Templated misfolding of Tau by prion-like seeding along neuronal connections impairs neuronal network function and associated behavioral outcomes in Tau transgenic mice
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Prion-like seeding and propagation of Tau-pathology have been demonstrated experimentally and may underlie the stereotyped progression of neurodegenerative Tauopathies. However, the involvement of templated misfolding of Tau in neuronal network dysfunction and behavioral outcomes remains to be explored in detail. Here we analyzed the repercussions of prion-like spreading of Tau-pathology via neuronal connections on neuronal network function in TauP301S transgenic mice. Spontaneous and GABAAR-antagonist-induced neuronal network activity were affected following templated Tau-misfolding using synthetic preformed Tau fibrils in cultured primary neurons. Electrophysiological analysis in organotypic hippocampal slices of Tau transgenic mice demonstrated impaired synaptic transmission and impaired long-term potentiation following Tau-seed induced Tau-aggregation. Intracerebral injection of Tau-seeds in TauP301S mice, caused prion-like spreading of Tau-pathology through functionally connected neuroanatomical pathways. Electrophysiological analysis revealed impaired synaptic plasticity in hippocampal CA1 region 6 months after Tau-seeding in entorhinal cortex (EC). Furthermore, templated Tau aggregation impaired cognitive function, measured in the object recognition test 6 months post-seeding. In contrast, Tau-seeding in basal ganglia and subsequent spreading through functionally connected neuronal networks involved in motor control, resulted in motoric deficits reflected in clasping and impaired inverted grid hanging, not significantly affected following Tau-seeding in EC. Immunostaining, biochemical and electron microscopic analysis in the different models suggested early pathological forms of Tau, including Tau-oligomers, rather than fully mature neurofibrillary tangles (NFTs) as culprits of neuronal dysfunction. We here demonstrate for the first time using in vitro, ex vivo and in vivo models, that prion-like spreading of Tau-misfolding by Tau seeds, along unique neuronal connections, causes neuronal network dysfunction and associated behavioral dysfunction. Our data highlight the potential relevance of this mechanism in the symptomatic progression in Tauopathies. We furthermore demonstrate that the initial site of Tau-seeding thereby determines the behavioral outcome, potentially underlying the observed heterogeneity in (familial) Tauopathies, including in TauP301 mutants.
KeywordsAD Tauopathies Tau Prion-like propagation Tau-pathology Neuronal network Synaptic Cognition Motoric deficits TauP301 heterogeneity
Tauopathies are a diverse group of neurodegenerative diseases, characterized by the presence of Tau aggregates composed of misfolded hyperphosphorylated Tau [7, 41, 62]. In Tauopathies, symptoms correlate strongly with the regional distribution of Tau-aggregates in the brain. In Alzheimer’s Disease, the most common Tauopathy, Tau aggregate formation progresses in a stereotypic pattern, along functionally connected neuroanatomical pathways, used for staging disease progression [2, 6, 34, 59]. In AD, Tau pathology starts in the entorhinal cortex (EC), progresses to the limbic regions such as subiculum, hippocampal cornu ammonis (CA) and amygdala (stages III and IV), and finally involves neocortical areas (stages V and IV), according to the Braak and Braak stages [6, 59]. Defects in neuronal function and neuronal loss of the affected or connected neurons are believed to contribute to the disease symptoms by affecting functionally critical neuronal networks, while the mechanism remains poorly understood [6, 15, 24, 58, 74].
Accumulating evidence indicates that Tau and related proteins linked to neurodegenerative proteinopathies display prion-like properties [12, 13, 14, 19, 22, 25, 26, 28, 32, 35, 37, 72]. In vitro studies have demonstrated that extracellular misfolded Tau can be taken up by cultured cells, can seed aggregation of endogenous soluble Tau and can propagate a fibrillar, misfolded state between co-cultured cells [20, 26, 32, 53]. Injections of brain lysates from neurofibrillary tangle (NFT) bearing transgenic mice into wild type Tau-expressing transgenic mice that do not develop NFTs, induce Tau pathology that spreads to brain regions remote from the injection site by connectivity rather than proximity [1, 13]. This has been recapitulated with brain extracts from patients with different Tauopathies, mimicking distinct forms of Tau-aggregation—in a cell-type and region specific way-reminiscent for the respective Tauopathies [5, 12]. Injection of pre-aggregated synthetic full length Tau and Tau fragments results in seeding and spreading of Tau-pathology in cells and in Tau transgenic mice, demonstrating that misfolded Tau per se, and not a different factor in the brain extracts, is sufficient to induce Tau-aggregation . Induction of Tau-aggregation by misfolded Tau has been demonstrated in non-transgenic mice , albeit at later age and to a more limited extent. Indefinite and stable maintenance of unique conformations—“strains”—in vivo that link structure to patterns of pathology was recently demonstrated, further corroborating prion-like properties of misfolded Tau [5, 53]. Although increasing dysfunction in neuronal networks is believed to explain the progression of behavioral disease symptoms, including memory loss [22, 28, 53, 58, 59], it remains to be demonstrated whether this can be caused by prion-like spreading of Tau-pathology. This is not a trivial question, as previous reports have indicated that NFTs per se may not disturb neuronal function and could be an off-pathway disease side effect [30, 38, 48, 52, 54, 61, 65, 66, 69].
In this work, we have addressed this question using in vitro, ex vivo and in vivo models with induction and spreading of Tau-pathology. Seeding of Tau pathology was shown to impair neuronal network function in primary neuronal cultures and in organotypic hippocampal slices. Furthermore, Tau-seeding caused prion-like spreading of Tau-aggregation through functionally connected neuronal networks and neuronal network dysfunction in TauP301S transgenic mice, leading to either cognitive or motoric deficits, depending on the initial site of Tau-seeding. Our data furthermore point to early pathological forms of Tau, including Tau oligomers, rather than somatic NFTs as culprits for the functional deficits.
