Fig. 5

Pathogenic tau species efficiently enter primary neurons in concentration- and time-dependent manner. a Representative image of neuronal uptake of extracellular human-derived AD tau (sarkosyl-insoluble 2p) fluorescently labelled with Alexa Fluor 488. Neurons (2–4 days in vitro) were incubated with 200 nM or 500 nM AD-tau Alexa Fluor 488 for 24 h. Images were obtained using LSM 710 confocal microscope (DIC grayscale, AD tau Alexa 488 green). Scale bar, 20 μm. Panel b shows intracellular localization of tau Alexa 488 protein aggregates (100 nM) 24 h after addition to primary neurons cultured for 4 days in vitro. Tau Alexa 488 oligomers (green) are partially engulfed in lysosomes stained with a fluorescent dye LysoTracker (75 nM) indicated by co-localization in merged image (appears in cyan signal). Cytoplasmic “free” AD tau (green) is indicated with white arrows. Scale bar 20 μm. c) Quantification of relative fluorescence intensity (arbitrary units) utilizing flow cytometry for internalized AD tau Alexa 488 (200 nM and 500 nM, 24 h) in primary neuronal cells. A right shift (blue - dark blue) in the distribution indicates a concentration-dependent increase in the amount of internalized AD tau in neurons (increase in fluorescence). Control neurons (no AD tau) indicated in grey. d Flow cytometry analysis of AD tau Alexa 488 neuronal uptake with increasing concentrations (200 nM; 500 nM after 24 h). Data are presented as mean ± SEM. *p < 0.001, difference between 200 nM vs 500 nM AD tau Alexa 488 treated neurons. Experiments were performed in triplicates from 2 separate neuronal preparations. e Time dependent effect of t-tau Alexa Fluor 488 (297–391; 100 nM) neuronal internalization quantified at indicated time point (3, 6 and 24 h) by flow cytometry showed as mean fluorescence intensity (arbitrary units). Results were obtained from 2 separate cultures and data are presented as mean ± SEM. *p < 0.001, difference between 3 h and 6 h; and between 6 h and 24 h after t-tau Alexa 488 treated neurons. In flow cytometry experiments a minimum of 10.000 events per sample was analyzed. f Neuronal uptake of AD tau 2p fraction in cells harvested at indicated time points (6 h and 24 h) was evaluated by a sandwich ELISA (DC25/DC190). Data were corrected for protein concentration in the sample. Increase in the amount of intracellular AD tau in cell lysate pellets in time was detected. Experiments were performed in 2 independent preparations. *p < 0.001, difference between 6 and 24 h