Antibodies
MC1 and Alz50 (provided by Peter Davies, Albert Einstein College) are conformation specific mouse monoclonal antibodies that recognize amino acids 7–9 and 313–322 (MC1) [10] or amino acids 5–15 and 312–322 (Alz50) [11] of tau and are specific for pathological tau. AT8 (Thermo Scientific, Rockford, IL) is a phosphorylation-dependent mouse monoclonal antibody that recognizes PHF-tau phosphorylated on dual sites Ser202 and Thr205. Other antibodies used in this study that recognize phosphorylated epitopes of tau include AT180 (pThr231; Thermo Scientific), AT270 (pThr181; Innogenetics), and PHF1 (pSer396/pSer404; provided by Peter Davies) [12]. Antibody TOC1 (tau oligomeric complex 1), which selectively labels tau dimers and oligomers, but does not label filaments [13] was kindly provided by Lester Binder (Michigan State University). Rabbit polyclonal 17025 is a pan-tau antibody recognizing total mouse and human tau raised against full length recombinant tau [14]. SMI31 (Covance, Princeton, N.J.) is a mouse monoclonal antibody that reacts with phosphorylated neurofilament H. The anti-actin mouse monoclonal antibody was obtained from the developmental studies hybridoma bank (dshb.biology.uiowa.edu).
Construction of Transgenic mice
[B6.Cg-Tg(Thy1-MAPT*)2652Gds]. The cDNA encoding the most abundant brain isoform (1N4R) of tau was cloned into the unique XHO I site in a mouse neuron specific expression vector, pThy1.2 [15]. Transgenic (Tg) mice were generated by pronuclear microinjection of the Thy1.2::Tau (1N4R) transgene at the University of Washington Nathan Shock Center Transgenic Animal Model Core (Warren Ladiges, PI). Founders were identified by PCR analysis of tail biopsies as described below. Founder mice were intercrossed with C57BL/6J mice to establish lines. The 2652 line was the focus of characterization due to its high level tau expression and robust phenotype. Mice from the Tau4RTg2652 line used in these studies were backcrossed 6–9 generations (incipient congenic) with the C57BL/6J strain. This mouse strain has been deposited with the Mutant Mouse Regional Resource Centers (MMRRC) and is available under the stock number MMRRC:036717 and the strain name of B6.Cg-Tg(Thy1-MAPT*)2652Gds.
Genotyping
Mice were genotyped using DNA prepared from tail clips from live mice. The presence of the Thy1.2::Tau (1N4R) transgene was detected by PCR analysis using primers yielding a 316 bp product for hemizygous Tg mice and no product for WT animals: cDNA trunc-F, AAGATCGGCTCCACTGAGAA; cDNA trunc-R, GGACGTGGGTGATATTGTCC.
Copy Number analysis
A custom TaqMan® copy number assay was designed by Applied Biosystems® (Assay #MAPT_CCRR9BJ). Primers are within exon 9 of MAPT: Forward primer sequence ACCCAAGTCGCCGTCTTC; reverse primer sequence CCGATCTTGGACTTGACATTCTTCA; Reporter sequence: CAGACAGCCCCCGTGCCCA. The TaqMan® Copy Number Reference Assay, Mouse Tert (part # 4458368) was used for an endogenous control. DNA was extracted from mouse tail samples using DNeasy ™ Blood and Tissue Kit (Qiagen). DNA was quantitated using the Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific), and DNA samples were diluted in water to a concentration of 1 ng/μL (to provide 4ng of DNA/reaction). This is less than the manufacturer’s recommended 5 ng/μ L, because of the exceptionally high copy number of the target DNA. TaqMan® Universal PCR Master Mix (Applied Biosystems) was used, in a 96 well plate format, according to the supplier’s protocol for TaqMan® Copy Number Assays; with the exception that primers for the reference assay (Tert) were not placed in the same reaction well as those for Exon 9, the target sequence. Primers failed to amplify Tert when duplexed in the same reaction with primers for the target, likely due to the overabundance of the target sequence relative to the reference sequence. Samples were run in quadruplicate. DNA from a single mouse from a different Tg mouse line [B6.Cg-Mapttm1HndTg(MAPT*)1Gds] was used as a calibrator sample for all assay plates. This mouse line has a low copy number of the human MAPT transgene. Reactions were run on a 7900HT Fast Real-Time PCR System instrument, using the amplification program: 50 °C for 2 min, 95 °C for 10 min, 95 °C for 15 s and 60 °C for 60 s (the last 2 steps repeated 40 times). Generation of standard curves showed that both Tert and MAPT PCR reactions approached 100 % efficiency and were within the linear range using 4 ng of DNA from Tau4RTg2652 mice. Results were analyzed using a CT threshold of 0.2. Because reference and target amplification reactions were performed in different wells, the comparative CT method (∆∆CT) of analysis was used to calculate copy number. In order to obtain an estimation of the absolute copy number of the MAPT cDNA transgene, the ∆ CT between Tert and MAPT reactions was calculated.