Materials and methods
Transgenic TauP301S mice (PS19)  expressing human Tau-P301S (1N4R), driven by the mouse prion protein promoter were backcrossed to C57B6 and used in this study. TauP301S mice bred and used in our lab, develop a neurodegenerative phenotype similar as previously reported, at ~10–11 months [35, 63, 73]. Stereotactic injections were performed at 3 months and age-matched littermates were used for analysis at different time points post-injection. All experiments were performed in compliance with protocols approved by the UCLouvain Ethical Committee for Animal Welfare.
Tau aggregation assays
Generation of Tau-PFFs from recombinant Tau Tau-PFFs (synthetic preformed fibrils) or Tau-seeds were generated as described [26, 35]. Briefly, truncated human Tau fragments (WT-Tau/P301L-Tau) containing the four repeat domain [K18; Q244-E372 (4RTau)], N-terminally Myc-tagged were produced in Escherichia coli. Tau-PFFs were obtained by incubation of Tau fragments (66 μM) at 37 °C for 5 days in the presence of heparin (133 μM) in 100 mM ammonium acetate buffer (pH 7.0). Following centrifugation (100,000g, 1 h, 4 °C), the pellet was resuspended in the same buffer (333 µM final) and sonicated before use. Tau fibrilization was confirmed using ThioT assay (Sigma-Aldrich, St. Louis, MO, USA) and immunoblotting. In vitro Tau aggregation assay in HEK293 cells PFFs induced Tau-aggregation in vitro was essentially performed as previously . Sonicated Tau-seeds (10 μM) were added to Bio-PORTER single use tubes (AMS Biotechnology, Milton, UK) and added to optiMEM-washed transiently transfected HEK293 (QBI) cells, 24 h post-transfection with Tau (P301L or WT). In vitro Tau-aggregation assay in primary neurons Primary cortical neuronal cultures (PNC) were generated as described previously , from P0 heterozygous TauP301S pups or non-transgenic (WT) littermates. Tau-seeds (10 nM) were added at DIV3 and DIV6, and primary neurons were used for calcium imaging at DIV13 and subsequently extracted or fixed for further analysis. Ex-vivo Tau-aggregation assay in organotypic hippocampal slices Organotypic hippocampal slices (OC) were generated using previously described protocols [21, 64]. Briefly, hippocampal slices were generated at P6 from heterozygous TauP301S mice and non-transgenic littermates. Tau-seeds (1 µL; 333 µM) were gently added on top of hippocampal slices at DIV3 and DIV6, and slices were analyzed electrophysiologically, biochemically and immunohistologically 10 days after seeding. Tau-seeded Tau-aggregation in vivo Sonicated pre-aggregated Tau-PFFs (5 µL; 333 µM) or vehicle without seeds (5 µL) were injected in 3 months old mice. Stereotactic injections were performed in the hippocampal CA1 region (A/P, −2.0; L, +1.4; D/V, −1.2), frontal cortex (A/P, +2.0; L, +1.4; D/V, −1.0), in entorhinal cortex (A/P, −4.8; L, −3.0; D/V, −3.7) and substantia nigra (A/P, −4.8; P/A, angle 16°; L, −1.1; D/V, −4.7) millimeter relative to bregma , using a 10 µL Hamilton syringe at a speed of 1 μL per min.
For immunoblotting analysis brain regions were dissected and snap-frozen in liquid nitrogen, and subsequently differential extraction of total homogenates, sarkosyl soluble and sarkosyl insoluble fractions was performed as previously described [67, 68] and in supplemental data. Similar extraction procedures were used for primary neurons and organotypic cultures as described in supplemental data. Proteins were quantified using BCA Protein Assay kit (Thermo Fisher Scientific, Waltham, MA, USA). For dot blotting equal amounts of total homogenates were spotted on a nitrocellulose membrane and subsequently immunoblotted using T22 [ABN454 Anti-Tau (T22) oligomeric antibody; EMD Millipore], against oligomeric Tau . Analysis under non-denaturating and non-reducing conditions was performed on 4-16 % Bis-Tris Native Page. For immunoblotting equal amounts of proteins were loaded on precasted gels [4–12 % (MOPS), 8 % Tris–glycine gel (Invitrogen, Life Technologies, Carlsbad, CA, USA)]. Immunoblotting was performed with anti-Tau P-S202/T205 (AT8; Thermo Fisher Scientific, Waltham, MA, USA), with anti-Tau P-S396/S404 antibody (AD2; Bio-Rad Laboratories Inc., Hercules, CA, USA), or with anti-total Tau antibody (HT7; Thermo Fisher Scientific, Waltham, MA, USA) and developed using ECL kit (PerkinElmer, Waltham, MA, USA).
Immunohistological and immunocytological analysis
Immunohistological analysis was performed as described previously [16, 17, 36, 49, 63]. Following transcardial flushing (2 min), brains were dissected and immersion fixed in 4 % paraformaldehyde in PBS for 24 h at 4 °C. Sagittal sections (40 µm) were cut on a vibrating HM650V microtome (Thermo Fisher Scientific, Waltham, MA, USA). Immunohistochemistry (IHC) with anti-Tau P-S202/T205 (AT8), anti-Tau P-S212/T214 (AT100) and anti-conformational specific Tau (MC1, Peter Davies) was done on 40 µm sagittal vibratome sections using appropriate Alexa coupled secondary antibodies (Invitrogen, Life Technologies, Carlsbad, CA, USA). Staining with Thioflavin S (ThioS; Sigma-Aldrich, St. Louis, MO, USA) and Gallyas silver staining were performed as previously described . Image acquisition was performed using a digital inverted fluorescence microscope EVOS-xl microscope (Life Technologies, Carlsbad, CA, USA), using a 4×, 10×, 20× or 40× lens, and standard light microscope. Image analysis was done using Image J (National Institutes of Health). Heat maps were generated using the HeatMap Histogram plugin for Image J. Briefly, the overview images of AT8 staining of a well-identified section of different mice (n ≥ 3 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. Immunocytochemistry (ICC) and IHC on HEK293 cells, primary neurons and organotypic cultures were performed similarly. Fixation was performed standard or under stringent extraction of soluble Tau (supplemental material). All other chemicals were from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA).
