Mice
Mice were housed in the SPF animal facility of Humanitas Clinical and Research Center in individually ventilated cages. Procedures involving animals handling and care conformed to protocols approved by the Humanitas Clinical and Research Center (Rozzano, Milan, Italy) in compliance with national (4D.L. N.116, G.U., suppl. 40, 18-2-1992) and international law and policies (EEC Council Directive 2010/63/EU, OJ L 276/33, 22-09-2010; National Institutes of Health Guide for the Care and Use of Laboratory Animals, US National Research Council, 2011). The study was approved by the Italian Ministry of Health (approval n. 6/2014). All the experimental procedures followed the guidelines established by the Italian Council on Animal Care and were approved by the Italian Government decree No. 27/2010. All efforts were made to minimize the number of subjects used and their suffering. Mice were housed in cages with free access to food and water at 22 °C and with a 12-h alternating light/dark cycle. Double transgenic APPswe/PSEN1dE9 (APP/PS1) mice were purchased from Jackson Laboratory [85]. C57BL/6 J-App (App−/−) mice were provided by Hertie Institute and Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE), Tübingen, Germany. BALB/c Mmp9 < tm1Tvu > (MMP9−/−) P1-P3 pups were provided by Istituto Nazionale dei Tumori, Milan, Italy.
Preparation of synthetic Aβ peptides
Synthetic human Aβ1–42 and Aβ1–24 were purchased from Bachem and prepared as previously described [86] to obtain oligomeric Aβ forms. Briefly, lyophilized Aβ 1–42 and 1–24 were dissolved in dimethyl sulfoxide (DMSO, Sigma) to a concentration of 2 mM and stored in small aliquots at−80 °C. Immediately prior to use, aliquots were quickly resuspended in 50 mM NaPi, 100 mM NaCl pH 7.4 buffer at a concentration of 40 μM, strongly vortexed, sonicated for 30 s and left at room temperature for 20 min before being further diluted for in vivo and in vitro experiments (the concentration used for each experiment is specified in the figure legend). For Aβ clearance experiments, Aβ42–488 (HILyte, AnaspeC) and Aβ42-FAM (Anaspect) were solubilised in NH4OH and stored in small aliquots at−80 °C.
Antibodies
Antibodies used for immunoblot (western/dot blot), immunoprecipitation, and immunofluorescence were as follows: monoclonal antibody 6E10 (1:2000; Covance), which recognizes residues 1–16 of human Aβ; rabbit anti-human beta amyloid 1–42 (1:1000; Alpha Diagnostic International) which recognizes C-terminal 6 aa peptide from human beta 1–42; 4G8 antibody (1:2000; Covance) directed against the central part of Aβ42 (aa 17–24); m3.2 (kindly provided by Prof. Paul Matthews) recognizes residues 10–15 of murine Aβ; rabbit anti-Iba1 antibody (1:500; Wako); ZO-1 (1:1000, clone R40.76, Millipore); Connexin-43 (1:400; C6219, Sigma); Claudin-5 (1:800; ABT45, Millipore). Secondary antibodies (1:200; Alexa Fluor®-conjugated, Molecular Probes).
Prediction of aggregation propensities
We used the AMYLPRED2 meta-predictor [87] to compare the aggregation profiles of the sequences of full Aβ42 peptide with respect to the truncated form including Aβ residues 1–24 (henceforth Aβ24). The meta-predictor identifies putative amyloigenic regions on the basis of the consensus between 11 methods considering a range of physico-chemical properties. Further analysis was carried out with the algorithms PASTA 2.0 [44] and PAGE/ABSOLUTERATE [88] in order to obtain quantitative predictions of putative aggregation propensities, rates, and the fibrillar’s beta-strand parallel versus antiparallel character. Putative three-dimensional fibrillar arrangements were obtained from the PDB database entries 2LMN, 2LMP, 2MXU [89].
