Distinct differences in prion-like seeding and aggregation between Tau protein variants provide mechanistic insights into tauopathies

Seeding propensity differs significantly among FTDP-17 Tau mutants

The ability of exogenously added preformed fibrils to seed Tau intracellular inclusions in cell culture has been demonstrated in a number of different cell lines and models (29–31, 46, 48). Using a human embryonic kidney 293T (HEK293T) cell Tau seeding model, we initially sought to assess and compare the propensity of 15 different FTDP-17 Tau mutants (Figs. 1 and 2) to be seeded to form inclusions. HEK293T cells were transfected with plasmids encoding WT full-length Tau (0N/4R) protein and FTDP-17 mutants thereof and inoculated with WT K18 Tau fibrils. Cells were harvested at 48 h post-transfection and fractionated into Triton-soluble and Triton-insoluble fractions. The accumulation of Tau in the Triton-insoluble pellets indicates the intracellular seeding and induction of intracellular Tau inclusion formation (48). Furthermore, we observed similar results using sarkosyl solubility fractionation (Fig. S1). Sharp differences among the first series of mutants analyzed was observed, with only P301L, P301S, and S320F Tau showing significant induced aggregation (Fig. 2, A and B; p < 0.01, p < 0.01, and p < 0.05, respectively). P301L and P301S Tau showed similar amounts of aggregation, whereas S320F Tau aggregated to a lesser extent. Furthermore, in the absence of WT K18 fibrils, S320F Tau transfected cells displayed a small amount of self-aggregation (p < 0.01), which was not observed for the other mutants or WT Tau (Fig. 2, A and C).

Next, because there is evidence to suggest that Tau pathologies could transmit in a “strain-dependent manner” (4, 33, 41, 42) and that Tau mutants can cause Tau to form conformationally distinct fibrils (43, 49), we sought to assess whether homotypic seeding may be more robust or necessary for some of the Tau mutants to aggregate. The previous experiment was thus repeated, this time comparing heterotypic seeding with WT K18 Tau fibrils to homotypic seeding with mutant K18 Tau fibrils. For the mutants that previously showed aggregation with seeding (P301L, P301S, and S320F), there was no difference in the level of Tau aggregation when WT or homotypic mutant K18 Tau fibrils were added (Fig. 3, A and B). Furthermore, the mutants that previously did not aggregate with WT K18 seeds also were not induced to aggregate with their respective mutant K18 fibrils (Fig. 3C; data not shown). To further compare the ability of these different Tau mutant fibrils to induce aggregation, P301L 0N/4R Tau-transfected cells were seeded with various mutant K18 fibrils and analyzed for their levels of aggregation. All mutant K18 fibrils were able to induce a similar level of aggregation of P301L Tau, with Δ280K fibrils causing slightly less aggregation (Fig. S2).

Next, we compared WT and P301 mutant Tau seeding in either the 0N/4R or 2N/4R Tau isoform, which can be resolved as discrete bands on Western blots because of their distinct molecular masses. Similar to 0N/4R Tau, WT 2N/4R Tau did not show a propensity to aggregate with seeding, whereas P301L 2N/4R Tau seeding–induced aggregation was observed (Fig. 4). Mutant P301L 2N/4R Tau was co-expressed with WT 0N/4R Tau and seeded with WT K18 fibrils to ascertain whether intracellular WT Tau can be recruited into the insoluble portion by intracellular P301L Tau aggregates, which can be discerned based on their different molecular masses. Even in the presence of intracellular P301L 2N/4R Tau aggregates, WT Tau was not recruited into the inclusions (Fig. 4).

P301 inhibition of Tau aggregation is relatively unique and context dependent

To further investigate the role and specificity of P301 in modulating the aggregation propensity of Tau in this cell model, proline residues located in each of the four microtubule binding repeats in Tau were mutated to leucine. Each proline amino acid is the first residue in a conserved PGGG motif located near the end of each MT-binding repeat and is thought to play an important role in tubulin binding, with mutations at these sites altering the binding ability of Tau (13–16, 50, 51). It is noteworthy that mutants P332S and P364S, which are relatively identical mutations to P301S within these PGGG motifs, did not influence the propensity of Tau to aggregate (Fig. 2). However, to specifically compare the effects of P to L mutations at each PGGG motif, P270L, P301L, P332L, and P364L 2N/4R Tau mutants were expressed with the addition of K18 seeds (Fig. 5A). Only the P301L Tau mutant demonstrated a propensity to be seeded in cells (Fig. 5B).

