Animal models
Studies were performed in C57BL/6J-wild type, Tau-P301L-transgenic, and Tau-P301S-transgenic mice. Tau-P301L mice were generated from JNPL3 (Tau-0N4R, P301L) mice purchased from Taconic, Inc. (NY, USA) and backcrossed to C57BL/6J over five generations. Tau-P301S mice were generated from PS19 (Tau-1N4R, P301S) mice purchased from Jackson Laboratories, Inc. (Bar Harbor, ME, USA) and backcrossed to C57BL/6J over five generations. All mouse genotyping was confirmed by PCR. The mice were caged (3 to 5 mice per cage) and kept on a 12h light/dark cycle at a constant ambient temperature (22±1 °C) with 40-60% humidity. Food and water were available ad libitum. All animal protocols were approved by Asan Institute for Life Science Animal Experimentation Committee.
Peptides
The peptide immunogen Tau 226-236 (phosphorylated tau threonine 231), Tau 275-286 (acetylated tau lysine 280), Tau 303-316 (acetylated tau lysine 311), and Tau 381-391 (cleaved tau glutamic acid 391) were synthesized at Anygen (Nam-myun, KOREA). N-terminal was conjugated with Keyhole Limpet Hemocyanin (KLH). The peptide was purified by Shimadzu HPLC 10AVP system (Purity 91.8%) with a 5-65% linear gradient of acetonitrile in 0.05% Trifluoroacetic acid (TFA).
Generation of hybridoma cells
Mouse hybridoma cells were manufactured by Young-In Frontier Co., Ltd. (Seoul, Korea). Briefly, the antigen was mixed with Complete Freund adjuvant (Sigma-Aldrich, St. Louis, MO, USA) and intraperitoneally injected into female BALB/c mice. After 2 weeks, serum was collected from mice and antibody titer was measured using ELISA. The antigen was mixed with PBS and injected into mice with a second injection for boosting. After 3 days, the spleen was extracted. After washing the tissue with culture medium, cells were separated. Myeloma cells (Sp2/0Ag14) were fused with cells separated from mice using polyethylene glycol (PEG) (Roche Life Science, Mannheim, Germany). Fused cells were cultured using 1XHAT culture medium (Sigma-Aldrich, St. Louis, MO, USA). Using a HT culture medium (Gibco Life Technologies, NY, USA), cells were pipetted into a 96-well plate. Hybridoma cells were cultured for 7-10 days. For positive clone screening, hybridoma supernatants were screened by ELISA for reactivity with K280-ac peptides. ELISA screens and the cloning process were repeated until the final clone was verified. The best clone was selected and termed ADEL-Y01 antibody.
Preparation of ADEL-Y01 antibody
Hybridoma cells were grown at 37°C in CD Hybridoma Medium (Gibco Life Technologies, NY, USA) supplemented with 8% glutamax at 1:25 dilution, 2% ultra-low IgG fetal bovine serum (FBS), and 1% penicillin streptomycin (Gibco). After 1 week, supernatant was collected and centrifuged at 13,000 rpm for 15 min at 4°C. Supernatant was filtered with 0.4 mm filter unit and 1 M Tris-pH 9.0 was added for neutralization. All experiments were performed on AKTA-start chromatography system (GE Healthcare Life Sciences, Piscataway, NJ, USA). The equipment was washed with distilled water, and the column was connected. The column was equilibrated with 1X Phosphate-Buffered Saline (PBS), and the sample was allowed to flow. Once the sample was exhausted, a wash with 1XPBS was performed, and the antibody was eluted using 0.1 M glycine (pH 2.7). The purified antibody was neutralized with 1 M Tris pH 9.0. The antibody was dialyzed with 1XPBS. The band was checked using Coomassie blue staining. The Y01 antibody was stored at -20°C.
Active immunization
At 3 months of age, male transgenic P301L mice and C57BL/6 mice were immunized with either peptide or Adju-Phos adjuvant (InvivoGen, San Diego, USA) only. Mice were intraperitoneally injected with 50 mg peptide mixed 1:1 (v/v) with Adju-Phos adjuvant (25 mg per mouse). After the first immunization, treatment was administered at intervals of 2 weeks. Injections were performed once a month. After the last injection, mice were subjected to behavioral tests and diffusion tensor imaging (DTI). All mice were sacrificed at 8 months.
Passive immunization
Intracerebroventricular infusion
At 8 months of age, C57BL/6 and P301L mice were administered intracerebroventricular (icv) injections of immunoglobulin G (IgG) control or Y01 antibody (1.9 mg/mL). The pump implantation surgery was performed according to the manufacturer’s instructions. Briefly, before the surgery, an L-shaped infusion cannula was attached to catheter tubing (Alzet, California, USA). A brain infusion kit was attached to a micro-osmotic pump (Alzet, California, USA). The assembly pump was implanted using a stereotactic apparatus (Harvard Apparatus, Massachusetts, USA) into the right lateral ventricle at 0.58 mm posterior to bregma, 1 mm lateral to the midline, and 2 mm from the skull surface. An osmotic pump was subcutaneously implanted into the back of each mouse. Each pump was filled with IgG control or Y01 antibody (1.9 mg/mL). The osmotic pump delivered antibodies continuously at 0.11 μL/h for 28 days. Reservoir volume was 100 μL. Behavioral analysis was performed during the last 3 weeks of infusion. At 9 months of age, all mice were sacrificed.
