Caspase-cleavage of tau is an early event in Alzheimer disease tangle pathology (original) (raw)
Tau is cleaved at D421 by executioner caspases. To determine which caspases cleave tau, recombinant human tau40 was treated with several active caspases. Incubation of tau with caspase-3 or -7, but not with caspase-1, -4, -5, -8, or -10, resulted in a 2-kDa electrophoretic shift by SDS-PAGE (Figure 1A, arrowhead). Analysis of the cleavage products by electrospray mass spectroscopy revealed two tau fragments with molecular weights of 45,900.00 Da (ΔTau) and 1,997.99 Da (C-terminus), corresponding to the theoretical molecular weights of tau cleaved after amino acid residue D421 (45,900.80 Da and 1,999.29 Da) (see supplemental data; available at http://www.jci.org/cgi/content/full/114/1/121/DC1). Cleavage after D421 was also confirmed by primary amino acid sequencing of the small fragment (data not shown).
Executioner caspases cleave tau at D421. (A) Recombinant human tau40 (Calbiochem) treated with various caspases was separated by SDS-PAGE and stained with Coomassie blue. Treatment with caspase-3 (C3) and caspase-7 (C7) resulted in a 2-kDa shift (arrowhead) relative to full-length tau (FL), while treatment with caspase-1 (C1), -4 (C4), -5 (C5), -8 (C8), or -10 (C10) did not (arrow). (B) Western blot analysis confirmed the specificity of polyclonal antibody (α-ΔTau) to D421 caspase-cleaved tau. α-ΔTau recognized caspase-3– and caspase-7–cleaved tau, but not uncleaved tau. (C) Executioner caspases remove the C-terminal epitope of tau that is recognized by antibody T46. Full-length and caspase-cleaved tau was probed with mAb T46.
To examine the role of ΔTau in NFT pathology, we generated a rabbit polyclonal antibody (α-ΔTau) directed against the caspase-cleaved carboxy-terminus of tau. The specificity of α-ΔTau was confirmed by Western blot analysis and was found to selectively recognize the D421-cleaved fragment generated by caspase-3 or -7 but not full-length tau (Figure 1B). In contrast, a previously generated antibody that recognizes the C-terminus of tau (T46; ref. 35, 36) detects full-length tau but not ΔTau (Figure 1C).
Specificity of the α-ΔTau antibody was further assessed, in vivo, using traumatic brain injury (TBI), a model that leads to neuronal caspase activation (37). Forty-eight hours after injury, immunohistochemical analysis confirmed caspase-3 activation within both wild-type (Figure 2A, inset) and Tau–/– brains (Figure 2B, inset). However, ΔTau immunoreactivity was observed within wild-type (Figure 2A) but not Tau–/– (Figure 2B) mice following TBI. In contrast, caspase-cleaved fodrin, another cytoskeletal target of caspases, was observed within both wild-type and tau knockout brains (see supplemental data). Therefore, the α-ΔTau antibody specifically detects caspase-cleaved tau and does not cross-react with other caspase-cleaved substrates in vivo.
The α-ΔTau antibody specifically recognizes caspase-cleaved tau in vivo. Wild-type (A) and Tau–/– (B) mice were subjected to TBI to induce caspase activation. Immunofluorescent labeling with an antibody to active caspase-3 confirmed caspase activation within both wild-type (A, inset) and tau knockout (B, inset) brains. ΔTau immunoreactivity was also detected within wild-type brains (A). In contrast, ΔTau immunoreactivity was not observed within tau knockout mice following TBI (B), which further confirms the specificity of the α-ΔTau antibody in vivo. Scale bar: 80 μm (A and B), 20 μm (insets).
Δ_Tau is prevalent within the AD brain and is inversely correlated with cognitive function_. To determine whether ΔTau is associated with AD neuropathology, AD and control hippocampal sections were immunolabeled with the α-ΔTau antibody. We observed extensive α-ΔTau labeling within AD hippocampus (Figure 3A). In contrast, only occasional ΔTau-positive cells were observed in controls (Figure 3B). Preadsorption of the α-ΔTau antibody with immunizing peptide led to a loss of all immunoreactivity (Figure 3A, inset).
