau and were shown to be involved in the initiation of tau aggregation (24von Bergen M. Friedhoff P. Biernat J. Heberle J. Mandelkow E.M. Mandelkow E. Assembly of tau protein into Alzheimer paired helical filaments depends on a local sequence motif (306VQIVYK311) forming β structure.Proc. Natl. Acad. Sci. U. S. A. 2000; 97 (10805776): 5129-513410.1073/pnas.97.10.5129Crossref PubMed Scopus (689) Google Scholar, 25von Bergen M. Barghorn S. Li L. Marx A. Biernat J. Mandelkow E.M. Mandelkow E. Mutations of tau protein in frontotemporal dementia promote aggregation of paired helical filaments by enhancing local β-structure.J. Biol. Chem. 2001; 276 (11606569): 48165-4817410.1074/jbc.M105196200Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar). Here, we attempted to identify aggregation-responsible sequences in the C-terminal region of tau using a cellular seed-dependent aggregation model of tau-CTF24. To determine the key sequences of the tau C-terminal region systematically, we prepared 10 deletion mutants of tau-CTF24 that were expressed in SH-SY5Y cells and treated with tau aggregates prepared from a patient's brain or recombinant tau. Then we identified Del 8 (353–368 aa) as a novel aggregation-responsible sequence, and this region overlapped with the core sequence of AD tau, which was recently determined by means of cryo-EM (26Fitzpatrick A.W.P. Falcon B. He S. Murzin A.G. Murshudov G. Garringer H.J. Crowther R.A. Ghetti B. Goedert M. Scheres S.H.W. Cryo-EM structures of tau filaments from Alzheimer's disease.Nature. 2017; 547 (28678775): 185-19010.1038/nature23002Crossref PubMed Scopus (748) Google Scholar). The role of this sequence in tau aggregation was tested in an in vitro aggregation assay using recombinant tau mutants or peptides. We found that this sequence also affected the morphology of recombinant tau fibrils, but the sequence itself had no aggregation properties. In addition, we further investigated the smallest sequences involved in tau aggregation within 353–368 aa. We found that the aggregation property can be attributed to a single amino acid, Asn-368, whose mutations affected tau aggregation differently, depending on the type of tau strains. To explore the sequences of the tau C-terminal region that is responsible for aggregation, we constructed a series of plasmids expressing deletion mutants of tau-CTF24 (243–441 aa) lacking 16-amino acid residues: Del 1 (Δ244–259 aa), Del 2 (Δ259–274 aa), Del 3 (Δ275–290 aa), Del 4 (Δ290–305 aa), Del 5 (Δ306–321 aa), Del 6 (Δ322–337 aa), Del 7 (Δ338–353 aa), Del 8 (Δ353–368 aa), Del 9 (Δ369–384 aa), and Del 10 (Δ385–400 aa) (Fig. 1A). Deletion sites of Del 5 (306–321 aa) and Del 3 (275–290 aa) are overlapped with PHF6 (306–311 aa) and PHF6* (275–280 aa), respectively, which have been shown to promote aggregation of tau (24von Bergen M. Friedhoff P. Biernat J. Heberle J. Mandelkow E.M. Mandelkow E. Assembly of tau protein into Alzheimer paired helical filaments depends on a local sequence motif (306VQIVYK311) forming β structure.Proc. Natl. Acad. Sci. U. S. A. 2000; 97 (10805776): 5129-513410.1073/pnas.97.10.5129Crossref PubMed Scopus (689) Google Scholar, 25von Bergen M. Barghorn S. Li L. Marx A. Biernat J. Mandelkow E.M. Mandelkow E. Mutations of tau protein in frontotemporal dementia promote aggregation of paired helical filaments by enhancing local β-structure.J. Biol. Chem. 2001; 276 (11606569): 48165-4817410.1074/jbc.M105196200Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar). SH-SY5Y cells were transfected with these tau-CTF24 mutants (Del 3 to Del 10) or WT (tau-CTF24 with no deletion) constructs and treated with tau seeds, which were mixed with a transfection reagent (Multifectam) for introduction into the cells. In this experiment, aggregates of recombinant 4R2N tau or lysates of AD, PSP, or CBD patients' brains were used as seeds (case descriptions and immunoblotting images are shown in Table S2 and Fig. S1). Del 1 and Del 2 were not included in the cellular experiments because these mutants were not expressed sufficiently in SH-SY5Y cells. Tau aggregation was evaluated