Synthesis of peptide, polymerization of the Bri peptide and assessment of conformation
This was done as previously published . In brief, the 13 residue peptide corresponding to the carboxyl terminus of ABri (Cys-Ser-Arg-Thr-Val-Lys-Lys-Asn-Ile-Ile-Glu-Glu-Asn) was synthesized on an ABI 430A peptide synthesizer (AME Bioscience, Chicago, IL) at the Keck peptide synthesis facility at Yale University, CT. Mass spectroscopy of the lyophilized end-product was used to verify the expected molecular weight. To make the 13-residue Bri peptide immunogenic and to have a stable oligomeric conformation, the peptide was subjected to controlled polymerization using the following protocol.
The peptide was dissolved at 3 mg/ml, in 100 mM borate buffer saline (BBS), pH 7.4. Fresh 1% glutaraldehyde in BBS was prepared and added to the peptide to a final 5 mM glutaraldehyde concentration and incubated in an Eppendorf block shaker at 800 rpm and 56°C for 16 h. The solution was then quenched with 0.5 M glycine to make the solution 100 mM in glycine. After 5 min, the solution was diluted 1:3 with BBS, dialyzed against 2 mM BBS overnight at 4°C, aliquoted and lyophilized. To determine the degree of aggregation the original monomeric Bri peptide and polymerized Bri (pBri) were electrophoresed on 12.5% sodium dodecyl sulfate (SDS)-polyacrylamide tris-tricine gels under reducing conditions. Western blots were performed with a rabbit anti-ABri polyclonal Ab  (1:1,000 dilution). The secondary antibody (1:2,000 dilution) was peroxidase-linked anti-rabbit IgG (Amersham Biosciences, Piscataway, NJ), and the immunoreactive material was visualized as chemoluminescence on X-ray film with the enhanced chemiluminescence (ECL) detection kit (Pierce). For electron microscope studies, the original and polymerized Bri peptides were incubated at 1 mg/ml in phosphate-buffered saline, pH 7.4. Next, 3 μl of sample was put onto a carbon-coated 400 mesh Cu/Rh grid (Ted Pella, Inc, Redding, CA) and stained with 1% uranyl acetate in distilled water (Polysciences, Inc, Warrington, PA), as previously published . For secondary structure analysis, aliquots of the original Bri peptide and pBri were reconstituted in 5 mM tris buffer (pH 7.0) to obtain a peptide concentration of 100 μM. Circular dichroism was measured on a Jasco J-720 spectropolarimeter (Easton, MD) equipped with a model CTC-344 circular temperature control system (Neslab Inc, Newington, NH).
Immunization of mice
Animal studies were approved by the New York University School of Medicine Institutional Animal Care and Use Committee and were consistent with the recommendations of the American Veterinary Association. Mice facilities were under a strict 12 h light/dark cycle. Triple transgenic (3xTg) mice (PS1M146V, tauP301L and APPK670N/M671L) are a model of combined tau- and amyloid-related pathology [19, 20]. To model vascular amyloid deposition, the well-characterized TgSwDI mouse model was used [21, 22]. This model expresses the APPE693Q/D694N (Dutch and Iowa) and APPK670N/M671L (Swedish) mutations. The pBri peptide was dissolved in sterile saline at 1 mg/ml and mixed 4:1 with aluminum hydroxide (alum) adjuvant (Brenntag Biosector, Denmark). Each mouse received a weekly subcutaneous injection of 100 μl of the preparation (50 μg of pBri) for 4 weeks, starting at the age of 3 months, followed by an inoculation a month later and two subsequent bimonthly injections. The last three inoculations used 25 μg of pBri per animal and the ratio of saline to alum was changed to 9:1. Both the 3xTg and TgSwDI mice were divided into two groups of 14 mice each. There were equal numbers of males and females in each experimental group. For each transgenic line, one group received pBri immunization while the second control group received vehicle injections (alum alone). The mice were subject to behavioral testing at the age of 15 to 16 months.