ΔTau is found predominantly within AD brain and is inversely correlated with cognitive function. Large numbers of ΔTau-immunoreactive neurons (red) were observed in the hippocampus and entorhinal cortex of AD brains (A). In contrast, only occasional ΔTau-immunoreactive cells were observed in age-matched control cases (B). Preadsorption of the α-ΔTau antibody with immunizing peptide resulted in no immunofluorescence (A, inset). Regression analysis of the number of hippocampal CA1 cells labeled with α-ΔTau versus AD Mini-Mental State Examination (MMSE) scores revealed a significant inverse correlation between ΔTau and cognitive function (P = 0.05, _r_2 = –0.72; (C). High-power confocal microscopy demonstrated distinct subcellular localization of ΔTau (red) and the C-terminal–specific antibody T46 (green) within cell bodies (D) and dystrophic neurites (E). Scale bar: 65 μm (A and B), 8 μm (D), 5 μm (E).
Next, we performed light-level immunohistochemistry to quantify the number of ΔTau-immunoreactive cells within CA1 of AD versus control hippocampus (data not shown). Significantly more ΔTau-immunoreactive cells were observed in AD than in control brains (AD, 28.2 ± 1.65; control, 4.25 ± 0.85; P < 0.05). Furthermore, the number of ΔTau-immunoreactive cells within CA1 of AD cases inversely correlated with cognitive function (Mini-Mental State Examination [MMSE] score) (Figure 3C). Thus, ΔTau may contribute to cognitive decline in AD.
To assess the relationship between caspase-cleaved tau and full-length tau pathology, fluorescent double labeling for ΔTau and the C-terminal–specific antibody T46 was performed (36). High-power confocal imaging revealed an intriguing difference in subcellular distribution between these two markers, suggesting that both full-length tau (T46, green) and cleaved tau (α-ΔTau, red) are present within the same NFTs (Figure 3, D and E). T46 immunoreactivity was frequently observed surrounding ΔTau immunofluorescence in both cell bodies (Figure 3D) and dystrophic neurites (Figure 3E). The C-terminal epitope recognized by T46 (35) is liberated by executioner caspases (Figure 1C). Therefore, the minimal subcellular colocalization of ΔTau and T46 further confirms the cleavage site specificity of α-ΔTau.
Tau becomes increasingly insoluble as AD progresses. Therefore, we performed sequential fractionation of control, mild cognitive impairment (MCI), and AD brain lysates into high-salt reassembly buffer (RAB) and detergent-soluble radioimmunoprecipitation buffer (RIPA) fractions to examine the solubility of ΔTau as AD progresses. We found that ΔTau recognized multiple caspase-cleaved tau isoforms in both MCI and AD cases but not controls within the high-salt soluble RAB fraction (Figure 4A). In the detergent-soluble RIPA fraction, ΔTau was present within AD cases and also in a more advanced MCI-AD transitional case (Figure 4A, asterisk), but not within MCI or control cases (Figure 4A). In addition, ΔTau appeared to be post-transationally modified (hyperphosphorylation or ubiquitination) within the RIPA fraction, as evidenced by smeared signal (Figure 4A). Therefore, ΔTau appears to become increasingly insoluble as AD advances.
ΔTau becomes increasingly insoluble with disease progression and is associated with tangle-like pathology at the ultrastructural level. Control (C), MCI, and AD temporal cortex samples were subjected to sequential fractionation into high-salt RAB and detergent-soluble RIPA fractions as previously described (50, 78) (A). ΔTau was detected in the soluble RAB fraction of MCI and AD but not control cases, suggesting that ΔTau production coincides with the early stages of cognitive decline in AD. In detergent-soluble RIPA fractions, ΔTau was only detected in higher-pathology MCI and AD cases, which suggests that ΔTau becomes more insoluble as AD progresses. *This patient was considered transitional between MCI and AD (see Methods). Immunogold electron microscopy demonstrated ΔTau at the ultrastructural level in association with early tangle-like pathology within a neuronal cell body (B) and a myelinated axon (C). Scale bar: 0.4 μm (B), 0.3 μm (C).
To further elucidate the subcellular distribution of ΔTau in the AD brain, immunogold electron microscopy was performed using the α-ΔTau antibody. A diffuse distribution of α-ΔTau conjugated gold particles was observed in association with tangle-like structures within neuronal somata (Figure 4B). In addition, gold particles were observed within structurally intact axons (Figure 4C), which indicates that caspase-cleavage of tau may precede neuritic pathology.
