Fig. 6

Therapeutic candidate antibody DC8E8 inhibits neuronal internalization of various extracellular diseased tau species. a Confocal microscope images of AD tau fluorescently labelled with Alexa 488 (100 nM, 24 h) in the presence of irrelevant control mAb DC51 or therapeutic DC8E8 antibody (both green). AD tau Alexa 488 in pre-formed complex with DC8E8 (15 min incubation, 37 °C) attenuated intracellular localization of AD tau in neurons in comparison to cells incubated with control mAb (AD tau Alexa 488 present intracellularly). Scale bar, 20 μm. b Flow cytometric quantification of mean fluorescent intensity (arbitrary units) of AD tau Alexa 488 (100 nM, 24 h post addition to neurons) in the presence of control and DC8E8 or AX004 antibody (168 μg/mL ~ 1 μM). A left shift (blue – red) indicates DC8E8-mediated inhibition of AD tau internalization into neurons. Control neurons with no AD tau are indicated in grey. c Neuronal internalization of AD tau at 6 and 24 h in combination with control or DC8E8 antibody (1 μM) analysed by a sandwich ELISA. Data were corrected for protein concentration in samples. Decrease in the amount of intracellular AD tau in neurons was measured with DC8E8 antibody. Experiments were performed in 2 independent cultures. *p < 0.001, difference in 6 and 24 h detected between AD tau with Control versus AD tau with DC8E8 antibody. d Quantification of neuronal uptake of various pathological tau species: recombinant truncated tau (dGAE, 297–391/4R) and human brain-derived sporadic and familial AD tau (100 nM for 24 h) in the presence of Control or DC8E8 antibody (both 1 μM). Experiments performed from 2 independent measurements are represented as mean fluorescence intensity normalized to % of the control antibody. For flow cytometry experiments a minimum of 10.000 events per sample was analysed. DC8E8 antibody strongly blocked the neuronal uptake of all three tau species (truncated tau, AD sporadic and AD familial tau) compared to tau species with Control antibody. Data are presented as mean ± SEM. Statistically significant change between groups stated as ***p < 0.001. e Comparison of AX004 antibody of isotypes IgG1 versus IgG4 (1 μM) in slowing neuronal internalization of AD tau Alexa 488 (100 nM, sonicated 2 min) after 24 h. Summary of data measured by flow cytometry from 5 independent experiments (n = 10). All data are presented as mean +/− SEM. Statistically significant change between AD tau with Control antibody in comparison to AD tau with AX004 of both isotypes (IgG1; IgG4). Statistically non-significant change shown as ns