Neuronal network activity-synchronized cytosolic calcium oscillations
Primary neurons from P0, TauP301S mice and non-transgenic littermates were used for calcium imaging, using incubation with fura-2 acetoxymethylester (Fura-2 AM; Calbiochem, Camarillo, CA, USA) 2 μM (final) in Krebs-HEPES buffer (10 mM HEPES, 135 mM NaCl, 6 mM KCl, 2 mM CaCl2, 1.2 mM MgCl2, 10 mM glucose, pH 7.4) for 30 min at RT. Coverslips were then washed in Krebs–HEPES buffer and mounted in a heated (37 °C) microscope chamber with 600 µL of buffer [44, 55]. After recording of basal spontaneous calcium oscillations, picrotoxin (PTX; Sigma-Aldrich, St. Louis, MO, USA) was added to the neurons to a final concentration of 100 µM for 3 min. Cells were alternately excited (1 or 2 Hz) at 340 and 380 nm for 100 ms using a Lambda DG-4 Ultra High Speed Wavelength Switcher (Sutter Instrument, Novato, CA, USA) coupled to a Zeiss Axiovert 200 M inverted microscope (20x fluorescence objective) (Carl Zeiss AG, Oberkochen, DE). Images were acquired with a Zeiss Axiocam camera coupled to a 510 nm emission filter and analyzed with the AxioVision software. Calcium concentration was evaluated from the ratio of fluorescence emission intensities excited at the two wavelengths, i.e. the ratio of F340/F380. Changes in the intracellular calcium fluorescence were expressed as ΔF/F 0 to represent the changes in the cytosolic calcium concentrations, relative to the resting fluorescence value F 0. Calcium oscillations were defined as variations of more than 10 % from F 0, occurring synchronously in several cells of the field.
Electrophysiology Electrophysiological analysis in organotypic hippocampal slices was performed on non-seeded (vehicle) or Tau-seeded cultured hippocampal slices (at DIV3 and DIV6) from WT or TauP301S P6 pups and analyzed after the indicated time post-seeding (2, 4, 10 days). Cultures were directly placed into the recording chamber and slices were kept in interface at 28 °C for 30 min before recordings. Electrophysiological analysis in acute hippocampal slices derived from seeded and non-seeded mice was performed essentially as described previously [17, 63]. Briefly, hippocampus was dissected and cut in 450 µm-thick slices with a tissue chopper. The slices were transferred into the recording chamber and kept in interface at 28 °C for 90 min. Electrophysiological recordings Hippocampal slices were perfused with artificial cerebrospinal fluid (ACSF) with the following composition: 124 mM NaCl, 5 mM KCl, 26 mM NaHCO3, 1.24 mM NaH2PO4, 2.5 mM CaCl2, 1.3 mM MgSO4, 10 mM glucose, bubbled with a mixture of 95 % O2 and 5 % CO2. The perfusion rate of ACSF was 1 mL/min. A bipolar twisted nickel-chrome electrode (50 µm diameter) was used to stimulate Schaffer’s collaterals. Extracellular field excitatory postsynaptic potentials (fEPSP) for acute slices or population spikes (PS) for organotypic slices were recorded in the stratum radiatum of the CA1 region with low resistance (2–5 MΩ) glass microelectrodes filled with ACSF. The slope of the fEPSP or the amplitude of PS was measured on the average of four consecutive responses. LTP was induced by applying one train of high-frequency stimulation (100 Hz, 1 s). Stimulation, data acquisition and analysis were performed using the WinLTP program 28 (website: http://www.winltp.com). For each slice, the fEPSP slopes or PS amplitudes were normalized with respect to the mean slope of the fEPSPs or the mean amplitude of the PS recorded during the 30 min period preceding induction of LTP. Statistical analysis of the data was performed using one-way ANOVA or Student’s t test in SigmaPlot 12.0 software (Systat Software Inc, Chicago, IL, USA). Data were expressed as mean ± SEM, and differences with p < 0.05 were considered significant. FDG-PET analysis Brain glucose metabolism was assessed using [18F]FDG (FDG) as described in detail in supplemental data .
The object recognition task The object recognition task was essentially performed as previously described [17, 51]. Briefly, following 10 min habituation to the open field box (60 × 60 × 50 cm), the mice were submitted the next day to a 10 min acquisition trial. During this trial, mice were placed in the open field in the presence of object A, and the time spent exploring object A was measured. During a 10 min retention trial performed 1 h later, a novel object B was added to the arena. The recognition index (RI), defined as the ratio of the time spent exploring the novel object B over the time spent exploring both objects [(tB/(tA + tB)) × 100] was used to measure nonspatial memory . Clasping scoring Scoring of clasping was performed using a scale between 5 and 1, with clasping score 5 representing no clasping (normal), score 4 representing initial signs of clasping, score 3 for intermediate clasping, score 2 for strong clasping, and score 1 representing very severe (maximal) clasping. Scores between 1 and 5 were assigned by an experimentator blinded for the experimental group. Inverted grid hanging task The inverted grid hanging test was used to assess the ability to grasp an elevated horizontal grid and to remain suspended for 2 min. The grid (40 cm × 20 cm/0.5 cm meshes) was positioned 50 cm above a flat, soft surface and the latency for the animal to drop off was measured.