Δ_Tau induces filament formation_. To determine whether ΔTau contributes to tangle pathology by promoting filament formation, we measured light scattering, which has previously been shown to be an effective means of monitoring tau filamentous aggregates (38). Tau was incubated in the presence of heparin, which accelerates tau aggregation in vitro, so that we could assay filament formation in real time (39). We found that caspase-3–cleaved tau (filled squares) demonstrated increased light scattering over time (indicative of filament formation) and aggregated more rapidly than full-length tau (filled diamonds, Figure 5). In addition, ΔTau accelerated filament formation of full-length tau, as the incubation of full-length tau in the presence of ΔTau resulted in a dramatic increase in the rate of scattered light (open squares, Figure 5). These results show not only that caspase-cleavage of tau leads to increased filament formation, but that ΔTau may also act to seed filament formation of full-length tau. These data further support a nucleation-dependent mechanism of tau filament assembly (40).
Caspase-cleavage of tau drives filament formation of full-length tau in vitro. Laser light scattering was used to assess tau filament formation in vitro. Equimolar (4 μM) amounts of ΔTau (filled squares), full-length tau (filled diamonds), or a 3:1 ratio of full-length tau to ΔTau (open squares) were monitored over 400 minutes. ΔTau (filled squares) scattered light (arbitrary units [AU]) more rapidly and to a greater degree than full-length tau (filled diamonds), indicating increased filament formation. Most interestingly, a 3:1 ratio of full-length tau to ΔTau (open squares) dramatically accelerated light scattering, suggesting that ΔTau may nucleate the assembly of full-length tau filaments.
Caspase-cleavage induces a conformational change in tau, which can be hyperphosphorylated by glycogen synthase kinase-3 β. The increased propensity of ΔTau to induce filament formation may ensue from a conformational change that follows liberation of the C-terminus. One of the earliest pathological alterations in tau during AD is a conformational change that is recognized by the antibody MC1 (12). Because the formation of the MC1 epitope occurs prior to paired helical filament (PHF) assembly (12), we hypothesized that ΔTau may likewise adopt this conformation. Immunoprecipitation with MC1 preferentially recognized ΔTau over full-length tau in three separate experiments (Figure 6A). Therefore, our results show that caspase-cleavage of tau induces an early conformational change recognized by MC1.
Caspase-cleavage of tau induces a conformational epitope that is recognized by the antibody MC1 and can be phosphorylated by GSK3-β. (A) MC1, a mAb that recognizes an early tau conformational change, was coupled to protein G and used to immunoprecipitate either full-length (lane 1) or caspase-3–cleaved recombinant tau (lane 2). Western blot analysis of immunoprecipitated proteins probed with an anti-tau polyclonal antibody revealed an increased affinity of the MC1 antibody for ΔTau over full-length tau. (B) In vitro phosphorylation of ΔTau: Either full-length or caspase-3–cleaved tau was phosphorylated with the kinase GSK-3β and then analyzed by Western blot with the phosphorylation-dependent antibody PHF-1. Immunoreactivity for PHF-1 demonstrates that, like full-length tau (FL), caspase-cleaved tau (ΔTau) can be hyperphosphorylated and suggests that caspase-cleavage of tau does not preclude subsequent hyperphosphorylation. Numbers over each lane indicate the number of hours that tau species were incubated with GSK-3β. MC1 and PHF-1 experiments were replicated three times, and representative immunoblots are shown.
Besides a conformational change, phosphorylation of specific residues is another important stage in the pathology of tau. Accordingly, we tested whether caspase-cleaved tau can be hyperphosphorylated in vitro. Recombinant full-length tau and caspase-cleaved tau were incubated at 37°C in the presence of ATP and glycogen synthase kinase-3β (GSK-3β), a kinase implicated in the hyperphosphorylation of tau (41). Western blot analysis with the tau phospho-epitope antibody PHF-1 revealed that both ΔTau and full-length tau were hyperphosphorylated in vitro by GSK-3β (Figure 6B). Although we assayed kinase activity over a 4-hour period, phosphorylation of ΔTau occurred rapidly within 1 hour. Therefore, caspase-cleavage does not preclude subsequent hyperphosphorylation of tau, which further supports the notion that ΔTau may occur early in the development of NFT pathology.
