Tau-proximity ligation assay reveals extensive previously undetected pathology prior to neurofibrillary tangles in preclinical Alzheimer's disease

Abstract

Background: Multimerization is a key process in prion-like disorders such as Alzheimer's disease (AD), since it is a requirement for self-templating tau and beta-amyloid amyloidogenesis. AT8-immunohistochemistry for hyperphosphorylated tau is currently used for the diagnosis and staging of tau pathology. Given that tau-tau interactions can occur in the absence of hyperphosphorylation or other post-translational modifications (PTMs), the direct visualization of tau multimerization could uncover early pathological tau multimers.

Methods: Here, we used bimolecular fluorescent complementation, rapamycin-dependent FKBP/FRB-tau interaction and transmission electron microscopy to prove the in vitro specificity of tau-proximity ligation assay (tau-PLA). We then analyzed MAPT KO and P301S transgenic mice, and human hippocampus and temporal isocortex of all Braak stages with tau-PLA and compared it with immunohistochemistry for the diagnostic antibody AT8, the early phosphorylation-dependent AT180, and the conformational-dependent antibody MC1. Finally, we performed proteinase-K treatment to infer the content of amyloidogenic beta-sheet fold.

Results: Our novel tau-proximity ligation assay (tau-PLA) directly visualized tau-tau interactions in situ, and exclusively recognized tau multimers but not monomers. It elicited no signal in MAPT KO mouse brains, but extensively labelled P301S transgenic mice and AD brain. Two groups of structures were detected, a previously unreported widespread small-sized diffuse pathology and large, neurofibrillary-like lesions. Tau-PLA-labelled diffuse pathology appeared from the earliest Braak stages, mostly unaccompanied by tangle-like tau-immunohistochemistry, being significantly more sensitive than any small-sized dot-/thread-like pathology labelled by AT180-, AT8- and MC1-immunohistochemistry in most regions quantified at stages 0-II. Tau-PLA-labelled diffuse pathology was extremely sensitive to Proteinase-K, in contrast to large lesions.

Conclusions: Tau-PLA is the first method to directly visualize tau multimers both in vitro and in situ with high specificity. We find that tau multimerization appears extensively from the earliest presymptomatic Braak stages as a previously unreported type of diffuse pathology. Importantly, in our study multimerization is the earliest detectable molecular event of AD tau pathology. Our findings open a new window to the study of early tau pathology, with potential implications in early diagnosis and the design of therapeutic strategies.

Keywords: AT8; Aggregation; Alzheimer’s; Early pathology; Multimer; Phosphorylation; Proximity-ligation assay; Tau.

Fig. 1

Tau-PLA detects tau-BiFC complexes. a Tau-BiFC constructs contain non-fluorescent halves of GFP fused to tau. GFP activity is detected only when tau molecules interact between them and the split GFPs fold together. b, c Expression of tau-BiFC constructs in HEK293 cells was analysed by fluorescent microscopy and western blot. Tau-BiFC constructs form oligomeric species, as shown by non-denaturing PAGE. Scale bar: 100 µm. d Green fluorescence in HEK293 cells expressing tau-BiFC constructs indicated tau–tau interactions. Tau-PLA signal (red) was co-localized with the BiFC signal (green) in transfected cells, indicating that tau-PLA detects tau–tau interactions. Nuclei were identified by DAPI staining (blue). Images are maximum projections of z-stacks. Scale bar: 10 µm. All experiments were performed in triplicate

Fig. 2

Tau-tau interaction is detected by tau-PLA. a Inducible homomeric tau complexes are detected by tau-PLA. Tau was fused to FKBP or FRB which form a ternary complex with rapamycin. Tau complexes are forced to conditionally associate in the presence of rapamycin. b HEK293 cells were transfected with both constructs and incubated with or without rapamycin for 1 h after transfection (left). Quantification of tau-PLA puncta (right). Tau-PLA puncta significantly increased in the presence of rapamycin, as determined by unpaired two-tailed Student’s t-test (*p < 0.05), indicating tau-PLA detects tau–tau interactions. Data are mean ± SEM, N = 20 cells per condition. Nuclei were identified by DAPI staining (blue). Images are maximum projections of z-stacks. Scale bar: 10 μm. All experiments were performed in triplicates. c Tau-PLA detects tau–tau interaction of recombinant tau but not monomeric tau. Aggregation of recombinant full-length unphosphorylated tau4R was induced by shaking in the presence of heparin. After 16 h of shaking, both amorphous and spherical/globular tau accumulations are seen on TEM analysis. After 360 h of shaking tau assembled into a mixed population of shaped species, including globular structures and filaments, mostly of short or intermediate length. Alpha- synuclein assembled into fibrils after 120 h of shaking (without heparin). The obtained recombinant protein preparations were subjected to TEM (all images at 18.500 x) and tau-PLA