Intraperitoneal injection
At 7 months of age, C57BL/6 and P301L mice were injected intraperitoneally (IP) with IgG control or Y01 antibody (50 mg/kg). Mice were subjected to behavioral tests. At 10 months of age, all mice were sacrificed. At 14 months of age, C57BL/6 and P301L mice were injected IP with IgG control or Y01 antibody (50 mg/kg). The mice were subjected to behavioral testing. At 17 months of age, all mice were sacrificed. At 5 months of age, C57BL/6 and P301S mice were injected IP with two different doses of Y01 antibody (5 and 50 mg/kg) or IgG control (50 mg/kg). At 8 months of age, all mice were sacrificed. All mice received injections weekly for 3 months. Behavioral analysis was performed during the last 4 weeks of injection.
Behavioral tests
Nest-building
Mice were isolated in home cages a day before testing. Cotton was placed in each cage for the scoring of nesting behavior. The next day, nesting behavior was scored according to the following guidelines: score 1, cotton rarely touching and >90% intact cotton; score 2, cotton partially ripped with 50-90% intact; score 3, cotton ripped with <50% intact and spread around the cage; score 4, cotton mostly ripped with <10% intact and gathered into side of the cage; score 5, cotton almost ripped and perfect nest shape.
Grip strength test
To measure strength, a weights test was performed. Animals were held by the base of the tail and lowered to the apparatus comprising a coiled wire ball for gripping. Apparatus weight was less than 3g. Time taken to drop the apparatus was measured. All tests were repeated three times.
Vertical grid test
All groups were subjected to a vertical test. Mice were placed on a wire net (horizontal, 18 cm; vertical, 30 cm). The wire net was turned over, and the time taken for mice to fall was measured. All tests were repeated three times.
Y-maze
Mice were subjected to a Y-maze test to assess short-term spatial memory. The apparatus was a Y-shaped plastic maze with three arms. Mice were habituated for 5 min. One arm (Y-shaped left arm) was open and the other arm (Y-shaped right arm) was blocked during a 5-min test. After 1 h, the other arm (Y-shaped right arm) was unblocked and behavior was analyzed for 5 min. Time in novel arm is the time to stay on the opened Y-shaped right arm.
Rotarod
Mice were placed on an accelerating rotarod, and exercise capacity was measured. The speed was slowly increased from 4 to 40 rpm. The time (sec) and speed (rpm) at which mice fell were measured. Mice were trained for 4 days before the test. On the fifth day, motor test was performed. All tests were repeated 3 times.
Water maze
Mice were subjected to a water maze(Vorhees, 2006). The water pool was divided into four quadrants, NE, North East; SE, South East; SW, South West; NW, North West quadrants. Briefly, all mice were trained to locate a visible platform for 4 days. Training was conducted three times a day and start positions were selected as NE, SE and SW quadrants. On the 5day, the platform was removed, and movement was analyzed, such as time taken to reach the target.
Contextual fear conditioning
Mice were placed in a context in which they received a foot shock (Hirotaka Shoji1, 2014). On day 1, all mice were habituated to the chamber for 2 min. The sound was presented for 30 seconds paired with a shock up to the last 2 s. This process was repeated three times. On day 2, after sufficient rest, mice were habituated to the context without any stimulation for 3 min. Then, the sound was presented without shock for 3 min, and behavior was analyzed.
Diffusion tensor imaging
Diffusion tensor imaging was performed in mouse after acquiring T2 weighted (T2w). All mice were anesthetized with 1-1.5% isoflurane in a mixture of oxygen and room air gases (1:2) delivered via a nose cone. Respiratory rate, electrocardiogram, and rectal temperature were monitored. Parameters and data processing were performed as previously described (H-J Kim, 2017). All mouse using a 4 shot spin-echo based echo planar imaging (EPI) sequence with TR/TE = 3750/46.22 ms, 96 × 96 matrix and an encoding scheme of 30 gradient directions at b-value = 1000 s/mm2 (H-J Kim, 2017). The hippocampus was selected as the ROI. Data were analyzed by fractional anisotropy (FA) and mean diffusivity (MD) values.
Blood sampling
Blood was collected from the facial vein 1 week before the start of immunization (T0), 1 week after the third injection (T1), 1 week after the fourth injection (T2), and at sacrifice (TF). Blood plasma was harvested by centrifugation, and antibody titer was analyzed.