To further investigate the influence of Pro³⁰¹ and its local surrounding molecular environment, we extended our studies to other FTDP-17 Tau mutants in this region. P301T also demonstrated a similar propensity to aggregate in cells under seeding conditions, but not the G303V, G304S, or S305N mutants (Fig. 6).

Given that all three pathogenic mutations at the Pro³⁰¹ site uniquely enhance aggregation of Tau, we decided to assess whether the loss of the proline residue in this region was sufficient to replicate this finding. Indeed, the deletion of Pro³⁰¹ (ΔP301) was sufficient to promote the aggregation of Tau under seeding conditions (Fig. 7A). Furthermore, mutating the neighboring residue, Gly³⁰², to a proline in P301L Tau (P301L/G302P double mutant) was sufficient to impair the propensity of P301L Tau to aggregate (Fig. 7B). Pro³⁰¹ is in close proximity to an established sequence of Tau termed “PHF6” (Fig. 1) that is important for both aggregation and, specifically, β-pleated sheet formation (37–40). Located in the beginning of the third microtubule binding repeat, PHF6 is adjacent to the Pro³⁰¹ site, and only nine residues away from the Ser³²⁰ site, which, when mutated, also showed robust aggregation with seeding. We assessed another proline residue near the PHF6 site, Pro³¹², to determine whether it affected Tau in a manner similar to Pro³⁰¹. However, the P312L mutation did not increase the propensity of Tau to aggregate in the seeded cell assay (Fig. 7C). We previously showed that P270L within 4R Tau did not promote Tau aggregation (Fig. 5). However, in 3R Tau, this Pro²⁷⁰ residue is located in a similar position to PHF6 as the Pro³⁰¹ residue in 4R Tau, because of the absence of the R2 MT-binding repeat (Figs. 1 and 7D). Thus Pro²⁷⁰ was changed to a leucine in 0N/3R Tau, and the seeding experiment was performed, but P270L 0N/3R Tau also did not demonstrate a propensity to be seeded by WT K18 fibrils (Fig. 7D). Because it is suggested that certain cross-seeding barriers between 3R and 4R Tau exist (52), this P270L 0N/3R Tau mutant was also seeded with K19 Tau fibrils (i.e. truncated Tau containing three rather than four MT-binding repeats) (Fig. 1). A small amount of Tau aggregation was observed upon treatment with K19 fibrils, compared with no aggregation when treated with K18 fibrils (Fig. 8, A and B). Interestingly, treatment with K18 fibrils was much more sufficient at seeding P301L 0N/4R Tau compared with K19 fibrils (Fig. 8, C and D).

The P301L/S320F double mutant results in robust aggregation even in the absence of exogenous Tau fibrils

To investigate whether the P301L and S320F mutations could have an additive or synergistic effect, the propensity of P301L/S320F Tau double mutant on Tau aggregation with or without exogenous WT K18 fibrils was assessed. Of note, both of these sites flank the PHF6 site. Expression of P301L/S320F Tau demonstrated robust aggregation without needing exogenous WT K18 seeds (Fig. 9, A–D). In fact, there was no discernable difference between the amount of Triton-insoluble Tau with or without WT K18 Tau fibril treatment. Amyloid inclusion formation by P301L/S320F Tau was further confirmed with thioflavin S staining that was not observed with WT Tau or the single Tau mutants (Fig. 10). To ensure that this effect was due to this specific combination of mutations, we also created the double mutants P301L/A152T and P301L/L266V. Unlike P301L/S320F, P301L/A152T and P301L/L266V Tau showed no aggregation without seeding (Fig. 9E). The double mutant P301S/S320F was also tested and aggregated without seeding at levels similar to P301L/S320F Tau (Fig. 9, F and G).

To assess whether the P301L/S320F double mutant could fibrillize in vitro without the use of a polyanionic inducer such as heparin (53), WT, P301L, S320F, and P301L/S320F 0N/4R Tau proteins were incubated with or without heparin with shaking at 37 °C for 8 days. Although thioflavin T analysis showed no significant amyloid formation for any of the groups without heparin at 8 days (Fig. 11A), electron microscopy showed sparsely populated Tau fibrils for the P301L and S320F single mutants and the P301L/S320F double mutant (Fig. 11B). However, these were rare compared with Tau proteins incubated with heparin (Fig. 11B).