Immunogold electron microscopy
Protein extracts from mouse brains, PNC, OC and HEK293 cells were applied on 300-mesh carbon-coated grids in drops of 3 µL for 5 min, blotted on a filter paper and air-dried before the immunogold procedure. Grids were blocked in PBS, 0.1 % cold water fish gelatin (PBS-CWFG) for 5 min before incubating with PBS-CWFG diluted primary antibodies (HT7 1:50, AT8 1:50, AT100 1:50) for 90 min at RT. After washing 5 times (2 min) in PBS-CWFG, grids were incubated for 60 min with secondary antibody (goat anti-mouse IgG) labeled with 10-nm gold particles (Aurion) diluted 1:30 in PBS-CWFG. Following washes in PBS and dH2O, grids were negatively stained with 2 % uranyl acetate for 1 min. The grids were examined with a JEOL JEM1400 transmission electron microscopy equipped with a Olympus SIS Quemesa 11 Mpxl camera, and images were taken at magnifications of 20× and 30× k (resp. pixel size = 0.72 and 0.48 nm). Immuno-gold electron-microscopy on brain slices is described in detail in supplemental data.
Statistical analysis was performed using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA) using one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test and Student’s t test. Data were expressed as mean ± SEM and differences were considered significant when p values <0.05.
Synthetic Tau-fibrils induce Tau-aggregation and altered neuronal network activity in primary neuronal cultures
Tau seeds extracted from brains of Tau transgenic mice with Tau pathology, from patients with different forms of Tauopathies or pre-aggregated synthetic Tau and Tau-fragments [12, 13, 35] induce Tau-aggregation in cultured cells and in vivo [19, 22, 26, 28, 53]. In this work, we used synthetic pre-aggregated Tau fragments, encompassing the four repeat domain of TauP301L, further referred to as “Tau-seeds”, to analyze the functional repercussions of Tau-aggregation in different in vitro, ex vivo and in vivo models. We first acquired and characterized Tau-seed induced Tau-aggregation in HEK293 cells, by administration of Tau-seeds to transfected TauP301L HEK293 cells using BioPORTER as previously shown . Biochemical analysis demonstrated the presence of detergent insoluble (Triton-insoluble (TX-100) and sarkosyl insoluble) Tau-aggregates phosphorylated at S202/T205 (AT8) (Fig. S1a) following Tau-seeding while absent in non-seeded cells. Tau aggregation was further confirmed by immunocytochemical (ICC) analysis using a stringent extraction protocol with TX-100, extracting soluble forms of Tau from the cells, while only aggregated forms of Tau remained detectable. Staining with anti-Tau P-S202/T205 antibody (AT8) demonstrated intense staining of Tau aggregates in seeded cells, absent in non-seeded cells (Fig. S1a). Since the AT8 epitope is situated outside of the Tau-seeding fragment, this demonstrates aggregation of full length Tau and not accumulation of Tau-seeds in the cells. To determine optimal conditions for Tau-aggregation, we used different combinations of wild type Tau and mutant TauP301L forms as inducer and receiver. Immunocytological and alpha-LISA analysis demonstrated that wild type Tau was markedly less prone to aggregate than TauP301L (recipient), when seeded with either wild type or P301 mutated pre-aggregated Tau-seeds (inducer) (Fig. S1b). Similarly, wild-type Tau seeds (inducer) were less efficient compared to mutated TauP301L seeds, in inducing Tau-aggregation (Fig. S1b, c). Hence, in this work, we analyzed the functional repercussions of Tau-aggregation in optimal conditions, using Tau mutated at Proline 301 acting both as inducer and recipient.
Synthetic Tau seed induced Tau-aggregation impaired basal synaptic transmission and synaptic plasticity in organotypic hippocampal slices
Tau aggregation following seeding with synthetic pre-aggregated Tau spreads to synaptically and functionally connected brain regions in TauP301S transgenic mice
Taken together, our results indicate that depending on the initial site of injection of Tau-seeds, i.e. cortical/hippocampal, basal ganglia or entorhinal cortex (Fig. 4a–d), different spreading patterns of Tau-pathology to functionally—and often symptomatically—connected brain regions was observed (Fig. 5). Spreading of Tau-pathology occurred by connectivity rather than proximity, raising the question and allowing us to analyze whether neuronal network function is affected by this process.
Tau-pathology induced by Tau-seeding impairs synaptic function in hippocampal CA1 region and cognition in TauP301S mice
Spreading of Tau aggregation along neuronal circuitries and impairment of associated behavioral outcomes is determined by the site of initial seeding in TauP301S mice
We next analyzed whether the site of initiation of Tau-aggregation determines the specific behavioral outcomes. We measured motoric performance 6 months after initiation of Tau pathology in basal ganglia and EC, respectively. Quantitative scoring of hindlimb clasping revealed significantly increased clasping following initiation of Tau-pathology in basal ganglia, compared to initiation of Tau-pathology in EC, which did not induce significant clasping (Fig. 6c). Furthermore, we measured inverted grid hanging performance, to assess motor coordination and grip strength. Performance in inverted grid hanging was severely impaired 6 months post-initiation of Tau-aggregation by Tau-seeding in basal ganglia, while not significantly affected 6 months post-seeding in EC (Fig. 6c). Intracerebral injections of buffer only in basal ganglia did not induce Tau-aggregation and did not affect clasping or inverted grid hanging (Fig. 6c). Taken together, our data indicate that prion-like seeding of Tau-aggregation along synaptic connections affects selective functional brain circuitries, thereby determining the behavioral dysfunction depending on the site of initiation of Tau-pathology.
