Short Fibrils Constitute the Major Species of Seed-Competent Tau in the Brains of Mice Transgenic for Human P301S Tau

Abstract

The interneuronal propagation of aggregated tau is believed to play an important role in the pathogenesis of human tauopathies. It requires the uptake of seed-competent tau into cells, seeding of soluble tau in recipient neurons and release of seeded tau into the extracellular space to complete the cycle. At present, it is not known which tau species are seed-competent. Here, we have dissected the molecular characteristics of seed-competent tau species from the TgP301S tau mouse model using various biochemical techniques and assessed their seeding ability in cell and animal models. We found that sucrose gradient fractions from brain lysates seeded cellular tau aggregation only when large (>10 mer) aggregated, hyperphosphorylated (AT8- and AT100-positive) and nitrated tau was present. In contrast, there was no detectable seeding by fractions containing small, oligomeric (<6 mer) tau. Immunodepletion of the large aggregated AT8-positive tau strongly reduced seeding; moreover, fractions containing these species initiated the formation and spreading of filamentous tau pathology in vivo, whereas fractions containing tau monomers and small oligomeric assemblies did not. By electron microscopy, seed-competent sucrose gradient fractions contained aggregated tau species ranging from ring-like structures to small filaments. Together, these findings indicate that a range of filamentous tau aggregates are the major species that underlie the spreading of tau pathology in the P301S transgenic model. Significance statement: The spread of tau pathology from neuron to neuron is postulated to account for, or at least to contribute to, the overall propagation of tau pathology during the development of human tauopathies including Alzheimer's disease. It is therefore important to characterize the native tau species responsible for this process of seeding and pathology spreading. Here, we use several biochemical techniques to dissect the molecular characteristics of native tau protein conformers from TgP301S tau mice and show that seed-competent tau species comprise small fibrils capable of seeding tau pathology in cell and animal models. Characterization of seed-competent tau gives insight into disease mechanisms and therapeutic interventions.

Keywords: aggregation; propagation; tau; tauopathy.

Figure 1.

A, Sucrose gradient centrifugation of TgP301S tau mouse brain lysates to separate soluble and insoluble tau. Western blot with anti-tau antibodies DA9, HT7 (both phosphorylation-independent), AT8 (phosphorylation at pS202/pT205), and AT100 (phosphorylation at pS212/pT214/pT217) of symptomatic (24.4 weeks) and presymptomatic (4.4 weeks) total-brain lysate following sucrose gradient fractionation for 4 h; gels loaded with 5 μg/well of each fraction. Representative blots from 3 independent sucrose gradient fractionations are shown. Filamentous tau runs at ∼64 kDa (high-molecular weight; HMW). Nonfilamentous tau runs at ∼55 kDa (low-molecular-weight; LMW). B, Representative Western blot of Sarkosyl-insoluble AT8-positive tau in sucrose gradient fractions of symptomatic TgP301S mouse brain. C, Seeding of tau aggregation with sucrose gradient fractions in a cell-based assay. Sucrose gradient fractions were used to seed aggregation of tau in HEK293T cells overexpressing 1N4R tau with the P301S mutation. The pellet from a 100,000 × g spin of the seeded cells was analyzed by Western blotting using anti-tau antibodies AT8 and DA9. A representative blot is shown; similar results were obtained in three separate experiments. Filamentous tau runs at ∼68 kDa (HMW). Nonfilamentous tau runs at ∼59 kDa (LMW). Positive control was seeding with Sarkosyl extracted tau from symptomatic TgP301S mice as described in Falcon et al. (2015) and the normalized positive control was seeding with Sarkosyl extracted tau from symptomatic TgP301S mice, normalized for DA9 tau levels to those of the symptomatic sucrose gradient fractions. Seeding ability correlated with the presence of the 64 kDa, hyperphosphorylated tau band in symptomatic (24.4 weeks) mice. No seeding was observed upon addition of the sucrose gradient fractions from presymptomatic (4.4 weeks) mice.

Figure 2.

A, The AT8 normalized sucrose gradient fractions (30–50% fractions) were tested for seeding efficiency in the cell-based assay. Western blots with AT8 and DA9 was used to detect seeding in the insoluble cell fraction following incubation with sucrose fractions. B, Quantification of the Western blot shown in A (left) and AlphaScreen analysis (right). The results are expressed as means ± SEM (n = 3). **p < 0.01, ****p < 0.0001 (one-way ANOVA + Dunnett's post hoc test relative to 40% fraction). The results confirmed that the 40% sucrose gradient fraction seeded significantly better than the 30 and 50% fractions.

Figure 3.

