Characterization of TauC3 antibody and demonstration of its potential to block tau propagation

Figures

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  Fig 1. High molecular weight tau from Alzheimer’s disease brain is C-terminally truncated. A. Western blot probed with Tau46 (C-terminal Tau antibody) and Tau13 (N-terminal antibody) showing the HMW fraction isolated from human AD brain tissue does not contain the full-length Tau (441 aa). B. Quantification of Tau13 and Tau46 from AD HMW fractions (n = 4 AD cases), showing that the amount of HMW tau that contains the C-terminal Tau46 epitope is significantly lower than the Tau13 N-terminal epitope. Dashed red lines indicate the same human case. Absolute amounts are calculated by normalization against a recombinant human full length tau standard. C. SDD-AGE analysis of a Control and AD case for total tau and TauC3 show that only the AD case has TauC3 signal in the High Molecular Weight (HMW) fraction. Error bars represent Mean +/- SEM. Two-tailed student t-test. *p<0.05.

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  Fig 2. Anti-TauC3 antibody is highly specific for TauC3 protein. A. Coomassie stained SDS—PAGE (left) and western-blot (right) probing recombinant Tau species (the Tau repeat domain, the N-terminal domain, TauC3, and full-length tau) using Anti-TauC3 antibody. B-C. Representative SPR binding curves of the Anti- TauC3 antibody to recombinant TauC3 (B) and full-length 4RTau (C).

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  Fig 3. Anti-TauC3 antibody is efficient at blocking tau seeding activity in HEK293 cells. (A). TauC3 positive AD lysate was pre-incubated with a PBS control, mid-domain tau (HT7), or TauC3 antibody and added to HEK293 cells that were transfected with the TauFRET2 construct. Cells were incubated with the lysate/antibody mixture for 60 hours and then analyzed on the flow cytometer. The relative tau seeding activity is the percent FRET positive cells multiplied by the median fluorescence intensity of the FRET-positive population, normalized to the PBS control mean. (B). Representative images of tau aggregates using the GFP channel. One-way ANOVA, Sidak post-hoc multiple comparisons. Error bars represent mean +/- SEM. ***p<0.001, ****p<0.0001.

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  Fig 4. Anti-TauC3 antibody recognizes a significant portion of tau seeding activity from human AD lysate in a neuronal aggregation assay. (A). Immunodepleted fractions were added to TauFRET2 DNA transfected neuronal-like murine N2A cells for 36 hours and analyzed for FRET activity. The relative tau seeding activity is the percent FRET positive cells multiplied by the median fluorescence intensity of the FRET-positive population, normalized to the IgG immunodepleted sample mean. (B). Representative images of tau aggregates in N2A cells on GFP channel. One-way ANOVA, Sidak post-hoc multiple comparisons. Error bars represent mean +/- SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.