Fig 1: Characterization of tau fibrils extracted from human AD subjects.A, immunoblots for total tau (HT7) and pTau-Thr231 (AT180) of the tau extracts used in this study (samples were as follows: C: control, A: brain A, B: brain B, AB: 1:1 mixture of brain A and B extracts, D: brain D, and Dconc: 10× concentrated brain D). B, overview of total protein and total tau concentration in each extract. Total protein concentration was evaluated by measuring absorption at 280 nm on a spectrophotometer. Total tau concentration was calculated by comparing densitometric quantifications of total tau (HT7) immunoblots to a dilution curve of recombinant tau (2N4R isoform). C, immunoblots for total tau (HT7) and pTau-Thr231 (AT180) for samples from brain A, B, and a 1:1 mixture of brain A and B (AB) to assess the tau isoform distribution and phosphorylation over a 60-h time course with measurements taken every 12 h. D, tau extracts were diluted in PBS to a concentration of 5 μg/ml, and 5 × 5 μm images were taken by atomic force microscopy (the scale bar represents 0.5 μm). Fibril length was quantified using Gwyddion. N(A) = 219 fibrils from two images, N(B) = 260 fibrils from three images, N(AB) = 494 fibrils from three images, N(D) = 116 fibrils from two images, and N(Dconc) = 225 fibrils from two images. E, primary mouse neurons were seeded at 50,000 to 75,000 density, treated with 0.25% (v/v) human tau extracts at DIV 7 and incubated until DIV 21 (the scale bar represents 50 μm). Cells were fixed in ice-cold methanol and stained for MAP2 and mouse tau (T49). Tau seeding was measured by quantifying the percent area occupied by aggregated mouse tau within MAP2-positive area using ImageJ. Statistics: Brown–Forsythe ANOVA test (F = 43.85; p < 0.0001) with Dunnett's multiple comparisons test to compare all groups to control-treated cells. N = 8 images per condition. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.005, and ∗∗∗∗p < 0.0001. AD, Alzheimer's disease; DIV, days in vitro.
Fig 2: Tau seeding correlates with oligomeric 3R and 4R tau levels.a Schematic diagram of the epitopes of tau antibodies used for western blot analysis. b, c Representative western blots of ITG b and PFC c lysates from twenty AD cases (subject no. 1–5, 11–15, 6–10, 16–20) developed with total tau (Tau13), anti-3R and 4 R tau antibodies. d, e Immunoblots were densitometrically quantified, normalized by total protein and HMW-3R and 4 R tau levels in the ITG d and PFC e were correlated with seeding. Two-tailed Spearman’s rank non-parametric correlation tests were used, and r coefficient and P values are indicated on the tables. f Correlation of the ratio of 3R-tau/4R-tau in the ITG from twenty AD cases with seeding is shown. A two-tailed Spearman’s rank non-parametric correlation test was used, and r coefficient and P values are indicated on the plot. g, h Quantification of HMW and LMW-total tau and isoform levels in the ITG of low and high seeders, represented as fold change of low seeders. Data are presented as mean ± SEM; n = 6 AD cases per group. *P < 0.05, **P < 0.01, ***P < 0.001 (t test). (i) Relative 3 R/4 R HMW-tau ratio in the ITG from six low and six high seeders was detected by immunoblots. Results represent the mean ± SEM; ***P < 0.001 (t test).
Fig 3: Tau seeding bioactivity correlates with cognitive decline in AD.a Graphs showing dementia progression in patients from Banner Sun Health Research Institute (cohort 1) and Mount Sinai ADRC (cohort 2). A line corresponds to a single patient and each dot to a visit at the research clinic. The patients were evaluated for their MMSE (cohort 1) or CDR-SOB (cohort 2) scores at each visit. b Schematic representation of the in vitro FRET-based tau seeding assay. c Representative 40× confocal images of FRET-biosensor cells showing no seeding, low and high tau seeding after treatment with a control and AD soluble brain lysates, with FRET-positive tau inclusions shown in bright green and nuclear DAPI staining in blue. Scale bar, 20 µm. d, e Graphs representing tau seeding activity (% FRET-positive cells) induced by ITG d and PFC e. Brain lysates from one cognitively unimpaired control and twenty patients were used. f, g Tau seeding induced specifically by ITG lysates f were significantly correlated with the rate of cognitive decline in both cohorts while the correlation between seeding by PFC lysates and cognitive decline was not significant g, determined using a two-tailed Spearman’s rank non-parametric test, P and r values are indicated on each plot.
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