Pro-aggregant Tau impairs mossy fiber plasticity due to structural changes and Ca++ dysregulation
- Jochen Martin Decker†1,
- Lars Krüger†1,
- Astrid Sydow1,
- Shanting Zhao4,
- Michael Frotscher4,
- Eckhard Mandelkow1, 2, 3 and
- Eva-Maria Mandelkow1, 2, 3Email author
© Decker et al.; licensee BioMed Central. 2015
Received: 30 January 2015
Accepted: 3 February 2015
Published: 3 April 2015
We used an inducible mouse model expressing the Tau repeat domain with the pro-aggregant mutation ΔK280 to analyze presynaptic Tau pathology in the hippocampus.
Expression of pro-aggregant TauRDΔ leads to phosphorylation, aggregation and missorting of Tau in area CA3. To test presynaptic pathophysiology we used electrophysiology in the mossy fiber tract. Synaptic transmission was severely disturbed in pro-aggregant TauRDΔ and Tau-knockout mice. Long-term depression of the mossy fiber tract failed in pro-aggregant TauRDΔ mice. We observed an increase in bouton size, but a decline in numbers and presynaptic markers. Both pre-and postsynaptic structural deficits are preventable by inhibition of TauRDΔ aggregation. Calcium imaging revealed progressive calcium dysregulation in boutons of pro-aggregant TauRDΔ mice. In N2a cells we observed this even in cells without tangle load, whilst in primary hippocampal neurons transient TauRDΔ expression alone caused similar Ca++ dysregulation. Ultrastructural analysis revealed a severe depletion of synaptic vesicles pool in accordance with synaptic transmission impairments.
We conclude that oligomer formation by TauRDΔ causes pre- and postsynaptic structural deterioration and Ca++ dysregulation which leads to synaptic plasticity deficits.
KeywordsCalcium dysregulation Long-term depression Mossy fiber pathway Presynapse Tau
Several neurodegenerative disorders are characterized by pathological aggregation of the axonal protein Tau into “neurofibrillary tangles” classifying them as tauopathies . The clinical signs of various tauopathies including Alzheimer disease (AD) and frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17) correlate well with the anatomical localization of Tau aggregates in the brain . Especially in AD, a correlation of hyperphosphorylated, aggregated Tau distribution and cognitive impairment is evident . So far, most electrophysiological studies investigated Tau-related postsynaptic changes in area CA1 of the hippocampus [4-6]. However, it became increasingly obvious from animal models expressing mutant Tau that Tau pathology appears prominently in the stratum lucidum (s.l.) of area CA3 where axons of granule cells form the mossy fiber path [7-9]. In transgenic mice expressing the aggregation-prone human Tau repeat domain with the FTDP-17 mutation ΔK280 (termed pro-aggregant TauRDΔ), exogenous mutant Tau co-aggregates with endogenous mouse Tau to assemble into neurofibrillary tangles in area CA3, whereas mice expressing anti-aggregant Tau (TauRDΔK280PP, termed TauRDΔPP, containing two additional prolines inhibiting ß-structure) do not show pathology . In Alzheimer disease pathological Tau is found within the hippocampal formation . Recent observations on human AD brains though demonstrated abnormal Tau phosphorylation in thorny excrescences, the specialized spine structures characteristic for CA3 pyramidal neurons which constitute the postsynaptic target of mossy fiber boutons . The density of thorny excrescences was decreased whereas their size was increased during the course of AD . Moreover, immunoreactivity with AD-diagnostic antibodies PHF1, Alz50, and TG3 [13-15] were found in DG granule cells of AD patients, pointing to a clinical relevance of the mossy fiber pathway to Tau-related disease progression.
In hibernating ground squirrels, Tau becomes highly phosphorylated and missorted from its normal axonal localization into the somatodendritic compartment, which leads to the retraction of mossy fibers from CA3 postsynapses . This process is reversible and suggests a physiological role of Tau in mossy fiber plasticity. In addition, Tau plays an important role in mossy fiber reorganization during development , and Tau is involved in the pathological sprouting of mossy fibers induced by kainate injection to generate epileptic seizures .
Thorny excrescences are innervated by “giant” mossy fiber boutons, which are characterized by their low basal transmitter release probability . This is maintained by the action of presynaptic adenosine (A1) receptors  and group II metabotropic glutamate receptors (mGluRII) . Long term depression (LTD) at the mossy fiber-CA3 synapse is expressed presynaptically as a reduction in neurotransmitter release  and depends on an activity-dependent rise in intracellular calcium (Ca++) concentrations .
In the present study we wanted to clarify to what extent pro-aggregant human TauRDΔ influences presynaptic plasticity in the hippocampus. For this purpose, we performed functional and morphological analyses of the mossy fiber synapse in Tau aggregation models and Tau knockout (TKO) mice. We found that aggregating Tau causes pre- and postsynaptic morphological changes at the mossy fiber-CA3 synapse and an impairment of LTD in pro-aggregant TauRDΔ. Endogenous Tau was necessary for basal synaptic transmission at that synapse. In vitro Ca++ imaging studies with organotypic hippocampal slices, primary hippocampal neurons and N2a cells expressing pro-aggregant TauRDΔ demonstrated dysregulation of neuronal calcium influx as a possible basis for plasticity impairment. Structural synaptic changes could be prevented partially by the inhibition of Tau aggregation.
