P4-028: Tau release from hTau mouse primary neuronal cultures

Studies describing the spreading of pathological tau in transgenic mouse models together with recent publications showing that tau is actively released from cell lines suggest that, under normal conditions, a significant amount of tau might be present in the extracellular environment. We sought to study tau release in a cellular model more closely related to human neurons, where exclusively the human isoforms of tau are expressed. Neurons were prepared from hTau and tauKO E18-P1 pups, and cultured in neurobasal serum free medium for 2–3 weeks. Total tau and p-tau levels were measured using the sensitive tau ELISAs established in our laboratory. We have characterized tau expression in hTau neurons during their maturation and found exclusive expression of the 3R isoforms of tau in this cellular system. After 14 days in culture, it was noted that the conditioned medium of the hTau neurons contained substantial levels of tau. Cultured neurons from tauKO mice did not show any evidence of a signal in the tau ELISAs. Further investigation of hTau cultures showed that even after removing the conditioned medium, washing the cells and adding fresh medium, tau concentrations rose rapidly within 1 hour at 37°C, while at 4°C there were low levels of tau in the medium. No LDH release was measurable over this 1h period, excluding that tau in the medium might reflect cell death. We were able to detect both total tau and p-tau (Ser202, ser396–404, Thr-231) in the medium of the hTau neurons. Immunoblotting medium and lysate from hTau neurons showed that the extracellular tau species ran a little faster on the gel than the major band present in the cellular lysate. This observation led us to think a truncated form of tau was released from the cells, possibly a caspase-cleaved tau form. Treating the cells with a pan-caspases inhibitor resulted in lower amounts of tau released in the extracellular space. Tau release is readily measurable from primary cultures of hTau mouse neurons, is extremely rapid and almost completely blocked by reduction of temperature to 4°C. Caspase cleavage may be necessary for release.