Additional file 1 of Tau-proximity ligation assay reveals extensive previously undetected pathology prior to neurofibrillary tangles in preclinical Alzheimer’s disease

Additional file 1. Fig. S1. Automated analysis and semi-quantitative scales used for analysis. A and B) Original images and images after analysis with quantification. Analysis performed as specified in Materials and methods. A) 3 sample images with low, middle and high density of diffuse small tau-PLA labelled structures were processed with ImageJ for the automated measurement of diffuse signal, insets show final counts. B) Samples from the 4 different immunolabellings were processed with ImageJ for the automated measurement of large labelled lesions, insets show final counts. C) Semi-quantitative scale used for semi-quantitative analysis of all brain regions. Fig. S2. Detection of tau–tau interactions in HEK 293 cells. Representative fields of view of the tau-PLA puncta quantification of Fig. 2B. Tau-PLA puncta are in red. Nuclei were identified by DAPI staining (blue). Scale bar: 10 μm. Fig. S3. In situ specificity of tau-PLA in mouse brain tissue. Top: Representative images from 6 months-old P301S transgenic, C57BL/6 wild-type and MAPT KO mice (N = 6 per genotype), stained with Tau-PLA and AT8. Brain histological analysis revealed an absence of tau-PLA signal in MAPT KO mouse brain tissue. C57BL/6 control mice showed almost negligible load of tau-PLA, whereas this signal appeared to be quite strong in the P301S mice. Tau-PLA revealed prominent tau multimerization in the CA1 region of hippocampus and striatum, anatomical areas negative for AT8. Bottom: Representative images from P301S transgenic animals at the age of 3 (N = 2), 6 (N = 6), and 9 (N = 2) months old. This analysis shows an age-dependent accumulation of tau-PLA signal. Scale bar 100 μm. Fig. S4. Tau-PLA detects endogenous human tau–tau interaction in situ. A) Tau-PLA recognized tau pathology in the CA4/dentate gyrus of Alzheimer’s disease (Braak V) as compared to Braak 0 controls. B) Tau-PLA was performed in samples of different Braak stages in the presence or absence of ligase. The absence of ligase in the PLA reaction resulted in the ablation of all PLA signal, indicating the staining is dependent on the ligation of two conjugates. C) 6xHisTag IHC and 6xHisTag-PLA performed in samples of Braak stage 0 and V. No signal was detected after performing immunohistochemistry with 6xHisTag antibody and 6xHisTag-PLA. Scale bar 50 μm. Fig. S5. A proportion of diffuse tau complexes locates to astrocytes and microglia. Representative co-immunofluorescence images (Braak IV) of indicated markers and tau-PLA. Arrowheads indicate astrocyte/microglial cells in top and bottom panels, respectively. Fig S6. Tau-PLA revealed that cases in Braak 0 group presented heterogenicity. A) Quantification of tau-PLA labelled small diffuse complexes in the different hippocampal regions, revealing the distribution of the population in Braak 0 group. ONE-WAY ANOVA (Tukey). AV; average. B) Representative images of EC stained with tau-PLA that illustrate the heterogenicity that characterize the Braak 0 group. Scale bar 50 μm. Fig. S7. Quantification of lesions in samples labelled with tau5 IHC. Samples immunolabelled with tau5 IHC were used for automated analysis. All groups were compared to control (Braak 0) through a ONE-WAY ANOVA (Dunnet). N = 7/7/7/4/6/6/6. AV; average. IHC: immunohistochemistry. Fig. S8. Early tau multimerization detection, prior to detection of tau hyperphosphorylation and misfolding across hippocampal regions and temporal isocortex – CA4. 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. 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. Scale bar 50 μm. B) Quantification of tau-PLA, AT8-, MC1-, and AT8- IHC labelled diffuse/thread 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. AV; average. IHC: immunohistochemistry. Fig. S9. Early tau multimerization detection, prior to detection of tau hyperphosphorylation and misfolding across hippocampal regions and temporal isocortex – Entorhinal Cortex. 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. 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 appearing 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. AV; average. IHC: immunohistochemistry. Fig. S10. Early tau multimerization detection, prior to detection of tau hyperphosphorylation and misfolding across hippocampal regions and temporal isocortex – CA1. 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. 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 appearing 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. AV; average. IHC: immunohistochemistry. Fig. S11. Quantification of diffuse pathology/threads and lesions labelled by AT180- and MC1- IHC in hippocampal regions and temporal isocortex. Quantification of diffuse pathology/threads and lesions labelled by AT180- and MC1- IHC in hippocampal regions and temporal isocortex. All groups were compared to control (Braak 0) through a ONE-WAY ANOVA (Dunnet). N = 11/12/12/9. AV; average. IHC: immunohistochemistry. Fig. S12. Quantification of lesions in samples labelled with 4G8 IHC. Clinically processed samples immunolabelled with 4G8 were used for automated analysis. All groups were compared to control (Braak 0) through a ONE-WAY ANOVA (Dunnet). N = 7/6/6/4/5/5/4. AV; average. IHC: immunohistochemistry. Table S1. Summary of patient information. AV; average. Table S2. Additional information of brain samples. PMI: post-mortem interval, CERAD: consortium to establish a registry for Alzheimer’s disease, MCI: mild cognitive impairment.