Heterotypic seeding of Tau fibrillization by pre-aggregated Abeta provides potent seeds for prion-like seeding and propagation of Tau-pathology in vivo
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Genetic, clinical, histopathological and biomarker data strongly support Beta-amyloid (Aβ) induced spreading of Tau-pathology beyond entorhinal cortex (EC), as a crucial process in conversion from preclinical cognitively normal to Alzheimer‘s Disease (AD), while the underlying mechanism remains unclear. In vivo preclinical models have reproducibly recapitulated Aβ-induced Tau-pathology. Tau pathology was thereby also induced by aggregated Aβ, in functionally connected brain areas, reminiscent of a prion-like seeding process. In this work we demonstrate, that pre-aggregated Aβ can directly induce Tau fibrillization by cross-seeding, in a cell-free assay, comparable to that demonstrated before for alpha-synuclein and Tau. We furthermore demonstrate, in a well-characterized cellular Tau-aggregation assay that Aβ-seeds cross-seeded Tau-pathology and strongly catalyzed pre-existing Tau-aggregation, reminiscent of the pathogenetic process in AD. Finally, we demonstrate that heterotypic seeded Tau by pre-aggregated Aβ provides efficient seeds for induction and propagation of Tau-pathology in vivo. Prion-like, heterotypic seeding of Tau fibrillization by Aβ, providing potent seeds for propagating Tau pathology in vivo, as demonstrated here, provides a compelling molecular mechanism for Aβ-induced propagation of Tau-pathology, beyond regions with pre-existing Tau-pathology (entorhinal cortex/locus coeruleus). Cross-seeding along functional connections could thereby resolve the initial spatial dissociation between amyloid- and Tau-pathology, and preferential propagation of Tau-pathology in regions with pre-existing ‘silent’ Tau-pathology, by conversion of a ‘silent’ Tau pathology to a ‘spreading’ Tau-pathology, observed in AD.
KeywordsAmyloid beta Tau Heterotypic seeding Prion-like Alzheimer’s disease
Brains of AD patients are characterized by the presence of neurofibrillary tangles and amyloid plaques composed of aggregated Tau and Aβ peptides, respectively [46, 47]. In AD, spreading of Tau-aggregation occurs in a very characteristic pattern (Braak stages) along functionally connected brain areas , strongly correlating with symptom progression, explaining its use as diagnostic criteria. Combined genetic, biomarker and histopathological data indicate increasing Aβ peptide concentrations as driving force for the conversion from preclinical to mild cognitive impairment (MCI) and finally AD [19, 25, 26, 53], strongly correlating with spreading of Tau-pathology beyond EC and the limbic system to the neocortex, by a poorly understood molecular mechanism. Noteworthy, a spatial dissociation between predominant amyloid- and Tau-pathology exists in (the initial stages of) AD. Amyloid-pathology accumulates predominantly in isocortex (Thal stages) , while Tau-pathology (Braak stages)  spreads from EC in a characteristic pattern along functionally connected regions.
While mechanistically unclear, Aβ-induced Tau-pathology has been demonstrated reproducibly in in vitro and in vivo models [2, 5, 13, 29, 36, 40, 49, 50]. Indeed, in a human neural stem cell derived 3D culture system, expression of EOFAD mutations induced aggregated Aβ and Tau filaments, with Tauopathy downstream of Aβ formation . Furthermore, different milestone papers have demonstrated amyloid-induced Tau-pathology in in vivo animal models, including following (1) crossing or co-expressing of mutant APP/PS1 with mutant Tau in transgenic mice [2, 29, 36, 40, 49] following (2) injection of pre-aggregated Aβ containing extracts derived from AD patients or APP/PS1 transgenic mice , or (3) following injection of synthetic pre-aggregated Aβ . Intriguingly, Aβ-induced Tau-pathology, following injection of pre-aggregated Aβ, induced not only Tau-pathology at the site of injection but also in brain regions remote from, but functionally connected to the injection site [2, 13].
