Conformation determines the seeding potencies of native and recombinant Tau aggregates

Benjamin Falcon, Annalisa Cavallini, Rachel Angers, Sarah Glover, Tracey K Murray, Luanda Barnham, Samuel Jackson, Michael J O'Neill, Adrian M Isaacs, Michael L Hutton, Philip G Szekeres, Michel Goedert, Suchira Bose, Benjamin Falcon, Annalisa Cavallini, Rachel Angers, Sarah Glover, Tracey K Murray, Luanda Barnham, Samuel Jackson, Michael J O'Neill, Adrian M Isaacs, Michael L Hutton, Philip G Szekeres, Michel Goedert, Suchira Bose

Abstract

Intracellular Tau inclusions are a pathological hallmark of several neurodegenerative diseases, collectively known as the tauopathies. They include Alzheimer disease, tangle-only dementia, Pick disease, argyrophilic grain disease, chronic traumatic encephalopathy, progressive supranuclear palsy, and corticobasal degeneration. Tau pathology appears to spread through intercellular propagation, requiring the formation of assembled "prion-like" species. Several cell and animal models have been described that recapitulate aspects of this phenomenon. However, the molecular characteristics of seed-competent Tau remain unclear. Here, we have used a cell model to understand the relationships between Tau structure/phosphorylation and seeding by aggregated Tau species from the brains of mice transgenic for human mutant P301S Tau and full-length aggregated recombinant P301S Tau. Deletion of motifs (275)VQIINK(280) and (306)VQIVYK(311) abolished the seeding activity of recombinant full-length Tau, suggesting that its aggregation was necessary for seeding. We describe conformational differences between native and synthetic Tau aggregates that may account for the higher seeding activity of native assembled Tau. When added to aggregated Tau seeds from the brains of mice transgenic for P301S Tau, soluble recombinant Tau aggregated and acquired the molecular properties of aggregated Tau from transgenic mouse brain. We show that seeding is conferred by aggregated Tau that enters cells through macropinocytosis and seeds the assembly of endogenous Tau into filaments.

Keywords: Aggregation; Neurodegenerative Disease; Prion; Protein Conformation; Seed-competent Aggregation; Tau Protein; Tauopathy.

© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Figures

FIGURE 1.
FIGURE 1.
A, Western blot with anti-Tau antibodies DA9 (phosphorylation-independent) and AT8 (Ser(P)202/Thr(P)205) of the total lysate and Sarkosyl-soluble and -insoluble fractions from HEK293T cells transiently transfected with or without P301S 1N4R Tau DNA. The cells were exposed for 3 h to increasing amounts of Sarkosyl-insoluble material from the brains of symptomatic TgP301S 0N4R Tau mice, followed by 3 days of incubation. A total lysate loading control for GAPDH is also shown. B, AlphaScreen showing levels of total Tau (DA9) (i and v), conformationally changed Tau (MC1) (ii and vi), and Tau phosphorylated at Ser235 (MC6) (iii and vii) or Ser202/Thr205 (AT8) (iv and viii) in HEK 293T cells transiently transfected with or without human P301S 1N4R Tau, exposed for 3 h to increasing amounts of Sarkosyl-insoluble material from the brains of symptomatic TgP301S 0N4R Tau mice, followed by 3 days of incubation. The results are the means ± S.D. (n = 3). Absolute quantification of the levels of Sarkosyl-soluble (top) and Sarkosyl-insoluble (bottom) Tau cannot be made because the magnitude of the signal is dependent on the aggregation state of Tau. C, PG5 (Ser(P)409)-positive Tau inclusions (green) in inducible HEK 293T cells expressing human P301S 1N4R Tau, following exposure to Sarkosyl-insoluble material from the brains of symptomatic TgP301S 0N4R Tau mice for 3 h followed by 3 days of incubation. Total Tau was visualized with an antibody specific for 1N Tau (phosphorylation-independent) (red).
FIGURE 2.
FIGURE 2.
A and B, time course of propagation (A) and seeding (B) following inoculation of inducible HEK293T cells expressing human P301S 1N4R Tau with the Sarkosyl-insoluble fraction from the brains of non-transgenic (wt) or TgP301S Tau mice. The samples were fractionated by centrifugation of total lysate at 100,000 × g, and the insoluble fraction was analyzed by Western blotting using anti-Tau antibodies DA9 (phosphorylation-independent) and AT8 (Ser(P)202/Thr(P)205). Representative blots from three separate experiments are shown. C, PG5 (Ser(P)409)-positive Tau inclusions in cells, as measured using high-content imaging, confirmed the time dependence of propagation (i–iii) and seeding (iv–vi). The results are the means ± S.D. (error bars) (n = 6); **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 versus unseeded (analysis of variance).
FIGURE 3.
FIGURE 3.
A, AlphaScreen assays showing the levels of DA9 (phosphorylation-independent)-, MC1 (conformationally changed)-, and AT8 (Ser(P)202/Thr(P)205)-positive Tau in the total lysate and the Sarkosyl-insoluble fraction from the brains of symptomatic TgP301S Tau mice. The results are the means ± S.D. (error bars) (n = 3). The levels of AT8- and MC1-positive Tau are expressed relative to those of DA9, taken as 1.0. B, Western blot showing DA9- and AT8-positive Tau in the Sarkosyl-insoluble fraction of HEK 293T cells expressing P301S 1N4R Tau, following the addition of either brain lysate or Sarkosyl-insoluble material (normalized for DA9-positive Tau) for 3 h, followed by 3 days of incubation.
FIGURE 4.
FIGURE 4.
A, cellular uptake of 500 nm fluorescently labeled monomeric recombinant P301S Tau (gray line) and 500 nm fluorescently labeled aggregated recombinant P301S Tau (black line) over 120 min as measured by flow cytometry. Uptake was not significantly different between the two groups. The results are the means ± S.D. (error bars) (n = 3). B, uptake of 500 nm fluorescently labeled monomeric (gray bars) or aggregated (black bars) P301S Tau after 30 min measured by flow cytometry. Incubation at 4 °C or with 100 μm EIPA significantly inhibited the uptake of both monomeric and aggregated Tau. The results are the means ± S.D. (n = 3); ****, p < 0.0001 versus controls (analysis of variance); 10,000 cells/well were analyzed by flow cytometry. RFU, relative fluorescence units. C, Western blot with anti-Tau antibody HT7 (phosphorylation-independent) of the Sarkosyl-insoluble fraction from HEK 293T cells expressing P301S 1N4R Tau, unseeded or seeded for 3 h with 500 nm monomeric or aggregated P301S Tau, followed by 3 days of incubation. A GAPDH loading control is also shown.
FIGURE 5.
FIGURE 5.
A, immunoelectron microscopy with anti-human Tau antibody HT7 (phosphorylation-independent) of the Sarkosyl-insoluble fraction from the brains of symptomatic TgP301S Tau mice and from recombinant P301S Tau assembled with heparin. Recombinant P301S Tau aggregates are shown following a 15-s sonication (Misonix, output 7). Scale bars, 400 nm. B, representative immunoblot with HT7 and quantification of the Sarkosyl-insoluble fraction of HEK 293T cells expressing 1N4R P301S Tau treated for 3 h with TgP301S Tau aggregates or with varying concentrations of recombinant Tau aggregates, followed by 3 days of incubation. At equal concentration (1×; equivalent to 7.2 nm, assuming complete assembly) recombinant P301S Tau did not seed. Over 10-fold higher concentrations of recombinant P301S Tau than TgP301S Tau aggregates were needed to give equivalent levels of seeding. The results are the means ± S.D. (error bars) (n = 3); **, p < 0.01; ***, p < 0.001 versus TgP301S Tau seeded (analysis of variance). C, immunofluorescence showing AT100 (Ser(P)212/Thr(P)214/Thr(P)217)-positive Tau inclusions and thioflavin S staining of HEK 293T cells expressing P301S 1N4R Tau, exposed to 1× TgP301S Tau aggregates or 50× recombinant P301S Tau aggregates. Nuclei were visualized by DAPI (blue). D, immunoelectron microscopy with anti-1N Tau antibody (phosphorylation-independent) of the Sarkosyl-insoluble fraction from HEK 293T cells expressing P301S 1N4R Tau treated for 3 h with 1× TgP301S Tau aggregates or 50× recombinant P301S Tau aggregates, followed by 3 days of incubation.