by biochemical analysis of sarkosyl-insoluble (insol.) fractions of cell lysates. After treatment with all the types of tau seeds, the amounts of ppt were significantly decreased in cells expressing the Del 5, Del 6, and Del 8 mutants (Fig. 2, A–D), regardless of the types of tau strains used as seed. There were no significant differences in the levels of soluble in the levels of soluble tau (sol.) detected irrespective of either deletion sites of mutants or the types of seeds used.Figure 4In vitro aggregation of recombinant tau-CTF24 deletion mutants. A, recombinant tau-CTF24 (Del 1 to Del 10) solutions were incubated at 37 °C on a shaker in the presence or absence of heparin (Hep). Tau aggregation was detected using ThS assays at the indicated times. Data are means ± S.D. (error bars) (n = 3). Data values at 96 h were analyzed statistically using Student's t test versus the value of WT at 96 h (***, p < 0.001). a.u., arbitrary units; n.s., not significant. B, after >7 days of incubation with heparin, aggregated recombinant tau-CTF24 deletion mutants were analyzed using TEM. Magnification level in the micrographs is ×30,000. Scale bar, 200 nm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Immunocytochemical analysis of intracellular tau aggregation. A, the representative images of confocal microscopic analysis of cellular aggregation of tau-CTF24 mutants after 2N4R seed treatment. Cells were stained with PHF-1 (green), anti-tubulin polyclonal antibody (red), and DAPI (blue). Tau aggregates are identified by white arrowheads, and nonaggregated tau are indicated by white arrows on the magnified images (magnified areas are indicated by the red square). Scale bar, 20 μm. B, total cells and cells with PHF-1–positive tau aggregates were counted, and the ratios of tau aggregate–positive cells were compared between WT + seed and D1–D9 + seed. The result for D10 was not included, because the PFH1 antibody did not stain either expressed D10 monomer or aggregates. Values are means ± S.D. (error bars) (n = 3). **, p < 0.01; ***, p < 0.001 by Student's t test versus WT + seed.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 2Aggregation assay of the cellular tau aggregation model expressing tau-CTF24 deletion mutants. A–D, SH-SY5Y cells transfected with tau-CTF24 deletion mutants and WT (tau-CTF24 without deletion) were treated with AD (A), PSP (C), and CBD (D) brain lysates and recombinant tau fibrils (2N4R) (B) as tau seeds. A68 buffer-soluble fraction (sol.) and sarkosyl-insoluble fraction (insol.) were detected with T46 antibodies, and relative intensity (versus WT + seed) is shown in the plot. Data are means ± S.D. (error bars) (n = 3). **, p < 0.01; ***, p < 0.001 by Student's t test versus WT + seed. a.u., arbitrary units; n.s., not significant.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 1Construction of a series of partial deletion mutants of tau C-terminal fragments. A, schematic diagrams of full-length 4R2N tau (1–441 aa) and the epitopes of tau antibodies used in this study. Deletion sites (1–10) were designed in the C-terminal region (244–400 aa) containing the microtubule-binding domain (M1–M4; 244–368 aa). B, schematic images of tau-CTF24 deletion mutants (Del 1–10). Each mutant possesses one of 10 deletions. The Del 1 to Del 8 mutants have deletions in the microtubule-binding domain (Del 1 (244–259 aa) and Del 2 (259–274 aa) on M1, Del 3 (275–290 aa) and Del 4 (290–305 aa) on M2, Del 5 (306–321 aa) and Del 6 (322–337 aa) on M3, and Del 7 (338–353 aa) and Del 8 (353–368 aa) on M4), and the deletion sites of Del 9 (369–384 aa) and Del 10 (385–400 aa) were designed to include the C-terminal neighboring region of this domain. PHF6 (306–311 aa) and PHF6* (275–280 aa) sites were located on Del 5 and Del 3 deletion sites, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The effects of deletion on seed-induced cellular aggregation of tau were also analyzed by fluorescence immunocytochemistry. SH-SY5Y cells expressing WT or deletion mutants were treated with recombinant 2N4Rtau seeds and the phosphorylated tau-specific antibody