These findings are not only reminiscent of the spatial dissociation between amyloid and Tau-pathology in the initial stages in AD patients, but also reminiscent of the prion-like spreading of Tau-pathology following Tau-seeding, along functional connections [1, 6, 22, 51]. Prion-like properties have been demonstrated for different proteins associated with neurodegenerative disorders [6, 8, 12, 16, 17, 22, 27, 28, 30, 33, 38, 39, 51, 56], including Tau. Prion-like seeding and propagation of Tau-pathology have been demonstrated in vitro and in vivo in different models with synthetic or patient-derived pre-aggregated Tau [6, 16, 17, 22, 28, 39, 44, 51]. Prion-like “seeding” using pre-aggregated Tau fibrils propagates Tau-aggregation by overcoming the rate-limiting nucleation step of Tau-aggregation. Once initiated Tau-pathology propagates rapidly in a prion-like way in in vivo models, and according to a pattern observed in AD, following initiation in EC Tau-pathology spreads to limbic system and cortex [7, 22, 27, 51]. Tau-pathology is thereby propagated to remote but functionally connected brain regions, affecting intrinsic functional neuronal networks, reminiscent of the networks affected in AD and related Tauopathies [1, 22, 51]. Although mechanistically not yet fully resolved, in terms of uptake and spreading of “seeds”, this process has been reproducibly recapitulated in different models. The strong similarity in induction of Tau-pathology by prion-like Tau-seeds and pre-aggregated forms of Aβ in in vivo models (i.e. in crosses of transgenic mice, following injection of pre-aggregated Aβ containing brain extracts from mice and AD patients, and pre-aggregated synthetic Aβ) has incited us to analyze whether pre-aggregated Aβ could directly cross-seed filamentous Tau-aggregation, leading to propagation of Tau pathology. Interestingly, cross-seeding between Tau and alpha-synuclein has been demonstrated previously in vitro and in vivo [11, 15]. Hence, in this work we have analyzed the potential of Aβ-seeds to induce Tau-aggregation and the potential of these Aβ-induced Tau-seeds to seed propagating Tau-pathology in vivo, as a potential mechanism underlying the propagation of Tau-pathology beyond EC in AD.
Materials and methods
In vitro generation of monomeric and pre-aggregated seeds
In vitro generation of pre-aggregated synthetic Aβ 1–42 peptides Aβ 1–42 peptides were purchased from Bachem (Bachem AG, Switzerland). Aβ monomers and Aβ aggregates (fibrils) were prepared as previously described . Briefly, lyophilized monomeric Aβ 1–42 was resuspended in HFIP (Sigma-Aldrich) and subsequently evaporated for 1 h in a SpeedVac (Thermo Fisher Scientific, Waltham, MA, USA) before its storage in single use aliquots at −20 °C. For Aβ 1–42 aggregation, the stored monomeric Aβ 1–42 was resuspended in DMSO (Sigma-Aldrich) at 5 mM, before dilution to 100 µM in 10 mM HCl solution and incubation 24 h at 37 °C. The aggregation nature of the preparations was assessed by thioflavin T (ThioT) assay, immunoblotting and immuno-EM. For all the experiments only freshly prepared sonicated fibrils (8 pulses of 30 % amplitude) were used.
In vitro generation of monomeric and pre-aggregated Tau-“Tau seeds” Tau seeds were generated as previously described [16, 22, 51]. The human truncated 4R Tau, encompassing the 4-repeat microtubule binding domain of Tau with the P301L mutation and a myc tag (K18-P301L; Q244-E372) was generated in Escherichia coli, further referred to as TauMono. Pre-aggregated synthetic Tau, “Tau seeds”, were obtained by incubation of TauMono (66.7 µM) with low molecular weight heparin [MP Biomedicals, Santa Ana, CA, USA; (ratio 1:2)] in 100 mM ammonium acetate buffer (pH 7) at 37 °C for 5 days. Before use, fibrillization mixture was centrifuged (100,000g for 1 h at 4 °C) and the resultant pellet resuspended in the same buffer without heparin to a final concentration of 333 µM and stored at −80 °C. Successful Tau fibrillization was confirmed by ThioT (Sigma-Aldrich, St. Louis, MO, USA) assay, immunoblotting and immuno-EM. For all experiments Tau seeds were sonicated (8 pulses of 30 % amplitude) before use.
In vitro generation of pre-aggregated synthetic amylin peptides Amylin peptides were purchased from Bachem (Bachem AG, Switzerland). Amylin aggregates were prepared as described [42, 43]. Briefly, amylin peptides were dissolved in DMSO to a final concentration of 20 mM and stored at −20 °C. For amylin aggregation, the stored monomeric peptides were dissolved in 25 µM KCl to a final concentration of 20 µM and incubated at 37 °C for 65 h. The aggregation nature of the preparations was assessed by ThioT assay and immunoblotting. For all the experiments only freshly prepared sonicated fibrils (8 pulses of 30 % amplitude) were used.