Materials and methods
Generation of TauRDΔ transgenic mice
Regulatable transgenic mice expressing the four-repeat domain of human Tau (TauRD, residues 244–372) were generated as described previously . The pro-aggregant form TauRDΔ carried the mutation ∆K280, the anti-aggregant form TauRDΔPP carried the mutations ∆K280/I277P/I308P where the additional prolines prevent aggregation through β-structure (for details see ). Transgene expression was monitored and quantified by bioluminescence of luciferin via the co-expressed luciferase (for details see [9,24]. The Tau knockout mouse was created by H. Dawson and colleagues by homologous recombination replacing exon 1 with a neomycin expression cassette . Non-transgenic littermates of pro- and anti-aggregant mutants were used as controls. Transgenic animals were fed with doxycycline-containing pellets for 3 weeks to shut off mutant Tau gene expression in the first postnatal phase to prevent developmental disturbances. All animals were housed and tested according to standards of the German Animal Welfare Act.
Hippocampal organotypic slice cultures
Hippocampal organotypic slice cultures were prepared following  with modifications . Briefly, 8 days old mice were decapitated, brains were rapidly removed, and hippocampi dissected at 4°C. A McIIwain tissue chopper (Gabler, Bad Schwabach, Germany) was used to prepare 400 μm thick transverse slices which were transferred to semi porous cell culture inserts (Millipore, Bedford, MA, USA; 0.4 mm). Inserts containing 6 slices were placed in 6-well culture trays containing 1 mL of culture media (50% Minimum Essential Medium (MEM), 25% Hank’s Balanced Salt Solution (HBSS), penicillin/streptomycin [all from PAA, Pasching, Austria], 25% horse serum, 4.5 mg/mL glucose [Sigma-Aldrich, St. Louis, MO, USA], pH 7.4). The culture medium was changed on the first day after preparation and afterward every third day.
Primary hippocampal cell culture
Primary hippocampal neurons were isolated from embryonic E18 Sprague Dawley rats and plated on poly-D-lysine-coated glass coverslips (50 g/ml) at a density of 50,000 cells per well in 24 well plates (Corning or Sarstedt) as described previously . Neurons were transfected with Lipofectamin (Invitrogen) at DIV 14–23. Per coverslip 0.6 μg of plasmid DNA was used either with pAAV (plasmid Adeno Associated Virus)/pro-aggregant TauRDΔ/IRES (Internal Ribosomal Entry Site)/hrGFP (human recombinant green fluorescent protein) or pAAV/IRES/hrGFP as a negative control plasmid. Transfections were carried out over a period of 72 h.
Inducible N2a cell-line
Tau expressing N2a cell-lines were generated previously . The stable N2a cell-lines N2a/Tet-ON/pro-aggregant TauRDΔ and N2a/Tet-ON/anti-aggregant TauRDΔPP were seeded on coverslips and grown confluently in Eagle’s minimum essential medium supplemented with 10% fetal calf serum, 2 mM glutamine, and 0.1% nonessential amino acids. The expression of constructs was induced by doxycycline and expression was continued for 4 days. On the fourth day expression was checked by bioluminescence. D-luciferin (Caliper Life Science) was added to the medium and bioluminescence was measured using an Ivis Spectrum imaging system (Caliper Life Science).
For electron microscopic analysis of mossy fiber boutons, 4 adult wild-type animals and 4 pro-aggregant TauRDΔ transgenic mice were used. The animals were deeply anesthetized with a lethal dose of sodium pentobarbital and then transcardially perfused, first with 5 ml of physiological saline, followed by approximately 150 ml fixative solution (1% glutaraldehyde and 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4). The fixative solution was perfused for approximately 15 min. Then, the brains were removed from the skulls and post-fixed in fixative solution over night at 4°C. After rinsing in 0.1 M phosphate buffer (pH 7.4), the brains were sectioned on a vibratome (Leica VT1000S) in the coronal plane at a thickness of 200 μm. Sections containing the dorsal (septal) hippocampus were processed for electron microscopy by placing the tissue in 1% OsO4 for 30 min. After careful washing (5 times, 5 min each) in distilled water, the sections were dehydrated in increasing concentrations of ethanol (block-staining the sections in 0.5% uranyl acetate in 70% ethanol for 1 hour). Thereafter, the sections were soaked in propylene oxide and finally embedded in Durcupan ACM resin (Fluca) and hardened at 56 ° C for at least 24 hours. The CA3 region was cut out and remounted on blank Durcupan blocks for thin sectioning. Thin sections were cut using an Ultracut (Leica) and a diamond knife. Sections were mounted on copper grids and post-stained with lead citrate. Electron micrographs of the stratum lucidum were taken at a magnification of × 5000. Ten mossy fiber boutons from each animal were used for quantitative analysis. Bouton area, number of vesicles, and the length of synaptic contact zones were measured using SIS software (Münster, Germany). Student’s t-test was used for statistical analysis.
Gallyas silver staining and immunohistochemistry - using antibodies: 12E8 (pSer262/pSer356, 1:2000, gift of Dr. P. Seubert, Elan, S. San Francisco), PHF1 (pSer396/pSer404, 1:50, gift of Dr. P. Davies, Albert-Einstein College, N.Y.), MC1 (conformational changes of tau, 1:10, Dr. P. Davies) - were performed on 5 μm paraffin sections according to published procedures [3,8].