Thioflavin T (ThT) assays
Thioflavin T (ThT) dye was purchased from Sigma Aldrich. For the ThT assays, 200 μL of Aβ42 (at 8 μM concentration), of Aβ24 (at 8 μM concentration), or of the equimolar mixture of the two peptides (at 4 μM concentration each) were incubated in 50 mM NaPi, 100 mM NaCl pH 7.4 buffer at 37 °C with ThT at 10 μM concentration. At different incubation times, the fluorescence emission spectra of the samples were collected after excitation at 450 nm [45] or at 270 nm [47] by the Cary Eclipse Spectrofluorimeter (Varian Australia Pty Ltd, Mulgrave VIC, Australia). Quartz cuvettes of 1 cm path length were employed. For time course experiments, the samples were kept at 37 °C and analyzed at each time point.
ATR-FTIR spectroscopy
For the ATR-FTIR measurements, 2 μL of Aβ42 (at 100 μM concentration), of Aβ24 (at 100 μM concentration), or of the equimolar mixture of the two peptides (at 50 μM concentration each) were deposed on the single reflection diamond element of the ATR device (Quest, Specac, UK). Spectra were recorded after solvent evaporation to allow the formation of an hydrated film as previously described [48]. FTIR measurements were performed using the Varian 670-IR spectrometer (Varian Australia Pty Ltd, Mulgrave VIC, Australia) under the following conditions: 1000 scan coadditions, 25 kHz of scan speed, 2 cm−1 of spectral resolution, triangular apodization, and a nitrogen-cooled Mercury Cadmium Telluride detector. Fourier self deconvolution was obtained with a full width at half height of 13.33 cm−1 and a resolution enhancement factor K = 1.5 [48] using the Resolutions-Pro software (Varian Australia Pty Ltd, Mulgrave VIC, Australia).
Intracranial injections
Stereotaxic intracranial injections in mice brains were made under a mixture of ketamine (100 mg/kg) and xylazine (10 mg/kg) anesthesia. After surgical exposure of dura mater, both the bregma and the skull surface served as the stereotaxic zero points. Using a Hamilton syringe, 4 μL of H-Aβ or vehicle (vehicle consists of physiological buffer−50 mM NaPi, 100 mM NaCl pH 7.4−) were injected into the neocortex (AP 1 mm, ML 2 mm, DV−2 mm) with a speed of 1.5 μL/min, the needle was kept in place for an additional minute before it was slowly drawn out. For each experiment 4 pmol of H-Aβ42, 4 pmol of H-Aβ24 and 2 pmol of H-Aβ42 plus 2 pmol of H-Aβ24 for the mix condition were injected, as described in [70, 90]. Different conditions were used for specific experiments as specified in the text. After suturing the incision, mice were maintained on a warm pad until recovery from the anesthesia, then returned to their cages. All procedures were conducted in accordance with institutional guidelines for the care and use of experimental animals.
Behavioral tests
Open field
Mice were placed in a multi-unit open field maze (ViewPoint instruments) with field chamber (25 cm long and 25 cm wide), and activity was recorded using ViewPoint video tracking software. Each quadrant was digitally divided into a peripheral and a central region using ViewPoint video tracking software. The central quadrants are collectively referred to as the center zone and peripheral quadrants are collectively referred to as the peripheral zone. Data were collected continually for 30 min and the distance traveled (cm), velocity (cm/s), and the distance traveled in the center zone versus the peripheral zone were all recorded and scored automatically. In the open field task ambulatory movements (valued as distance traveled and movement speeds) and anxiety-like behaviors (as the as distance traveled in the center zone versus the peripheral zone) can be assessed in response to a novel environment.
Social interaction task
The social interaction test was used to measure how mice respond to a social partner during a 5-min test following isolation housing. Since isolation housing potentiates expression of innate territorial defensive responses, in this test we evaluated the reaction of mice to the presence of a new animal in free conditions in the same arena. For this test, mice were placed for 30 min alone into the open field arena to familiarize themselves with the new environment. After this time a new female was introduced and the record started. We used the ViewPoint system to count the number of time the tested mouse made contact with the new females. The decision to use females was made to avoid any occurring of an aggressive behaviour simply caused by a normal intermale instinct.