“Early” pathological forms of Tau rather than fully mature NFTs as causal culprits for neuronal dysfunction
We next analyzed the presence of these different forms of Tau following Tau-seed induced Tau-aggregation in primary neurons (PNC) and organotypic cultures (OC) (Fig. 7). Tau-seeding induced more robust and more abundant AT8 staining in organotypic hippocampal slices than in primary neurons, due to higher concentrations of Tau-seeds and topical application of the seeds on the slice. In primary neurons Tau-seed induced Tau-aggregation was less robust than previously published  since lower concentrations of Tau-seeds were used. Immunological analysis revealed the presence of pathological forms of Tau reflected by AT8, AT100 and MC1 positive signals in both models, OC and PNC (Fig. 7a). EM analysis revealed the presence of Tau fibrils in OC following Tau seeding, albeit less abundant than in brains (Fig. S5). Fibrils were not detected by EM analysis in primary neurons, paralleled by the lack of ThioS staining in PNC (results not shown). This indicates that fully mature Tau-fibrils were absent or present in very low quantities below detection limit of the assay (EM and ThioS) in PNC. Oligomeric Tau forms were increased in primary neurons and organotypic cultures as demonstrated by dot blotting with the Tau oligomer-specific T22 antibody (Fig. 7b). In both models, Tau-seeding increased AT8-positive Tau-multimers detected in Native PAGE (Fig. S6). To further analyze early pathological forms of Tau in PNC, we performed a more sensitive AT8 staining, by omitting the stringent extraction of soluble forms of Tau, thereby staining both aggregated and soluble forms of AT8-stained Tau. This revealed marked Tau-seed induced diffuse and punctate AT8-staining, in addition to the somatic Tau-aggregation as previously detected (Fig. 7c). As indicated above, strong somatic NFT-like Tau-staining of aggregated Tau was only observed in 5–10 % of the neurons in PNC. In contrast, the punctate and more diffuse staining pattern was robustly observed throughout the culture (Fig. 7c), and was markedly induced by Tau-seeding in Tau-expressing neurons and absent in wild-type neurons (Fig. S7). This punctate and diffuse staining pattern was also observed in neurons not yet displaying strong NFT-like somatic AT8 signal, hence representing an early form of pathological AT8 positive Tau. In view of the detection of somatic aggregated Tau in only very limited number of neurons, and the inability to detect fibrillar Tau in primary neurons following Tau-seeding using EM and ThioS, the combined data point to early pathological forms of Tau, encompassing non-NFT AT8-positive Tau and oligomeric Tau, rather than fully mature fibrillary NFTs, as culprit for Tau-seed induced neuronal dysfunction in PNC. The clearcut presence of these early pathological Tau-forms in brains and organotypic hippocampal slices following Tau-seeding, further supports their potential role in neuronal dysfunction in all models. Taken together, our data suggest that early pathological forms, encompassing diffuse and punctate AT8 stained Tau, hyperphosphorylated, misfolded (MC1) and oligomeric Tau forms (stained with T22 or AT8), rather than fully mature NFT, correlate with the neuronal dysfunction induced by Tau-seeded Tau-misfolding.
Accumulating evidence has demonstrated that Tau displays prion-like properties and that prion-like spreading of Tau-pathology occurs through connectivity rather than proximity . This process has been proposed to underlie the stereotyped progression along unique brain circuitries of Tau-pathology and symptoms in Tauopathies [22, 28, 50, 53, 58, 74]. In this work, we demonstrated—for the first time—that templated Tau-misfolding induced by Tau-seeds through functionally connected neuro-anatomical pathways, impairs neuronal network function and associated behavioral outcomes, dependent on the initial site of seeding. More precisely, we demonstrated that small amounts of pre-aggregated synthetic Tau fragments, focally injected, without changing expression-level of Tau but only inducing Tau-misfolding, are sufficient to propagate Tau aggregation and impair neuronal network function and behavioral outcomes 6 months post-injection, dependent on the initial site of injection. These findings emphasize the potential relevance of this intensively studied process in the stereotypic progression of symptoms in Tauopathies. We have further extended our analysis to highlight early pathological forms of Tau, rather than fully mature fibrillar NFTs as causal culprits for the neuronal network dysfunction.
We demonstrated that templated Tau-misfolding by Tau-seeds affected neuronal network activity in primary neuronal cultures. Primary cortical neurons display synchronized calcium oscillations in culture, which were previously shown to be modulated by PTX, a GABAA receptor antagonist . Exposure of cortical neurons to PTX renders calcium transients to exhibit increased amplitude, more regular frequency and higher synchronicity . A refractory period has previously been identified which may play a role in increased synchronization. Our data indicate a significant dysregulation of spontaneous and PTX-induced calcium oscillations, indicating that Tau-seeded Tau-misfolding affects neuronal network activity. Our findings suggest that Tau-seeding impairs GABA-ergic synaptic transmission, based on the similarity of the effect of PTX and Tau-seeds on spontaneous oscillations. As we further demonstrated that PTX elicited calcium transients are NMDA- and AMPA-dependent as shown previously , their reduced amplitudes in Tau-seeded neuronal cultures indicate impaired glutamatergic, NMDA- and AMPA-dependent signaling. We further extended our analysis in models with conserved network architecture, i.e. to organotypic hippocampal slices . In organotypic cultures, we demonstrated impaired population spike amplitude measured in hippocampal CA1 region following stimulation of Schaffer collaterals. This basal synaptic transmission is predominantly generated by excitatory glutamatergic (NMDA/AMPA-mediated) neurotransmission, further corroborating data obtained in primary neuronal cultures. Furthermore, measurement of LTP revealed that synaptic plasticity is impaired in organotypic slices by Tau-seed induced Tau misfolding. Notably, NMDA- and AMPA-receptor functions are critically involved in hippocampal LTP in CA1. Our data were further corroborated in vivo. Electrophysiological analysis in acute hippocampal slices revealed that LTP was affected 6 months post-induction of Tau-pathology by Tau-seeding in vivo, although basic synaptic transmission was not. The latter may be explained by the fact that effects of Tau-aggregation can be more easily compensated in vivo than in vitro, or that the aggregation process occurred more rapidly in vitro, or that organotypic cultures are more sensitive than the intact hippocampus in vivo. It must be noted that electrophysiological analysis in acute hippocampal slices was performed in hippocampal CA1 region, while initial Tau-seeding was performed in entorhinal cortex, indicating that neuronal function remote from the initial seeding site was affected. We further demonstrated conserved presynaptic parameters including paired pulse ratio, synaptic fatigue and SV2 staining, pointing towards a postsynaptic mechanism. Together with the results obtained in primary neurons, indicating impaired NMDA/AMPA-dependent neuronal network function following Tau-seeding, our data are in line with a role of pathological Tau forms in postsynaptic dysfunction, including NMDA/AMPA dysfunction as previously shown [8, 60, 70]. We further assessed behavioral effects, and demonstrated that cognition, measured in an object recognition task was impaired in bilaterally injected mice, 6 months post-injection. Together our data indicate that Tau-seed induced misfolding of Tau, leading to Tau-aggregation through connectivity, affects neuronal function, neuronal network activity and behavioral outcome, and hence could contribute to progression of the symptoms in Tauopathies.