Sensorimotor and cognitive testing
Sensorimotor and cognitive testing were done as previously described [16, 20, 22]. Prior to testing, the mice were adapted to the room with lights on for 15 min. The main objective of performing these sensorimotor tasks was to verify that any treatment-related effects observed in the cognitive tasks could not be explained by differences in sensorimotor abilities.
A Hamilton-Kinder Smart-frame Photobeam System was used to make a computerized recording of animal activity over a designated period of time. Exploratory locomotor activity was recorded in a circular open-field activity chamber measuring 70 cm in diameter. A video camera mounted above the chamber automatically recorded horizontal movements in the open field in each dimension. Total distance was measured in centimeters traveled and is defined as the sum of sequential movement between interruptions of the animal. The duration of the behavior was timed over 15 min. Results were reported based on distance traveled (cm), mean resting time and maximum speed of the animal.
This task tests balance and general motor coordination and function integration. Mice were assessed by measuring their ability to traverse a graded narrow wooden beam to reach a goal box. This specifically examines hind limb function. The mice were placed on a 1.1 cm wide beam, 50.8 cm long, supported 30 cm above a padded surface on two identical columns. A shaded goal box was attached at each end of the beam. Mice were placed on the beam in a perpendicular orientation to habituate, and were then monitored for a maximum of 60 sec. The number of foot slips each mouse made before falling or reaching the goal box was recorded for each of three successive trials. The average number of foot slips for all three trials was calculated and recorded. Errors are defined as foot slips and were recorded both numerically and using Feeney scores. To prevent injury from falling, a soft foam cushion was always kept underneath the beam. Animals that fell off were placed back in the position they were in prior to the fall.
Each animal was placed onto a rod (diameter 3.6 cm) apparatus to assess differences in motor coordination and balance by measuring fore- and hindlimb motor coordination and balance (Rotarod 7650 accelerating model; Ugo Basile, Biological Research Apparatus, Varese, Italy). This was designed to assess motor behavior without a practice confound. The animals were habituated to the apparatus by receiving training sessions of two trials, which was sufficient to reach a baseline level of performance. The mice were tested a further three times, with increasing speed. During habituation, the rotor rod was set at 1.0 rpm, which was gradually increased every 30 sec to a maximum speed of 40.0 rpm. The rod was wiped clean with 30% ethanol solution after each session. A soft foam cushion was placed beneath the apparatus to prevent potential injury from falling. Each animal was tested for three sessions, with each session separated by 15 min. The latency to fall or invert (by clinging) from the top of the rotating barrel was recorded.
Radial arm maze
Spatial learning was evaluated using an eight-arm radial maze with a water well at the end of each arm, as we have previously published [16, 20, 22], for both TgSwDI and 3xTg mice. For each group of transgenic mice, an age-matched control group of non-transgenic mice (n = 10) were also tested in the radial arm maze. Clear Plexiglas guillotine doors, operated by a remote pulley system, controlled access to the arms from a central area from which the animals entered and exited the apparatus. After 4 days of adaptation to the maze, water-restricted mice (only given access to water 2 h per day) were given one training session per day for 10 consecutive days. We use this relatively long adaptation period as we have found that these transgenic (Tg) AD mice tend to be anxious and will not run the maze well without adaptation [16, 20, 22]. Prior to each day’s testing, the mice were adapted to the room with the lights on for 15 min. For each session, all arms were baited with saccharine-flavored water, and the animals were permitted to enter all arms until the eight rewards had been consumed. The number of errors (entries to previously visited arms) and time to complete each session were recorded.