Behavioral analysis
All mice were bred and housed at the University of Pennsylvania (Penn) animal facility, and all experiments were approved by the IACUC committee. Mice were housed on a 12:12 light cycle in static microisolators cages, with rodent chow and water available ad libitum. Nestlets (Animal Specialties and Provisions, Quakertown, PA) were added to cages for environmental enrichment in accordance with Penn IACUC mouse care guidelines. All behavioral experiments were conducted during the light phase. Subjects were male and female Tau4RTg2652 animals and their non-Tg littermates. Two separate groups of animals were tested at each of three ages, to eliminate the potential effects of repeated testing. The group sizes were: 3 month group A, 14 Tg and 11 WT; 3 month group B, 12 Tg and 12 WT; 6 month group A, 9 Tg and 11 WT; 6 month group B, 10 Tg and 10 WT; 11 month group A, 7 Tg and 8 WT.
Open field testing was conducted in a 16 × 16 inch square clear plastic arena. All animals were moved into the testing room 30–40 min prior to start of testing. The arena was cleaned with 70% ethanol between animals. Total activity, vertical rearing, and activity in the center of the arena were measured for 10 min using a San Diego Instruments Photobeam Activity System.
The Barnes maze (San Diego Instruments) is a circular white plastic platform 36 inches in diameter with 20 holes around the perimeter. Nineteen holes were occluded with shallow, off-target boxes and one hole leading to a target escape box. Latency to enter the escape box was recorded. Placards with unique monochromatic patterns (vertical lines, horizontal lines, large circles, etc.) were placed around the room to be used as landmarks to find the escape box. The placards were approximately 12" by 14" and were placed at various heights. In addition to these placards a stuffed frog, a tether ball, and a car license plate were also suspended in various areas of the room. The light level on the platform was 375 lux. Mice are placed in a box in the center of the maze for 30 s at the beginning of each trial, so the direction each mouse faces at the trial start is random. When the start box is removed, each animal has a maximum of 2 min to locate the escape box. If the animal locates the hole, it is allowed to shelter there for 1 min before being returned to the home cage. If the animal fails the task, it is gently guided to the escape box by the investigator and allowed to shelter there for 1 min. All animals were moved into the testing room 30–40 min prior to start of testing, and the maze was cleaned with 70% ethanol between animals. Training trials were conducted twice daily for four consecutive days.
Two tests of grip strength were used: ability to hang from an inverted wire grid, and ability to grip a wire bar when pulled by the tail. For the inverted grid test, animals were placed onto a standard wire cage top and encouraged to grip it by gently shaking it. Then the wire top was slowly inverted and suspended approximately 30cm over a padded surface. The amount of time each animal was able to grip the wire grid was recorded, up to a maximum of 2 min. Each animal received three trials on the same day, with a minimum of 30 min rest between trials. Data presented are the average of the three trials for each animal. The T-bar pull-test was performed using a IITC Life Science Grip Strength Meter (Woodland Hills, CA) force gauge with a metal T-bar attached. Animals were held by the tail and allowed to grip the T-bar with forelimbs only, then gently pulled away from the grid to measure maximum grip strength. Each animal received five trials on the same day, with approximately 20 min rest between trials. Data are the average of the five trials for each animal.
Rotorod testing was conducted using an accelerating UGO Basile Rota Rod. The rod surface is covered with ridged plastic, located 16cm above the floor. All mice were transferred to the testing room 30–40 min prior to the start of testing, and were tested on four consecutive days. On the first three days, all mice received three training trials separated by approximately 35 min rest time. For all training trials, mice were placed onto a slowly moving rotorod (3 RPM). Once all mice in a testing group were on the rotorod, the apparatus was accelerated up to 16.75 RPM over 2 min. On the fourth day, mice also received three trials (45–50 min of rest between trials), but the rod was accelerated up to 39 RPM over 5.5 min to test maximum motor coordination abilities.