Novel object recognition (NOR)
The test apparatus consisted of an open field box measuring 50 cm × 25 cm and all sessions were video-recorded. The first day the animal was allowed to explore the empty field arena for a 10-min time period (habituation session) before being exposed to a 10-min period of familiarization session in the presence of identical objects (A/A). This familiarization session was followed by 1 h and 24 h delays during which the animals were returned to their home cages. After the delay the animals performed 10-min of test session (A/B) in which one object was kept as during the familiarization session (A) and another was changed (B). The objects were made of hard plastic and had previously been counterbalanced to control for any object preference bias. The total amount of time spent with each object was recorded and scored using fully automated ViewPoint video tracking software. The time spent around each object was defined as the time in which the animal directed its nose to the object at a distance <2.0 cm and/or by the animal touching the object with its nose. Data are shown as the total amount of time that animals spend exploring the novel object during both the 1 and 24 h delay.
Microglia cell culture
Primary microglia were obtained from mixed cultures prepared from the cerebral cortex of mice at the postnatal day 1 (P1-3). Microglia cells were isolated by shaking flasks for 45 min at 230 rpm at day 10 after plating. Cells were then seeded on poly-L-ornithine (Sigma) pre-coated wells at the density of 1,5×105 cell/mL in DMEM containing 20 % heat-inactivated fetal bovine serum (FBS) and incubated at 37 ° C in a humidified atmosphere of 5 % CO2 and 95 % air. Where indicated, cells were pre-treated for 45 min with protease inhibitors and EDTA 2.5 mM (Roche) which were directly added to the cell culture medium. N9 cells were maintained in IDMEM (Gibco Laboratories, USA), supplemented with 10 % FBS. For siRNA transfection, N9 cells were plated at a concentration of at 1×105 cells/mL into either 96 or 24 multiwell plate. Specific siRNAs were diluted at a final concentration of 20 nM siRNA. Cells were used within 48–72 h after transfection. When not differently indicated, for in vitro experiments microglia were treated with H-Aβ42 400 nM.
Endothelial cells culture and BBB model
Mouse brain endothelial cells (bEnd.3) [BEND3] (ATCC® CRL2299™) were used as a representative BBB model. bEnd.3 cells were cultured at 37 °C, 5 % CO2/saturated humidity in DMEM supplemented with 10 % heat-inactivated fetal bovine serum (FBS), 1 % penicillin-streptomycin.
Transendothelial cell electrical resistance (TEER) assay
bEnd.3 cells were seeded at a concentration of 30000 cells/cm2 onto geltrex-coated (thin layer; Gibco) transwell inserts (polycarbonate, 12 mm diameter, 3 μm pore size; Costar) until a monolayer was established. TEER was assessed using a Voltohmeter (Millicell Electrical Resistance System, Millipore). Background resistance from cell-free matrix-coated transwells was subtracted from recorded values to determine absolute TEER values and corrected for the area covered by the cell monolayer. TEER was measured once a day to monitor cell confluence and development of tight junctions. Change in absolute TEER from T0 for each individual transwell was recorded over time and then averaged for each day before treatment.