Antibody levels were determined in duplicate on 1:100 dilutions of plasma using ELISA as described previously [16, 20, 22], in which 50 μg/plate aggregated Aβ1-42, aggregated Aβ1-40, pBri or purified human paired helical filaments (PHF) was coated onto Immulon 2HB 96-well microtiter wells (Thermo, Waltham, MA). The human PHF was prepared and characterized for purity by Western blotting with PHF1 and electron microscopy, as previously published . The bound antibodies were detected by a horseradish peroxidase-labeled goat anti-mouse IgG (Amersham Biosciences, Piscataway, NJ) or a peroxidase conjugated goat anti-mouse IgM (Sigma; A8786). The color developing substrate was tetramethylbenzidine (TMB) (Pierce, Rockford, IL) and the readings were made at 450 nm.
The mice were anesthetized with sodium pentobarbital (150 mg/kg, intraperitoneally), perfused transaortically with phosphate buffer, and the brains processed as described previously [16, 20, 22]. The right hemisphere was immersion-fixed in periodate-lysine-paraformaldehyde, whereas the left hemisphere was snap-frozen for measurements of Aβ levels in both transgenic lines and phosphorylated tau in the 3xTg mice. Serial coronal sections (40 μm) were cut (30 to 40 sections in total), and every fifth section was stained with a mixture of 4G8 and 6E10, which are monoclonal antibodies that recognize Aβ and stain both pre-amyloid and Aβ plaques [23, 24]. In 3xTg mice, the degree of tau-related pathology was determined with anti-abnormally phosphorylated tau monoclonal antibodies PHF1 (which recognizes phosphorylated serine in position 396 and 404)  and AT8 (which recognizes tau phosphorylated at both serine 202 and threonine 205) ; these antibodies were kindly provided by Dr Peter Davies from the Albert Einstein College of Medicine, Bronx, NY.
Glial fibrillary acidic protein (GFAP) is a component of the glial intermediate filaments that forms part of the cytoskeleton and is found predominantly in astrocytes. CD45 is a protein tyrosine phosphatase, commonly used to detect microglial activation at the later stages of plaque development [27, 28]. Two series of sections were immunostained with anti-GFAP (Dako, Carpinteria, CA) and anti-CD45 (Abd Serotec, Raleigh, NC) antibodies.
Immunostaining was performed as described previously [16, 20, 22]. Briefly, sections were incubated in 6E10/4G8 each at a 1:1000 dilution in PBS-T for 3 h. A mouse-on-mouse immunodetection kit (Vector Laboratories, Burlingame, CA) was used. The sections were incubated first with biotinylated anti-mouse IgG secondary antibody for 1 h at a 1:2000 dilution and later with the avidin-peroxidase complex for 30 min at the same dilution. The sections were then reacted in 3,3-diaminobenzidine tetrahydrochloride with nickel ammonium sulfate (Mallinckrodt, Paris, KY) color intensification solution. Immunohistochemistry of 6E10/4G8 immunolabeled tissue sections was quantified with a Bioquant image analysis system (BIOQUANT Image Analysis Corporation, Nashville, TN), and unbiased sampling was used . All procedures were performed by an individual blinded to the experimental conditions of the study.
The cortical area analyzed was dorsomedial from the cingulate cortex and extended ventrolaterally to the rhinal fissure within the right hemisphere. The area of the grid was 800 × 800 μm2, and the Aβ deposit load was measured for 20 cortical frames per mouse (640 × 480 μm2 each) chosen randomly. The threshold of the Aβ immunoreactive areas was set so that areas of <5 μm in diameter are not counted. This was done so that small artifactual areas of staining were not counted and the intra-neuronal immunoreactivity was also not counted. With the latter caveat, the Aβ burden was defined as the percentage of the area in the measurement field occupied by the reaction product. GFAP-staining (polyclonal, 1:1000; 3 h, Dako, Denmark) was performed with a primary antibody diluent composed of 0.3% triton X-100, 0.1% sodium azide, 0.01% bacitracin, 1% bovine serum albumin (BSA) and 10% normal goat serum in PBS, and a secondary biotinylated goat anti-rabbit antibody (Vector Laboratories, Burlingame, CA) reacted for 1 h at 1:1000 dilution. The analysis of the CD45 immunohistochemistry (rat anti-mouse, 1:500; 3 h, Serotec) was performed like the GFAP-staining except that the secondary antibody was goat anti-rat (Vector Laboratories, Burlingame, CA) diluted 1:1000.
