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Keywords:

  • Alzheimer disease;
  • cleavage;
  • Down syndrome;
  • early event;
  • phosphorylation;
  • tau

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Author contributions
  9. References

Aims

Phosphorylation, conformational changes and cleavage of tau protein have been widely suggested to contribute to abnormal tau processing in the pathogenesis of Alzheimer's disease, as well as in other tauopathies. Consistently, many phosphorylated sites, such as Ser199–202–Thr205 and Ser396–404, have been associated with this pathological processing. The present study examined the chronological appearance of phosphorylation during the neurofibrillary tangle (NFT) evolution in Alzheimer disease (AD) and Down syndrome.

Methods

Immunohistochemistry for modified tau [phosphorylated at Ser199–202–Thr205 (AT8) and Ser396–404 (PHF-1) or truncated at D421 (TauC3) and E391 (MN423)] was performed on paraffin-embedded human brain sections. Double immunofluorescence for phosphorylated and truncated tau was used to detect intensity and distribution of tau immunoreactivity, and provided detailed characterization of NFT pathology.

Results

Phosphorylation at sites Ser396–404 was significantly increased when compared with phosphorylations at sites Ser199–202–Thr205. Around 50% of the total structures containing phosphorylation at sites Ser396–404 were found as early phospho-tau aggregates with a well-preserved neuronal soma. Phosphorylation of tau protein at sites Ser396 coexists with early and late truncation events. Tau abnormal processing in Down syndrome consistently showed similar alterations as observed in AD.

Conclusion

Phosphorylation of tau protein at the carboxyl terminus may be among the earliest tau events, and it occurs prior to the apparition of the classical fibrillar structure. Finally, these data validate PHF-1 as an efficient marker for AD cytopathology following the progression of tau aggregation into NFT.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Author contributions
  9. References

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 [1]. However, this approach has not proven successful [2, 3]. Identifying early events that lead to aggregation therefore becomes crucial [4]. 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 [5]. 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 [24]. 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 [8], 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 [28]. Recently, we have found that tau protein has a physiological function at the synaptic terminal that is regulated by tau phosphorylation at different sites [29]. 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) [30]. 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.

Material and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Author contributions
  9. References

Human brain tissue

The brain tissues were collected, stored and used for research following approval from the institutional ethics committee and written informed consent from close legal relatives of the subjects. We studied brains (ages 56–91 years) received from the Case Western Reserve University Brain Bank (Cleveland, OH, USA). All of the patients had a clinical diagnosis of either AD or DS. All of the pathological cases stained for phosphorylated tau and exhibited Alzheimer pathology, NFTs and senile plaques. The mean duration of illness was 9.1 years (range 1–20 years) for the AD cases. The mean post mortem interval in these cases averaged 15 h (± 8). Further, control brains, with no evidence of clinical dementia or other neurological diseases, were examined and were found to be negative for the presence of tau atrophy. The control group showed negative or low staining when stained with PHF-1, an antibody that recognizes the early stages of a NFT. Brain hippocampal tissue was fixed in routine formalin, dehydrated and embedded in paraffin, 6-μm sections were placed on saline-coated slides.

Immunohistochemistry

After rehydration through xylene and graded ethanols, sections were treated with 3% H2O2, for 30 min to reduce endogenous peroxidase activity and blocked with 10% normal goat serum (NGS; Sigma, St. Louis, MO, USA) in Tris-buffered saline (TBS) (50 mM Tris, 150 mM NaCl, pH 7.6) for 45 min. Primary antibodies were incubated overnight at 4°C and followed the peroxidase anti-peroxidase method using 3,3-diaminobenzidine (DAB) and 0.015% H2O2 as cosubstrate. Adjacent serial sections were used to directly compare pathological structures recognized by antibodies listed in Table 1.