Ca++ imaging of mossy fiber boutons
To load hippocampal slice cultures with Oregon Green 488 BAPTA 1-AM (OGB-1 AM), we added 4 μl 20% Pluronic F-127 in DMSO (Invitrogen) and 36 μl DMSO to a 50 μg vial of OGB-1 AM (Molecular Probes). The solution was added to slice culture medium to a final concentration of 10 μM. Slice cultures were submerged and incubated for 60 min at 37°C. Slices were washed with slice culture medium and then transferred to HEPES-buffered saline (HBS; 130 mM NaCl, 5.4 mM KCl; 10 mM HEPES, 25 mM glucose, 1.8 mM CaCl2, 1 mM MgCl2; ph 7.4) and allowed to recover for 30 min prior to an experiment. Imaging was performed with a Zeiss LSM 700 laser-scanning confocal microscope equipped with a 20x1A objective (Zeiss, Jena, Germany). Light from an argon laser (488 nm) was used for excitation and a FITC/GFP filter set for emission. Ca++ transients at individual mossy fiber boutons were evoked by applying 572 mM potassium chloride (KCl). The relative Ca++ increment is calculated as ΔF/F0, where F0 is resting fluorescence intensity of OGB-1 after subtraction of background fluorescence, and ΔF is the fluorescence increment from F0.
Calcium imaging in cell-culture
Prior to calcium imaging experiments N2a cell cultures were induced with doxycycline (1 μg) for 4 days. N2a cell-lines and primary hippocampal neurons were washed at least 3 times with HEPES buffered saline. Thereafter cells were loaded with 10 μM Fura 2-AM (Molecular Probes) for 30 minutes at 37°C and 5% CO2 in HEPES-buffered saline. The Fura2-AM solution was removed and cells were washed three or more times and left for a few minutes in the incubator to allow complete de-esterification of the Fura dye. After washing, cultures were transferred to a submerged imaging chamber of an Examiner A1 microscope (Zeiss, Jena, Germany). Fura-2 fluorescence was imaged at room temperature (RT) in HEPES-buffered saline, using a 40× water immersion objective (Zeiss). The emission of Fura-2-loaded cells was collected at 510 nm after excitation at 340 and 380 nm respectively with a Sutter DCIV shutter (Sutter Instrument Co, Navato, CA, USA). Images were taken at a rate of 1 Hz. For baseline, intracellular Ca++ levels were recorded for at least 30 seconds subsequently cells were stimulated with a high potassium solution (286 mM KCl) by manual application and recorded for another 90 seconds. In primary hippocampal cultures, neurons and astrocytes were distinguished on the basis of their morphology and delayed intracellular Ca++ peak (~30 sec after high KCl for neurons and ~90 sec for astrocytes).
To obtain preserved mossy fiber tracts, acute, transverse hippocampal slices were prepared from 13(±1) month-old female mice as described before . ACSF was adjusted by increased Ca++ concentrations (126 mM NaCl, 26 mM NaHCO3, 3 mM KCl, 2.5 mM CaCl2, 1.3 mM MgSO4, 1.25 mM NaH2PO4 and 10 mM glucose, saturated with 95% O2/5% CO2). After 1.5 h recovery period in a homemade interface chamber, slices were transferred to a submerged recording chamber at 30°C. The mossy fiber tract was visualized by infrared (IR)/differential interference contrast (DIC) microscopy. As stimulation electrode, a patch-clamp pipette was used (1–2 MΩ) to stimulate mossy fibers (mf) in the region of the hilus. Recording electrodes (2.5-3 MΩ) were filled with ACSF and separated constantly 200 μm from stimulation electrode. fEPSPs were 10 × preamplified (custom made preamplifier) filtered at 1 kHz and sampled at 10 kHz using a HEKA double patch-clamp EPC 10 USB amplifier (HEKA Elektronik Dr. Schulze GmbH).
Stimulation protocols and recording procedures
Constant current pulses were elicited every 20 s (0.05 Hz) with a pulse width of 0.1 ms. Input output (I/O) curves were generated using stimulus intensities ranging from 10–100 μA with an increment of 10 μA. In the same slice paired pulse facilitation (PPF) was evoked by a 50 ms inter-stimulus interval (ISI). In a next step frequency facilitation (ff) of the mossy fiber pathway was measured by switching stimulation frequency from 0.1 Hz to 1 Hz. Only slices which showed robust ff and PPF of approximately 1.5 were used further on for mf-LTD recordings. LTD was induced by 1 Hz stimulation for 15 min (900 pulses) at 50% of the maximum stimulus intensity as revealed initially during the I/O recording. At the end of each recording 5 μM of the mGluRII receptor antagonist DCGIV (Tocris Biosciences) diluted in ACSF was washed in for at least 5 min to test for mossy fiber specificity. In case the fEPSP slope and amplitude were not reduced to more than 50% of the initial value, slices were excluded from further mossy fiber analysis.
Brains from adult 13 month-old mice were fixed for 48 h in 4% PFA at 4°C and horizontal, hippocampal free-floating sections with a thickness of 80 μm were prepared with a vibratome (Leica VT 1200S, Wetzlar, Germany). Sections were rinsed with 0.4% TritonX in 5% horse serum for 1 h at RT. After washing with PBS first antibodies (PHF1 (pSer396/pSer404, Dr. P. Davies): 1:250; MC1 (Dr. P. Davies): 1:20) were incubated at 4°C over night. Secondary antibodies (donkey anti-mouse Alexa 488, Dianova) were incubated for 1:100 for 2 h at RT.