Δ_Tau is observed throughout the evolution of NFT pathology in the AD brain_. Because ΔTau adopts a conformation that is recognized by the MC1 antibody and can be phosphorylated to form the PHF-1 epitope, we performed triple-labeling experiments with α-ΔTau (red), MC1 (green), and fluorescently labeled PHF-1 (blue) to determine whether ΔTau colocalized with early (MC1) or late (PHF-1) tau pathological alterations in AD (Figure 7). In the AD hippocampus and entorhinal cortex, we found both neurons (Figure 7, A–D) and dystrophic neurites (Figure 7, E and F) that labeled with ΔTau, MC1, and PHF-1. We observed all three labels in pretangle (Figure 7A), diffuse tangle–bearing (Figure 7B), and mature tangle–bearing neurons (Figure 7, C and D). Interestingly, we did not observe any cells that expressed MC1 and PHF-1 without ΔTau, which strengthens our assertion that ΔTau is important in tangle formation. Finally, triple labeling of plaque-associated dystrophic neurites revealed numerous permutations of the three tangle markers, suggesting that dystrophic neurites also show varying degrees of AD pathological progression (Figure 7F).
ΔTau is present throughout the evolution of NFT pathology. Triple immunofluorescence was used to examine the association of ΔTau (red) with the conformation-sensitive tau antibody MC1 (green) and the tau phosphoepitope antibody PHF-1 (blue) in hippocampal NFT pathology. ΔTau (red) was coincident with MC1 and PHF-1 throughout the evolution of NFT pathology within pretangle neurons (A), diffuse tangle–bearing neurons (B), mature tangle–bearing neurons (C and D), and dystrophic neurites (E and F). Scale bar: 8 μm (A, C, and D), 12 μm (B), 4 μm (E), 10 μm (F).
Active caspase-3 colocalizes withΔTau within pretangle neurons and dystrophic neurites of the AD brain. To further examine whether caspase activation and caspase-cleavage of tau occur early in the formation of tangles, we performed fluorescent double labeling for active caspase-3 and ΔTau. We observed colocalization of active caspase-3 with ΔTau, specifically within pretangle neurons of CA1 (Figure 8A) and dystrophic neurites (Figure 8B). In contrast, within mature extracellular tangles, active caspase-3 was not observed, possibly because of loss of membrane integrity and cell death (Figure 8C). Taken together, these data suggest that caspase activation and caspase-cleavage of tau occur early in tangle development. However, caspase activation appears to be lost with cell death, while ΔTau persists within extracellular NFTs, perhaps as insoluble accumulations.
Caspase activation and intracellular Aβ colocalize with ΔTau in the AD brain. Fluorescent confocal imaging demonstrated that ΔTau (red) and active caspase-3 (green) colocalized within pretangle neurons (A) and dystrophic neurites (B) of the AD hippocampus. In contrast, mature extracellular tangles contained ΔTau without evidence of caspase activation (C). Intracellular Aβ (green), a known initiator of caspases, colocalizes with ΔTau (red) within AD neurons (D and E). In addition, ΔTau-immunoreactive dystrophic neurites (red) were frequently associated with extracellular amyloid plaques (F). Scale bar: 5 μm (A), 1.5 μm (B), 6 μm (C), 3 μm (D and E), 25 μm (F).
Evidence thatΔTau is induced by A β. Aβ activates caspases in vitro (42–44). Therefore, we sought to examine whether ΔTau colocalized with Aβ in the AD brain. We found that ΔTau (red) colocalized with granular intraneuronal Aβ deposits (green) within CA1 (Figure 8, D and E). Similar results were observed with two different Aβ-specific antibodies (Aβ1–42 and 4G8). ΔTau-immunoreactive dystrophic neurites were also frequently observed in association with Aβ-immunoreactive plaques (Figure 8F). Combined, these data suggest that both intracellular and extracellular Aβ may induce caspase-cleavage of tau.
