Fig. 1

Effect of r-mTBI on the elimination of exogenous tau species from the brain. A The time course of tau elimination from the brain was established by examining biotin-labeled tau (n = 6) and 10 kDa LyD (n = 5) levels in the brain at various time points following intracranial injection into naïve wild-type mice (9 months of age). Biotin-labeled tau content was analyzed using an ELISA and LyD was analyzed via fluorescence. The half-life for both biotin-labeled tau and LyD were determined using nonlinear regression and a one phase decay fit. B Following intracortical injection in r-sham (n = 5–8) and r-mTBI mice (n = 4–8) (12 months post-injury), the amount of exogenous biotin-labeled tau species residing in the brain was determined at 2 h post-injection. For each injection, biotin-labeled tau was co-injected with LyD. Biotin-labeled tau content was analyzed using an ELISA while LyD was analyzed via fluorescence. Values represent mean ± SD (n = 4–8) and are expressed as pg of tau per μg of LyD. **P < 0.01, ***P < 0.001 as compared to r-sham as determined by a two-way ANOVA and Bonferroni post-hoc test. C–E In characterizing each tau species, biotin-labeled tau (4.35 µM) was incubated with heparin (1 µM) for 6 h at 37 °C to induce aggregation. The aggregates were separated using a 100 kDa molecular weight cutoff filter into aggregate enriched (tau > 100 kDa) or seed competent (tau < 100 kDa) fractions. C The conformational status of the seed competent (50 ng/ml) and aggregate enriched (50 ng/ml) biotin-labeled tau fractions were compared to monomeric (50 ng/ml) biotin-labeled tau via dot blot using an MC1 antibody. D Quantitative analysis of MC1 immunoreactivity in the dot blot. E Heparin-induced in vitro aggregation of monomeric biotin-labeled tau (4.35 µM) was assessed using the indicator dye ThS. ThS fluorescence was measured in the presence of monomeric biotin-labeled tau (4.35 µM) immediately after the addition of heparin (1 µM) or vehicle (0 h), and again after 6 h. Values represent the change in ThS fluorescence (excitation 450 nm, emission 510 nm) over the 6 h period