Prion like induction of Tau-pathology—a process which raised considerable scientific interest—has been elegantly demonstrated and analyzed in exquisite detail for different aspects [1, 12, 13, 14, 19, 20, 22, 26, 28, 32, 35, 37, 40, 53, 72]. The functional repercussions of this process remained however, to be analyzed. This question is important to evaluate its relevance in the pathogenetic process of Tauopathies. In AD, Tau-pathology is known to progress according to stereotypical and predictable patterns reflected in its incorporation in criteria for the neuropathological diagnosis of AD [6, 34]. The first neurons to be affected are in entorhinal cortex (EC) (stage I), the neurons that give rise to the perforant pathway, the major projection connecting cerebral cortex with the hippocampus [23, 33]. Next NFTs spread to the CA1 region of the hippocampus and accumulate in limbic structures such as the subiculum (stage II–III) and the amygdala, thalamus, and claustrum (stage IV). Finally, NFTs spread to isocortical areas (stage V–VI). Symptoms in AD are strongly correlated with this characteristic appearance of NFTs. In this work, we mimic spreading of Tau-pathology following seeding in entorhinal cortex of TauP301S mice, similar as observed in AD. Tau pathology starts first in the entorhinal cortex, subsequently in CA1, the subiculum and CA3-4 and later in the DG, and finally in neocortical areas (stages V and IV), according to the Braak and Braak stages. Noteworthy, minor discrepancy between spreading of Tau-pathology in AD [EC, subsequently CA1/subiculum/CA3-4 and later dentate gyrus (DG)] and functional connectivity (EC, subsequently DG/CA3-4 and then CA1/subiculum) may be ascribed to additional factors determining selective vulnerability or protection in these regions, which appear to be recapitulated in our model, as the AD pattern was mimicked following seeding in EC. It must be noted however that in contrast to AD and sporadic Tauopathies, characterized by aggregation of wild type Tau, we used in the current work recipient Tau and inducer Tau-seeds mutated at Proline 301, as this combination results in the highest Tau-aggregation efficiency. Previous work also indicated that mutation of Proline 301, rather than its substitution by Serine or Leucine, is determining aggregation efficiency . However, wild type Tau acting as recipient, in combination with either wild-type or mutant Tau seeds as inducer has been demonstrated to be capable of prion-like Tau seeding, albeit much less efficient. The potential of wild type Tau to undergo prion-like Tau seeding allows a (cautious) extrapolation of our data in the context of AD or sporadic Tauopathies, characterized by aggregation of wild type Tau. Hence, neuronal network dysfunction and associated behavioral dysfunctions induced by prion-like seeding and spreading of Tau-pathology along functionally connected circuitries could relate to symptomatic progression in AD and related Tauopathies.
Besides in AD patients, spreading of Tau-aggregation according to a characteristic pattern along functionally connected brain circuitries is also observed in argyrophilic grain disease, a different Tauopathy. Other Tauopathies are very heterogeneous between individuals with identical mutations, including for patients with Tau mutated at Proline 301 (P301L) . This heterogeneity hampers the delineation of a specific progression pattern. However, symptoms progressively affect certain behaviors driven by a particular brain circuitry. We here demonstrate for the first time that the initial site of Tau-seeding determines the behavioral outcomes, resulting in different behavioral outcomes depending on the initiation site. Initiation of prion-like seeding in transgenic mice expressing mutant TauP301S, in entorhinal cortex resulted in spreading of Tau-pathology along functionally connected circuits, resulting in impaired LTP in hippocampal CA1 neurons. Cognition measured in an object recognition task was demonstrated to be impaired following Tau-seeded Tau-aggregation. In contrast, prion-like seeding in basal ganglia resulted in spreading to brain regions involved in motor control, thereby resulting in motor impairments, 6 months after Tau-seeding. Motoric impairments were not observed following injection of Tau-seeds in EC. These findings are reminiscent of the heterogeneity of symptoms in TauP301 patients, where the initial site of Tau seeding by environmental, genetic or accidental factors, may determine the symptomatic outcome (cognitive symptoms or Parkinsonism).
