Monoclonal antibody AT8 recognizing a phosphorylated tau pS202-pT205-pS208 and tau monoclonal antibody HT7 recognising the epitope 159–163 of human tau protein were purchased from Thermo Fisher Scientific, antibody DC190 mapping tau 368–376 was purified from hybridoma supernatant and conjugated to horse radish peroxidase. Antibodies DC8E8  and DC25 (epitope 347–353) were affinity purified from serum-free hybridoma supernatant. Indiferent monoclonal antibody DC51 (IgG1 isotype) was used as unrelated control (specifically binds to surface glycoprotein of rabies virus) . Monoclonal anti-tau antibodies AX004/IgG1 and AX004/IgG4, humanized version of anti-tau monoclonal antibody DC8E8  were produced in Expi-CHO cells (Thermo Fisher Cat. No. A29133). The activity of purified antibodies was verified by ELISA, Western blot and immunohistochemistry.
DC8E8 vaccine administration
DC8E8 vaccine was administered in 2 weeks intervals starting at the age of 6 weeks and continuing until the age of 6 months. Purified antibody DC8E8 prepared in PBS was injected intraperitoneally (1 mg of antibody/per dose/200 μl) into transgenic mice R3m/4 expressing human truncated tau (151–391/3R) under the control of the mouse Thy1 promoter, with tau pathology predominantly located in the brainstem . An additional set of R3m/4 animals was injected in the same vaccination scheme (timing and dosing) with unrelated antibody DC51 (anti-rabies IgG1), thus representing the control. Then animals were sacrificed and their brainstems were subjected to immunohistochemical and biochemical quantification of tau pathology. The experiment was carried out in accordance with the Slovak and European Community Guidelines, approved by the State Veterinary and Food Administration of the Slovak Republic.
Quantitative immunohistochemistry of mouse brain tissue samples
Mouse were anesthetized and perfused intra-cardially for 1 min with phosphate-buffered saline (PBS), followed by 3 min perfusion with 4% paraformaldehyde (PFA) in PBS (4% PFA, pH 7.2). Brain were transferred for post fixation for 24 h in 4% PFA, then transferred to PBS, embedded in paraffin and serially cut using Leica RM 2255 microtome into 8 μm thick sagittal brain sections. Immunostaining was performed using the standard immunohistochemistry staining procedure. Briefly, brain sections were treated with 80% formic acid (40 s) followed by heat pretreatment for 20 min in antigen retrieval solution (Retrieval 2100, Aptum, Southampton, UK). The sections were blocked with Aptum section block (Aptum, Southampton, UK) followed by incubation with mouse monoclonal primary antibody (Anti -human phospho tau AT8, 1:1000, ThermoScientific, IL, USA) overnight at 4 °C and were immunostained using the standard avidin-biotin-peroxidase method (Vectastain ABC kit) with VIP as chromogen (VIP kit, Vector Laboratories, Burlingame, CA, USA). After mounting, sections were evaluated using Olympus BX51 microscope. Neurofibrillary tangles were counted in two parallel sections from each mouse brain. Quantification of structures was carried out by investigators blinded to the treatment status of the mice.
Preparation of sarkosyl-insoluble tau protein (2p fraction)
Brainstems of transgenic animals R3m/4 were subjected for biochemical analysis of sarkosyl-insoluble tau proteins. The sarkosyl-insoluble fraction was extracted according to published protocol . Frozen tissues were homogenized for 30 s in tenfold weight excess of ice-cold extraction buffer (20 mM Tris, pH 7.4, 800 mM NaCl, 1 mM ethylene glycol tetraacetic acid), 1 mM ethylenediaminetetraacetic acid, 10% sucrose, containing protease inhibitor (Roche Diagnostics) and phosphatase inhibitor cocktail (Sigma Aldrich). The homogenates were centrifugated at 20,000 g for 20 min at 2 °C. The supernatants designed 1 s were transferred into clean tubes. Next, solid sarkosyl (N-lauroylsarcosine sodium salt; Sigma-Aldrich) was added to the 1 s supernatant to achieve 1% concentration and then stirred for 1 h at room temperature (RT). Samples were then centrifuged at 100,000 g for 1.5 h at RT. Pellets (designated 2p) were gently rinsed with 1 ml of the sarkosyl extraction buffer and spun at 100,000 g for 20 min at RT. The pellets (sarkosyl-insoluble tau fractions) were resuspended in 6 M Guanidine for ELISA or in PBS for Western Blotting analysis to a final volume representing the 1/25 volume of the 1 s fraction followed by 5 min sonication and stored at − 20 °C. The sarkosyl-insoluble 2p tau fractions were also isolated from human AD brain tissue using the same procedure as described above. Human brain samples (transentorhinal cortex, NFTs rich Braak stage VI, AD sporadic and AD familial with a missense mutation of PSEN1 (Thr116Asn) ) were obtained from Newcastle Brain Bank and Slovak Brain Bank in accordance with ethical approval.