Fig. 3

Tau-PLA highlights different staining patterns. Images taken from a stage V patient. a Tau-PLA labels (i) neurofibrillary tangles (CA1). Note also unlabelled neurons. (ii) Tau-PLA labels neuropil threads (entorhinal region). Arrow in inset shows alignment of tau-PLA dots onto elements with axonal morphology. (iii) Tau-PLA labels neurites within neuritic plaque (CA1). (iv) Tau-PLA labels morphologically intact neurons with accumulation of tau complexes (CA1). Note these appear in areas lacking any AT8 immunohistochemistry labelling and are different to pre-tangle neurons. (v) Tau-PLA labels diffuse accumulation of tau–tau complexes in the neuropil (CA4). (vi) Tau-PLA labels the perikarya of occasional oligodendrocytes (arrows, white matter). Scale bar 50 μm. b Neurofibrillary tangle maturity characterized by tau-PLA, from left to right examples of tau-PLA staining for normal, diffuse, pretangle, mature and ghost pathology. Scale bar 50 μm. c Tau-PLA distribution through the different Braak stages. Scale bar 2 mm

Fig. 4

Early tau multimerization detection, prior to detection of tau hyperphosphorylation and misfolding across hippocampal regions and temporal isocortex. Representative images of selected hippocampal regions are shown here. FFPE sections of posterior hippocampus at the level of lateral geniculate nucleus from Braak 0 to VI were stained with tau-PLA and AT8-immunohistochemistry. Minimal PLA and immunohistochemistry signal is seen in Braak 0. Tau-PLA reveals abundant pathology in Braak stages I to III and onwards, while AT8-immunohistochemistry is absent or relatively low at initial stages, with the signal mostly appearing after Braak III. Scale bar 50 μm

Fig. 5

Quantification of tau-PLA labelled diffuse pathology and tau-PLA and AT8 IHC labelled large perikaryal neurofibrillary-type lesions in hippocampal regions. a–c Automated quantification of a tau-PLA diffuse signal, b tau-PLA lesions and c AT8-IHC lesions in different brain regions. All groups were compared to control (Braak 0) through a ONE-WAY ANOVA (Dunnet). N = 11/12/12/9/7/8/8. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. TC temporal isocortex, EC entorhinal cortex, SO stratum oriens, AV average

Fig. 6

Early tau multimerization detection, prior to detection of tau hyperphosphorylation and misfolding across hippocampal regions and temporal isocortex–temporal isocortex. a Representative images of selected hippocampal regions are shown here. FFPE sections of posterior hippocampus at the level of lateral geniculate nucleus from Braak 0 to III were stained with tau-PLA and AT180-, AT8-, MC1-immunohistochemistry. Minimal PLA and immunohistochemistry signal is seen in Braak 0 stage. Tau-PLA reveals abundant pathology in Braak stages I to III, while AT180-immunohistochemistry shows a modest increase in signal in Braak stages I to III. AT8- and MC1-immunohistochemistry is absent or relatively low at initial stages, with signal starting to appear in Braak III. Scale bar 50 μm. b Quantification tau-PLA, AT8-, MC1-, and AT8- IHC labelled diffuse pathology. All groups in each Braak stage were compared to tau-PLA through a ONE-WAY ANOVA (Dunnet). N = 11/12/12/9/7/8/8. *p < 0.05, **p < 0.01, ***p < 0.001. AV average, IHC immunohistochemistry

Fig. 7

Tau self-interaction as one of the earliest events occurring during neurofibrillary tangle maturity. Schematic representation and images of the dynamic lifespan of NFT. Representative images of the hippocampal region are shown here. FFPE sections of posterior hippocampus at the level of lateral geniculate nucleus, stained with tau-PLA and AT180-, AT8-, MC1-immunohistochemistry. Tau-PLA reveals that multimerization is one of the earliest detectable events occurring during the development of tangles. AT180 positive staining can be detected even before the formation of pretangles, occurring straight after tau self-interaction appears. Hyperphosphorylation recognized by AT8 and conformational changes by MC1 occur early in the maturity of tangles as they are present in both pretangles and mature NFT. Ghost tangles appeared to be positive for AT180 and AT8, but not for MC1 (and possibly tau-PLA)

Fig. 8

Tau-PLA labelling is very sensitive to PK digestion which has a differential effect on small diffuse complexes and larger neurofibrillary-type lesions. Samples were treated for 0/10/60/120 s with PK at 37 °C and then stained for tau-PLA. Tau-PLA signal was very sensitive to PK treatment. Signal form diffuse small complexes was depleted by 10 s of PK treatment, while larger neurofibrillary-type lesions (neuritic and perikaryal lesions) labelled with tau-PLA required 1 min of PK digestion for complete depletion. NFT: neurofibrillary tangles