Statistical analysis was performed using Statistica (StatSoft). For all measures, sex and testing group were included as factors in the ANOVA. If either sex or group did not have a significant effect on a particular measure, then the analysis was repeated without that factor. For repeated measures (rotorod and Barnes testing), ANOVA was followed up with planned comparisons of the two genotypes on each testing day.
Electroencephalographic analysis
Homozygous 4RTauTg3652 mice were anesthetized with isofluorane and implanted bilaterally with electrodes—one with bilateral skull screw electrodes and two with one skull screw and contralateral depth electrodes in the cortex and hippocampus. Following recovery mice were placed in recording cages for 24/7 video EEG recording for 4 to 13 days. EEG was filtered (high pass 0.1 Hz, low pass 600 Hz) and digitized at 2000 Hz.
Immunoblot analysis
We conducted a sequential extraction of tau protein using buffers of increasing solubilizing strength as previously described [14]. Mouse brains from three non-Tg and nine Tau4RTg2652 mice at varying ages were isolated and snap frozen on liquid nitrogen prior to extraction. Mouse brain hemispheres were homogenized in high salt re-assembly buffer (RAB-High Salt [0.1 M MES, 1 mM EGTA, 0.5 mM MgSO4, 0.75 M NaCl, 0.02 M NaF, 0.5 mM PMSF, 0.1 % protease inhibitor cocktail, pH 7.0]) and ultra-centrifuged at 50,000x gravity yielding the soluble fraction (supernatant) and an insoluble pellet. Next, myelin was floated by resuspending the pellet in RAB/1M sucrose and centrifuging as above. To extract detergent soluble tau, the RAB insoluble material was re-extracted with an ionic and non-ionic detergent containing RIPA buffer [50 mM Tris, 150 mM NaCl, 1% NP40, 5mM EDTA, 0.5 % DOC, 0.1 % SDS, 0.5 mM PMSF, 0.1 % protease inhibitor cocktail, pH 8.0] and centrifuged as above yielding abnormal tau in the supernatant. Finally, the detergent insoluble pellet was re-extracted with 70 % Formic Acid (FA) to solubilize detergent insoluble tau. Total protein fractions containing ~15 ug of protein per lane were boiled 5 min and loaded onto 10 % pre-cast SDS-PAGE gels (Biorad, Hercules, CA). Subsequent RIPA and FA extracted fractions were normalized according to the levels of total protein in the total fraction. For semi-quantitative immunoblotting, we detected mouse and human tau using the pan tau antibody 17025 at a dilution of 1:3000 as described previously [16] and anti-actin antibody at 1:1000 (DSHB). Densitometry measurements were performed using Adobe Photoshop to determine total tau levels in non-Tg and Tg mice.
Immunohistochemistry and histological stains
Tg mice at three months (n = 9) and 1 year (n = 12) were examined by immunohistochemistry with antibodies MC1 and AT8. A subset of these brains was also immunostained with Alz50, AT180, AT270, PHF-1, TOC1 and SMI31. Mice were anesthetized and fixed by transcardial perfusion with 4% paraformaldehyde. Brains and spinal cords were removed and paraffin embedded for sectioning. Coronal sections from the forebrain, hippocampus, and brainstem were cut at 10 μm thickness and stored at 4 °C until use. Sections were deparaffinized and rehydrated through alcohols, and an antigen retrieval step consisting of heat pretreatment by microwave in citrate buffer was used when necessary. Sections were treated for endogenous peroxidases with 3 % hydrogen peroxide in PBS (pH 7.4), blocked in 5 % non-fat milk in PBS, and incubated with primary antibody overnight at 4°C followed by biotinylated secondary antibody for 45 min at room temperature. Finally, sections were incubated in an avidin-biotin complex (Vector’s Vectastain Elite ABC kit, Burlingame, CA) and the reaction product was visualized with 0.05 % diaminobenzidine (DAB)/0.01% hydrogen peroxide in PBS. Negative controls with secondary antibody alone did not immunostain tissue sections (data not shown). The presence of neurofibrillary tangles (NFTs) was assessed by Gallyas silver and Thioflavin-S staining using standard methods [17]. Bielschowsky silver stain was utilized to assess axonal pathology [18].
Photomicrography and figure preparation
Photomicrographs were taken with a digital camera and imported into Adobe Photoshop for mounting. To optimize visualization of staining, photomicrographs were modified when necessary by adjusting brightness and contrast.