To assess the phosphorylated status of the tau protein recognized by PHF-1, we performed double labelling, combining PHF-1 in green colour and thiazin red (TR) in red colour (Figure 3a, in colour scale yellow colour represents equal contribution of both markers). TR is an analogue of naphthol-based azo structures whose functional

characteristic is to bind β-pleated sheet structures [34]. None surprisingly, well defined NFTs detected by PHF-1 were found in a fibrillar state as revealed by the yellow tonality (Figure 3c). Similar results were observed in additional pathology like NFTs in earlier state were coexisting events were found, named phosphorylation and TR labelling (Figure 3b). Interestingly, some early aggregates labelled by PHF-1 were found with little fibrillar structure, as revealed

by the intense green tonality (Figure 3a, white arrows). Note check details that the presence of nuclei reveals the early state of the NFT-like structure (Figure 3a, blue). Overall, PHF-1 marker is able to detect the classical NFT structure in fibrillar conformation, but more importantly is also capable of detecting early phospho aggregates that are not yet in fibrillar conformation. Our group previously Crizotinib purchase reported that cleavage of tau protein is sequential starting by the carboxyl terminus, with cleavage at D421 being an early event and cleavage at E391 occurring latter in AD [8, 24, 32]. Taking advantage of this finding, we wanted to evaluate if phosphorylation Pregnenolone of tau protein was present in coexistence with both cleavage events (Figure 4Ai,Aii), and more importantly, if phosphorylation suffers any changes during the tau abnormal processing. In this regard we found coexistence of phosphorylation at site Ser396 with either, D421 or E391 truncated tau (Figure 4c blue arrow and stars, and 4f arrows and stars). Some NFT pathology was found with nothing but phosphorylation at site Ser396 (Figure 4a,c white arrows). Interestingly,

some NFT pathology (population A) showed an elevated level of phosphorylation at site Ser396 with lowest levels of E391 truncated tau (Figure 4d–f white arrow), while, some others (population B) showed an increased level of E391 truncated tau with lowest levels of phosphorylation at site Ser396 (Figure 4d–f blue arrows). Relative expression analysis confirmed the two populations; one with significantly elevated presence of phosphorylation (Figure 4B,D, population A) and the other with significantly elevated presence of cleavage at E391 (Figure 4C,D, population B). Further analysis of both cleavage events (D421 and E391) revealed that almost all the structures containing truncated tau also comprised phosphorylated tau (Figure 4E). In summary, phosphorylation of tau protein appears as single event and remains during early and advanced proteolytic events.