Tau mammalian expression plasmids

The cDNAs encoding the full-length 0N/4R or 2N/4R human Tau isoforms were cloned in the mammalian expression vector pcDNA3.1(+). The different missense MAPT mutations were created through QuikChange site-directed mutagenesis (Agilent Technologies, Santa Clara, CA) using mutant-specific oligonucleotides. All mutations and the absence of errors throughout the entire length of the Tau cDNA was confirmed by Sanger sequencing performed as a service by the Interdisciplinary Center for Biotechnology Research at the University of Florida.

Expression and purification of recombinant Tau

The cDNA corresponding to the human Tau K18 fragment (residues Gln²⁴⁴–Glu³⁷² in 2N/4R human Tau) with an added methionine residue at the N terminus cloned in the bacterial expression plasmid pRK172 was used for expression in BL21 (DE3)/RIL Escherichia coli (Agilent Technologies). The K19 Tau fragment, which does not code the R2 repeat (Fig. 1), was similarly generated. pRK172 plasmids expressing the various missense Tau mutations within the K18 fragment were created by site-directed mutagenesis as described above. K18 Tau proteins were purified as previously described for Tau proteins (77). Protein concentrations were determined using the bicinchoninic acid assay (Pierce) and BSA as the standard.

Assembly of recombinant Tau fibrils

Recombinant K18, K19, or 0N/4R Tau proteins (1 mg/ml) were assembled into filaments by incubation at 37 °C in sterile PBS with 50 μm heparin (53, 78) while shaking at 1050 rpm with an Eppendorf Thermomixer R for at least 48 h. Tau amyloid fibril formation was confirmed by K114 or thioflavin T fluorometry as previously described (48, 79, 80). For the thioflavin T assays, 5 μl of each sample were diluted into a 50 μm working concentration of thioflavin T in 0.1 m glycine, pH 8.6. To remove the heparin, Tau fibrils were centrifuged at 100,000 × g and resuspended in sterile PBS, and the resulting protein concentrations were determined using bicinchoninic acid assay. Tau filaments were fragmented into shorter Tau “seeds” by bath sonication for 60 min as previously described (48).

Cell culture and transfection

HEK293T cells were maintained with Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS), 100 units penicillin/ml, and 100 μg streptomycin/ml, at 37 °C and 5% CO₂. The cells were plated on polystyrene 6- or 12-well plates and transfected with the various plasmids expressing 0N/4R or 2N/4R human Tau using calcium phosphate precipitation at ∼30–50% confluency. For transfection per 2 ml of cell culture media, 3 μg of plasmid DNA was diluted into 37.5 μl of 1 m CaCl₂ and stepwise added to an equal volume of 50 mm N,N-bis(2-hydroxymethyl)-2-aminoethanesulfonic acid, 280 mm NaCl, 1.5 mm Na₂HPO₄, pH 6.96. This mixture was incubated at room temperature for 15–20 min before adding dropwise to the media in each well. For cellular Tau seeding studies, K18 or K19 Tau fibrils (final concentration, 1 μm) were added 1 h following the beginning of the transfection (48). The cells were either harvested for biochemical fractionation or fixed for immunofluorescence staining 48 h thereafter.

Biochemical cellular fractionation

The cells were washed with PBS and harvested in 400 μl for 6-well plates and 200 μl for 12-well plates of CSK buffer (25 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1 mm EDTA, 1% Triton X-100, 20 mm NaF) and a mixture of protease inhibitors (1 mm phenylmethylsulfonyl fluoride and 1 mg/ml each of pepstatin, leupeptin, N-tosyl-l-phenylalanyl chloromethyl ketone, N-tosyl-lysine chloromethyl ketone, and soybean trypsin inhibitor). The samples were sedimented at 100,000 × g for 30 min at 4 °C, and the supernatants were collected. To ensure complete removal of the supernatant, the pellets were washed with 400 μl (6-well plates) or 200 μl (12-well plates) of CSK buffer and underwent 100,000 × g centrifugation. The supernatants were completely removed, and the pellets were resuspended in 400 μl (6-well plates) or 200 μl (12-well plates) of CSK buffer. 5× SDS sample buffer (final concentration of 10 mm Tris, pH 6.8, 1 mm EDTA, 40 mm DTT, 0.005% bromphenol blue, 0.0025% pyronin yellow, 1% SDS, 10% sucrose) was then added to both the CSK buffer–soluble and –insoluble fractions (referred to as the Triton-soluble and -insoluble fractions), and the samples were heated at 100 °C for 10 min and probe-sonicated for the Triton-insoluble samples. For sarkosyl fractionation, the cells were washed with PBS and harvested from 6-well plates in 400 μl of lysis buffer containing 1% sarkosyl, 10 mm Tris-HCl, 150 mm NaCl, 1 mm EGTA, and 5 mm EDTA, pH 7.4, with protease inhibitor as previously described (81). The cell suspensions were syringe-sheared with a 27-gauge needle, followed by incubation on ice for 15 min, bath sonication two times for 2 min, and incubation at room temperature for 20 min. The samples were sedimented at 100,000 × g for 1 h. The supernatants were saved as the sarkosyl-soluble fractions, whereas the sarkosyl-insoluble pellets were resuspended in 1% sarkosyl with 5× SDS sample buffer, heated for 10 min at 100 °C, and probe-sonicated.