The tau burden was analyzed based on the extent of immunostaining with two anti-tau antibodies, PHF1 and AT8, as previously described . PHF1 was analyzed at 10× magnification in both the hippocampus and cortex. Approximately six sections were analyzed per animal. AT8 was analyzed at 10× magnification for the hippocampus (AT8 was not analyzed in the cortex as the cortical AT8 immunolabeling was minimal). Approximately eight cortical sections and six hippocampal sections were analyzed per animal. The rating was based on the number of reactive neuronal bodies and processes. Reactive astrocytosis (GFAP immunoreactivity) was rated on a scale of 0 to 4. The rating was based on a semiquantitative analysis of the extent of GFAP immunoreactivity (the number of GFAP immunoreactive cells and complexity of astrocytic branching), as we have previously published [16, 20, 22]. The assessment of the CD45 immunostained sections was based on a semiquantitative analysis of the extent of microgliosis (0, no microglia; 1, a few resting microglia; 2, a few ramified and/or phagocytic microglia; 3, moderate number of ramified/phagocytic microglia; 4, numerous ramified/phagocytic microglia), as we have previously reported [16, 20, 22].
Perl’s iron staining was performed for the TgSwDI mice on another set of sections to detect cerebral microhemorrhages, as we have previously reported . Sections were stained in a solution containing 5% potassium ferrocyanide and 10% hydrochloric acid for 40 min.
Tissue homogenization and sandwich ELISA assay for soluble Aβ levels
Aβ was extracted from brain tissue as described  for both TgSwDI and 3xTg mice. Brains were weighed and homogenized (20% w/v) in a homogenization buffer, 20 mM Tris, 250 mM sucrose, 1 mM Ethylenediaminotetraacetic acid disodium salt (EDTA), 1 mM Ethylene glycol tetraacetic acid tetrasodium salt (EGTA) with freshly prepared 100 mM phenylmethylsulfonyl fluoride, 5 μg/ml pepstatin A and a protease inhibitor cocktail (Complete, Roche Diagnostics, Indianapolis, IN). Subsequently, 400 μl of the solutions was spun at 100,000 g for 1 h at 4°C, aliquoted, flash-frozen on dry ice and stored at -80°C until used for both Aβ and tau measurements in ELISA and Western blots.
The total and soluble Aβ levels were measured using a combination of mouse monoclonal antibody 6E10 (specific to an epitope present on amino acid residues 1 to 16 of Aβ) and two different rabbit polyclonal antibodies specific for Aβ40 (R162) and Aβ42 (R165), in a double-antibody sandwich ELISA as described previously [23, 24]. The optical density (OD) was measured at 450 nm. The relation between OD and Aβ peptide concentration was determined by a four-parameter logistic log function. Non-linear curve fitting was performed with the KinetiCalc program (Biotek Instruments, Inc, Winooski, VT) to convert the OD of plasma to estimated concentrations. The assay was performed by an investigator blinded to group assignment. The levels of Aβ species are presented as micrograms of Aβ per gram of wet brain, taking into account dilution factors introduced by multiple steps throughout the assay (brain homogenization and extraction procedures).