Cell culture and Tau aggregation assay
Human kidney-derived QBI-293 (QBiogene, Carlsbad, CA, USA) were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % (v/v) heat inactivated FBS, 1 % Pyruvate (10 mM), 1 % Penicillin–Streptomycin (PenStrep) and l-glutamine (20 mM). Cells were maintained at 37 °C in humidified atmosphere containing 5 % CO2. One day prior to transfection, 80 % confluent cells were trypsinized and then seeded in 10 cm2 dishes at 1.5 × 106 cells per well. The growth medium was renewed directly before transfection. DNA mixture containing 2.5 µg pcDNA6-TR and 2.5 µg 2N4R-TauP301L-GFP-pcDNA4/TO was diluted in 500 µL OptiMEM and 15 μL FuGENE® 6 transfection reagent diluted in 500 µL OptiMEM was added. The mixture was incubated for 15 min at room temperature (RT), then added to the cells. After incubation for 24 h, the growth medium was removed and replaced with a new one containing 5 µg/mL blasticidin and 200 µg/mL Zeozin. The cells were cultured until selection was complete. Monoclonal lines were generated by limited dilution. Cells were then grown in full media (DMEM, 10 % FBS; Invitrogen, Life Technologies, Carlsbad, CA, USA) supplemented with penicillin/streptomycin, l-glutamine and pyruvate. For the Tau aggregation assays, cells were plated at 100,000 per well in 12-well plates in medium containing doxycycline (Westburg, Leusden, Netherlands). On the day of aggregates transfection, Tau-seeds and Aβ-seeds were diluted in the respective buffers to a 10 µM solution and sonicated with 8 pulses/30 % amplitude; 80 µL of this solution was added to single use BioPORTER tubes (AMS Biotechnology, Milton, UK), vortexed at low speed for 5 s and incubated 10 min at RT. During the seeds-BioPORTER complexes formation, cells were washed with OptiMEM (Invitrogen) and placed in 250 µL of OptiMEM. 420 µL of OptiMEM was added to the BioPORTER tubes, and 250 µL of the resultant solution was added to the cells. The cells were incubated with the seeds for 4 h, and subsequently 500 µL medium was added (complete medium containing 20 % FBS) and further incubated for 3 days. For experiments using 24-well plates, the volumes were adjusted accordingly. For the experiments using Aβ seeds as catalyst of pre-existing Tau aggregation, two “BioPORTER transfections” were performed. On the first day of the experiment, Tau-seeds were transfected to the cells followed by the transfection of Aβ-seeds 24 h later. For the control condition BioPORTER without Aβ-seeds was added. The cells were further incubated for 2 days.
For total cell extracts, cells were washed and scraped in OptiMEM and centrifuged at 1000g for 5 min at 4 °C. Cell pellets were then resuspended in Triton lysis buffer (1 % TX-100, 50 mM Tris, 150 mM NaCl, pH 7.6) containing proteases and phosphatases inhibitors (F. Hoffman-La Roche AG, Basel, CH) before storage at −20 °C. For the separation of soluble and insoluble Tau, the Triton/SDS method was used as previously described [16, 22, 51]. Briefly, cells were washed with PBS, scraped into Triton lysis buffer containing protease and phosphatase inhibitors and incubated on ice for 15 min. Cell extracts were sonicated and centrifuged at 100,000g for 30 min at 4 °C. The supernatants yielded the Triton-soluble fraction (soluble proteins), while the pellets were resuspended, sonicated and vortexed in SDS lysis buffer (1 % SDS, 50 mM Tris, 150 mM NaCl, pH 7.6) and stored as Triton-insoluble fraction, after centrifugation at 100,000g for 30 min at 28 °C. For all samples proteins were quantified using BCA Protein Assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Samples were run in precast 4–12 % Bis–Tris gels (MOPS running; Invitrogen) and immunoblotting was performed with anti-Tau P-S202/T205 (AT8 1:500; Thermo Fisher Scientific), anti-total Tau antibody (HT7 1:500; Thermo Fisher Scientific) and anti-actin antibody (1:500; Sigma-Aldrich) and developed using ECL kit (PerkinElmer, Waltham, MA, USA).
Cytological and immunocytological analysis
Cells were fixed under stringent conditions for the removal of soluble Tau proteins using fixation/extraction buffer (4 % PFA, 4 % sucrose, 1 % TX-100 in PBS) for 10 min. After washing with PBS, cells were blocked (5 % FCS or milk in 0.1 % TX-100 PBS) for 30 min. The specific anti-Tau P-S202/T205 (AT8 1:100) and anti-conformational specific Tau (MC1 1:100, Peter Davies) were incubated for 2 h at RT or overnight at 4 °C followed by staining with Alexa Fluor 568 goat anti-Mouse IgG1 (1:500) or Alexa Fluor 647 goat anti-Mouse IgG1 (1:500) for 1 h at RT. DAPI staining to label nuclei was performed in a PBS washing step. The GFP bleaching experiments were performed as previously described with minor modifications . Briefly, a 5 µM acid-denatured GFP solution (pH 1.5) was prepared by mixing equal amounts of solution A (50 mM Tris–HCl pH 7.5) with solution B (125 mM HCl) and incubated with cells 1 min at RT. Image acquisition was performed using a digital inverted fluorescence microscope (EVOS-xl auto microscope). The 10×, 20× and 40× coverslip corrected lens were used in this study.