Aβ42 permeability of the endothelial barrier
To assess Aβ42 exchange across the BBB model, bEnd.3 cells were cultured upside-down on a transwells system as described above, until steady-state TEER had been reached. 1 μM FAM-labeled Human Beta-Amyloid (1–42) or FAM-labeled scrambled Beta-Amyloid (1–42) or HiLyte Fluor™ 488-labeled Beta-Amyloid (1–42) (Anaspec Peptide, Eurogentec) in the presence or absence of Human Beta-Amyloid (1–24) (Bachem) were pre-incubated for 20 min in FluoroBrite™ DMEM (Gibco) medium at 37 °C, and added to the apical (brain) side corresponding to the donor compartment. The basolateral (blood) side of the transwells represented the receiver side and was exposed to medium alone. Alternatively, primary microglial cells were incubated for 24 h at 37 °C in FluoroBrite™ DMEM medium containing Human Beta-Amyloid (1–42) (Bachem) in the presence or absence of protease inhibitors (Roche). Microglia conditioned medium was recovered and spun for 2 min at max speed. 1 μM HiLyte FluorTM 488-labeled Beta-Amyloid (1–42) was pre-incubated for 30 min at 37 °C with microglia conditioned medium, and added to the apical compartment. Following 120 min of incubation at 37 °C with slow mixing, samples were collected from the upper and lower chambers to assess the movement of fluorescently-labelled Aβ42 across the bEnd.3 monolayer (apical to basolateral). The level of fluorescence in the media collected was measured for 488-Aβ42 (λex = 503 nm and λem = 528 nm) or for FAM-Aβ42 (λex = 492 nm and λem = 518 nm) using a Synergy™ H4 Hybrid Multi-Mode Microplate Reader (BioTek). Relative fluorescence units were converted to concentration according to prepared standard, and were corrected for background fluorescence. The amount of fluorescently-labelled Aβ42 was calculated as apparent permeability (Papp) coefficient [ref. Zhao Z 2015 Nat Neurosci; Keaney J 2015 Sci Adv] and expressed as fold change of control. Briefly, Aβ42 volume cleared (ΔVc) was calculated using the equation ΔVc = Clower × Vlower/Cupper, where Cupper and Clower are fluorescently-labeled Aβ42 concentrations in the donor and receiving compartments, respectively, and Vlower is the volume on the basolateral side. The volume cleared (ΔVc) was plotted against assay time. Permeability coefficients (P) were calculated by dividing against the surface area of the filter (1.12 cm2).
Immunocytochemistry and cells imaging
Cells were fixed for 15 min at room temperature in a 4 % (w/v) PFA, 4 % (w/v) sucrose, 20 mM NaOH and 5 mM MgCl2 in PBS, pH 7.4. Cells were permeabilized and blocked for 30–60 min at room temperature in 15 % (w/v) goat serum, 0.3 % (v/v) Triton X-100, 450 mM NaCl, 20 mM phosphate buffer, pH 7.4 and incubated at 4 °C overnight with primary antibodies diluted in blocking buffer. Coverslips were mounted onto slides with PBS containing 70 % glycerol and 1 μM DAPI. Representative images were taken using a confocal microscope Fluoview FV1000 Olympus IX81 (Center Valley, PA, USA) with an oil immersion objective (×40 or × 60 × 1.4 NA Plan-Apochromat; Olympus) using laser excitation at 405, 488 or 594 nm, and processed using Fiji [91]. Alternatively bEnd.3 cells were stained with Diff Quick (Dade Behering, BioMap) and acquired with inverted microscope Olympus IX53 (Center Valley, PA, USA).
Brain homogenate preparation and western blot analysis
Animals were anesthetized and perfused with PBS before brains were removed, weighted and homogeneted in a mild hypotonic buffer (50 mM Tris pH 8, 150 mM NaCl, 5 mM EDTA supplemented with phosphatase and protease inhibitor, EDTA-free, Roche). Supernatants were then centrifuged for 1 h at 3000 × g to pellet insoluble material, including insoluble Aβ species. Samples were either frozen on dry ice or LDS sample buffer (Invitrogen) containg 5 % β-mercaptoethanol was immediately added. Pellets were directly resuspended in 20 μl of LDS sample buffer. 10 μl sample were loaded onto a Bolt 4–12 % Bis-Tris Plus Gels (Thermo Fisher) for Western Blot evaluation. Samples were separated using MES buffer and transferred onto a 0.22 μM nitrocellulose membrane (Bio-Rad). Membranes were washed in TBS-Tween (150 mM NaCl, 50 mM Tris and 0.1 % (v/v) Tween-20) and incubated for 45 min at room temperature in blocking solution (5 % milk or 2.5 % serum bovine albumin in TBST). Membranes were subsequently probed overnight at 4 °C with primary antibodies diluted in TBS-T buffer. Membranes were washed extensively and incubated for 45 min at room temperature with horseradish peroxidase-conjugated (HRP) secondary antibody diluted in TBST buffer. Antibody-specific signals were detected using enhanced chemiluminescence reagents (Clarity Western ECL substrate, Bio-Rad).