Biochemical quantification of sarkosyl-insoluble tau using sandwich ELISA
Sarkosyl-insoluble tau fractions (2p) were subjected for biochemical quantification using sandwich ELISA assays (AT8/DC190 for transgenic animal efficacy study; DC25/DC190 for neuronal internalization in vitro experiments). A 96-well ELISA plate (Nunc Medisorp, Denmark) was coated with 2 μg/ml AT8 antibody (efficacy studies) or DC25 antibody (AD tau neuronal internalization) in PBS overnight (16–18 h) at 4 °C. The plate was washed five times with PBS buffer supplemented with 0.075% Tween 20 (PBST) followed by blocking with PBST buffer for 1 h at RT. Guanidine hydrochloride samples of sarkosyl-insoluble 2p fractions were diluted 50-fold with PBST buffer (in duplicates). For standard curves, recombinant pathogenic tau 151–391/4R (in vitro phosphorylated, affinity purified) was used, in 2 fold dilution steps in PBST buffer. The plate with tau standard and sarkosyl-insoluble tau fractions was incubated 90 min at 37 °C, followed by washing with PBST for five times. As detection antibody DC190-HRP at 1: 15000 dilution in PBST (0,3 μg Ab/ml) was used and incubated for 60 min at 37 °C, followed by washing with PBST for five times. Next, substrate Colorburst Blue (TMB/peroxide substrate, ready to use; Alerchek USA) was added to the plate and incubated for 20 min in dark. The reaction was stopped by adding of 0.25 M H2SO4. The 450 nm absorbance was measured and plotted against the protein concentration of tau 151–391/4R standard. Concentrations of AT8-positive (efficacy study) or DC25-positive tau (neuronal internalization) in samples was obtained based on extrapolation from the standard calibration curve.
Expression, purification and fibrillization of recombinant tau protein
Truncated tau 297–391/4R, dGAE (t-tau; numbering according to the longest human tau isoform 2N4R) was expressed in Escherichia coli strain BL21(DE3) (Sigma-Aldrich, St. Louise, Missouri, United States) from a pET-17 expression vector and purified from bacterial lysates as described previously , except the anion-exchange chromatography step was omitted and size-exclusion chromatography was performed in PBS-argon (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4) (AppliChem GmbH, Darmstadt, Germany). Purified tau protein was stored in PBS-argon in working aliquots at − 70 °C. The purity of tau protein was subsequently verified by gradient SDS gel electrophoresis (5 to 20% gel), Coomassie blue staining and Western blot analysis with DC25 antibody (AXON Neuroscience SE, Bratislava, Slovakia), recognizing residues 347–354 of the longest human tau isoform 2N4R). In vitro fibrillisation of recombinant truncated tau protein (100 μM) was carried out using heparin (Sigma-Aldrich, St. Louis, Missouri, United States) as an inducer at a concentration 240 μM tau + 60 μM heparin in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4). The reaction was performed for 24 h at 37 °C. After incubation, tau aggregates were sonicated for 2 min at 20% power output using an MS72 probe of a Bandelin Sonopuls Sonifier (Bandelin, Berlin, Germany). Subsequently, 1 μM aliquots were stored at − 70 °C. The oligomerization of the tau protein was verified by non-reducing SDS-PAGE gel electrophoresis and quantitative thioflavin T (ThT) fluorescence spectroscopy with excitation at 450 nm and emission at 510 nm.