In vitro Tau aggregation assay (cell-free assay)
To assess the ability of Aβ seeds to directly cross-seed Tau aggregation, in vitro Tau fibrillization experiments were performed. TauMono (66.7 µM) was incubated with heparin (133 µM), pre-aggregated synthetic Tau seeds (6.67 µM; buffer A), Aβ seeds (6.67 µM; buffer B), amylin seeds (6.67 µM), albumin (6.67 µM), or with buffers alone (diluted as used in the respective seeding conditions). The fibrillization reaction was performed for 3 days at 37 °C with constant agitation at 150 rpm. Aliquots of all reactions were collected at different time points and successful fibrillization of Tau was verified by ThioT assay, sedimentation test and immunogold electron microscopy.
The assay was performed as previously described [10, 55]. Briefly, samples were incubated with ThioT solution (5 µM ThioT in 100 mM ammonium acetate buffer pH 7.0) in 96-well plates at RT. Fluorescence was measured with a PerkinElmer Victor X3 (PerkinElmer) with an excitation filter of 440 nm and an emission filter of 486 nm.
The sedimentation analysis was performed as previously described with minor modifications . Briefly, samples were ultracentrifuged (100,000g for 1 h at 4 °C) and the resultant pellet was resuspended in a volume of buffer corresponding to the volume of supernatant. Western Blot analysis was performed using an anti-total Tau antibody (1:2000; DAKO, Glostrup, Denmark).
Immunogold electron microscopy
Immunogold labeling was performed as previously described . Pure protein samples of the in vitro Tau fibrillization experiments were applied on 200-mesh carbon-coated grids in drops of 2 µL for 5 min, blotted on filter paper and air-dried before immunogold labeling. Grids were blocked in PBS 0.1 % cold water fish gelatin (PBS-CWFG) for 5 min before incubation with PBS-CWFG diluted primary antibody (Polyclonal Total Human Tau 1:50) for 90 min at RT. After washing five times (2 min) in PBS-CWFG, grids were incubated for 30 min with protein A secondary antibody labeled with 10 nm gold particles (Aurion, Wageningen, Netherlands) diluted 1:50 in PBS-CWFG. Following washes in PBS and ddH2O, grids were negatively stained with 2 % uranyl acetate for 45 s. Grids were examined with a JEOL JEM1400 transmission electron microscopy equipped with an Olympus SIS Quemesa 11Mpxl camera, and images were taken at magnifications of 10×, 20× and 30× k. For co-staining, grids were sequentially incubated for 90 min with the primary mouse anti-Aβ antibody (WO2 1:50) followed by rabbit anti-total human Tau antibody (DAKO Tau 1:50). Subsequently the grids were incubated with secondary antibodies at RT in PBS-CWFG. The secondary antibodies used were, respectively, 10 nm gold coupled goat anti-mouse antibody (1:50 in PBS-CWFG) and 5 nm gold coupled goat anti-rabbit antibody (1:50 in PBS-CWFG). The grids were negatively stained with 2 % uranyl acetate for 45 s and were examined as previously described.
In vivo seeding experiments
Animals TauP301S mice (PS19)  expressing the Tau isoform with one N-terminal insert and four microtubule binding repeats (1N4R) with the P301S clinical mutation, driven by the prion protein promotor, were backcrossed with C57B6 mice and used in this study. The TauP301S mice used in our lab develop similar neuropathology to previously reported studies, at around 11 months [22, 50, 58]. Animals were housed on a 12 h light/dark cycle in specific pathogen-free (SPF) facilities with access to food and water ad libitum. Stereotactic injections were performed in 4 months old mice and age-matched littermates were used for analysis at 3 months post-injection. All experiments were performed in compliance with protocols approved by the UCLouvain Ethical Committee for Animal Welfare.
Stereotactic injections For stereotactic surgery, 4 months old mice were deeply anesthetized with a mixture of ketamine/xylazine (Ketalar/Rompun). Unilateral (right hemisphere) stereotactic injections were performed in the hippocampal region (A/P, −2.0 mm; L, +1.4 mm; D/V, −1.4 mm) and frontal cortex (A/P, +2.0 mm; L, +1.4 mm; D/V, −1.0 mm); all coordinates are expressed relative to bregma . Sonicated pre-aggregated homotypic Tau-induced Tau-aggregates (66.7 µM), Aβ-induced Tau-aggregates (66.7 µM), non-aggregated or monomeric Tau controls (66.7 µM), Aβ seeds (333 µM) and buffer controls were injected using a 10 µL Hamilton syringe at a speed of 1 μL per min. After injection, the needle was kept in place for additional 5 min before gentle withdrawal.
