Ethics statement

All animals were cared for and procedures performed in accordance with the Institutional Guidelines for the care and use of laboratory animals in Kyoto University and National Institute of Radiological Science (NIRS), and all experiments were approved by the Animal Experiment Committees of Kyoto University and NIRS.

SeV vector

SeV vector deleted fusion gene (SeV/ΔF) to eliminate transmission was obtained from ID Pharma (Tsukuba, Japan). SeV vector carrying the cDNA of human tau isoform with one N-terminal 1N4R with the P301S mutation fused to a signal sequence for directing gene products to a secretory pathway was constructed. The signal sequence was obtained from the cDNA of the N-terminal in APP. SeV vector expressing EGFP (SeV-EGFP) for use as a gene-expressing control was also constructed to check the gene expression induced by SeV vector in nasal mucosa. The nasal cavity was removed one week after the infection of SeV-EGFP, fixed in 4% formaldehyde at room temperature for 2 h, decalcified by 0.24 M EDTA solution at room temperature for 5 days, and transferred into 30% sucrose overnight. The tissue was then embedded in Tissue Mount (Chiba Medical, Chiba, Japan), sectioned at 10-μm thickness by a microtome, and observed by fluorescence microscopy.

To confirm the secreted tau protein expression, we transfected SeV vector carrying human P301S (1N4R) tau protein gene with a secretion signal to HEK293T cells. Transfected tau gene expression was confirmed by immunoprecipitation.

Immunoblotting

Tau protein levels were determined by homogenizing brains in 2 ml/g tissue of ice-cold high-salt reassembly buffer (RAB-HS) (0.1 M MES, 1 mM EGTA, 0.5 mM MgSO 4 , 0.75 M NaCl, 0.02 M NaF, 1 mM PMSF) and protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland), followed by centrifugation at 50,000×g for 40 min at 4 °C in a Beckman TLA-55 ultracentrifuge. Protein concentrations were determined, and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) followed by western blot analysis was performed as described52,53. The following primary antibodies were used: phosphorylated tau (AT8) (1:1,000, #. MN1020, Thermo Scientific, Waltham, MA) and β-actin (1:5,000, #. 5441, Sigma). Uncropped images of the original blots were supplied as a supplementary figure (Supplementary Fig. 5).

Dot blot analysis

HEK293T cells were infected with SeV vector carrying control-v or tau-v. After 24 h, the medium was changed to serum-free medium containing DMEM/F12 (Life Technologies) and Neurobasal (Life Technologies) mixed at a 1:1 ratio, 1% N 2 supplement (Life Technologies), 2% B27 (Life Technologies), and the cells were cultured for an additional 48 h. The cells were then harvested and lysed in TBS containing protease inhibitor and phosphatase inhibitor. After sonication and centrifugation at 13,000 × g for 15 min, each of the lysate samples (1.2 µg/spot) was loaded on a nitrocellulose membrane (0.45 µm pore size, GE Healthcare, Chicago, IL). The cell culture medium was collected and centrifuged at 200 × g for 3 min to remove debris. The same amount of supernatant from each sample was concentrated from 500 to 50 µl using Vivaspin (GE Healthcare), and its molecular weight cut-off for the filtration membrane was 10 kDa. 2 µl of each concentrated sample was loaded onto nitrocellulose membranes. Membranes were blocked with 5% skim milk, hybridized with the appropriate antibodies, and visualized using Western Lightning Plus-ECL (PerkinElmer, Inc.). Images were acquired on ImageQuant LAS 4000 (GE Healthcare). The following antibodies were used: mouse monoclonal antibody against misfolded tau (TOC1, gifted by Dr. Binder) and mouse monoclonal antibody against human tau (Tau12, #. MAB2241, Millipore, Burlington, MA).

Animals

We previously established transgenic mice expressing mutant (P301S) human T34 isoform tau (1N4R) on a B6C3H/F1(C3H) background30, and we used a congenic strain created by 10× backcrossing the transgenic mice and wild-type offspring.

SeV vaccine administration

SeV vectors (5 × 107 cell-infectious units/head) were administered weekly to each 3-month-old mouse intranasally in a 20-µl volume with PBS.

Tissue-tau immunoreactive antibody assay

P301S brain sections fixed in 4% paraformaldehyde were permeabilized in PBS containing 0.2% Triton X-100 for 10 min at room temperature, followed by blocking for 30 min with 2% BSA and 10% horse serum. The serum of mice 6 months after vaccination was diluted 30×, 100×, 300×, 1,000×, and 3,000×, and then applied to P301S brain sections. After incubation with the diluted serum overnight at 4 °C, the brain sections were washed three times with PBS-T and incubated with anti-mouse IgG secondary antibodies for 1 h at room temperature, followed by incubation with streptavidin–biotin-peroxidase. The maximal dilution of plasma that gave positive staining was estimated as the tangle immunoreactivity titer54.