The fluorescently tagged tau protein (after fibrillization) was prepared by labelling with Alexa Fluor™ dyes (Invitrogen, Carlsbad, California, United States) according to the manufacturer’s recommendations. The labeling was carried out at pH 6.5 for preferential labeling of N-terminus. Succinimidyl esters of Alexa Fluor™ 488 and Alexa Fluor™ 594 (ThermoFisher) were dissolved in anhydrous DMSO (Molecular probes) and mixed with tau proteins (in PBS) in 5:1 M ratio. The mixture was incubated at RT for 1 h with 400 rpm shaking. The unreacted dye was subsequently separated from the labeled tau protein using Zeba 7 k MWCO desalting spin columns (Thermo Fisher Scientific) equilibrated with PBS. Labeled truncated tau (297–391/4R) and human brain-derived AD Tau (2p sarkosyl-insoluble fraction) were incubated at 37 °C with 700 rpm shaking for 2 days. The size distribution was monitored using DLS with DynaProNanoStar (Wyatt technologies). The sample was centrifuged for 5 min at 5000 g prior to DLS measurement.
Dynamic light scattering (DLS)
Tau protein samples (10 μl) were centrifuged at 5 000 g for 5 min at 25 °C, transferred into a 4 μl disposable cuvette (Wyatt Technology) and measured in a DynaproNanoStar instrument controlled by Dynamics software v. 188.8.131.52 (Wyatt Technology). Measurements were performed in 1 second acquisition time averaged 10-times except for truncated tau dGAE monomer, which was measured using 10 s acquisition time. Data from at least five individual measurements of dynamic light scattering (DLS) per sample were evaluated by the Dynamics software v. 184.108.40.206. To cull the acquisitions influenced by dust or irregular particles, an automatic filtering of autocorrelation functions was applied with an individual limit for baseline threshold and maximal allowed sum-of-squares (SOS) error for cumulants fit. After filtering, at least 65% of original data remained for analysis. To determine the size distribution of protein preparations, DLS autocorrelation data were subjected to a regularization analysis by Dynals algorithm. Final graphs were prepared in Prism 6 software (GraphPad).
Infrared spectra were collected on ThermoScientific Nicolet iS50R Research FTIR Spectrometer equipped with a DTGS detector (Thermo Fisher Scientific). The instrument and sampling accessory were continuously purged with water and CO2 free air. One μl of sample in PBS was loaded into a ConcentratIR2 Multiple Reflection ATR (Harrick Scientific Products) adopting a silicon element with a nominal incident angle of 30° and eleven reflections. The sampling plate was sealed and the sample drop dried under flow of dry air. After vanishing of liquid water absorption bands, the flow of dry air was stopped and 32 scans were collected at 4 cm− 1 resolution within a spectral range of 650–4000 cm− 1 wavenumbers. Spectra were collected using zero-filling factor 2, Happ-Genzel apodization, Mertz phase correction, aperture 160, samples gain 4, optical velocity 0.4747 cm.s− 1. Reference spectra were recorded under identical conditions with empty ATR sampling plate and were subtracted from the protein-sample spectra. Spectra were further baseline-corrected and processed with ATR advanced correction as implemented in OMNIC software v. 9 (Thermo Fisher Scientific).
Kinetics of truncated tau (297–391/4R) aggregation – Thioflavin T (ThioT) fluorescence spectrometry
Detection of tau filaments assembly-aggregation of recombinant tau protein was monitored by ThT fluorescence. 40 μl of 350 μM truncated-tau 297–391 (dGAE) with 20 μM ThioflavinT (Sigma) was incubated in black solid polystyrene 384 wells Greiner BioOne plate in the absence and presence of heparin (tau-heparin concentration ratio 4:1). The fluorescence was measured using the Fluoroskan Ascent FL (Labsystems) every 10 min with excitation at 450 nm and emission at 510 nm. The plate was shaken at 720 rpm and incubated at 37 °C. The aggregation kinetics was monitored for 4 days.