Immunohistological analysis Immunohistological analysis was performed as previously described [41, 50]. Animals were transcardial perfused with ice cold PBS (2 min) by transcardial flushing and brains were dissected and fixed in 4 % PFA in PBS for 24 h at 4 °C. 40 μm thick sagittal sections were cut in a vibrating HM650V microtome (Thermo Fisher Scientific, Waltham, MA, USA). Immunohistochemistry (IHC) was performed on these sections with anti-Tau P-S202/T205 (AT8), anti-Tau P-S212/T214 (AT100) and anti-conformational Tau (MC1, Peter Davies) primary antibodies followed by appropriate Alexa-coupled secondary antibody, as previously described . Staining with Thioflavin S (ThioS; Sigma-Aldrich, St. Louis, MO, USA) and Gallyas silver (all chemicals from Sigma-Aldrich, St. Louis, MO, USA) staining were performed as previously described [50, 51] and were used to demonstrate the “amyloid” nature of Tau aggregates in brain sections. Image acquisition was performed using a digital inverted fluorescence microscope (EVOS-xl auto microscope), using the 4×, 10× and 20× objectives. Image analysis was performed using Image J (National Institutes of Health). Heat maps were generated using the HeatMap Histogram plugin for Image J, as previously described . Briefly, the overview images of AT8 staining of a well-identified section of different mice (n = 6 per group) were grouped. Stacked images representing averaged intensities were generated using the Image J stacking tool with the average intensities outcome option. Finally a Gaussian Blur filter of 5.0 was applied.
Statistical analysis was performed using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA). Normality testing was performed for all the data using D’Agostino and Pearson omnibus normality test when possible. Kolmogorov–Smirnov tests were used for smaller sample sizes. According to the results of normality tests appropriate statistical tests were used, including one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test, two-way ANOVA followed by Bonferroni post hoc test and Kruskal–Wallis test followed by Dunn’s multiple comparison post hoc test. The tests used are indicated in the legends of the figures. Data were expressed as mean ± SEM and differences were considered significant when p values <0.05.
Pre-aggregated Aβ-seeds induce Tau-aggregation in a well-characterized cellular Tau aggregation assay
Pre-aggregated Aβ-seeds accelerate incipient Tau-aggregation in a cellular Tau aggregation assay
Pre-aggregated Aβ induces Tau fibrillization, in a cell-free assay, providing direct proof of heterotypic seeding
Heterotypic Aβ-seeded Tau provides potent seeds for induction and propagation of Tau-pathology in vitro and in vivo
Together, our data demonstrate heterotypic seeding of filamentous Tau-aggregation by pre-aggregated Aβ. Aβ-seeds thereby provide seeds for Tau-aggregation, strongly accelerating the lag-phase for Tau-aggregation. These newly identified heterotypic Aβ-seeded Tau-seeds provide potent seeds for induction and propagation of Tau-pathology in vivo.
In this work we have demonstrated (1) direct seeding of Tau-fibrillization by pre-aggregated Aβ in a cell-free assay, demonstrating filamentous Tau-aggregation by heterotypic seeding with Aβ-seeds. We furthermore demonstrate (2) induction of Tau-aggregation and acceleration of incipient Tau-aggregation by Aβ-seeds in a well-characterized cellular assay of Tau-aggregation and (3) finally we analyzed the repercussions of these heterotypic Aβ-seeded Tau in vitro and in vivo. We thereby demonstrate that heterotypic seeded Tau by Aβ provides potent seeds to induce and propagate Tau-aggregation in vivo. Taken together, we demonstrate that pre-aggregated Aβ can template filamentous Tau aggregation by cross-seeding, providing potent seeds for induction and propagation of Tau-pathology in a prion-like way in vivo. This presents a compelling molecular mechanism of Aβ-induced propagation of Tau-pathology in AD.