Table 1. Antibodies employed
StateAntibodyClassEpitopeReference
Phosphorylation dependentSer262IgGpSer262Biosource, USA
AT8IgGpSer199, pSer202 Thr205[32]
PHF-1IgGpSer396, pSer404[36]
Ser396IgGpSer396[37]
Truncation dependentTau-C3IgGTruncation D421[17]
MN423IgGTruncation E391[38]

Double-label immunofluorescence

For double-label immunofluorescence, sections were blocked with 10% NGS (Sigma) in TBS for 30 min. Double-labelling experiments were conducted by combining two of the primary antibodies listed in Table 1. Bound monoclonal antibodies were detected with FIT-C or TRIT-C conjugated goat anti-mouse IgG (γ-specific) and anti-mouse IgM (μ-specific) (Jackson Immuno-Research laboratories, Bar Harbor, ME, USA). In all experiments, incubation with primary antibodies was done overnight at 4°C, followed by 2 h at room temperature with the appropriate secondary antibodies. The sections were mounted in antiquenching medium (Vectashield, Vector Laboratories, Inc., Burlingame, CA, USA).

Confocal microscopy

Labelled brain sections were viewed with a 40× Plan-Apochromat on a TCP-SP2 Leica (Heidelberg, Germany) laser scanning-confocal microscope. Additional high power lenses (60× and 100×) were used to critically evaluate colocalization in single optical sections. Confocal images were obtained as single sections and the stack of images was projected as individual two-dimensional extended focus images. Resulting images were analysed using the software included with the microscope and Image J (Image Processing and Analysis in Java) software.

Statistical analyses and morphometry

Using the peroxidase technique, NFTs were counted in the area of interest (see Table 2). Morphometric quantification in the areas was assessed on three microscopic fields from randomly chosen regions in the area of interest. Observations were conducted by bright-field microscopy (Nikon FN1, Melville, NY, USA). Identification and counting of pathological structures was conducted using 10× and 20× objective lenses and values expressed per mm2 as previously described [33]. Relative expression intensity was measured in neurones by using Image J software (Image Processing and Analysis in Java). Values represent relative surface area expression. Student's t-test was applied when counts were compared between different groups. Statistical analysis was conducted in Excel. Bar diagrams represent the experimental mean; the error bars represent the standard error. For statistical analysis we used the Student's t-test with the significance set a P-value of 0.05.

Table 2. Clinical and neuropathological characteristics of groups
GroupsnAge at death (year)Clinical severityBraak stagesBrain region
Control367.33 ± 6.11Nondemented casesHippocampus with adjacent temporal cortex
AD485.5 ± 5.32Severe dementia (more than 5 years)(I–V)Hippocampus with adjacent temporal cortex
AD381 ± 7.02Mild to moderate dementia(I–IV)Hippocampus with adjacent temporal cortex
AD/DLBD280.5 ± 2.5Mild to moderate dementia(I–IV)Hippocampus with adjacent temporal cortex
Down syndrome367 ± 1.41Clinical diagnosisCortical area

Anatomical areas

As mesocortices and the hippocampal formation are the most vulnerable brain areas to NFTs, they were the focus of this study. Mesocortices include entorhinal cortex, perirhinal cortex while the hippocampal formation contains parasubiculum, presubiculum, subiculum, CA1, CA2, CA3, CA4, and dentate gyrus. The same groups of neurones were compared with regard to morphological and cytopathological observations of NFTs for the different tau antibodies. For example, entorhinal layer II was compared in each case with all the tau antibodies. Furthermore, NFTs were compared across areas within each case. Different degrees of neuropathological changes among cases afforded the opportunity to compare morphological subtypes of NFTs in the context of the overall stage of neuropathological change. The same criteria were used to examine cortical areas.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Author contributions
  9. References

Immunoreactive NFTs labelled by PHF-1 were observed during early and late Braak stages