Immunoprecipitation of native, sarkosyl-insoluble AD tau with DC8E8 antibody
The sarkosyl resistant 2p tau fraction was isolated from 1 g of AD human brain sample using the procedure outlined above for mice brain tissue. The brain was examined by immunohistochemistry and tau pathology found to correspond to Braak stage VI. The 2p pellet fraction was re-suspended in 1 ml of PBS (supplemented with 50 mM NaF, 1 mM Na3VO4 and the cocktail of protease inhibitors Complete® without EDTA (Roche)) by sonication for 2 min on ice using a Bandelin Sonopuls HD2200/UW2200 equipped with a MS72 probe, at 20% duty cycle with the output set at 20% (Bandelin Electronic, Germany). The resulting suspension was split into two 500 μl portions and each portion received 25 μg of one of two purified antibodies: either DC8E8 or a control antibody DC51. The suspensions were incubated with the antibodies with head-over-tail rotation at 6 °C for 2 h. In order to isolate the antibody-disease tau complexes, 50 μl of the 50% suspension of Protein G Mag Sepharose beads (GE Healthcare), equilibrated in PBS+ 0.01% Igepal CA-630 (SIGMA), were added to each of the two mixtures of sarkosyl-insoluble AD tau and antibodies. The immunoprecipitation mixtures were further incubated at 6 °C for 1 h. The beads in each of the incubation mixtures with bound antibody-tau complexes were harvested using magnet and washed three times with 150 μl of PBS (supplemented with 50 mM NaF, 1 mM Na3VO4, 0.02% IGEPAL CA-630 (SIGMA) and the cocktail of protease inhibitors Complete® without EDTA (Roche)) and once with 150 μl of PBS only. The bound antibody complexes were eluted from the beads by three consecutive 5-min incubations in 100 μl of 200 mM formic acid pH 2.7. The eluates were pooled, lyophilized, the proteins dissolved in SDS-PAGE sample loading buffer, separated on 12% SDS-PAGE gels, transferred onto nitrocellulose membranes and the tau proteins detected by incubation with the pan tau antibody DC25 conjugated to HRP (Kementec, Denmark). The blots were developed with a SuperSignal West Pico Chemiluminescent Substrate system (Pierce, U.S.A) and the signals detected using a LAS3000 imaging system (FUJI Photo Film Co., Japan).
Cortico-hippocampal neurons, viability, viral transduction and cellular physiological functions
Rodent cortico-hippocampal neurons were prepared and cultured as described, with minor modifications . Neocortes were isolated from pregnant female mice C57BL6N on embryonic day 16–18 using 10% Ketamidor and 10% Xylariem as lethal anesthesia. Brains of embryos were dissected out, placed in ice-cold sterile L-15 medium free of L-glutamine (PAA) and meninges were removed. Cerebral cortices with hippocampi were isolaed, chopped into small pieces and incubated with 0.25% Trypsin-EDTA for 10–15 min at 37 °C. After incubation, trypsinisation was inhibited by adding media with inactivated serum. The cells were dissociated by gentle pipetting with subsequent centrifugation at 300 g for 5 min. Cortico-hippocampal neurons were triturated with glass Pasteur pippete in fresh plating media (DMEM supplemented with 10% (v/v) fetal calf serum, 2 mM L-glutamine and 100 units/mL penicillin/streptomycin (all from Life Technologies Invitrogen, Carlsbad, California, United States). Cells were plated onto Poly-D-lysine coated 6 well plates or glass Nunc Lab-Tek Chambers (Thermo Fisher Scientific) at a density of 2.105 cells/cm2 (unless indicated otherwise) and and cultivated at 37 °C, 5% CO2 in a water-saturated atmosphere. After 24 h plating media was exchanged for Neurobasal Media containing 2% B27, 2 mM L-glutamine and gentamycin 10 μg/mL. Experiments were carried out after 2 ± 5 days in vitro (DIV). Transfection of primary neurons with fluorescently labelled antibody DC8E8 Alexa Fluor 546 (30 μg) was performed with MaxCyte Flow Electroporation™ Technology according to the manufacters instructions and cells were examined for viability/nuclear morphology (Hoechst 33258 Sigma, 1 μg/ml; Trypan blue Sigma) and physiological functions (ENLITEN ATP Assay System Bioluminescence Detection kit; Promega) 24 h later. Alternatively, primary neurons 1–2 DIV were transduced with virus containing human truncated tau - AAV9-hSynapsin1-tau (151–391)-P2A-mCherry WPRE (Vector Biolabs). Two to three days after AAV viral transduction (9.1 × 1013 GC/ml) neurons were examined for t-tau (151–391/4R) protein expression with immunocytochemistry and western blotting. All procedures involving animal work were in accordance with ethical standards and approval from the State Veterinary and Food Committee of Slovak Republic and the number of sacrifised animals recorded.