In this work, we demonstrate direct seeding of Tau-fibrillization by pre-aggregated Aβ in a cell-free assay. Prion-like seeding mechanisms have gained important interest as pathogenetic mechanism in a variety of neurodegenerative proteinopathies [6, 8, 12, 16, 17, 22, 27, 28, 30, 33, 38, 39, 51, 56]. Homotypic seeding and prion-like properties have been demonstrated for different proteins associated with these neurodegenerative disorders [6, 8, 12, 16, 17, 22, 27, 28, 30, 33, 38, 39, 51, 56] including Tau. Heterotypic seeding or cross-seeding has been demonstrated for Tau and alpha-synuclein, and for different combinations of amyloidogenic proteins in vitro [11, 14, 15, 35]. Direct cross-seeding between pre-aggregated Aβ and Tau is supported by demonstrating direct binding between Aβ peptides and Tau  and in silico models for Aβ-induced beta-sheet formation of Tau-fragments . However, we here demonstrate direct induction of Tau fibrillization by pre-aggregated Aβ-seeds, in an unequivocal way using ThioT assay, sedimentation analysis and immuno-EM. Having demonstrated the induction of Tau-fibrillization in a cell-free assay, we further extended this analysis in a well-characterized cellular Tau aggregation assay. In this assay, the nucleation-dependent initial lag phase for Tau-fibrillization in cells is proposed to be by-passed by the addition of preformed pre-aggregated synthetic Tau-seeds, which accelerate fibrillization of monomeric Tau. This demonstrated that pre-aggregated Aβ was able to seed Tau-aggregation, similarly as Tau-seeds, albeit less efficient. Previous data demonstrated the occurrence of homotypic seeding for Aβ, alpha-synuclein, Tau and huntingtin in a cellular Tau aggregation assay, while heterotypic cross-seeding was not observed . The observed difference in our current data-set may rely on the fact that homotypic seeding is much more efficient compared to heterotypic seeding, thereby masking the effect of heterotypic seeding in the previous study which was focused on homotypic seeding, or may rely on the properties of the Aβ-seeds used or assay sensitivity. In favor of this is the demonstration of heterotypic seeding of alpha-synuclein and Tau, in a similar Tau-aggregation assay and in primary neurons [15, 57], further corroborating the potential of heterotypic seeding between prion-like proteins in cellular assays. Clearly, in our assay heterotypic seeding was much less efficient compared to homotypic seeding. Interestingly, Aβ-seeds potently induced Tau-aggregation in the cellular Tau-aggregation assay, when incipient Tau-aggregates were present. This finding is reminiscent and intriguing in the context of AD, as outlined below. Furthermore, inflammatory processes or neuron-specific mechanisms can be mechanistically excluded for Aβ-induced Tau-aggregation in this assay, since a non-neuronal cell line is used. Our assay suggests that pre-aggregated Aβ-seeds provide seeds for templating Tau-aggregation, bypassing the required nucleation phase for Tau-aggregation. Neither monomeric Aβ nor monomeric Tau forms induced Tau-aggregation in this assay, further strengthening templated seeding as the mechanism involved. Finally, we demonstrated that heterotypically aggregated Tau by Aβ, provided potent and efficient seeds for induction and propagation of Tau-aggregation in vivo in Tau transgenic mice. Tau-pathology induced by Aβ-seeded Tau was characterized by immunostaining with antibodies recognizing Tau phosphorylated at pathologically relevant epitopes, i.e. AT8, AT100 and recognizing conformationally altered Tau. Tau aggregates were stained by Gallyas silver staining and ThioS staining, demonstrating the true fibrillary nature of these aggregates. Tau pathology propagated efficiently from the injected brain hemisphere to the contralateral side, in a similar pattern as previously observed [39, 51]. Our data thereby demonstrate the strong potential of heterotypic Aβ-seeded Tau to seed and propagate Tau-pathology in vivo, strengthening the relevance of these newly identified seeds.
Heterotypic seeding of Tau-pathology by Aβ-seeds yielding potent seeds for propagation of Tau pathology in vivo, as demonstrated in this work, strongly fits with published genetic, pathological, clinical and biomarker data from patients. It presents as a compelling mechanism for Aβ-induced Tau-pathology in AD and fits some peculiar findings in AD patients. Tau-pathology in EC and LC is present already early in life in most healthy people, but this early pathology does not spread beyond EC and LC and is not associated with clinical symptoms [3, 4, 19, 25, 26, 31, 46, 47, 53, 54]. Hence, this Tau-pathology can be considered as rather “silent” or “non-aggressive” (yet a form of pre-existing pathological Tau). In AD, pathological analysis, clinical analysis and biomarker data indicate that following accumulation of a high β-amyloid load in isocortical regions, Tau-pathology is propagated beyond EC/LC in a more aggressive way and with associated symptoms [3, 4, 19, 25, 26, 31, 46, 47, 53, 54]. Heterotypic seeding of Tau-pathology by Aβ-seeds thereby provides a compelling molecular mechanism of Aβ-induced propagation of Tau-pathology in AD, capable of resolving some peculiar findings in AD as further outlined.