The clearcut demonstration of impaired neuronal network function and behavior by prion-like seeding raises questions about the causal culprit, in terms of pathological forms of Tau. The combination of our functional analysis with biochemical and immunological analysis points to early pathological forms of Tau, including pathological hyperphosphorylated and misfolded Tau and Tau oligomers rather than fully mature NFTs as potential pathological culprits. Notably, strong somatic AT8 aggregation was only observed in 5–10 % of neurons following Tau-seeding in primary neurons. Furthermore, the presence of fully mature fibrils was below detection limit using electron microscopy and ThioS staining was not observed in PNC. It must be noted that Tau-seeding in primary neurons in this work was less robust than previously published , probably due to lower concentration of Tau-seeds used. We have however, demonstrated the abundant presence of “early” AT8-positive forms of Tau detected as diffuse staining and punctated synaptic staining in the current model. This staining was present throughout the primary neuronal culture and robustly induced following Tau-seeding. Furthermore, oligomeric forms of Tau stained with T22 or with AT8 were increased following Tau-seeding in all models. Although an approximately, twofold increase may seem rather low, the spatio-temporal characteristics (e.g. at the synapse) of this increase may underlay its pronounced effect on neuronal function. Taken together our data indicate that either a few neurons displaying strong somatic AT8 staining are responsible for impaired neuronal network activity, or most parsimoniously, that early forms of aggregated Tau or “pre-tangle stage” pathological forms of Tau, are responsible for neuronal network dysfunction. This is further supported by their abundant induction in brains and in organotypic cultures following Tau-seeding. Taken together our data suggest that early pathological forms of Tau rather than fully mature NFTs may represent the causal culprits. Final proof can only be delivered by selective elimination of different pathological forms of Tau and analysis of the functional repercussions. Our observations fit however with previous observations using elegant approaches to determine the repercussions of Tau-pathology, and more particularly of NFTs on neuronal function in vivo [18, 30, 38, 48, 52, 54, 61, 65, 66, 69]. This demonstrated either no effects of NFTs in neurons on calcium or Arc responses [38, 52]. Furthermore, using inducible expression of Tau or pro-aggregant Tau, reversibility of synaptic deficits despite the continued presence of NFTs was demonstrated, indicating association of synaptic defects with the aggregation process and early pathological Tau forms, rather than with the full-blown NFTs [3, 30, 54, 61, 65, 66, 69]. These data are in line with clear demonstration of detrimental effects of Tau-oligomers on neuronal function and behavior, as demonstrated following acute injection of oligomeric Tau and immunization with anti-Tau oligomeric antibodies [10, 11, 39, 40]. Our findings are furthermore in line with previous reports demonstrating that Tau overexpression or Tau-alterations affect neuronal function or function of connected neurons [29, 31, 42, 47, 48, 56, 57, 63, 73]. It must be noted that in the current work, Tau-expression is not affected, but only misfolding of Tau is induced by templated seeding by focal administration of small amounts of seeds. We demonstrate that this templated Tau-misfolding is sufficient to propagate neuronal network dysfunction and behavioral outcomes, which correlate with the presence of early pathological forms of Tau, including Tau oligomers, rather than with the presence of fully mature Tau fibrils.
In summary, our data indicate for the first time and unequivocally, using in vitro, ex vivo and in vivo approaches that (i) prion-like Tau-seed induced Tau-aggregation with NFT formation—a mechanism under intensive investigation—causes synaptic and neuronal network dysfunction, resulting in behavioral impairments. Our data thereby indicate that prion-like spreading of Tau-pathology may contribute to progression of disease symptoms in Tauopathies by affecting intrinsic functional critical networks. Furthermore, our data indicate unequivocally that (ii) behavioral outcomes following seeding in Tau P301 transgenic mice, are determined by the initial site of Tau seeding. Hence, motoric problems were demonstrated following Tau-seeding in basal ganglia (substantia nigra), while absent following injection in entorhinal cortex, resulting in cognitive defects. These findings are reminiscent of the heterogeneity of clinical symptoms in familial cases with P301 mutations, in which initial seed formation by environmental, genetic or accidental factors, may determine the behavioral outcomes. Finally, (iii) our results support a role of early pathological forms of Tau, including oligomeric Tau rather than fully mature NFTs as pathogenic culprits of prion-like induced spreading of neuronal dysfunction. Our findings provide a basis to further identify the molecular mechanisms involved in Tau-seed induced synaptic dysfunction and provide a model to develop novel therapeutic strategies targeting Tau-seed induced neuronal dysfunction, and to analyze repercussions on neuronal function induced by different Tau-“strains”.
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 Interuniversity Attraction Poles Programme-Belgian State-Belgian Science Policy, The Belgian Fonds de la Recherche Scientifique Médicale, by the Institute for the Promotion of Innovation by Science and Technology (IWT) in Flanders (IWT O&O), Belgium.
Conflict of interest
The authors have no competing interest. E.P., A.B., T.O., and D.M. are employees of Janssen Pharmaceutical Companies of Johnson and Johnson. D.T. is employee of reMYND.
- 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 127:667–683CrossRefPubMedCentralPubMedGoogle Scholar
- 3.Berger Z, Roder H, Hanna A, Carlson A, Rangachari V, Yue M, Wszolek Z, Ashe K, Knight J, Dickson D, Andorfer C, Rosenberry TL, Lewis J, Hutton M, Janus C (2007) Accumulation of pathological tau species and memory loss in a conditional model of tauopathy. J Neurosci 27(14):3650–3662CrossRefPubMedGoogle Scholar
