Fig. 2

Monoclonal antibody DC8E8 requires no uptake into primary neuronal cells with pathogenic truncated tau. a Characterization of primary cortico-hippocampal neurons transduced with pathogenic AAV human truncated tau linked to mCherry (red). Forty eight hours after AAV viral transduction neurons were examined for truncated tau expression (151–391/4R) by immunocytochemistry with anti tau antibody (DC190 recognising epitope 368–376, green). Merged image confirmed substantial co-localization between the AAV transduced t-tau and additional pan tau staining. b Three days after AAV transduction primary neurons were screened for t-tau protein expression by Western Blot utilizing 2 different anti tau antibodies (HT7; DC25 recognising 347–353). SDS-PAGE and immunoblotting in both cases show expression of the 25 kDa human truncated tau protein in neuronal cells as indicated (arrows). c Primary neurons cultured on glass with confirmed AAV transduced t-tau protein were examined for viability with trypan blue. Subsequently, neurons were subjected to addition of DC8E8 antibody. Immunocytochemical labelling of DC8E8 showed that therapeutic antibody was detected and localized only in trypan blue positive neurons (dying neurons) as indicated by arrows. d Human neuroblastoma cells SHY5Y expressing human truncated tau protein (151–391/3R), were cultured on glass Labtec dishes and stained live for condensed nuclei (Hoechst 33358 1 μg/mL). Next, cells were subjected to an addition of fluorescently labelled humanized version of DC8E8/AX004 Alexa Fluor 546 (red) in media (1 μM ~ 168 μg/mL) and monitored over time with LSM 710 confocal microscopy for up to 4 h. AX004 antibody was not internalized in neuronal cells with t-tau expression, only in dying cells with condensed nuclei (Hoechst) as indicated with arrows. Experiments were repeated 3 times with similar results obtained. Scale bars, 10 μm