Neuronal tau internalization assay
Truncated tau (297–391/4R) and human brain-derived AD tau (transentorhinal cortex from two sporadic AD patients and one familial AD case , both NFT rich Braak stage VI) fluorescently labelled with Alexa Fluor 488 or Alexa Fluor 594 were diluted in neuronal conditioned media at the concentration corresponding to 100–200 nM for monomeric tau protein in combination with the following antibodies: unrelated control antibody DC51 , mouse monoclonal DC8E8 (mDC8E8), humanized DC8E8 (AX004/IgG1; AX004/IgG4) and their isotype controls IgG1/IgG4 (BioLegend), all of concentration of 1 μM and incubated for 30 min at 37 °C. Pre-formed tau-antibody complexes were added to neurons for 24 h. For experiments where heparin was used (Tinzaparin sodium, Sigma-Aldrich, which is a LMWH), fluorescently labelled tau aggregates were preincubated with heparin (5 μM) for 30 min and heparin-AD tau complexes were applied to neurons for 16–20 h. Neurons were washed three times with pre-warmed PBS, followed by mild trypsinisation (0.06% trypsin-EDTA 2–3 min) to remove cell surface bound tau and dissociate neurons into single cells. Next, neurons were processed live for flow cytometry measurements to quantify the amount of fluorescently labelled internalized tau (BD LSRFortessa™ II cell analyzer). Neurons were gated using forward and side scatter to remove cellular debris. Measurements were recorded as mean fluorescent intensity of Alexa Fluor 488 or Alexa Fluor 594. Alternatively, neurons grown on glass Nunc Lab-Tek Chambers (Thermo Fisher Scientific) were imaged for fluorescence AF488 labelled tau protein (excitation at 488 nm and emission at 525 nm) with a 20× objective LSM 710 confocal microscopy and examined for intracellular localisation by fluorescent labelling of lysosomes with fluorescent dye LysoTracker (75 nM, 45 min Thermo Fisher Scientific). Typically, experiments were performed in triplicates from minimum of three independent experiments and 10 000 cells for each well were quantified by flow cytometry. Data were analysed using Prism Software (GraphPad).
Rodent cortico-hippocampal neurons were cultured on cover glass pre-coated with Poly-D-lysine at a density of 1 × 106 cells per milliliter and cultivated for 48 h. Neurons were treated with fluorescently labelled (Alexa Fluor 488 or Fluor 594) sarkosyl-insoluble AD tau (2p, 100 nM) only or in combination with control and DC8E8/AX004 antibody (1 μM) and cultivated for 20 h in conditioned Neurobasal media at 37 °C, 5% CO2. Next day neurons were washed with pre-warmed PBS and mild trypsin (0,06% trypsin-EDTA, 3 min), fixed with 4% PFA-PHEM, pH 6.9 (60 mM PIPES, 25 mM HEPES, 10 mM EGTA, 2 mM MgCl2, PFA) for 12 min. Neurons were permeabilized with 0.1% Triton X100 in PBS (PBS-T) and blocked with 5% BSA in TBS-T. The cells were then incubated with anti-heparan sulfate antibody (10E4, AMSBIO 1:100) for 1 h or, in a different experiment with HT7 antibody (epitope against human tau, MN1000, Thermo Fisher Scientific 1:500), washed and incubated with secondary antibody goat anti-mouse Alexa Fluor 488 (Invitrogen). The samples were mounted in FluoroshieldTM medium with DAPI (Sigma-Aldrich). Images were captured by LSM 710 confocal microscope (Zeiss, Jena, Germany).
Western blot analysis of fibrillized and sarkosyl-insoluble tau
Samples of fibrillized truncated tau 297–391/4R (1 μg) and human-derived AD tau fractions (6 μl) from sarkosyl isolations (2p, 1 s and 2 s) were mixed with SDS sample loading buffer and heated at 95 °C for 5 min. Each sample (6 μl) was then loaded onto 5–20% gradient SDS polyacrylamide gels and electrophoresed in a Tris-glycine-SDS buffer system for 40 min at 25 mA. Proteins were transferred to nitrocellulose membrane and, after blocking in 5% fat-free dry milk in PBS for 1 h at room temperature, the membrane was incubated for 1 h with pan-tau mAb DC25 and with therapeutic antibody DC8E8. After washes, HRP-conjugated goat anti-mouse Ig (Dako Denmark) diluted 1:3,000 in PBS was used as a secondary antibody. Blots were washed and developed using ECL chemiluminescence detection (GE Healthcare), detected with SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology), and imaged using a FujiFilm LAS-3000 imaging system (Fuji).
Data are presented as means ± SEM. To compare two groups, the Mann–Whitney U test or an unpaired t-test was applied. For statistical comparison of more groups, one-way analysis of variance ANOVA and post hoc Tukey’s test were used using Prism software v. 7 (GraphPad Software, Inc., San Diego, USA). Differences were considered significant at the level of p < 0.05.