Dot blot analysis
Aliquots of supernatant samples (200 μL) were loaded on nitrocellulose membrane Trans-Blot Transfer Medium (0.22 μm, Bio-Rad), by vacuum deposition on the Bio-Dot SF blotting apparatus (Bio-Rad). Serial dilution curves of Aβ42 synthetic protein were preliminarily run to obtain non-saturating condition of immunodetection. DB dots images were analysed by Image Lab™ software (Bio-Rad).
ELISA
Aβ42 levels were determined using specific ELISA kit (Amyloid-beta (x-42) ELISA IBL, International) following manufacturer’s instructions. Briefly, 100 μL of sample was added into the pre-coated plate and was incubated overnight at 4 °C. After washing each well of the pre-coated plate, 100 μL of labeled antibody solution was added and the mixture was incubated for 1 h at 4 °C in the dark. After washing, chromogen was added and the mixture was incubated for 30 min at room temperature in the dark. After the addition of stop solution, the resulting color was assayed at 450 nm using a microplate absorbance reader (Synergy H4 Synergy™ H4 Microplate Reader, BioTek).
Histological and immunohistochemical analyses
Mice were anesthetized and perfused with 0.9 % saline, followed by 4 % PFA. The 30 μm cryosections of brain were blocked in PBS containing 10 % goat serum and 0.1 % Triton X-100 for 1 h at room temperature before being incubated overnight at 4 °C with primary antibodies. The following day, the slides were rinsed in PBS and incubated at room temperature for 1 h with secondary antibody. The slides were processed using the ABC detection kit (Vector Laboratories). The presence of the antigens was revealed using the DAB (diaminobenzidine) (brown) peroxidase substrate kit (Vector Laboratories). Immunofluorescence and ThT staining were performed on brain sections. Brain slices were washed three times in PBS and incubated for 1 h at room temperature in blocking solution (3 % BSA). Subsequently, the slides were washed 3 times and incubated overnight with specific antibodies. The next day, sections were washed in PBS and incubated for 2 h at room temperature with specific fluorochrome-conjugated secondary antibodies diluted in 3 % BSA in the dark. ThT solution was prepared as described above. Final solution was added to free floating slides for 1 min and very quickly washed with 80 % methanol followed by 3 washes with distilled water. Images were acquired by Virtual Slides microscope (VS120, Olympus). For quantification, both diffuse-plaques and dense-core plaques were considered, as described in [92]. Small dots and deposits at the slice edge were not counted. The entire surface of the slice was examined. For congo red histological staining, slices were air-dried on glass overnight, then stained with a 0.2 % congo red solution according to [93]. Specimens were acquired by transmittance polarized light microscopy, FITCH filtered epifluorescence. For quantification, bright field images were analyzed by color segmentation plugin-ImageJ software (NIH, Bethesda, MD). The entire area of deposits was considered.
Statistical analysis
Statistical analysis was performed with PRISM software (Graph-Pad Software, San Diego, CA, USA). Data are expressed as mean ± SEM. Comparisons between two groups were performed using Student’s t test or by non-parametric two-tailed Mann–Whitney U test. For the comparison of more than two groups, two-way ANOVA followed by Bonferroni’s post hoc test was used. Differences were considered significant at *P < 0.05, **P < 0.01, ***P < 0.001.