- 4.Bird TD, Nochlin D, Poorkaj P, Cherrier M, Kaye J, Payami H, Peskind E, Lampe TH, Nemens E, Boyer PJ, Schellenberg GD (1999) A clinical pathological comparison of three families with frontotemporal dementia and identical mutations in the tau gene (P301L). Brain 122:741–756CrossRefPubMedGoogle Scholar
- 5.Boluda S, Iba M, Zhang B, Raible KM, Lee VM, Trojanowski JQ (2015) Differential induction and spread of tau pathology in young PS19 tau transgenic mice following intracerebral injections of pathological tau from Alzheimer’s disease or corticobasal degeneration brains. Acta Neuropathol 129(2):221–237CrossRefPubMedCentralPubMedGoogle Scholar
- 8.Burnouf S, Martire A, Derisbourg M, Laurent C, Belarbi K, Leboucher A, Fernandez-Gomez FJ, Troquier L, Eddarkaoui S, Grosjean ME, Demeyer D, Muhr-Tailleux A, Buisson A, Sergeant N, Hamdane M, Humez S, Popoli P, Buée L, Blum D (2013) NMDA receptor dysfunction contributes to impaired brain-derived neurotrophic factor-induced facilitation of hippocampal synaptic transmission in a Tau transgenic model. Aging Cell 12:11–23CrossRefPubMedGoogle Scholar
- 10.Castillo-Carranza DL, Gerson JE, Sengupta U, Guerrero-Munoz MJ, Lasagna-Reeves CA, Kayed R (2014) Specific targeting of Tau oligomers in Htau mice prevents cognitive impairment and Tau toxicity following injection with brain-derived Tau oligomeric seeds. J Alzheimers Dis 40:S97–S111PubMedGoogle Scholar
- 11.Castillo-Carranza DL, Sengupta U, Guerrero-Munoz MJ, Lasagna-Reeves CA, Gerson JE, Singh G, Estes DM, Barrett ADT, Dineley KT, Jackson GR, Kayed R (2014) Passive immunization with Tau oligomer monoclonal antibody reverses tauopathy phenotypes without affecting hyperphosphorylated neurofibrillary tangles. J Neurosci 34:4260–4272CrossRefPubMedGoogle Scholar
- 12.Clavaguera F, Akatsu H, Fraser G, Crowther RA, Frank S, Hench J, Probst A, Winkler DT, Reichwald J, Staufenbiel M, Ghetti B, Goedert M, Tolnay M (2013) Brain homogenates from human tauopathies induce tau inclusions in mouse brain. Proc Natl Acad Sci USA 110:9535–9540CrossRefPubMedCentralPubMedGoogle Scholar
- 16.Dewachter I, Filipkowski RK, Priller C, Ris L, Neyton J, Croes S, Terwel D, Gysemans M, Devijver H, Borghgraef P, Godaux E, Kaczmarek L, Herms J, Van LF (2009) Deregulation of NMDA-receptor function and down-stream signaling in APP[V717I] transgenic mice. Neurobiol Aging 30:241–256CrossRefPubMedGoogle Scholar
- 17.Dewachter I, Reverse D, Caluwaerts N, Ris L, Kuiperi C, Van den Haute C, Spittaels K, Umans L, Serneels L, Thiry E, Moechars D, Mercken M, Godaux E, Van LF (2002) Neuronal deficiency of presenilin 1 inhibits amyloid plaque formation and corrects hippocampal long-term potentiation but not a cognitive defect of amyloid precursor protein [V717I] transgenic mice. J Neurosci 22:3445–3453PubMedGoogle Scholar
- 32.Holmes BB, DeVos SL, Kfoury N, Li M, Jacks R, Yanamandra K, Ouidja MO, Brodsky FM, Marasa J, Bagchi DP, Kotzbauer PT, Miller TM, Papy-Garcia D, Diamond MI (2013) Heparan sulfate proteoglycans mediate internalization and propagation of specific proteopathic seeds. Proc Natl Acad Sci USA 110:E3138–E3147CrossRefPubMedCentralPubMedGoogle Scholar
- 34.Hyman BT, Trojanowski JQ (1997) Consensus recommendations for the postmortem diagnosis of Alzheimer disease from the National Institute on Aging and the Reagan Institute Working Group on diagnostic criteria for the neuropathological assessment of Alzheimer disease. J Neuropathol Exp Neurol 56:1095–1097CrossRefPubMedGoogle Scholar
- 45.Paxinos G, Franklin KBJ (2001) The mouse brain in stereotaxic coordinates. Academic, San DiegoGoogle Scholar
- 46.Poisnel G, Herard AS, El Tannir El TN, Bourrin E, Volk A, Kober F, Delatour B, Delzescaux T, Debeir T, Rooney T, Benavides J, Hantraye P, Dhenain M (2012) Increased regional cerebral glucose uptake in an APP/PS1 model of Alzheimer’s disease. Neurobiol Aging 33:1995–2005CrossRefPubMedCentralPubMedGoogle Scholar
- 48.Polydoro M, Dzhala VI, Pooler AM, Nicholls SB, McKinney AP, Sanchez L, Pitstick R, Carlson GA, Staley KJ, Spires-Jones TL, Hyman BT (2014) Soluble pathological tau in the entorhinal cortex leads to presynaptic deficits in an early Alzheimer’s disease model. Acta Neuropathol 127:257–270CrossRefPubMedCentralPubMedGoogle Scholar
- 49.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 LF, Fahrenholz F (2004) A disintegrin-metalloproteinase prevents amyloid plaque formation and hippocampal defects in an Alzheimer disease mouse model. J Clin Invest 113:1456–1464CrossRefPubMedCentralPubMedGoogle Scholar
- 54.SantaCruz K, Lewis J, Spires T, Paulson J, Kotilinek L, Ingelsson M, Guimaraes A, DeTure M, Ramsden M, McGowan E, Forster C, Yue M, Orne J, Janus C, Mariash A, Kuskowski M, Hyman B, Hutton M, Ashe KH (2005) Tau suppression in a neurodegenerative mouse model improves memory function. Science 309:476–481CrossRefPubMedCentralPubMedGoogle Scholar
- 57.Schindowski K, Bretteville A, Leroy K, Begard S, Brion JP, Hamdane M, Buee L (2006) Alzheimer’s disease-like tau neuropathology leads to memory deficits and loss of functional synapses in a novel mutated tau transgenic mouse without any motor deficits. Am J Pathol 169:599–616CrossRefPubMedCentralPubMedGoogle Scholar
- 63.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 28:2620–2631CrossRefPubMedGoogle Scholar
- 66.Sydow A, Van der Jeugd A, Zheng F, Ahmed T, Balschun D, Petrova O, Drexler D, Zhou L, Rune G, Mandelkow E, D’Hooge R, Alzheimer C, Mandelkow EM (2011) Tau-induced defects in synaptic plasticity, learning, and memory are reversible in transgenic mice after switching off the toxic Tau mutant. J Neurosci 31:2511–2525CrossRefPubMedGoogle Scholar
- 67.Terwel D, Lasrado R, Snauwaert J, Vandeweert E, Van Haesendonck C, Borghgraef P, Van Leuven F (2005) Changed conformation of mutant Tau-P301L underlies the moribund tauopathy, absent in progressive, nonlethal axonopathy of Tau-4R/2N transgenic mice. J Biol Chem 280(5):3963–3973CrossRefPubMedGoogle Scholar
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