Measurement of anti-tau antibody titer

Anti-tau antibody titer in brain lysates and serum was evaluated by ELISA assay. The brain samples and serum of FTLD-tau mice were collected 6 months after vaccination. Frozen brain samples were dissolved in TBS buffer containing protease inhibitor (Roche) by homogenization and sonication. Samples were centrifuged at 13,000 × g for 15 min at 4 °C, and protein concentrations in the supernatants were determined with bicinchoninic acid (BCA) assay kit (Thermo Fisher Scientific). Total protein extracts of brain lysates were diluted to 500 μg/100 μl. Serum samples were diluted to 1:50. 96-well plates (Greiner) were coated with 1 μg/ml P301S tau recombinant protein in 0.05 M sodium carbonate buffer (pH 9.4) at 4 °C overnight. After washing and blocking with 1% BSA–PBS-T for 1 h, 100 μl of brain lysates or serum was applied to the plates and incubated for 2 h at room temperature. For detection, the plates were incubated with sheep anti-mouse IgG F(ab)’2 fragment linked to a horseradish peroxidase (GE Healthcare) at 1:5,000 dilution for 1 h at room temperature, followed by incubation with tetramethyl benzidine solution (BD) for 15 min. The absorbance at 450 nm was measured by VersaMax (Molecular Device, Sunnyvale, CA).

Volumetric MRI assays

MRI studies of both control and vaccinated mice were conducted at 24 weeks after the initiation of the vaccine treatment using a 7.0-T MRI scanner (magnet, Kobelco and JASTEC, Kobe and Tokyo Japan; console, Bruker Biospin, Ettlingen, Germany) with a volume coil for transmission (Bruker) and a two-channel phased-array coil for reception (Rapid Biomedical, Wuerzburg, Germany). Rectal temperature was continuously monitored by an optical fiber thermometer (FOT-M, FISO, Canada) and maintained at 36.5 ± 0.5 °C using a heating pad (Rapid Biomedical) and warm air. The first imaging slices were carefully set at the rhinal fissure with reference to the mouse brain atlas. Signal excitation and detection were performed by a 25-mm resonator. T2-weighted 3D spin-echo rapid acquisition with relaxation enhancement (RARE) MRI scans were performed. The imaging parameters were as follows: TR/effective TE = 4200/36 ms, Fat-Sup = on, NA = 4, RARE factor = 8, number of slices = 13, and scan time = 6 min 43 s. Frequency selective saturation pulses and crusher magnetic field gradients were used for fat suppression. An anatomical volume of interest (VOI) was manually defined on the hippocampus of the MRI images using PMOD® software (ver 3.6, PMOD Technologies Ltd, Zurich, Switzerland), and VOIs were measured.

PET imaging

PET scans were performed using a microPET Focus 220 animal scanner (Siemens Medical Solutions, Erlangen, Germany)55. Control and vaccinated mice were anesthetized with 1.5% (v/v) isoflurane, and a 30-G needle connected to a 1-ml polypropylene syringe via a length of polyethylene tubing was inserted into the tail vein. After transmission scans for attenuation correction using a 68Ge–68Ga point source, emission scans were acquired for 60 min in 3D list mode with an energy window of 350–750 keV, and the intravenous injection of [11C]Ac5216 (13.26 ± 14.27 MBq) or [11C]PBB3 (30.28 ± 22.34 MBq) was performed immediately. Summation images from 0 to 60 min after [11C]Ac5216 or [11C]PBB3 injection were reconstructed with maximum a posteriori reconstruction, and dynamic images were reconstructed with filtered back-projections using a 0.5 mm Hanning filter. To analyze the [11C]Ac5216-PET data, VOIs were placed on the hippocampus and striatum using PMOD image analysis software (PMOD Group) with reference to the MRI template at 0 and 3 weeks after initiation of the treatment and to individual MRI data at 24 weeks. Tracer uptake in each VOI was estimated as the percentage of injected dose per tissue volume (%ID/ml). To analyze the [11C]PBB3-PET data, VOIs were defined on the brain stem and cerebellum with reference to the MRI template at 24 weeks.

