The relationship between truncation and phosphorylation at the C-terminus of tau protein in the paired helical filaments of Alzheimer's disease

Abstract

We previously demonstrated that, in the early stages of tau processing in Alzheimer's disease, the N-terminal part of the molecule undergoes a characteristic cascade of phosphorylation and progressive misfolding of the proteins resulting in a structural conformation detected by Alz-50. In this immunohistochemical study of AD brain tissue, we have found that C-terminal truncation of tau at Asp-421 was an early event in tau aggregation and analyzed the relationship between phospho-dependent tau epitopes located at the C-terminus with truncation at Glu-391. The aim of this study was to determine whether C-terminal truncation may trigger events leading to the assembly of insoluble PHFs from soluble tau aggregates present in pre-tangle cells. Our findings suggest that there is a complex interaction between phosphorylated and truncated tau species. A model is presented here in which truncated tau protein represents an early neurotoxic species while phosphorylated tau species may provide a neuroprotective role in Alzheimer's disease.

Keywords: Alzheimer's disease; PHFs; neurofibrillary tangles; neurotoxicity; tau oligomers; tau protein; truncation.

Figure 1

Tau protein domains and location of antibody epitopes. The schematic representation shows three domains of tau protein and the location of epitopes for the antibodies used in this study. It is depicted as the longest tau isoform in the central nervous system of 441 amino acids (grey). Inserts from alternatively spliced exons are denoted in pink. There are two in the N-terminus and one in the tandem repeat domain; the other 3 repeat domains are shown in green. Non-phospho-dependent epitopes are shown above the drawing, whereas phospho-dependent epitopes are indicated below. Details for antibodies are listed in Table 2. In green are depicted the repeat domains of tau protein and gray is the tau protein amino acid sequence.

Figure 2

Double and triple immunolabeling of tangles with antibodies. Intracellular NFTs were detected by both pS396 and AD2 (A). Extracellular NFTs, however, displayed intense immunoreactivity with pS396 but only sparse and granular staining with AD2 (B, arrowheads). Thiazin red detected some PHF bundles located at the center of both intra- and extra-cellular NFTs (A,B, arrows). Double immunolabeling with TauC3 and pS396 identified two tangle subtypes: one in which both antibodies were co-localized (C) and another that was reactive only with pS396 (D). TauC3 immunoreactivity was restricted to short, thin neurites located in the vicinity of NFTs (arrowheads). Triple immunolabeling with pS396 and Alz-50 with either Tau-12 (E,F) or T46 (G,H); and double immunolabeling with pS396 and AD2, counterstained with thiazin red (A,B); and pS396 with TauC3 (C,D). Two subtypes of tangles were observed. (i) The first was recognized by pS396 (green), but not by Alz-50 (blue) either with Tau-12 (red) (E) or with T46 (red) (G). (ii) A second subtype was defined as the one which displayed immunoreactivity with all three markers (F,H).

Figure 3

Double and triple immunolabeling with pS396 and mAb 423, and Alz-50 in neuronal cells with tau degeneration in Alzheimer's disease. (A) pS396 immunoreactivity was observed in the intracellular early tangles, being present in the perinuclear area (arrows) and closely associated with lipofuscin (arrow, red channel). The mAb 423 failed to identify these structures (red channel, B, C, D) In tissue pre-treated with Pronase/formic acid, pS396 and mAb 423 antibodies co-localized in early tangles (B, arrows), confluent tangle bundles (C, arrows), and NFT (D, arrow). (E, F) Triple immunolabeling with mAb 423, pS396, and Alz-50 in Pronase/formic acid treated brain tissue, show all three antibodies co-localized in the intracellular tangle (E). An extracellular tangle (F) displayed immunoreactivity with only mAb 423. ^* lipofuscin granules.

Figure 4

Triple immunolabeling with antibodies mAb 423, pS396, and Alz-50 of pre-tangle cells after Pronase/formic acid treatment. (A–D) Triple labeling with mAb 423 (green), Alz-50 (blue) and pS396 (red). A pre-tangle cell displayed diffuse granular cytoplasmic immunolabeling with mAb 423 in the green channel (arrows) the majority of which was also detected by Alz-50 in the blue channel (B; arrows). However, pS396 failed to detect such granular structures in the red channel (B). Autofluorescent lipofuscin granules were also observed (^*). N, nucleus.

Figure 5

A model for the stages of tau assembly into PHFs in pre-tangle neurons in Alzheimer's disease. Unaffected neurons (Stage 0) will have normal tau (neither truncated or hyperphosphorylated) that is associated predominantly with axonal microtubules. Stages 1–3 represent early stages that precede the appearance of fibrillary inclusions. Stage 1 is characterized by the appearance of the PHF core, namely a fragment of 93–95 amino acids in length corresponding to the repeat domain of tau. Stage 2 is defined by the cytoplasmic aggregation of tau molecules resulting from the binding of PHF-core fragments with full-length tau and that is reactive with the N-terminal phospho-dependent antibody. This stage corresponds to the diffuse granular structures seen by confocal microscopy. The PHF-core tau is not evident at this stage, but can be exposed after Pronase/formic acid treatment. Stage 3 corresponds to a further stage in which C-terminal truncation at Asp-421 appears. By Stage 4, intracellular PHFs and fibrils are present, which are phosphorylated at both N- and C-termini and showed variable immunoreactivity to mAb 423. The fibrillar nature of the tangles was confirmed by labeling with TR. As the tangle develops intracellularly in Stage 5, the N-terminal portions are removed and proteolysis reveals the epitope recognized by mAb 423. By Stage 6, the plasma membrane has been disrupted and the extracellular “ghost” tangles are evident, which are comprised of highly insoluble tau, reactive with mAb 423 but with only occasional pS396 epitopes remaining. See text for more detailed description.