Because we found that Aβ colocalized with ΔTau in the AD brain, we determined whether Aβ might induce caspase-cleavage of tau in cultured rat primary cortical neurons. Using immunofluorescent triple labeling for ΔTau (red), the neuronal marker MAP-2 (green), and DAPI (blue), we found a significant increase in ΔTau-positive neurons following fibrillar Αβ1–42 treatment (Figure 9). Quantification of cell number (see Methods) revealed a significant increase in ΔTau-positive cells following Αβ1–42 treatment (F (7, 31) = 43.28, P < 0.0001). After 6 hours of treatment, a twofold increase in ΔTau-immunoreactive cells was observed (control, 1.0 ± 0.33 cells; treated, 2.13 ± 0.35 cells; P = 0.012). However, by 24 hours, the number of ΔTau-immunoreactive cells was increased almost ninefold over that of untreated cells (control, 0.75 ± 0.37 cells; treated, 6.63 ± 0.38 cells; P < 0.0001) (Figure 9D). The number of ΔTau-immunoreactive cells was not significantly affected after only 1 hour (P = 0.560) or 4 hours (P = 0.086) of Αβ1–42 treatment. The majority of ΔTau-bearing neurons (91.5%) in control and Αβ1–42-treated cultures contained apoptotic nuclei, as assessed by DAPI (Figure 9, A–C, blue). Furthermore, we discovered that ΔTau labeled striking tangle-like structures in dying neurons (Figure 9, B and C). Therefore, Aβ1–42 triggers the production of ΔTau in vitro.
Treatment of cortical neurons with Aβ leads to increased ΔTau immunoreactivity. Fluorescent triple labeling of rat primary cortical neurons for ΔTau (red), MAP-2 (green), and DAPI (blue) following Aβ treatment (A–C). A significant increase in ΔTau-positive cells (red) was observed after 6 hours (*P = 0.012) and 24 hours (**P < 0.0001) of Aβ treatment (D). The great majority of ΔTau-positive cells (91.5%) contained apoptotic nuclei as revealed by DAPI (A–C). In addition, ΔTau immunolabeling also revealed striate tangle-like morphology in association with apoptotic nuclei (B and C). Scale bar: 9 μm (A), 5 μm (B), 7.5 μm (C).
Δ_Tau is detected early in the progression of tangle pathology in a triple-transgenic model of AD_. To better assess the role of caspase-cleaved ΔTau in the pathogenesis of AD, we determined whether ΔTau was present in the brains of transgenic mice. We used a recently characterized triple-transgenic mouse (3xTg-AD) that recapitulates many salient features of AD: progressively developing intraneuronal Aβ, deficits in long-term potentiation, amyloid plaques, and, subsequently, NFTs (45). We examined brains from 6- (with only Aβ deposits but no NFT pathology), 12- (in the initial stages of NFT pathology), and 18-month-old mice (in which both plaque and NFT were well established). At 6 months of age, we detected intracellular Aβ, but not ΔTau immunoreactivity (data not shown). By 12 months of age, we found that ΔTau (red) colocalized with intraneuronal Aβ immunoreactivity within CA1 neurons of the hippocampus (Figure 10A). In older mice (18 months), Aβ and ΔTau colocalized within both CA1 neurons (Figure 10B) and the neocortex (Figure 10C). This finding closely mimics the progression of NFT pathology that has previously been reported in 3xTg-AD mice (45). Next, we investigated whether ΔTau colocalized with MC1 in this transgenic model. Colocalization of ΔTau (red) and MC1 (green) was first observed within CA1 pyramidal neurons at 12 months of age (Figure 10D) and persisted in 18-month-old mice (Figure 10, E and F). Interestingly, the subcellular distribution of ΔTau evolved with increasing age. At 12 months, the great majority of ΔTau-immunoreactive cells displayed punctate cleavage product within the soma and proximal dendrites of neurons (Figure 10, A and D). However, by 18 months of age, many ΔTau-bearing neurons exhibited aggregated filamentous structures that appeared strikingly similar to AD tangle pathology (Figure 10, C and F).
ΔTau colocalizes with pathological features of a triple-transgenic model of AD. Fluorescent double labeling of 3xTg-AD mice demonstrates that intraneuronal Aβ (green) colocalizes with ΔTau (red) within CA1 pyramidal neurons of 12-month-old (A) and 18-month-old (B) 3xTg-AD mice and within 18-month-old neocortex (C). ΔTau immunoreactivity (red) also colocalizes with an early tangle marker, MC1 (green), within CA1 pyramidal neurons at both 12-month (D) and 18-month time points (E and F). Scale bar: 30 μm (A, D, and E), 20 μm (B), 4 μm (C and F).