Rabbit antibody for TRPA1 (NB110-40763) was from Novus (Littleton, CO, USA). Goat antibody for Akt (sc-1619), rabbit antibodies for PP2B (sc-9070), mouse antibodies for glial fibrillary acidic protein (GFAP, sc-166481), goat anti-rabbit FITC-conjugated (sc-2012), goat anti- mouse Texas red-conjugated (sc-2781) and FITC-conjugated (sc-2010) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse antibodies for GFAP (MAB360), NeuN (MAB3770), ionized calcium-binding adapter molecule 1 (IBA-1, MABN92), von Willebrand factor (vWF, MAB7356), and the cellular PP2B activity kit were from Millipore (Darmstadt, Germany). Rabbit LDLR-related protein 1 (LRP-1, L2170), mouse antibody for α-tubulin (T-9026), bovine serum albumin (BSA), phosphatase inhibitor cocktails 1 and 2, HC030031, allyl isothiocyanate (AITC), ethylene glycol tetraaceticacid (EGTA), ethylenediaminetetraacetic acid (EDTA), cyclosporine (CsA) and fenvalerate (Fen) were from Sigma-Aldrich (St. Louis, MO, USA). Mouse antibody for Aβ (SIG-39320-200) was from Covance (Dedham, MA, USA). Rabbit antibody for β-APP C-terminal fragment (βCTF, 802801) was from BioLegend (San Diego, CA, USA). Mouse antibody for ATP-binding cassette transporter A1 (ABCA1, ab18180), IL-4 (ab9622) and IL-10 (ab9969) were from Abcam (Cambridge, MA, UK). Rabbit antibody for apolipoprotein E (apoE, 1930-5) was from Epitomics (Burlingame, CA, USA). Mouse anti-phosphor-Akt (587 F11) was from Cell Signaling (Danvers, MA, USA). Retrieval buffer was from Biocare Medical (Concord, CA, USA). The mounting medium with DAPI was from Vector Laboratories (Burlingame, CA, USA). TurboFect was from Fermentas (Glen Burnie, MD, USA). The ELISA kit for NF-κB activity was from Cayman Chemical (Ann Arbor, MI, USA) and for NFAT activity was from Active Motif (Carlsbad, CA, USA). ELISA kits for IL-1β, IL-4, IL-6 and IL-10 and mouse antibodies for IL-1β (AF-401-NA) and IL-10 (AF-406-NA) were from R&D systems (Minneapolis, MN, USA). Quest™ Fluo-8 NW calcium assay kit was from AAT Bioquest (Sunnyvale, CA, USA).
The investigation conformed to the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, eighth edition, 2011), and all animal experiments were performed in accordance with the approved guidelines by the Animal Care and Utilization Committee of the National Yang-Ming University (#1031269). B6.Cg-Tg(APPswe, PSEN1dE9)85Dbo/J (APP/PS1 Tg) mice and TRPA1−/− mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA) and were backcrossed to C57BL mice for at least 10 generations to ensure genetic homogeneity. For APP/PS1 Tg/TRPA1−/− mice, TRPA1−/− mice were crossed with the APP/PS1 Tg background, and the genotypes were confirmed by PCR of genomic DNA. Mice were housed in barrier facilities, maintained on a 12-h/12-h dark cycle. Temperature (22 °C) and humidity (40-60 %) of the vivarium were tightly controlled. Mice were group-housed 3–4 per cage and fed a regular chow diet, which contained 4.5 % fat by weight (0.02 % cholesterol) (Newco Distributors, Redwood, CA, USA). At the end of the experiment, mice were euthanized with CO2, then brains were harvested for histological analysis and stored at −80 °C. The isolated brains were homogenized and lysates were subjected to western blot analysis.
Western blot analysis
Cells and brain tissues were lysed in immunoprecipitation lysis buffer (50 mmol/L Tris pH 7.5, 5 mmol/L EDTA, 300 mmol/L NaCl, 1 % Triton X-100, 1 mmol/L phenylmethylsulfonyl fluoride, 10 μg/mL leupeptin and 10 μg/mL aprotinin). Aliquots of brain lysates or cell lysates were separated on SDS-PAGE, transferred to membranes and immunoblotted with primary antibodies (1:1000), then horseradish peroxidase-conjugated secondary antibody (1:1000). Bands were revealed by use of an enzyme-linked chemiluminescence detection kit (PerkimElmer, Waltham, MA) and density was quantified by use of Imagequant 5.2 (Healthcare Bio-Sciences, Philadelphia, PA).
