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Alzheimer's disease (AD) continues to be a poorly managed disease, in which an aggregated state of proteins, Aβ and tau, proposed as possible causes of the disease, remains as an important therapeutic target . However, this approach has not proven successful [2, 3]. Identifying early events that lead to aggregation therefore becomes crucial . One of the aggregated structures that characterized AD, the neurofibrillary tangles (NFTs) emerge in nearly every Down syndrome (DS) individual by the time they are in their 40s . Not surprisingly, both diseases are clinically defined by cognitive decline [6, 7].
The formation of NFT during AD involves phosphorylations, conformational changes and cleavage of tau protein [8-22]. We have reported that this pathological entity is thought to proceed through phosphorylation, conformational changes and cleavage in a chronological order, all showing the characteristic β-sheet conformation [8, 23]. Additionally, our group has proposed that the cleavage around the Glu391 (E391) site is probably the latest event during tau pathological processing . Besides this cleavage labelled by MN423 [15, 25], a new cleavage event around Asp421 (D421) labelled by TauC3 has been described [17, 22]. Opposite to the E391 event, we reported that cleavage at D421 is an event that happens during the early stages of AD , and therefore, contributes to the pathological processing and aggregation of the protein into NFTs.
Like cleavage, phosphorylation of tau protein is another important event suggested to be responsible for the tau pathological processing during AD in addition to contributing to the aggregated state [26, 27]. Nonetheless, the specific role of phosphorylation remains under extensive study . Recently, we have found that tau protein has a physiological function at the synaptic terminal that is regulated by tau phosphorylation at different sites . Tau has phosphorylation sites located in the proline-rich region (P-region) (residues 172–251) and the C-terminal tail region (C-region) (residues 368–441) . The sites located at both regions such as those labelled by AT8 (Ser199–202–Thr205) and PHF-1 (Ser396–404) seem to cause: (a) abnormal folding and (b) protein cleavage, which together could lead to tau deposition [8, 31]. In previous work, we reported that phosphorylation of tau protein can be found alone and also coexisting with truncation of tau at D421 and the conformational change labelled by Alz-50 [8, 32], therefore suggesting that tau pathology may begin with misfolded and abnormally phosphorylated tau protein, pretangle (NFT-like structures), in the somatodendritic compartment of involved cells. Clearly, the different phosphorylations sites affect protein processing in different ways; therefore the chronology of these events becomes crucial in order to further elucidate the mechanism of abnormal tau processing that could lead to deposition.
Here, by using moderate and severe AD cases, we found that AD markers AT8 and PHF-1 have different chronological appearance in relation to pathology severity, with AT8 correlating with more severe stages. Conversely, we observed that PHF-1 was able to recognize more tau pathology when compared with the AT8 marker at all AD stages. Furthermore, phosphorylation at Ser396 was found closely related to early tau pathological events such as cleavage at site D421, as well as to the late E391 cleavage, validating PHF-1 as neuropathological markers of AD progression.
To further analyse our findings, we evaluate the processing of tau protein in DS. Here we found that tau pathological processing mimics what is seen during early stages of AD. In other words, our data showed a well-defined pathway with phosphorylation at sites Ser396–404 as the earliest event, followed by phosphorylation at sites Ser199–202–Thr205 and cleavage at site D421.
Taken together, the data suggest that phosphorylation of tau protein at those sites labelled by PHF-1 precedes the phosphorylation at sites labelled by AT8, and PHF-1 phosphorylation is present even before the classical aggregate in β-sheet conformation.
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In mild AD cases we found considerable cytopathology around the affected areas, that is, tau early aggregates, mature NFTs and neurites, all of them comprising phosphorylated tau at the Ser396–404 and Ser199–202–Thr205 sites (Figure 1). Such pathology was also present in severe AD cases (Figure 1). Interestingly, in mild and severe AD cases, phosphorylation at sites Ser396–404 was found in higher density when compared with phosphorylations at sites Ser199–202–Thr205 (Figure 2). More importantly, 50% of the total structures containing phosphorylation at sites Ser396–404 were found as early phospho-tau aggregates with a well-preserved neuronal soma (Figures 2 and 3). Importantly, this early aggregated state does not showed fibrillar conformation as revealed by TR labelling (Figure 3). Similar findings were reported by using AD2 antibody that also labels Ser396–404 .