Immunoelectron microscopy of sucrose gradient fractions from the brains of presymptomatic (4.4 weeks) and symptomatic (24.4 weeks) TgP301S tau mice. Anti-tau antibody HT7 (phosphorylation-independent) was used to detect tau. A, For presymptomatic mice, only sparse labeling was observed in the Top, 10–50% sucrose gradient fractions. B, For symptomatic mice, tau filaments were present in in the 50% (250 ± 15 nm, n = 25) and 40% fractions (179 ± 12 nm, n = 25). The 30% fraction contained globular, ring-like tau-positive structures. The 20% fraction comprised a mixture of sparse labeling and small, ring-like tau-positive structures. The Top and 10% fraction showed only sparse tau labeling. A typical experiment is shown; similar results were obtained in three separate experiments. Scale bar, 50 nm.

Figure 4.

Nondenaturing gradient gel electrophoresis of sucrose gradient-fractionated TgP301S tau mouse brain lysates. Four to 22% nondenaturing gels of presymptomatic (A) and symptomatic (B) TgP301S sucrose gradient fractions was performed (5 μg/well of protein normalized fractions). Western blots with phosphorylation-independent anti-tau antibody DA9, phosphorylation-dependent antibodies AT8 and PHF1, and an antibody specific for tau nitrated at Y29 (nY29). In the presymptomatic mice, the Top, 10%, and 20% fractions, tau species were recognized by the phosphorylation-independent pan tau antibody, DA9. Some of these tau species showed low-level reactivity with AT8 and nY29 antibodies but were PHF1-negative. In symptomatic mice, in the Top, 10%, and 20% fractions a seeding-incompetent tau species of ∼400 kDa was recognized by AT8 and nY29 (black arrow).In the 30–50% fractions, tau species were unable to migrate into the gel (HMW species; red arrow) and were recognized by all four antibodies. Representative blots from three independent experiments shown.

Figure 5.

Seeding efficiency correlates with level of 64 kDa hyperphosphorylated tau. A, Western blot with AT8 or DA9 antibodies and (B) quantification of brain lysates from symptomatic TgP301S tau mice (24.4 weeks) following immunodepletion with phosphorylation-independent anti-tau antibody DA9 or phosphorylation-dependent antibodies AT8, PG5, and PHF1. IgG1 served as the control. A representative blot from three separate experiments is shown, each with three replicates per condition. GAPDH was used as the loading control. Immunodepletion reduced levels of the 64 kDa (HMW) AT8-positive band by all antibodies to varying degrees (AT8 > PHF1 > PG5 = DA9); only DA9 significantly reduced the 55 kDa (low-molecular-weight; LMW) tau species. The quantification results are the mean ± SEM (n = 6); 64 kDa band quantified for AT8 blot, and the sum of 64 and 55 kDa bands quantified for DA9. C, Western blot and quantification (D) with anti-tau antibodies DA9 and AT8 of the insoluble cell fraction, following seeding with immunodepleted samples. Positive control was Sarkosyl extracted tau from symptomatic TgP301S mice as described by Falcon et al. (2015). A representative blot from three separate experiments is shown. D, Results are the means ± SEM of three separate experiments; 68 kDa band quantified for AT8 blot, and the sum of 68 and 59 kDa bands quantified for DA9. Statistical analysis: one-way ANOVA with Dunnett's post hoc test. *p < 0.05, ****p < 0.0001, compared with IgG1. E, The levels of 64 kDa, AT8-positive tau in the brain lysate correlated with the seeding ability measured by Western blotting. No correlation was observed with DA9-positive tau as the input. Linear regression was performed to determine the relationships between input and output tau (r² = 0.8531, p < 0.0001, n = 55 for AT8-positive tau).

Figure 6.

Tau-species in the 40% sucrose gradient fraction from the brains of symptomatic TgP301S tau mice (24.4 weeks) are key mediators of seeding and spreading. A, Unilateral infusion of brainstem lysate (positive control) from symptomatic TgP301S mice into the hippocampus of asymptomatic TgP301S mice resulted in the robust accumulation and spread of PG-5-positive tau pathology in the infused (∨) and contralateral (∧) hippocampus, respectively. Mice infused with brainstem lysate from wild-type (negative control) mice had minimal tau pathology in the infused or contralateral hippocampus. Infusion of the 40% sucrose gradient fraction also showed substantial induction and spread of PG-5-positive tau pathology, whereas mice infused with the 10% sucrose gradient fraction were indistinguishable to wild-type lysate infused mice. Infusing a preparation of TgP301S brainstem lysate that was normalized to have comparable DA9 levels (total tau) to the 10 and 40% sucrose gradient fractions did not show robust tau propagation. B, Quantification of PG-5-positive immunoreactivity in the infused hippocampus showed significantly higher levels of tau pathology in mice infused with TgP301S brainstem lysate compared with all other groups and a significant increase associated with the 40% sucrose gradient fraction when compared with wild-type and 10% sucrose groups. C, Analysis of the contralateral hippocampus showed a similar pattern albeit tau pathology milder than the infused hippocampus. Images shown in A are representative of the group mean. Scale bar, 500 μm. Graphs in B and C display group mean ± SEM. Statistical analyses: one-way ANOVA and least significance difference post hoc test; *p <0.05, **p <0.01, #p <0.01 for all groupwise comparisons.