Enzyme-linked immunosorbent assay
For measuring plasma titers of antibodies, indirect ELISA was performed. Briefly, 96-half well plates (Costar, Corning Life Sciences, Massachusetts, USA) were coated with either 250 ng/well immunizing peptides diluted in capture solution overnight at 4°C. After blocking for 1 h, plasma samples were diluted 1:500 in blocking solution and incubated for 2 h. After 2 h of incubation at room temperature, horseradish peroxidase (HRP)-conjugated secondary antibody was added to each well for 2 h. All steps were followed by three washes with wash solution. Then, samples were processed with 3,3′,5,5′-tetramethylbenzidine (TMB) and a microtiter plate reader MAX190 (Molecular Devices, California, USA) calibrated to 450 nm.
Western blot
Mouse tissues were homogenized in Pro-prep, a protein extraction solution (Intron Biotechnology, Seongnam, Korea), according to the manufacturer’s instructions. The suspension was centrifuged, and the supernatant was collected. Concentrations were measured using the Bradford Assay. Proteins were mixed with 4X sample buffer (60 mM Tris-HCl [pH 6.8], 2% [w/v] sodium dodecyl sulfate [SDS], 25% [v/v] glycerol, 14.4 mM [v/v] b-mercaptoethanol, and bromophenol blue). For semi-denaturation conditions, mouse tissue or cell lysate samples were mixed with 2X laemmli sample buffer without -mercaptoethanol. Protein was resolved by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidenedifluoride membranes (BioRad, California, USA). After 1 h incubation in blocking buffer, blots were incubated with primary antibodies overnight at 4°C. Membranes were washed in washing buffer and incubated with horseradish peroxidase-conjugated anti-IgG (Vector Laboratories, California, USA). Membranes were visualized using enhanced chemiluminescence reagents (Thermo Fisher Scientific, Rockford, IL, USA) and x-ray film. Band intensities were measured and analyzed with ImageJ software (NIH, Bethesda, MD).
RAB-RIPA-formic acid extraction
A three step extraction protocol was performed as described previously(Kaoru Yamada and Holtzman, 2015). Briefly, cortex was homogenized with RAB buffer (0.1 M MES, 1 mM EDTA, 0.5 mM MgSO4, 750 mM NaCl, 20 mM NaF, 1 mM Na3VO4, protease/phosphatase inhibitors) and centrifuged at 50,000 ´ g for 20 min. The RAB insoluble pellet was solubilized with RIPA buffer (0.15 M NaCl, 50 mM Tris, 0.5% deoxycholic acid, 1% Triton X-100, 0.5% SDS, 25 mM EDTA, pH 8.0, protease/phosphatase inhibitor) and centrifuged at 50,000 ´ g for 20 min. The RIPA insoluble pellet was solubilized with 70% formic acid.
Immunoprecipitation
Brain lysates of immunized mice were incubated with 100 mL of protein G-agarose bead (GE Healthcare Life Sciences, Piscataway, NJ, USA) at 4°C overnight. The samples were collected by centrifuging at 6,000 rpm for 1 min at 4°C and washed three times with 0.1% Triton X-100 in PBS. The samples were boiled in SDS sample buffer and processed by western blot.
Immunohistochemistry
Frozen mouse brain blocks were sectioned and attached on coated glass slides. Sectioned brain tissues were washed with 1XPBS and tissues were permeabilized with 1XPBS containing 0.1% Triton X-100 for 10 min at RT. The tissues were washed and blocked with PBS containing 3% bovine serum albumin for 1 h at RT. The tissues were then washed with 1XPBS and incubated with primary antibody at 4°C overnight. After washing with 1XPBS, for DAB staining, a biotinylated secondary antibody (Vector Laboratories, California, USA) was added for 1 h at RT. Tissues were washed and incubated in an avidin-biotin-peroxidase complex (Vector Laboratories, California, USA). For visualization, tissues were incubated for 10 min in diaminobenzidine (DAB) solution. Tissues were washed and mounted with Canada balsam (Sigma-Aldrich, St. Louis, MO, USA). DAB-reacted images were obtained using a Zeiss microscope (Carl Zeiss, Oberkochen, Germany) and processed using the AxioVision Imaging System.
Antibodies
In this experiment, following antibodies were used: Y01 antibody (ADEL, Inc.), anti-Tau5 (Invitrogen, AHB0042), anti-AT8 (Thermo, MN1020), anti-pSer396 (Thermo, 44-752G), anti-acetyl-K280 (Anaspec, AS-56077), anti-pT231 (Thermo, MN1040), anti-PSD95 (Abcam, ab2723), anti-NMDAR (Chemicon, AB1548), anti-synapsin-1 (Chemicon, MAB355), anti-synaptophysin (Sigma, S5768), anti-Control IgG (Bioxcell, BE0083), and anti-β-actin (Sigma, A5441).
Statistical analysis
All data were analyzed with GraphPad Prism v.5. Data were analyzed using one-way ANOVA (Tukey’s post hoc test) or Student's t-test. A p-value < 0.05 was considered statistically significant.