Immunohistochemistry of slice cultures
Slice cultures were left attached on the Millicell membrane and stained as free-floating sections in 6-well plates. Cultures were first fixed with 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) (PAA, Austria) for 2 h at 4°C. After washing with PBS, slices were permeabilized by 0.1% TritonX-100/PBS for 90 min at RT. Slices were then blocked with 5% bovine serum albumin (BSA) for 2 h and afterwards incubated with primary antibody diluted in PBS for 3 days at 4°C. After washing three times with PBS, slices were incubated with secondary antibody for 2 days at 4°C. Finally, slices were incubated with the nuclear counterstain and dead cell indicator TO-PRO3 (Molecular Probes, US) for 15 min and washed three time with PBS before getting mounted with Permafluor mounting solution (Beckman Coulter, Paris, France. The following primary antibodies were used: pan-Tau antibody K9JA (Dako, Hamburg, Germany, Nr. A0024 (1:1000)), 12E8 (1:1000) for phosphorylated S262/S356 Tau (gift from Dr. P. Seubert, Elan Pharma, South San Francisco, CA, US) and Synaptophysin (1:1000; Sigma). All fluorescent (goat anti-rabbit/mouse cyanine 2 and 3)-labeled secondary antibodies were from Dianova (Hamburg, Germany) (1:1000).
DiI labeling of organotypic slice cultures
Hippocampal slice cultures were fixed at day 10 in vitro (DIV 10) in 4% PFA for 2 hours, still attached to the semi-porous cell culture inserts. A DiI crystal was placed on the dentate gyrus followed by an incubation of 10 days at room temperature in 4% PFA to allow diffusion. Mossy fiber boutons and dendritic spines were imaged by confocal microscopy using tetramethyl rhodamine isothiocyanate (TRITC)-filter settings. Image J (NIH, USA) was used to measure morphological parameters such as spine density, mf bouton diameter and filopodia from resulting Z-stacks. For 3D surface determination, boutons were reconstructed by Imaris software (Version 7.6.4; Bitplane, Switzerland). For structural analysis at least 10 slices were prepared from 5 different animals per group.
Slice cultures were treated with compound bb14, a rhodanine-based Tau aggregation inhibitor identified in previous screens [31,32]. Compound bb14 was dissolved in 100% DMSO and added to the culture media at a final concentration of 15 μM (0.15% (v/v) DMSO). Control slices were treated with the same amount of DMSO alone. In all experiments treatment was done for the entire cultivation period starting at DIV 1. During treatment, the compound was replenished once a day after preparation and later on every 3rd day.
Throughout the study error bars denote standard error of the mean (SEM) except for ultrastructural analysis where errors denote standard deviation. Statistical hypothesis testing was done by using Prism5 (GraphPad, La Jolla, CA, USA), Statistica (Statsoft, Europe GmbH, Hamburg) and OriginPro8.5 (OriginLab cooperation, Northhampton, MA, USA). Evaluation of data was performed either by Student’s t-test or One way ANOVA followed by Tukey’s post-hoc test for morphological measurements in organotypic slices and multiple way repeated measure ANOVA was used for comparisons in electrophysiological recordings. Moreover, when groups did not have normal distribution the paired sample Wilcoxon signed rank test was used as a nonparametric test. p-values are indicated as follows: * p < 0.05, ** p < 0.01, and ***p < 0.001. Additional file 1: Supplemental experimental procedures.
Endogenous Tau becomes phosphorylated and aggregated in area CA3 of the hippocampus due to expression of the human Tau repeat domain ΔK280
Since Tau is known to be hyperphosphorylated in several tauopathies [33,34] we looked for Tau’s phospho-status by using different phosphorylation-dependent antibodies (for a complete list of antibodies see Additional file 2: Table S1). Using the phospho-dependent antibody 12E8 (pSer262/pSer356) we detected abnormally phosphorylated and mislocalized Tau in area CA3 a-c and DG in sagittal hippocampal sections from 13 ± 1 month-old mice (Figure 1b1). 12E8 immunoreactivity was detected in cell somata in the stratum pyramidale (s.p., Figure 1b1 and b3) but not in control littermate slices (Figure 1b2). We also used the phospho-dependent antibody PHF1 (pSer396/pSer404), a marker of pathological phosphorylation of endogenous Tau. PHF1 showed exclusively axonal immunoreactivity of mossy fibers in the stratum lucidum (Figure 1c1, Additional file 3: Figure S1b and e) but not in control littermates (Figure 1c2, Additional file 3: Figure S1a). Indeed detailed inspection of PHF1 immunoreactivity in area CA3 demonstrated that apical dendrites of pyramidal cells were not PHF1 positive (Figure 1c3, white arrows). We confirmed the presence of pathologically, phosphorylated endogenous Tau in the mossy fiber tract with the antibodies PHF1 and AT-8 (pSer202/Thr205; Additional file 3: Figure S1a-d) by immunofluorescence. Moreover we used immunofluorescence to trace single mossy fiber axons with PHF-1 immunoreactivity projecting from granule cells towards CA3 pyramidal cells (Additional file 3: Figure S1e). Finally, human Tau expression was confirmed by western blotting from CA3 hippocampal homogenates in pro-and anti-aggregant mice (Additional file 4: Figure S2a). The phosphorylation sites AT180 and AT8 were detected by western blotting in pro- and anti-aggregant mice but not in control littermates and Tau knockout mice (Additional file 4: Figure S2b-c) and we found weak immunoreactivity against the MC1 epitope demonstrating conformational change of Tau in that region (Additional file 4: Figure S2d).