These data clearly suggest that phosphorylation at sites Ser396–404 is an early phenomenon, which could be happening in tau protein even before phosphorylations at sites Ser199–202–Thr205, or conformational modifications. In addition, our data open a new perspective in terms of chronology and pathogenesis as both events are present in different sites of the molecule, suggesting that phosphorylation at the carboxyl terminal could be crucially related as pivotal events for further processing and aggregation of tau protein. To further develop our hypothesis we studied the association of this particular phosphorylation to early and late tau processing events, cleavage at the D421 and E391 sites respectively. Here we found that phosphorylation is strongly coincident with both cleavage events (Figure 4). Interestingly, when we analysed the relationship between phosphorylation at Ser396 and the early cleavage at site D421 we found mainly two NFT populations; one containing just phosphorylation and the other containing phosphorylation and cleavage (Figure 4). These data suggest that phosphorylation at this particular site does not require cleavage at site D421 to be present. Conversely, the majority of structures comprising cleavage at site D421 were found in coexistence with phosphorylation events, suggesting that cleavage requires phosphorylation in order to be present. When phosphorylation was studied in relationship to the late cleavage at E391 we found two populations as well, one with significantly elevated phosphorylation and the other with significantly elevated cleavage at E391 (Figure 4). These data suggested a sequential pattern, where phosphorylation appears as the earliest insult probably promoting early cleavage and remaining into the NFT maturation until events like cleavage at E391 take place. But, why is the remaining fragment not longer labelled by pS396? Here we believed that the small tau fragment containing this epitope could be undergoing degradation (Figure 4).
It also should be noted that the same tau protein could not hold all the events at the same time, namely phosphorylations at Ser396–404 and cleavage at the E391 site, therefore oligomeric tau structures with different tau molecules at different processing stages must coexist during the process and maturation of NFTs.
Interestingly, this is not the case for the phosphorylation at sites Ser199–202–Thr205. Using the AT8 marker, we found that the total number of structures does not show differences when reaching advanced AD stages, suggesting that at some point during the tau processing this phosphorylation reaches a stable level (Figure 5); conversely PHF-1 during advanced stages remains significantly increased (Figure 5). Again, these data show important differences between events in the carboxyl terminus vs. the middle of the molecule, suggesting that the carboxyl terminus is exposed to phosphorylation events from early to advanced processing stages.
To further evaluate the role of phosphorylation of tau protein at sites Ser396–404, we studied the abnormal processing of tau protein in DS. In this study, we found that hyperphosphorylated tau protein at sites Ser199–202–Thr205 and Ser396–404 is present in the cytopathology found in DS. Here again, PHF-1 detected both early aggregates (iNFT) and mature NFTs (Figure 6), and finally, the density of structures displaying phosphorylation at sites Ser396–404 was significantly increased compared with those phosphorylated at Ser199–202–Thr205 or Ser262 (Figure 6). According to these data, we propose that phosphorylation at Ser396–404 is followed by phosphorylation at sites Ser199–202–Thr205 and possibly some other phosphorylations like Ser262 (Figure 6). However using the same criteria, we cannot rule out the possibility that phosphorylation at site Ser262 is also an early event, mainly due to the fact that most of the structures comprising this event were found in a pretangle like stage (Figure 6). Here, we suggest that abnormal aggregation of this protein, in a different tau disease, is conducted by common mechanisms promoting its hyperphosphorylated state. To further analyse if processing of tau protein was similar to what we saw during AD, we studied the presence of cleavage events at sites D421 and E391. Some NFT pathology showed a considerable level of cleavage at site D421 and small amount of pathology with of the E391 truncated tau (Figure 6). These data show a clear difference between AD and DS. Tau protein does not seem to reach late stages of abnormal processing during DS (Figure 7). Despite this finding, the presence of E391 truncated tau in DS may suggest that NFTs during DS are exposed to proteolytic events and processed similarly to intracellular NFTs during AD. In sum, like in AD, in DS phosphorylated tau was observed in a nonfibrillar state suggesting again that phosphorylation at the carboxyl terminus could be critically related to the pathogenesis of the disease.
Figure 7. Maturation of NFTs comprises phosphorylations of tau protein at Ser396–404 during early stages in AD and DS. Over the course of the tau pathological processing, tau protein suffers phosphorylation events at ending sites, amino and carboxyl (a), being the carboxyl the earliest ones. Such early events could promote the protein to the following processing that comprises early and late cleavage (b). Maturation of tau protein during DS showed a similar processing of tau protein when compared with AD (c), being the phosphorylation at sites Ser396–404 also an early event. However DS showed an important difference when compared with AD, the latest photolytic events labelled by MN423 are barely present in DS cytopathology (c).
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In normal conditions, neurones in the hippocampus process sensory and motor cues to form a cognitive map encoding spatial, contextual and emotional information, which they transmit throughout the brain. However, during neurodegeneration function could be dramatically altered by the aggregation of phosphorylated tau protein. Interestingly, prior to formation of NFT alterations, neurone functioning could be compromised. Here, we believed that the study of pretangle like structures could become a more suitable research model in order to find the pathogenesis of such complex tau diseases.
Overall, our findings document a well-defined pattern of phosphorylation and sequential or simultaneous cleavage of tau at D421 in both AD and DS, with phosphorylation at sites Ser396–404 being one of the earliest events. Finally, these data validate PHF-1 as an efficient marker for AD cytopathology following the progression of tau aggregation into NFT.