PHF-1 (27Greenberg S.G. Davies P. Schein J.D. Binder L.I. Hydrofluoric acid-treated tau PHF proteins display the same biochemical properties as normal tau.J. Biol. Chem. 1992; 267 (1370450): 564-569Abstract Full Text PDF PubMed Google Scholar, 28Otvos Jr., L. Feiner L. Lang E. Szendrei G.I. Goedert M. Lee V.M. Monoclonal antibody PHF-1 recognizes tau protein phosphorylated at serine residues 396 and 404.J. Neurosci. Res. 1994; 39 (7534834): 669-67310.1002/jnr.490390607Crossref PubMed Scopus (396) Google Scholar) to visualize intracellularly formed tau aggregates but not recombinant 4R2N tau seed itself (not phosphorylated) remaining on the cell surface even after washing. Aggregated tau was detected as cytoplasmic inclusion-like structures (Fig. 3A, white arrowhead), which were morphologically distinct from diffusely expressed nonaggregated tau (Fig. 3A, white arrow) The results with Del 10 were not included because the PHF-1 antibody did not stain either expressed Del 10 monomer or aggregates. This was probably because the PHF-1 epitope (pSer-396/404) overlapped with the Del 10 deletion sequence (Δ385–400 aa). The ratio of aggregate-positive cells was significantly decreased in cells expressing Del 3, Del 5, Del 8, and Del 9 compared with WT (Fig. 3B). The Del 6 mutant, which exhibited a significant decrease in tau aggregation in the biochemical analysis (Fig. 2, A–D), showed only a moderate reduction in tau aggregation in this setting. It is possible that the Del 6 mutation may affect the tau aggregation, ability more mildly than Del 8 and Del 9, and thereby the detectable amount of Del 6 aggregates was preserved through the immunocytochemistry staining process but easily solubilized and monomerized by sarkosyl extraction. We also tried to analyze cells treated with other tau strains, but the seeds extracted from tauopathy patients' brains were highly phosphorylated so that PHF-1 antibody strongly stained the seeds themselves, which remained on the coverslips or cell surface, and intracellular tau aggregates were indistinguishable from them. Collectively, Del 5 (harboring the aggregation-prone PHF6 sequence), Del 8, and Del 9 showed significant decreases in tau-CTF24 aggregation using both biochemical and histochemical analyses. To further analyze the effect of deletion in vitro, we conducted ThS fluorescence assays using a series of recombinant tau-CTF24 deletion mutants. We successfully purified recombinant tau-CTF24 mutants except for Del 4, because most of the bacterially expressed Del 4 mutant was captured in inclusion bodies, and therefore, we were unable to prepare sufficient amounts of recombinant Del 4. Tau-CTF24 WT and deletion mutants (Del 1–10) were incubated at 37 °C on a shaker in the presence or absence of heparin. Consistent with the results of cultured cells, Del 5 and Del 8 showed a significant decrease in ThS fluorescence compared with the WT after incubation for 96 h, suggesting that the deleted regions in Del 5 and Del 8 are critical for tau aggregation (Fig. 4A). Del 3 also exhibited a slight reduction in fluorescence, and Del 6 showed significantly decreased aggregation at 24 and 48 h, but ThS fluorescence elevated rapidly and showed almost identical levels to WT at 72 and 96 h. Other mutants, including Del 9, showed almost the same or higher ThS fluorescence compared with WT. In particular, Del 2 exhibited significantly higher ThS fluorescence levels than WT. We were not able to clarify the mechanisms for the increased aggregation of Del 2 and Del 4 (inclusion bodies in bacteria), but considering the fact that the deletion sites of these mutants (Del 2, 259–274 aa; Del 4, 290–305 aa) were N-terminally adjacent to PHF6* (275–280 aa) or PHF6 (305–311 aa), we speculated that these sequences might be an obstacle to the aggregation-prone motif. All of the mutants and WT showed no increased ThS fluorescence in the absence of heparin. Recombinant tau-CTF24 aggregates were negatively stained with phosphotungstate and observed by transmission EM (TEM). Other than Del 5 and Del 8, TEM observations of tau-CTF24 aggregates