Western blot and Meso Scale Discovery electrochemiluminescence analysis of phosphorylated tau for 3xTg mice
For Western immunoblot analysis, 20% w/v brain homogenates from the 3xTg mice were centrifuged at 25,000 g for 10 min at 4°C, and the supernatants were transferred to clean tubes and stored as previously described [23, 24]. The total protein concentration in the supernatant was determined using the bicinchoninic acid assay (Pierce, Rockford, IL). Samples (40 μg of total protein) were mixed with an equal volume of tricine sample buffer (BioRad, Hercules, CA), electrophoresed on 12.5% SDS-tris-tricine polyacrylamide gels under non-reducing conditions and transferred to nitrocellulose membranes. To assess whether there was equal protein loading in each lane, the membranes were stained with reversible 0.1% Fast Green FCF (Fisher Scientific, USA) in 25% methanol destained in 25% methanol and transferred to distilled water. The blots were then blocked with 5% non-fat dry milk in 50 mM tris buffer saline- 0.1% Tween 20 (TBS-T), pH 8.3, for 2 h at room temperature, then incubated with PHF1 diluted 1:500 in TBS-T, 0.1% BSA for 2 h at room temperature. Bound antibodies were detected after 1 h incubation with horseradish peroxidase-conjugated goat anti-mouse IgG 1:8000 (Pierce, Rockford, IL) and the ECL detection system (Pierce, Rockford, IL). The specificity of the PHF1 band was confirmed by Western blots using homogenates of non-transgenic mouse brains. Only the bands detected by PHF1 in 3xTg transgenic mouse homogenates and not in the non-transgenic mouse homogenates were quantitated. Densitometric analysis of the PHF1 specific bands was performed with the NIH Image J software version 1.34.
The same 20% brain homogenates containing only soluble forms of tau were centrifuged at 100,000 g. Total tau and phosphorylated tau (Thr231) were quantified using a Meso Scale Discovery (MSD) (Rockville, MD) system that utilizes electrochemiluminescence analysis. The multi-array phospho (Thr231) tau assay kit, which measures both total human tau and phosphorylated Thr231 tau, was used with the MesoQuickPlex SQ 120 system, following the manufacturer’s instructions. In brief, samples were diluted 1:125 with the provided standard diluent buffer and 100 μl aliquots were seeded in each well. The plates were incubated for 2 h at room temperature, then washed four times for 25 sec each, before the Hu Aggregated Aβ biotin conjugate was added and incubated for 1 h. The plates were again washed four times and the Streptavidin-HRP working solutions was added. The plates were covered to block the light and incubated for 25 min before the reaction was stopped with the stop solution. The plates were read on the MSD system at 450 nm. All data were recorded and calculations made using the software provided with the MSD system.
Quantitation of aggregated/oligomeric Aβ
Aggregated/oligomeric Aβ levels were determined using the Human Aggregated Aβ ELISA kit (Invitrogen, Camarillo, CA), following the manufacturer’s instructions. In brief, the levels of aggregated/oligomeric Aβ in each sample were measured against a standard containing aggregated Aβ. Next 20% w/v brain homogenates were thawed, diluted 1:4 with the diluents buffer and applied to the ELISA plates. The samples were then incubated for 2 h at room temperature allowing the N-terminal portion of the Aβ to bind the pre-coated capture monoclonal antibody, followed by extensive washing and incubation for 1 h at room temperature with biotin conjugated detection antibodies (same as the capture antibody), which binds only to the immobilized aggregated Aβ. After removal of excess antibody, horseradish peroxidase-labelled streptavidin was added. The sample was incubated for 30 min, followed by washing and TMB substrate incubation to produce color. The intensity of this colored product is directly proportional to the concentration of aggregated/oligomeric Aβ in the sample. The standards produced a linear curve and the best-fit lines determined by linear regression were used to calculate aggregated Aβ concentrations in the samples.
Data from the accelerating rotor rod and locomotor test were analyzed using two-tail, Student’s t-tests. The data collected from the radial arm maze test was analyzed using two-way ANOVA, and also by one-way ANOVA followed by Newman–Keuls post hoc tests. Differences in total amyloid burden, levels of extracted Aβ, levels of Aβ aggregates/oligomers, tau burden, astrogliosis and microgliosis between the two groups were analyzed using two-tailed t-tests. All statistical tests were performed using Prism 6.0 (Graphpad, San Diego, CA).