Single-labelled immunohistochemistry in mild and severe AD cases (BST II and V respectively) was performed by using PHF-1 marker (phosphorylation at sites Ser396–404). A substantial NFT pathology around the affected areas (see Table 2 and methods) of mild AD cases was observed (Figure 1a). In a similar way, in severe AD cases with advanced cognitive deficit, substantial NFT pathology was found (Figure 1b). We divide tau pathology in two groups; NFT-like structure (iNFT) that comprises all kind of phospho-tau aggregates (Figure 1c–e) and NFTs that comprises a well-defined and mature NFT, a densely immunoreactive set of phospho-tau fibrils in the shape of a neuronal cell body (Figure 1f–h). We included cells containing diffuse phospho-tau positive staining within the cytoplasm, sometimes comprising small punctate regions (Figure 1c); in this stage the nucleus was detectable and the general cell morphology appeared normal. No condensed inclusions were noted (Figure 1c). On the other hand, intermediate-NFTs are defined by their presence of aggregated filamentous structures within the cytoplasm that are positive for phospho-tau. These groups were included into the NFT group (Figure 1f). The nucleus was frequently displaced by the inclusion (Figure 1f–h). In summary, in both severe and mild AD cases, the immunoreactivity of PHF-1 is present and, more importantly this marker is able to detect all kinds of aggregates during AD progression, from early aggregates (iNFTs) to mature aggregates (NFTs).

figure

Figure 1. Phosphorylation of tau protein at sites Ser396–404 is present during NFT evolution. Phosphorylation at sites Ser396–404 labelled by PHF-1 was found around the affected areas of both mild (a) and severe AD cases (b), scale bar 100 μm. Panel c represent pretangles (iNFT) with punctuate regions. Panel dh displays NFTs at different stages, being the panel h the most typical NFT that appear thicker with no clear nucleus, scale bar 20 μm.

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Phosphorylation at sites Ser199–202–Thr205 showed greater preference for mature NFTs than early NFT-like structures when compared with phosphorylation at sites Ser396–404

The main difference between phosphorylation at sites labelled by AT8 and PHF-1 is that they are located in different sites of the molecule (Figure 2a). The PHF-1 sites are situated close to the carboxyl terminus whereas the AT8 sites are located close to the middle of the molecule (Figure 2a). We evaluated the presence of all events labelled by AT8 and PHF-1 respectively. Here we found that all events were present in different cases around the affected areas (Figure 2b,c). Both markers displayed the typical AD pathology, NFTs and neurites (Figure 2b,c). However, by taking a closer look, we observed a major difference in the patterns of both markers; PHF-1 seemed to label more iNFT than the AT8 marker (Figure 2d). Indeed, when we analysed the total amount of lesions in mild and severe cases, we found that PHF-1 immunoreactive structures per mm2 were significantly higher when compared with AT8 immunoreactive structures (Figure 2e). Interestingly, for the PHF-1 marker, around 50% of the total numbers of structures were iNFTs and 50% NFTs, whereas in the case of the AT8 marker, 30% were iNFTs and 70% were NFTs (Figure 2f). Overall, both markers are capable of detecting both stages of the NFT evolution, the pretangle and the tangle; however, the PHF-1 marker showed higher affinity for both stages, which was not the case for the AT8 marker.

figure

Figure 2. Phosphorylation of tau protein at sites labelled by PHF-1 was preferentially found in early phospho-tau aggregates (iNFT). Phosphorylated sites labelled by AT8 and PFH-1 markers respectively (a). Immunostaining with antibodies PHF-1 (Ser396–404) and AT8 (Ser199–202–Thr205) (a) revealed the classical tau pathology comprising NFTs, neurites and iNFT (b and c). The immunostaining with PHF-1 marker showed affinity for iNFT (d), scale bar 100 μm. The total density of phosphorylated structures per mm2 labelled by PHF-1 showed a significantly increased number of pathology when compared with AT8 cytopathology (e) (P < 0.05). No significant differences were found in the total density of phosphorylated iNFT when compared with NFT with PHF-1 marker (f). Significantly higher number of NFTs were observed when compared with iNFTs with AT8 marker (f) (P < 0.05). Error bars represent the standard error, n = 9.