showed straight filamentous structures of 10–15-nm width. Del 5 showed shorter fibrils gathering each other, and Del 8 indicated a characteristic tangled string–like shape that clearly differed from WT or other mutants (Fig. 4B). However, there were inconsistent results between cellular biochemical analyses and the in vitro aggregation assay of Del 6 and Del 9. Previous studies have demonstrated that structural differences exist between heparin-induced tau filaments and those from brain tissues and that different seeding properties were observed between tau filaments seeded by AD tau and those assembled by heparin (29Zhang W. Falcon B. Murzin A.G. Fan J. Crowther R.A. Goedert M. Scheres S.H. Heparin-induced tau filaments are polymorphic and differ from those in Alzheimer's and Pick's diseases.eLife. 2019; 8 (30720432): e4358410.7554/eLife.43584Crossref PubMed Scopus (129) Google Scholar, 30Falcon B. Cavallini A. Angers R. Glover S. Murray T.K. Barnham L. Jackson S. O'Neill M.J. Isaacs A.M. Hutton M.L. Szekeres P.G. Goedert M. Bose S. Conformation determines the seeding potencies of native and recombinant Tau aggregates.J. Biol. Chem. 2015; 290 (25406315): 1049-106510.1074/jbc.M114.589309Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 31Morozova O.A. March Z.M. Robinson A.S. Colby D.W. Conformational features of tau fibrils from Alzheimer's disease brain are faithfully propagated by unmodified recombinant protein.Biochemistry. 2013; 52 (24033133): 6960-696710.1021/bi400866wCrossref PubMed Scopus (60) Google Scholar). Hence, we speculated that tauopathy seed-induced cellular aggregation and heparin-induced in vitro aggregation occurred in a different manner, so that inconsistent results were obtained, even using the same tau deletion mutants. We further tested the effects of 306–321 (Del 5) and 353–368 (Del 8) aa deletions on the aggregation of full-length tau (2N4R and 2N3R isoforms). Cellular and in vitro aggregation experiments revealed that the 353–368 aa deletion in 2N4R and 2N3R tau caused decreases in tau aggregation and formation of abnormal fibrous structures (Fig. 5). These data showed that deletion of 353–368 aa affects the aggregation of tau, regardless of the length (C-terminal region or full length) and the presence or absence of microtubule-binding repeat 2 (2N4R or 2N3R). The results in Figure 2, Figure 3, Figure 4 are summarized in Fig. 6A. This schematic diagram indicates that the deletions of 306–321 aa (Del 5) and 353–368 aa (Del 8) in tau showed decreased aggregation in all experimental conditions. Moreover, TEM observation of Del 8 fibrils revealed abnormal structures, which were clearly distinct from WT or other mutant fibrils. Focusing on the core regions of each tau strain (Fig. 6A, solid and broken lines), the Del 5 sequence overlapped with the all of the core regions, but the Del 8 sequence was not included in the recombinant 4R2N core. The cause of the different filament morphology is not clear, but it is possible that sequences outside of the core region also can participate in the molecular process of tau aggregation or that the remarkable ultrastructural versatility of 4R2N fibrils (Zhang et al. distinguished at least four types of them: snake, twister, jagged, and hose (29Zhang W. Falcon B. Murzin A.G. Fan J. Crowther R.A. Goedert M. Scheres S.H. Heparin-induced tau filaments are polymorphic and differ from those in Alzheimer's and Pick's diseases.eLife. 2019; 8 (30720432): e4358410.7554/eLife.43584Crossref PubMed Scopus (129) Google Scholar)) may contribute to this result.Figure 6The characteristics of candidate sequences involved in tau aggregation. A, the results of Figs. 2–4 are summarized as a schematic diagram in the left panel. The deletion mutants, which showed significantly decreased tau aggregation, are marked with check symbols in the table. The core regions of AD (red), 4R2N recombinant (blue), PSP (green), and CBD (purple) tau fibrils are indicated by solid (determined by the cryo-EM method (26Fitzpatrick A.W.P. Falcon B. He S. Murzin A.G. Murshu
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