Figure 6: Neuronal uptake of PBS-soluble HMW tau derived from human AD brain.

(a,b) Primary neurons were incubated with AD or control brain extracts (cases were matched for age and postmortem interval (Supplementary Table S1)) and immunostained at day 2 (a). (b) Quantification of fluorescence intensity of human tau staining. One-way ANOVA and a subsequent Tukey-Kramer test. (c,d) Tau uptake (c) and seeding activity (d) assay in HEK-tau-biosensor cells. (Mann–Whitney U-test) (e) Subcellular localization of human tau taken up by neurons (PBS-3,000g, 500 ng ml⁻¹ human tau). (f) Neuron-to-neuron transfer of tau in a 3-chamber microfluidic device. AD brain extract (PBS-3,000g, 500 ng ml⁻¹ human tau) was added to the 1st chamber. Human tau positive neurons were detected in both the 1st and 2nd chamber at day 7 (arrow). (g,h) Quantification of total-tau (g) and phospho-tau (h) levels in AD and control brain extract (ELISA). Unpaired t-test. (i) Brain extracts were immunoblotted with phospho-tau specific antibodies recognizing different epitopes. Representative immunoblot and quantification of phospho-tau levels at each epitope. Unpaired t-test. (j,k) SEC analysis of PBS-soluble tau from AD and control brain. (j) Representative graph of total tau levels (ELISA) in SEC-separated samples. Small peaks for HMW fractions were detected in both groups (right panel). (k) Mean total tau levels of HMW SEC fractions. (l) Tau uptake from each SEC fraction (5 or 500 ng ml⁻¹ human tau) by primary neurons. (m) Phospho-tau levels in each SEC fraction (ELISA). Unpaired t-test. Scale bar, 25 μm. *P<0.05, **P<0.01.