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Phosphorylation at sites Ser396–404 is present in fibrillar and nonfibrillar state

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 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.

figure

Figure 3. Phosphorylation of tau protein at sites Ser396–404 is present in fibrillar and nonfibrillar state. Early NFT-like structures showed zones that are mainly composed by phosphorylated tau protein at sites Ser396–404 (a, white arrows). Mature NFTs are characterized by phosphorylated tau protein at sites Ser396–404 in fibrillar conformation as revealed by coexistence of both markers PHF-1 and TR (b and c).

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Phosphorylation at sites Ser396 relates to early and advanced tau abnormal processing events

Our group previously 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 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.

figure

Figure 4. Phosphorylation of tau protein at sites Ser396 coexists with early and late events. Cleavage events labelled by TauC3 (A-i) and MN423 (A-ii) respectively. Double-labelled immunofluorescence showed the presence of phosphorylated tau at site Ser396 (a, red channel) close related to truncated tau at site D421 (TauC3, b green channel) in the same NFT pathology (c, asterisk and blue arrow in the merge channel). Phosphorylation at site Ser396 was the only event found as an isolated event (a and c, white arrows). Phosphorylated tau at Ser396 (d, red channel) was also found coexisting with truncated tau at site E391 (e, green channel) in some NFT pathology (f, asterisks). NFTs with low density of phosphorylation at site Ser396 and higher density of cleavage (E391) were found (d–f, blue arrow), as well as the opposite pattern (d–f, white arrow) scale bar 20 μm. Analysis of expression confirmed the two population existence, one with significantly higher expression of phosphorylation when compared with cleavage at site E391 (B and D), and the other with the opposite pattern (C and D). No significant differences were found in the total number of NFTs comprising phosphorylation in association with cleavage at either D421 or E391 (E) (P < 0.05). Error bars represent the standard error, n = 3.

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PHF-1 immunoreactivity remains increased in severe AD cases when compared with AT8 immunoreactivity

To further analyse the chronology of phosphorylation at sites Ser199–202–Thr205 and Ser396–404 during late AD stages, we examined cases that presented an elevated number of NFT pathology comprising cleavage at site E391 (Figure 5c). Here, extracellular NFTs, a densely immunoreactive set of truncated-tau fibrils in the shape of a neuronal cell body were detected (Figure 5c, superior corner). Again, phosphorylation markers where able to detect a considerable number of phospho-NFT pathology, that is, NFTs and neurites around the affected areas (Figure 5a,b). When we quantified the total amount of structures per mm2 we observed an interesting fact, in advanced AD cases phosphorylation at sites Ser396–404 remains significantly increased when compared with phosphorylation at sites Ser199–202–Thr205 (Figure 5d). While the total number of structures labelled by AT8 does not showed significant differences when compared with structures labelled by MN423 (Figure 5d). These data suggest that at some point the phosphorylation of tau protein at the sites Ser199–202–Thr205 stabilizes, while phosphorylation at the sites Ser396–404 remains dynamic.

figure

Figure 5. Phosphorylation of tau protein at sites Ser396–404 remains strongly present in late AD stages. Severe AD cases were found characterized by presence of phosphorylated tau protein at sites labelled by PHF-1 (a), AT8 (b) and elevated number of truncated tau at site labelled by MN423 (c, see magnification in superior corner), scale bar 100 μm. Total numbers of NFTs per mm2 labelled by PHF-1 were found significantly increased when compared with NFTs labelled by AT8 and MN423 (d) (P < 0.05). Error bars represent the standard error, n = 4.