Finally, Gallyas silver staining was applied to monitor Tau aggregation in our model (Figure 1d1). Similar to 12E8 immunoreactivity, Tau pathology occurred predominantly in somata of CA3 a-c pyramidal neurons of stratum pyramidale (Figure 1d1 and 1d3, white arrows), whereas there was no aggregation in control littermates (Figure 1d2).
Tau is missorted to dendrites and somata in area CA3 of pro-aggregant TauRDΔ mice
The above observations prompted us to investigate the mossy fiber pathway in more detail. We stained horizontal hippocampal slices with the pan-Tau antibody K9JA. In 13 month-old control littermate mice, Tau was restricted to axons in mossy fiber bundles in s.l. and there was neither dendritic nor somatic Tau immunoreactivity throughout CA3 a-c (Additional file 5: Figure S3a). By contrast, in pro-aggregant TauRDΔ mice we observed missorting of Tau (endogenous and transgenic) into somato-dendritic compartments (Additional file 5: Figure S3b). Under these circumstances Tau was detectable in all three hippocampal layers (s.r., s.l. and s.p., Additional file 5: Figure S3b, arrows). To better visualize mossy fibers and their projection targets (thorny excrescences) we used the lipophilic tracer DiI in combination with pan-Tau immunohistochemistry. This method highlighted Tau in mossy fiber boutons and postsynaptic, dendritic compartments in area CA3 a-c of pro-aggregant mice (Additional file 5: Figure S3f-h) whereas in control littermates pan-Tau immunoreactivity was consistently restricted to axonal and presynaptic “giant” bouton structures (Additional file 5: Figure S3c-e).
Presynaptic pro-aggregant TauRDΔ impairs basal synaptic transmission, mossy fiber plasticity and reduces synaptic markers
Next we investigated short-term plasticity measured by paired pulse facilitation (PPF). Pro-aggregant mice revealed a significant reduction of mf-PPF compared to control littermates (paired pulse ratio control: 1.46 ± 0.1, n = 24; paired pulse ratio pro-aggregant: 1.25 ± 0.03, n = 34; t-test: p = 0.023; Figure 2b), whereas anti-aggregant mice and Tau knockout mice differed not significantly from control littermates (anti-aggregant: 1.33 ± 0.06, n = 22, t-test: p = 0.296; TKO: 1.32 ± 0.06, n = 33, t-test: p = 0.222; Figure 2b). 2 month-old pro-aggregant mice however displayed no impairment of PPF compared to control littermates (paired pulse ratio control: 1.53 ± 0.12, n = 19 and paired pulse ratio pro-aggregant: 1.45 ± 0.14; n = 15; data not shown). Mossy fiber frequency facilitation (mf-ff) was decreased in pro-aggregant mice (last 20 stimulation pulses displayed in graphs: 167.4 ± 4% of baseline, n = 11 compared to control littermates: 208.4 ± 6% of baseline, n = 6; multiple way repeated measure ANOVA: group effect p = 0.019, Figure 2c). In anti-aggregant mice, mossy fiber frequency facilitation was scarcely and not significantly reduced confirming previous results (177.9 ± 5% of baseline, n = 4, Figure 2d; multiple way repeated measure ANOVA: group effect p = 0.455, ). TKO mice displayed frequency facilitation well comparable to control mice (199.3 ± 8% of baseline, n = 5, Figure 2e). Finally when comparing 2 month-old control littermates with pro-aggregant siblings we could not detect any differences in mf-ff (229.4 ± 7.7% of baseline, n = 10 and 240 ± 12.1% of baseline, n = 7; data not shown).
Mossy fiber long-term depression (mf-LTD) is expressed at the presynaptic site  and depends on proper neuronal Ca++ dynamics . In control littermate mice, LTD induction by low frequency stimulation (LFS) led to an average synaptic depression of −31.1 ± 0.5% (n = 8, Figure 2f) of the initial baseline mf-fEPSP slope, whereas in pro-aggregant mice LTD was induced in the first 15 minutes after LFS, but was no longer present during the last 10 minutes of recording (reduction only −2.2 ± 0.4%; n = 7; Figure 2f). Therefore mf-LTD was significantly impaired in pro-aggregant mice compared to control littermates (paired sample Wilcoxon signed rank test, p = 0.0019, last 10 minutes of recording). In contrast, the same LTD protocol evoked a LTD expression closely comparable to control mice in anti-aggregant and TKO mice (Figure 2g and 2h respectively). Moreover comparing young cohorts of pro-aggregant mice and non-transgenic siblings we found no differences in LTD expression (last 10 minutes of recording, control littermates: −23.6 ± 1.2% of baseline recording and pro-aggregant: −31.8 ± 1.3% of baseline, Figure 2i). Consistently we found in CA3 homogenates dissected from pro-aggregant TauRDΔ mice a reduction of the presynaptic vesicle protein synapsin1 and synaptophysin (Additional file 3: Figure S2e and f). This was accompanied by a loss of the postsynaptic markers PSD95 and the NMDA receptor NR1 subunit (Additional file 3: Figure S2e and f).
Pro-aggregant TauRDΔ leads to pathological Tau accumulation in presynapses and impairs synapse morphology
In organotypic slices we detected pan-Tau immunoreactivity in mossy fiber boutons from control littermates (Figure 5a1 and c1) and TauRDΔ (Figure 5b1 and d1) at DIV 5 and 10. We confirmed such Tau containing boutons by co-localization with presynaptic marker protein synaptophysin (Figure 5c7-c9 and d7-d9). In pro-aggregant TauRDΔ slices we observed strong 12E8 immunoreactivity in mf-boutons at DIV 10, which was not seen in control cultures either at DIV 5 or 10 (compare Figure 5d4-d6 with Figure 5a2 and c2). At DIV 5, 12E8 phosphorylation in TauRDΔ slices (Figure 5b1-b3) was weaker when compared to DIV 10 and in particular present in the postsynaptic compartment. In contrast, 12E8 immunoreactiviy was found in pre-and postsynaptic compartments at DIV 10 (Figure 5d4-d6).
