Total RNA was extracted and qPCR analysis of the complementary DNA (cDNA) product was carried out using primers against the transgenic human tau construct. The qPCR data clearly show that the neurons that were human tau protein-positive and RNA-negative by FISH indeed did not express detectable levels of the tau transgene (Figure 3G), in contrast to robust detection of tau mRNA in neurons positive for tau mRNA by FISH. Taken together, these data strongly suggest that the human

tau protein may be undergoing neuron-to-neuron transmission. The above experiments strongly suggest the spread of human tau protein from neuron to neuron, which could cause seeding of misfolding and aggregation of tau. It has been shown in cell culture experiments that extracellular tau aggregates could be internalized DNA Damage inhibitor transmitting tau misfolding from the outside to the inside of the cell, where these aggregates could seed fibril formation of recombinant tau monomer. Moreover, the same study showed that tau aggregates were transferred between cocultured cells (Frost et al., 2009). Another recent study reported that brain extracts from neurofibrillary tangle-bearing mouse brain injected in wild-type tau-expressing mice induces seeding of tau fibrils

in neurons (Clavaguera et al., 2009). To determine whether mouse tau is recruited by human tau to aggregate, we performed immunohistochemical analysis using an antibody specific for mouse

tau that revealed that mouse tau HA-1077 indeed accumulates in the somatodendritic compartment of MEC neurons of 24-month-old rTgTauEC mice (Figure 4A). Age-matched control mice have diffuse axonal staining with the mouse tau antibody, and tau knockout mice show no immunoreactivity, as expected. Human however AD cases also have no immunoreactivity to mouse tau, indicating that the observed immunoreactivity is not due to human tau becoming reactive to the mouse tau antibody during pathological changes. Results from double labeling using Alz50 and mouse tau antibodies showed that Alz50 and mouse tau staining colocalized in neuronal cell bodies of the MEC, which is further evidence for mouse tau recruitment into aggregates in the rTgTauEC mouse model (Figure 4B). Immunoblotting using the mouse tau-specific antibody also revealed that mouse tau increased with age in rTgTauEC mice (Figure 4C), indicating that it may accumulate in tangles. In confirmation of this idea, sarkosyl-insoluble and -soluble fractions both contain endogenous mouse tau (Figure 4D). The specificity of the mouse tau antibody was confirmed by western blot analysis (Figure 4E), which revealed mouse tau (mTau) reactivity in rTgTauEC and control mouse brain but not in tau knockout mouse brain or human AD brain.