Antibodies

Total Tau antibodies used in these experiments were H150 (Santa Cruz, Dallas, TX), a rabbit polyclonal antibody raised against amino acids 1–150 of human Tau, and rabbit polyclonal antibody 3026 raised against full-length recombinant 0N/3R human Tau (82). Additionally, AT8 (Thermo Fisher) is a mouse monoclonal antibody that reacts with Tau phosphorylated at Ser²⁰² and Thr²⁰⁵ (83).

Western blot analysis

Equal volumes of Triton-soluble and Triton-insoluble protein fractions or sarkosyl-soluble and sarkosyl-insoluble protein fractions were loaded onto 10% polyacrylamide gels and resolved by SDS-PAGE, followed by electrophoretic transfer onto nitrocellulose membranes. The membranes were blocked in 5% dry milk/TBS (50 mm Tris, pH 7.5, 150 mm NaCl) and incubated overnight at 4 °C with primary antibody diluted in 5% dry milk/TBS or 5% BSA/TBS for the phospho-specific AT8 antibody. After washing, membranes were incubated in goat anti-rabbit or anti-mouse secondary antibodies conjugated to horseradish peroxidase (Jackson ImmunoResearch Laboratories) diluted in 5% dry milk/TBS for 1 h, and immunoreactivity was detected using Western Lightning-Plus ECL reagents (PerkinElmer) followed by chemiluminescence imaging (PXi; Syngene, Frederick, MD). ImageJ software was then used to quantify the signal of the resulting protein bands, and the results are represented as the percentages of Triton-insoluble Tau over total Tau, as previously described (48). Statistical analysis was done in GraphPad Prism using either a Student's t test or one-way analysis of variance (ANOVA), with post hoc analysis using Dunnett's test to compare each “group” (i.e. mutant) to our WT control.

Immunofluorescence analyses

For immunofluorescence staining, the cells were plated onto poly-d-lysine–coated glass coverslips. The cells were rinsed with PBS and fixed with 4% paraformaldehyde for 10 min, followed by PBS washes. The cells were blocked with PBS, 2% FBS, 0.1% Triton for 30 min, followed by the application of primary antibody diluted in PBS, 2% FBS for 1 h at room temperature. The cells were washed with PBS before adding the secondary antibodies, conjugated to Alexa fluorophores 488 or 594 (Thermo Fischer Scientific, Waltham, MA). The cells were washed with PBS, stained with 4′,6′-diamidino-2-phenylindole, and mounted using Fluoromount-G (Southern Biotech, Birmingham, AL). For thioflavin S staining, a stock solution of 1% thioflavin S (Sigma–Aldrich) in 50% ethanol in PBS was filtered through a 0.2-μm filter and kept foil-wrapped. The cells were rinsed with PBS and fixed with 4% paraformaldehyde for 10 min. Millipore autofluorescence reagent (Millipore, Billerica, MA) was applied to cells for 5 min, followed by washing with 40% ethanol. The cells were incubated with 0.0125% thioflavin S in 50% ethanol, PBS for 3 min in the dark, followed by sequential washing with 50% ethanol and PBS. The cells were then blocked, and the primary and secondary antibodies were sequentially applied as described above, keeping the samples in the dark when possible. The coverslips were similarly stained with 4′,6′-diamidino-2-phenylindole and mounted using Fluoromount G (Southern Biotech). The images were captured using an Olympus BX51 fluorescence microscope mounted with a DP71 digital camera (Olympus, Center Valley, PA). Cell count analysis was performed with ImageJ software.

Electron microscopy imaging

The samples were adsorbed onto 300-mesh carbon coated copper grids (Electron Microscopy Sciences, Hatfield, PA), negatively stained with 1% uranyl acetate, and visualized with a Hitachi 7600 transmission electron microscope at 50,000× magnification.