The expression of pro-aggregant TauRDΔ led to a 42% increase in diameter of “giant” mf-boutons, comparable to hippocampal slices from adult mice, which was prevented when the aggregation inhibitor bb14 was added to the preparation (control: 3.20 ± 0.13 μm; pro-aggregant: 4.54 ± 0.16 μm; pro-aggregant + bb14: 3.86 ± 0.20 μm; F(2/83) = 20.66; p < 0.0001; Figure 5e and f). In agreement with this, the 3D surface area of pro-aggregant boutons was larger (~74%) than in control littermate slices (control: 37.97 ± 2.69 μm2; pro-aggregant: 66.24 ± 6.30 μm2; pro-aggregant + bb14: 50.16 ± 3.41 μm2; F(2/56) = 10.67; p < 0.0001; Figure 5g). We measured the average distance between mossy fiber boutons as a function of total excitation strength and observed an increased by ~51% in pro-aggregant TauRDΔ slices (185.4 ± 22.3 μm) compared to control littermates (122.9 ± 18.5 μm, Figure 5h). Again compound bb14 prevented this effect on mf-boutons (131.3 ± 6.7 μm, Figure 5f).
As a measure of feedforward inhibitory drive we determined the length and number of filopodia per bouton. Filopodia were defined as described elsewhere  as emanating from the main body of the bouton, with lengths of 1–5 μm. The filopodia number increased somewhat (~21%, control: 1.56 ± 0.17; pro-aggregant: 1.89 ± 0.17; pro-aggregant + bb14: 1.77 ± 0.23; F(2/83) = 0.9031; p = 0.4092; Figure 5i) and correspondingly filopodia lengths were reduced to a similar extent so that the total filopodia length remained roughly the same (~17%, control: 1.66 ± 0.16 μm, pro-aggregant: 1.39 ± 0.08 μm, pro-aggregant + bb14: 1.69 ± 0.24 μm; F(2/134) = 1.656, p = 0.2207; Figure 5j).
The synaptic vesicle density from pro-aggregant mice was severely reduced in mossy fiber “giant” boutons
Ca++ influx is attenuated due to pro-aggregant TauRDΔ expression in mossy fiber boutons before tangle formation
The reduction of Ca++ influx after membrane depolarization is exacerbated with increasing aggregation of pro-aggregant TauRDΔ in N2a cells
To test if aggregation directly contributes to the observed reduction in Ca++ influx after activity induction we made use of an inducible Tet-ON N2a cell line expressing pro-aggregant TauRDΔ and the reporter gene firefly luciferase (Figure 8f-i) . Induction of pro-aggregant TauRDΔ expression by doxycycline led to stable expression of the construct, tested via bioluminescence (data not shown). After 4 days of expression ~5% of the cells developed Tau aggregates, which was visualized by Thioflavine-S (ThS) fluorescence (Figure 8g and h). N2a cells expressing pro-aggregant TauRDΔ displayed a severely reduced KCl induced Ca++ influx (−222%) compared to non-induced control cells, even before aggregates became detectable (pro-aggregant TauRDΔ: 370.4 ± 26% of baseline, n = 16; non-induced control cells: 592.07 ± 42% of baseline, n = 38, t-test p < 0.0017; Figure 8i). This effect of pro-aggregant TauRDΔ was almost doubled (−436%) in N2a cells with ThS detectable tangle load (156.83 ± 8% of baseline; Figure 8h,i). As an additional control we performed experiments with N2a cells expressing anti-aggregant TauRDΔPP, in which evoked Ca++ influx was not different from non-induced control cells (657.08 ± 28% of baseline, n = 60; Figure 8i).
We conclude that the pathophysiological effect of Tau is tightly coupled to its propensity for aggregation.
Several authors have investigated the influence of pathological Tau on electrophysiological parameters in the hippocampus of transgenic mice. So far these studies were focused on postsynaptic functions in area CA1 [4,6,42,43]. However, in mature neurons Tau is largely restricted to axons, suggesting a presynaptic role [44,45]. In the present study we therefore focused on the physiological and pathophysiological role of axonal Tau and the impact of Tau aggregation on presynaptic morphology and function.