Firstly, in AD Aβ and Tau-pathology progress in remarkably distinct spatio-temporal patterns according to Thal and Braak staging [3, 54]. Nevertheless, increasing Aβ load in remote brain regions is generally accepted to drive propagation of Tau-pathology. Heterotypic induced Tau-aggregation by Aβ-seeds is in line with this (1) spatial paradox, since this seeding process can occur along functional connections. Hence, Aβ-seeds, strongly accumulating in isocortical regions can via functional connections cross-seed Tau-pathology in EC to spread beyond EC. Prion-like seeding and propagation of Tau-aggregation has been demonstrated to occur between functionally connected brain regions [1, 6, 16, 17, 22, 28, 39, 44, 51], reminiscent of the progression of Tau-pathology in AD [1, 3, 7, 22, 27, 51]. Interestingly, in vivo models of Aβ-induced Tau-pathology display features reminiscent of this prion-like seeding process [2, 13, 40, 50].
Secondly, it is intriguing that in AD brains, despite accumulation of high β-amyloid plaque load in isocortical brain regions, these regions remain (2) relatively spared from Tau-pathology in earlier stages of the disease [3, 54]. And instead, there is a (3) preferential propagation of Tau-pathology out of regions with pre-existing Tau-pathology, resulting in the very characteristic Braak staging. Intriguingly, we here demonstrate that Aβ-seeded Tau-aggregation efficiently accelerates pre-existing Tau-pathology. This is also in line with findings in preclinical models, where no induction of Tau-aggregation is demonstrated in models expressing APP/PS1, or following injection of pre-aggregated Aβ without overexpression of Tau on the murine Tau background, in contrast to models overexpressing mutant Tau .
Thirdly, heterotypic seeding of Tau by Aβ could explain (4) conversion to more potent and aggressively propagating Tau-pathology beyond EC, following cross-seeding by Aβ-seeds. And finally, since (5) homotypic prion-like seeding occurs preferentially, initially Aβ load will increase preferentially and exponentially in isocortical regions . This exponentially increasing Aβ load may thereby increase dramatically the probability of cross-seeding, inducing propagation of Tau-pathology, associated with the conversion to AD.
Taken together, heterotypic seeding of filamentous Tau-aggregation by Aβ presents as a compelling mechanism for Aβ-induced Tau-pathology, capable of resolving some peculiar AD patient data. However, we do not exclude the potential involvement of other molecular and cellular processes in Aβ-induced Tau-pathology, particularly neuronal signaling pathways and inflammation. The respective contribution of these mechanisms to Aβ-induced Tau-pathology in preclinical AD models and AD patients remains to be further analyzed.
In conclusion, Aβ-induced Tau-pathology is based on genetic data, biomarker data, clinical data and data from in vitro and in vivo models to be considered as a key initiating mechanism in the pathogenesis of AD, more particularly in the conversion from preclinical stages to MCI and AD [3, 4, 19, 25, 26, 31, 46, 47, 53, 54]. Unraveling the exact molecular mechanisms which can actually contribute to Aβ-induced Tau-pathology is required for design of novel therapeutic venues to halt or slow down conversion to AD. In this work we have proven the occurrence of heterotypic seeding of filamentous Tau-aggregation by Aβ and further demonstrated the seeding and propagating potential of these newly identified species in vivo. We believe this presents a very compelling mechanism for Aβ-induced Tau-pathology in preclinical models of AD, and AD patients, which is in line with and capable of resolving some characteristic findings in AD patients.
This work was supported by the Belgian Fonds National pour la Recherche Scientifique—Fonds de la Recherche Scientifique (FNRS-FRS; Qualified Researcher, Impulse Financing, Research Credits), by the Stellar project (Johnson and Johnson pharmaceutical companies), by Interuniversity Attraction Poles Programme-Belgian State-Belgian Science Policy, The Belgian Fonds de la Recherche Scientifique Médicale, by the Fondation Médicale Reine Elisabeth (F.M.R.E.), by the Institute for the Promotion of Innovation and by Science and Technology (IWT) in Flanders (IWTO&O), Belgium.
Compliance with ethical standards
Conflict of interest
The authors have no competing interest. A. Buist, K. Van Kolen, A. Verheyen and D. Moechars are employees of Janssen Pharmaceutical Companies of Johnson and Johnson.