Immunohistochemistry

Mice were deeply anesthetized and transcardially perfused with 15 ml phosphate-buffered saline (PBS). The brains were removed, immersion-fixed for 24 h with 4% paraformaldehyde in PBS, and then transferred to 20% sucrose solution at 4 °C for at least 4 days. After cryoprotection, the brains were rapidly frozen by heat exchange from vaporized CO 2 gas (−70 °C), and then sections (12 μm) were cut with a cryostat. The brains were immunostained using streptavidin–biotin-peroxidase52,53. The following antibodies were used: mouse monoclonal antibody against phosphorylated tau (AT8, 1:1,000, #. MN1020, Thermo Fisher Scientific), rabbit polyclonal antibody against Iba1 (1:1,000, #. 019-19741, WAKO, Osaka, Japan), goat polyclonal antibody specific for TSPO (1:1,000, #. sc-30920, Santa Cruz, Santa Cruz, CA), rabbit polyclonal antibody specific for GFAP (1:1,000, #. Z033401, DAKO, Santa Clara, CA), and hamster polyclonal antibody specific for CD3e (1:1,000, #. 553058, BD Biosciences, Franklin Lakes, NJ).

ELISA

Hippocampal human phosphorylated tau protein was evaluated by ELISA assay using anti-phosphorylated antibody (AT8, #. MN1020, Thermo Fisher Scientific). Microtiter plates were coated with 3 µg/ml AT8 antibody in 0.05 M sodium carbonate buffer (pH 9.4) at 4 °C overnight. After washing and blocking with TBS-T containing 1% BSA, 100 µl of diluted mouse hippocampus RAB-HS soluble fractions (the protein concentration of each sample was adjusted) was added, and the incubation was carried out for 2 h at room temperature. Serial dilutions of collected FTLD-tau mouse brain cortical lysates ranging from 1:100 to 1:102,400 were used as positive control. For detection, the plates were incubated with 2 μg/ml rabbit anti-human tau protein antibody (#. A0024, DAKO), followed by sheep anti-rabbit IgG F(ab)′2 fragment linked to horseradish peroxidase (GE Healthcare) at 1:3000 dilution. After incubation with tetramethylbenzidine solution (BD Bioscience) at room temperature for 30 min, absorbance at 450 nm was read on an automated plate reader (Model 353; Thermo Fisher Scientific). The amount of phosphorylated tau protein of positive control was defined as 1000 units, and unknown titers of samples were determined by interpolation from a standard curve generated by positive control standards of known dilution. All samples were analyzed in duplicate.

Behavioral analysis

For the behavioral analysis, mice were housed 3 or 4 per cage in a room with a 12-h light/dark cycle (lights on at 7:00 a.m.) with access to food and water ad libitum. Behavioral testing was performed between 9:00 a.m. and 6:00 p.m. After the tests, the equipment was cleaned with super hypochlorous water to prevent any bias due to olfactory cues. All behavioral testing procedures were approved by the Animal Care and Use Committee of Kyoto University Graduate School of Medicine.

The neuromuscular strength test was performed with 28-week-old male mice, using both the grip strength test and wire hang test. A grip strength meter (O’Hara & Co., Tokyo, Japan) was used to assess forelimb grip strength. Mice were lifted and held by their tail so that their forepaws could grasp a wire grid. The mice were then gently pulled backward by the tail with their posture parallel to the surface of the table until they released the grid. The peak force applied by the forelimbs was recorded in Newtons (N). Each mouse was tested three times, and the greatest value was used for statistical analysis. In the wire hang test, the mouse was placed on a wire mesh that was then inverted and waved gently, so that the mouse gripped the wire. Latency to fall (in seconds: s) was recorded, with a 60-s cut-off time. Our 28-week-old FTLD-tau mice showed significantly lower body weight, and tended to show lower body temperature, lower grip strength, and longer duration in the wire hang test.

The other tests were performed as previously described34. In the elevated plus-maze test in a novel environment (one-chamber social interaction test), a genotypic mismatch was found in two pairs (one FTLD-tau mouse/control-v pair and one FTLD-tau mouse/tau-v pair), and they could not be analyzed.

The applications used for the behavioral studies (Behavioral Image EP, Behavioral Image SI, Behavioral Image BM, and Image FZ) were based on the public domain NIH Image program (developed at the U.S. National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image/ website) and ImageJ program (http://rsb.info.nih.gov/ij/ website).

Statistical analysis

Statistical analysis was conducted using StatView (SAS Institute, Cary, NC), Kyplot (Kyens Lab, Tokyo, Japan), and SPSS version 21.0 (IBM SPSS Statics, Chicago, IL). Data were analyzed by Student’s t-test, Mann–Whitney’s U-test, paired t-test, and for multiple comparisons, one-way ANOVA, repeated measures ANOVA, or Kruskal–Wallis test, with successive post hoc analyses. Statistical significance was defined as p-value < 0.05. Values in graphs are expressed as means ± SEM.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.