The Tau mutation ΔK280 was first described in a patient with FTDP-17  and later in a case of Alzheimer disease . The mutation affects Tau splicing and is unusual in that it enhances the fraction of 3R-Tau (where repeat R2 is excluded) compared to 4R-Tau, in contrast to many other mutations where 4-R Tau is enhanced. In theory, the mutation of the exon splicer enhancer element in exon 10 would be expected to cause complete exclusion of repeat R2 so that there would be no protein containing the ΔK280 mutation , but in the patients a substantial amount of 4R-Tau with the ΔK280 mutation is present in the hippocampus and cortex . Since the mutant protein has a much greater propensity for aggregation compared to wild type Tau , and since Tau aggregation is a nucleation-dependent process, nuclei formed by ΔK280 Tau could be elongated even from the pool of normal endogenous Tau and thus poison the entire Tau population . Thus even a small amount of 4R-Tau would be sufficient to initiate pathophysiological events (gain of toxic function). The toxic potential of the mutation is supported by cell and animal models with anti-aggregant Tau (where the propensity for ß-structure is eliminated by insertion of prolines). Mice expressing anti-aggregant TauRDΔPP show some weak Tau phosphorylation and mislocalization as well as slightly reduced frequency facilitation in the mossy fiber pathway [7,8]. But overall these changes are not sufficient to cause a behavioral phenotype in these mice. Alterations are likely due to an increase in the overall Tau level which may disturb the balance of kinases/phosphatases in the neuron as described previously . In previous studies of the Tau-ΔK280 mutation we had noticed pronounced aggregation, but it was not clear in what pathway the major effect of mutant Tau was located. Here we demonstrate that it is the mossy fiber pathway in the hippocampus that is pronouncedly affected by degeneration. We describe a specific impairment of morphology of Tau-containing presynaptic mossy fiber boutons, which corresponds to an impaired synapse physiology and plasticity. Pronounced pathological phosphorylation, aggregation and missorting occurs in area CA3 in aged mice expressing pro-aggregant TauRDΔ, particularly in the stratum lucidum. Tau becomes enriched in mossy fiber giant boutons and reveals the pathognomonic MC-1 epitope in axons and presynapses, arguing for misconformational Tau in the mossy fiber presynapse due to co-aggregation of mutant and endogenous Tau. The mossy fiber boutons displayed a pathological increase of size, observable both in middle- aged mouse brains and in organotypic slices.
The organotypic slice model was further used to analyze the presynaptic structural changes in detail. Giant boutons on granule cell axons were spaced further apart in slices from pro-aggregant TauRDΔ mice, and at the same time the bouton diameter and 3D surface became larger (~74%). This corresponds to a “condensed presynapse” pathological phenotype when considering the granule cell axon as a whole. We found less but bigger mossy fiber boutons at such axons. Taken together, the excitatory drive of granule cells is decreased due to “condensed presynapses” in pro-aggregant mice, which is reflected in a severely reduced basal synaptic transmission. By contrast, in anti-aggregant animals there was no impairment in basal synaptic transmission or in bouton diameter although endogenous Tau became phosphorylated at AT8 and AT180 site. Looking at the inhibitory site of transmission, we found that filipodia which are known to innervate inhibitory interneurons were slightly increased in number but decreased in length which could account for a compensatory effect. We conclude that aggregation-prone Tau is causal for the reduction in synaptic transmission at the mf-CA3 synapse. Consistent with this, treatment with the aggregation inhibitor bb14  prevented pathological pre- and postsynaptic morphological changes in area CA3 of pro-aggregant TauRDΔ slices. Data from organotypic slice cultures enabled us to present a comparison of pathological effects on postsynaptic structures (loss of dendritic spines in area CA3: −20%) and the effect of pro-aggregant TauRDΔ on the presynapse-site (bouton diameter increase: +42%) in parallel. Moreover ultrastructural analysis demonstrated severe reduction of vesicle density in the pathologically modified mossy fiber boutons. Although we cannot rule out the possibility that reduced vesicle density is simply due to an increase in bouton size the finding correlates well with the dramatic effect of pro-aggregant TauRDΔ on basal synaptic transmission of the mossy fiber pathway (Figure 2a).
Unexpectedly, the depletion of Tau in the TKO mice  also led to a pronounced increase in bouton diameter and a concomitant reduction in basal synaptic transmission (Figure 2a), comparable to the phenotype observed in pro-aggregant mice. In pro-aggregant mice, it is likely that an accumulation of aggregating Tau (as visualized by MC1 antibody) in presynaptic terminals leads to an overall increase of the mossy fiber bouton diameter. In contrast, the phenotype observed in TKO mice is more ambiguous. A role of Tau during axon growth and maturation  was previously described and primary neurons obtained from the Tau knockout strain showed reduced axon length . This illustrates that Tau reduction can cause structural changes in axons. Why this does not lead to impairment in synaptic plasticity at 13 months remains to be investigated. Nevertheless, the reduction of basal synaptic transmission in the mossy fiber pathway implies a physiological role of Tau as a regulator of neuronal excitability at the DG-CA3 synapse. This might in turn explain how a genetic reduction of Tau could lead to a reduction of seizure susceptibility in hAPP mouse models when crossed with TKO mice  and even in non-AD related epilepsy models [53,54]. It has been speculated that the dentate gyrus might act as a “shield” protecting from spreading of epileptiform activity from cortical regions into the hippocampus because of the low intrinsic excitability of dentate granule cells compared with pyramidal cells [55,56]. On the basis of this hypothesis increased seizure susceptibility in hAPP models would be ameliorated by decreased synaptic transmission at the DG-CA3 synapse due to Tau depletion.
Since we did not detect any plasticity impairments in our TKO mice at the mossy fiber-CA3 synapse we conclude that Tau reduction at that synapse could counteract hyperexcitability effects in hAPP mouse models without affecting learning and memory. However a recent study reports plasticity deficits in a different experimental paradigm due to Tau reduction , but in contrast to our study deficiencies were found in area CA1.