- 1.Ahmed Z, Cooper J, Murray TK, Garn K, McNaughton E, Clarke H, Parhizkar S, Ward MA, Cavallini A, Jackson S, Bose S, Clavaguera F, Tolnay M, Lavenir I, Goedert M, Hutton ML, O’Neill MJ (2014) A novel in vivo model of tau propagation with rapid and progressive neurofibrillary tangle pathology: the pattern of spread is determined by connectivity, not proximity. Acta Neuropathol 127:667–683CrossRefPubMedPubMedCentralGoogle Scholar
- 2.Bolmont T, Clavaguera F, Meyer-Luehmann M, Herzig MC, Radde R, Staufenbiel M, Lewis J, Hutton M, Tolnay M, Jucker M (2007) Induction of tau pathology by intracerebral infusion of amyloid-beta-containing brain extract and by amyloid-beta deposition in APP × Tau transgenic mice. Am J Pathol 171:2012–2020CrossRefPubMedPubMedCentralGoogle Scholar
- 5.Choi SH, Kim YH, Hebisch M, Sliwinski C, Lee S, D’Avanzo C, Chen J, Hooli B, Asselin C, Muffat J, Klee JB, Zhang C, Wainger BJ, Peitz M, Kovacs DM, Woolf CJ, Wagner SL, Tanzi RE, Kim DY (2014) A three-dimensional human neural cell culture model of Alzheimer’s disease. Nature 515:274–278CrossRefPubMedPubMedCentralGoogle Scholar
- 25.Jack CR Jr, Knopman DS, Jagust WJ, Petersen RC, Weiner MW, Aisen PS, Shaw LM, Vemuri P, Wiste HJ, Weigand SD, Lesnick TG, Pankratz VS, Donohue MC, Trojanowski JQ (2013) Tracking pathophysiological processes in Alzheimer’s disease: an updated hypothetical model of dynamic biomarkers. Lancet Neurol 12:207–216CrossRefPubMedPubMedCentralGoogle Scholar
- 33.Meyer-Luehmann M, Coomaraswamy J, Bolmont T, Kaeser S, Schaefer C, Kilger E, Neuenschwander A, Abramowski D, Frey P, Jaton AL, Vigouret JM, Paganetti P, Walsh DM, Mathews PM, Ghiso J, Staufenbiel M, Walker LC, Jucker M (2006) Exogenous induction of cerebral beta-amyloidogenesis is governed by agent and host. Science 313:1781–1784CrossRefPubMedGoogle Scholar
- 37.Paxinos G, Franklin K (2001) The mouse brain in stereotaxic coordinates. Academic, San DiegoGoogle Scholar
- 39.Peeraer E, Bottelbergs A, Van Kolen K, Stancu I-C, Vasconcelos B, Mahieu M, Duytschaever H, Ver Donck L, Torremans A, Sluydts E, Van Acker N, Kemp JA, Mercken M, Brunden KR, Trojanowski JQ, Dewachter I, Lee VMY, Moechars D (2015) Intracerebral injection of preformed synthetic tau fibrils initiates widespread tauopathy and neuronal loss in the brains of tau transgenic mice. Neurobiol Dis 73:83–95CrossRefPubMedPubMedCentralGoogle Scholar
- 40.Pooler AM, Polydoro M, Maury EA, Nicholls SB, Reddy SM, Wegmann S, William C, Saqran L, Cagsal-Getkin O, Pitstick R, Beier DR, Carlson GA, Spires-Jones TL, Hyman BT (2015) Amyloid accelerates tau propagation and toxicity in a model of early Alzheimer’s disease. Acta Neuropathol Commun 3:14CrossRefPubMedPubMedCentralGoogle Scholar
- 41.Postina R, Schroeder A, Dewachter I, Bohl J, Schmitt U, Kojro E, Prinzen C, Endres K, Hiemke C, Blessing M, Flamez P, Dequenne A, Godaux E, van Leuven F, Fahrenholz F (2004) A disintegrin-metalloproteinase prevents amyloid plaque formation and hippocampal defects in an Alzheimer disease mouse model. J Clin Investig 113:1456–1464CrossRefPubMedPubMedCentralGoogle Scholar
- 44.Sanders DW, Kaufman SK, DeVos SL, Sharma AM, Mirbaha H, Li A, Barker SJ, Foley AC, Thorpe JR, Serpell LC, Miller TM, Grinberg LT, Seeley WW, Diamond MI (2014) Distinct tau prion strains propagate in cells and mice and define different tauopathies. Neuron 82:1271–1288CrossRefPubMedPubMedCentralGoogle Scholar
- 50.Stancu IC, Ris L, Vasconcelos B, Marinangeli C, Goeminne L, Laporte V, Haylani LE, Couturier J, Schakman O, Gailly P, Pierrot N, Kienlen-Campard P, Octave JN, Dewachter I (2014) Tauopathy contributes to synaptic and cognitive deficits in a murine model for Alzheimer’s disease. FASEB J Off Publ Fed Am Soc Exp Biol 28:2620–2631Google Scholar
- 51.Stancu IC, Vasconcelos B, Ris L, Wang P, Villers A, Peeraer E, Buist A, Terwel D, Baatsen P, Oyelami T, Pierrot N, Casteels C, Bormans G, Kienlen-Campard P, Octave JN, Moechars D, Dewachter I (2015) Templated misfolding of Tau by prion-like seeding along neuronal connections impairs neuronal network function and associated behavioral outcomes in Tau transgenic mice. Acta Neuropathol 129:875–894CrossRefPubMedPubMedCentralGoogle Scholar
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