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Phosphorylation and cleavage of tau in DS parallel the early processing of tau found in Alzheimer pathology

To further evaluate our finding of phosphorylation at sites Ser396–404 as one of the earliest events, we studied DS, which is also characterized by phosphorylated tau protein. Here, in a similar way to AD, we found a large population of NFTs comprising phosphorylated tau (Figure 6a,b). The total number of NFTs per mm2 expressing phosphorylation at sites Ser396–404 was around 110 structures per mm2 (Figure 6h), a number quite similar to that seen during AD. Those structures were composed of tau phosphorylated at many sites; Ser396–404, Ser199–202–Thr205 and Ser262 (Figure 6a–d). To assess the status of C-termini of tau in those structures, single labelling using antibodies specific to early truncated tau (TauC3) and late truncated tau (MN423) was performed, and again, a considerable numbers of NFTs were detected with the cleavage at the D421 site (Figure 6e), whereas very few NFTs were detected with the cleavage at the E391 site (Figure 6f). In a similar way to the processing of tau protein during AD, PHF-1 immunoreactivity was able to detect early aggregates ‘NFT-like structures’ (Figure 6g, i and ii) as well as mature NFTs (Figure 6g, iii). Quantification analysis of all those structures revealed a similar pattern of events as seen during AD. The majority of NFTs were mainly composed of tau phosphorylated at sites Ser396–404, followed by phosphorylation at sites Ser199–202–Thr205. Sequentially followed by cleavage at site D421 (Figure 6h). To evaluate whether the evolution of the tangle was similar to what was seen during AD, we analysed the morphology of the NFTs seen during DS in terms of early aggregates and mature aggregates (criteria described earlier). Here we found that 80% of the NFTs labelled by pS262 were intracellular, while pSer396 and PHF-1 showed around 50% of iNFT and 50% of NFTs (Figure 6i). Again and similar to AD, AT8 marker showed that close to 70% of the structures where mature NFTs (Figure 6i). In sum, when compared with AD, these data suggest similar processing of tau protein during early stages of DS.

figure

Figure 6. NFT cytopathology during Down syndrome shows phosphorylation and cleavage of tau protein. Immunolabelling of tau protein during DS revealed the presence of phosphorylation at the Ser396–404 sites labelled by pS396 and PHF-1 respectively (a and b); phosphorylation at the Ser199–202–Thr205 sites labelled by AT8 (c); phosphorylation at the Ser262 site labelled by pS262 (d); cleavage at the D421 site labelled by TauC3 (e) and cleavage at the E391 site labelled by MN423 (f), scale bar 10 μm. Panel g represent pathology revealed by PHF-1; iNFT with punctuate regions (i and ii) as well as NFT that appear thicker with no clear nucleus (iii), scale bar 20 μm. Total number of NFT per mm2 labelled by AT8, S262, TauC3 and MN423 were found significantly decreased when compared with NFTs labelled by PHF-1 and pS396 (h), (P < 0.05). Total numbers of iNFT per mm2 labelled by S262 were found significantly increased when compared with NFTs labelled by the same marker (i). PHF-1 and pS396 did not showed significant difference in labelling iNFT when compared with NFT (i) (P < 0.05). Total number of NFTs where found significantly higher when compared with iNFTs with AT8 marker (i) (P < 0.05). Error bars represent the standard error, n = 3.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Author contributions
  9. References

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 [35].

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

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.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Author contributions
  9. References

We thank to Peter Davis for PHF-1 antibody donation. We thank Katarina Stojkovic for critical comments. Work in the authors' laboratories is supported by Consejo Nacional de Ciencia y Tecnología (Conacyt), Mexico; Canadian Institutes of Health Research (CIHR), Canada and Fonds de la recherche en santé du Québec (FRSQ), Québec, Canada. This project was supported by grants from the National Center for Research Resources (5 G12RR013646-12), the National Institute on Minority Health and Health Disparities (G12MD007591) from the National Institutes of Health, and from the Research Centers in Minority Institutions (RCMI). S.M.-R. was awarded with a postdoctoral scholarship support FRSQ, Canada.

Author contributions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Author contributions
  9. References

Conceived and designed the experiments: S.M.-R. Performed the experiments: S.M.-R. and J.L.-M. Analysed the data: S.M.-R., G.P. and M.C.A.-A. Contributed reagents/materials/analysis tools: G.P., M.C.A.-A. and S.W. Wrote the paper: S.M.-R. Financial support: G.P. and S.W. All authors read and approved the final manuscript.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Author contributions
  9. References
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