Subsequently to synaptic transmission we measured mossy fiber short-term plasticity (by paired pulse facilitation and frequency facilitation) and long-term depression (by low frequency stimulation). In contrast to the results on basal synaptic transmission we found that endogenous Tau was indeed not essential for normal mossy fiber short-term plasticity and long-term depression in the mossy fiber pathway. TKO mice were indistinguishable from controls in both parameters in spite of the pathological mossy fiber bouton size increase. However, in middle-aged pro-aggregant mice we observed reduced paired pulse facilitation, frequency facilitation and disrupted LTD expression. This phenotype of impaired mf-plasticity was not present in 2 month-old mice expressing pro-aggregant TauRDΔ confiming a “phenotype progression” in our mouse model. Paired pulse facilitation is known to depend on presynaptic calcium dynamics . In AD patients a colocalization of A1 receptors with Aß plaques and with tangle bearing pyramidal cells in the hippocampus has been described . Moreover the activation of A1 receptors led to the phosphorylation of Tau and its translocation towards particulate fractions from SH-SY5Y neuroblastoma cells . Given that frequency facilitation is closely correlated with LTD  in vivo and since proper A1 receptor function is essential for mossy fiber plasticity , any modifications of A1 receptor function or transport inhibition might partly contribute to reduced basal synaptic transmission, reduced frequency facilitation and impaired LTD in pro-aggregant animals.
Since the mossy fiber synapse is essential for memory recall, storage and pattern completion [61,62], the described loss of plasticity at this synapse should have deleterious consequences on memory and cognition. This assumption is consistent with previous behavioral observations [7,8] and should therefore be extended in future studies to mossy fiber specific pattern-separation behavioral tests .
The major and general cellular prerequisite for bidirectional plasticity is an intact Ca++ homeostasis and activity-dependent Ca++ influx [64-66]. In particular, at the mf synapse an activity-dependent rise in Ca++ levels is necessary for proper LTD expression . We detected a strong reduction of Ca++ influx after membrane depolarization at the mossy fiber presynapse, which argues that this is the reason for the pathophysiological effect of TauRDΔ on presynaptic plasticity. Notably this effect occurs at a time point (DIV 10) where no tangles are present in organotypic slices from pro-aggregant mice, suggesting a soluble, “pre-tangle” form of aggregating Tau that is sufficient for this pathological effect. At an earlier time point (DIV 5) there was only a slight decrease in Ca++ influx which underscores that there is a pathological progression in the slice model system. Similarly, two previous studies reported early presynaptic deficits in the enthorinal cortex of Tau transgenic mice [67,68]. Using primary hippocampal cell culture, we found that pro-aggregant TauRDΔ also led to severe attenuation of Ca++ influx in the soma of transiently transfected neurons. This was true even in the absence of Tau aggregates. To investigate if this effect is exacerbated by Tau aggregates, we next used inducible N2a cells expressing the same pro-aggregant TauRDΔ. N2a cells display a prominent Ca++ influx mediated by L-type voltage-gated calcium channels (VGCC) as a response to membrane depolarization . Like the primary hippocampal neurons, the N2a cells showed a reduction of Ca++ influx due to pro-aggregant TauRDΔ, even before the appearance of ThS-stained Tau aggregates. This effect occurs only with pro-aggregant but not with anti-aggregant TauRDΔPP and may be explained again by “pre-tangle” oligomeric aggregates which are known to impair cognition in transgenic Tau mice before full-blown tangles appear [9,70,71]. Interestingly, in N2a cells the impairment of Ca++ influx was further worsened when ThS-positive Tau aggregates appeared intracellularly. This leads us to propose the activity dependent dysfunction in neuronal Ca++ regulation as a cellular patho-mechanism of the observed pre-and postsynaptic tauopathy phenotypes.
In summary we suggest that the pathophysiological effects of pro-aggregant TauRDΔ on calcium influx and the reduction of synaptic vesicle density causes the impairment of transmission and presynaptic plasticity at the mf-synapse, which will in turn lead to behavioral and cognitive deficits in AD and other tauopathies (Additional file 6: Figure S4).
- anti-aggregant TauRDΔPP = TauRDΔK280PP:
Tau repeat domain with mutation ΔK280 and two additional prolines
- Ca++ :
Frontotemporal dementia with parkinsonism linked to chromosome-17
Low frequency stimulation
Long term depression
- pro-aggregant TauRDΔ = TauRDΔK280:
Tau repeat domain with mutation ΔK280
Tau knockout strain
We thank the following lab members for their expert help and advice: Olga Petrova and Anne Hofmann (Hamburg) for help with some of the brain sections and biochemical analysis; Julia Lüdtke (Bonn) for preparation of hippocampal cell cultures, Stefanie Koenen (Bonn) for help with statistical analysis, Dr. Katja Hochgräfe (Hamburg) for monitoring gene expression by bioluminescence, Dr. Marcus Pickhardt and Dr. Jacek Biernat (Bonn) for help with N2a cell-lines, Dr. Marcus Pickhardt (DZNE) and Dr. Bruno Bulic (Humboldt University, Berlin) for aggregation inhibitor compound bb14. We owe special thanks to Dr. Andreas Haemisch and his team at the animal facility at the University of Hamburg Medical School for their continuous help in mouse breeding. Furthermore we thank the team of the animal facility of Caesar (headed by Dr. Dagmar Wachten) and the team of the animal facility of DZNE Bonn (headed by Dr. Ylva Mende & Dr. Michaela Möhring). Reagents are gratefully acknowledged from Prof. Dr. E. Kandel (Columbia University, New York, NY; CaMKIIa-tTA transgenic mice), Dr. P. Seubert (Elan Pharma, South San Francisco, CA; 12E8 antibody), and Dr. Hana Dawson for generously providing the Tau KO mice. This research was supported by MPG, DZNE, Wellcome Trust/MRC and Tau consortium. MF is Senior Research Professor supported by the Hertie Foundation.
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