FIGURE 6.
FIGURE 6.
A, confocal imaging of HEK293T cells exposed to 500 nm TgP301S Tau aggregates or 500 nm recombinant P301S Tau aggregates for 3 h, followed by immunostaining with anti-Tau antibody HT7 (phosphorylation-independent). Punctate, often perinuclear, inclusions of internalized Tau aggregates can be distinguished. B, uptake of 500 nm fluorescently labeled immunopurified TgP301S Tau aggregates and recombinant P301S Tau aggregates after 30 min measured by flow cytometry. Incubation with 100 μm EIPA or 300 nm latrunculin A significantly inhibited the uptake of both TgP301S Tau and recombinant Tau aggregates. The results are the means ± S.D. (error bars) (n = 3); ****, p < 0.0001 versus untreated. RFU, relative fluorescence units; 10,000 cells/well were analyzed by flow cytometry. C, confocal imaging of HEK293T cells exposed to 50 μg/ml fluorescent dextran plus 500 nm TgP301S Tau aggregates or recombinant P301S Tau aggregates for 1 h, followed by immunostaining with HT7. Internalized Tau aggregates co-localized with dextran in large vesicles resembling macropinosomes.
FIGURE 7.
FIGURE 7.
A, black triangles show the aggregation of soluble recombinant P301S Tau in the presence of 5% (v/v) Sarkosyl-insoluble fraction derived from the brains of mice transgenic for human mutant P301S Tau, as measured by thioflavin T binding over time. Heparin was not used. Blue circles and green squares show the thioflavin T traces for soluble recombinant P301S Tau and 5% (v/v) TgP301S Tau aggregates, respectively. RFU, relative fluorescence units. B, Western blots with anti-Tau antibodies DA9 (phosphorylation-independent) and AT8 (Ser(P)202/Thr(P)205) of the insoluble fraction from inducible HEK293T cells expressing P301S 1N4R Tau seeded for 3 h with TgP301S Tau aggregates (at a concentration equivalent to Fig. 5B, 1×); equivalent concentration of recombinant P301S Tau seeded with 5% (v/v) TgP301S Tau aggregates; equivalent concentration of heparin-assembled recombinant P301S Tau aggregates; or the 5% (v/v) TgP301S Tau aggregate component alone followed by 3 days of incubation. C, quantification of the AT8-positive insoluble fraction from B. The values are the means ± S.D. (error bars) (n = 3). Seeding with TgP301S Tau aggregates and recombinant P301S Tau seeded with (5%, v/v) TgP301S Tau aggregates were not significantly different.
FIGURE 8.
FIGURE 8.
A, Western blot with anti-Tau antibodies BR134 (phosphorylation-independent; top panel), Ser(P)422 (panel 2), AT100 (Ser(P)212/Thr(P)214/Thr(P)217; panel 3), and AT8 (Ser(P)202/Thr(P)205; panel 4) of 0N4R P301S recombinant Tau treated with (+ kinases) or without (− kinases) PKA and SAPK4. Sarkosyl-insoluble TgP301S Tau is shown as a control. B, Western blot with anti-Tau antibody HT7 (phosphorylation-independent) of Sarkosyl-insoluble recombinant P301S Tau following treatment with (+ kinases) or without (− kinases) PKA and SAPK4 followed by assembly with heparin. C, Western blot with anti-Tau antibody HT7 of the total lysate (top) and Sarkosyl-insoluble fraction (middle) from unseeded cells (no seed) or cells seeded for 3 h with either TgP301S Tau aggregates (+ TgP301S), unphosphorylated recombinant P301S Tau aggregates (+ unphosphorylated), or hyperphosphorylated recombinant P301S Tau aggregates (+ hyperphosphorylated), followed by 3 days of incubation. GAPDH loading control is shown (bottom).
FIGURE 9.
FIGURE 9.
A, Western blot with anti-Tau antibody BR135 (phosphorylation-independent) of TgP301S Tau aggregates and heparin-assembled recombinant P301S Tau aggregates after digestion with increasing concentrations of proteinase K (PK). Recombinant P301S Tau aggregates were more resistant to proteinase K digestion than TgP301S Tau aggregates. B and C, Western blots with anti-Tau antibody HT7 (phosphorylation-independent) of heparin-assembled recombinant P301S Tau aggregates (P301S Tau + heparin; red squares); hyperphosphorylated, heparin-assembled recombinant P301S Tau aggregates (hyperphosphorylated P301S Tau + heparin; green diamonds); TgP301S Tau aggregates (blue circles); and recombinant P301S Tau seeded with 5% (v/v) TgP301S Tau aggregates (P301S Tau + 5% (v/v) TgP301S Tau aggregates, no heparin; orange triangles) following treatment with increasing concentrations of guanidine hydrochloride (GdnHCl) and centrifugation at 100,000 × g to pellet remaining Tau aggregates. Unphosphorylated and hyperphosphorylated recombinant P301S Tau aggregates were resistant to treatment with up to 6 m guanidine hydrochloride; TgP301S Tau aggregates and recombinant P301S Tau seeded with TgP301S Tau aggregates were partially solubilized with as little as 2 m guanidine hydrochloride, and their solubilization increased in a concentration-dependent manner. C, quantification of B. The results are the means ± S.D. (error bars) (n = 3; *, p < 0.05; ***, p < 0.001 versus P301S Tau + heparin (analysis of variance)).
FIGURE 10.
FIGURE 10.
A, schematic diagram of the 0N4R Tau constructs used. B, Western blot with anti-Tau antibody BR133 (phosphorylation-independent) of HEK293T cells expressing full-length 0N4R Tau or 0N4R Tau 1–187, which were seeded for 3 h with TgP301S Tau aggregates, followed by 3 days of incubation. C, Western blot with anti-Tau antibody BR134 (phosphorylation-independent) of HEK293T cells expressing full-length 0N4R Tau or 0N4R Tau 188–383, which were seeded for 3 h with TgP301S Tau aggregates followed by 3 days of incubation. D, Western blot with BR134 of HEK293T cells expressing either 0N4R Tau or 0N4R Tau ΔPHF6/6*, which were seeded for 3 h with TgP301S Tau aggregates, followed by 3 days of incubation.

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