The brain sections were fixed in 4 % paraformaldehyde and 15-μm cross sections were prepared. Sections were incubated with retrieval buffer for 10 min, blocked with 2 % BSA for 60 min and incubated with primary antibody (1:100) overnight at 4 °C, then FITC- or Texas red-conjugated secondary antibody (1:400) for 1 h at 37 °C. Antigenic sites were visualized under a Nikon TE2000-U microscope (Tokyo) with QCapture Pro 6.0 software (QImaging, BC, Canada).
Fibrilization of Aβ1–42
Aβ1–42 was purchased from American Peptide Co. (Sunnyvale, CA, USA), solubilized in sterile water (1 mg/mL) and incubated 1 to 7 days at 37 °C for fibrillation as described [31–33]. The level of Aβ fibrilization on a 16.5 % tricine gel was examined by western blot analysis (Additional file 1: Figure S1).
The primary culture of astrocytes and neurons was prepared as described [24, 34]. Briefly, the cortex and hippocampus were isolated from pups on postnatal 1 day and loosely homogenized by use of a sterile razor blade in DMEM/F12 (HyClone, Logan, UT). Tissues were digested with 0.01 % trypsin and incubated at 37 °C for 25 min; the cell suspension was titrated by use of a 70-μm nylon mesh. Isolated cells were seeded onto 75-mm flasks and incubated for 7 days in DMEM/F12 supplemented with 10 % FBS, 100 U/mL penicillin and 100 μg/mL streptomycin at 37 °C. Cells were re-suspended, followed by orbital shaking at 180 rpm for 24 h to remove microglia and oligodendrocytes. The purified astrocytes that tightly adhered to the bottom of the flasks were then detached with trypsin and seeded onto culture dishes and incubated for an additional 7 days to return to a resting state. The primary neuron culture was prepared as follows: the cortex was isolated from pups on postnatal 1 day and loosely homogenized by use of a sterile razor blade in DMEM (HyClone, Logan, UT). Isolated cells were seeded onto 3.5-cm dishes and incubated in Neurobasal media supplemented with 2 % B-27 supplement, 0.25 % GlutaMAX, 10 % FBS, 100 U/mL penicillin and 100 μg/mL streptomycin (Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C. Cells were treated with cytosine-1-β-D-arabinofuranoside (10 μM) from day 2 and half the medium was changed every 3 days for 14 days’ culture. Human embryonic kidney 293 (HEK293) cells and mouse brain microvascular endothelial cells (BMECs), bEnd.3 cells, were cultured in DMEM supplemented with 10 % FBS, 100 U/mL penicillin and 100 μg/mL streptomycin. The growth media was replaced every other day.
Plasmid construction and transient transfection
The coding region for the human TRPA1 DNA fragment was cloned into a pCMV5 N-Flag vector with MluI and HindIII restriction sites. The sequence of isolated DNA fragments was confirmed by sequence analysis. TurboFect was used for transient transfection experiments according to the manufacturer’s instructions. Briefly, 1 μg of vector or TRPA1 plasmid was transfected into HEK293 cells. Transfected cells were used in further experiments.
Detection of Ca2+ influx
Primary astrocytes or HEK293 cells were pretreated with Fluo-8NW dye for 1 h, then medium was replaced with fresh medium containing test compounds. The intensity of fluorescence was evaluated by fluorometry (Molecular Devices, Sunnyvale, CA, USA) with 490-nm excitation and 525-nm emission. Images were captured under a TE2000-U fluorescence microscope and quantified with use of QCapture Pro 6.0.
Measurement of PP2B activity
The activity of PP2B in primary astrocytes or fresh brain lysates was measured by use of a cellular PP2B activity kit.
Measurement of inflammatory cytokines
The concentrations of inflammatory cytokines including IL-1β, IL-4, IL-6 and IL-10 in culture medium or brain lysates were measured by use of ELISA kits.
Measurement of DNA-binding activity on NF-κB and NFAT
The DNA-binding activity of NF-κB and NFAT in primary astrocytes and brain was measured by use of ELISA kits.
Primary astrocytes were fixed with 4 % paraformaldehyde for 30 min, blocked with 2 % BSA for 30 min and incubated with primary antibodies (1:100), then FITC- or Texas red-conjugated secondary antibodies (1:400). Cellular images were viewed under a TE2000-U fluorescence microscope and quantified with use of QCapture Pro 6.0.
Open field activity
The locomotor activity of mice was assessed in a cage (length × width × height: 28.5 × 28.5 × 30 cm). Mice were placed in the central of the cage and allowed to explore the open field for 5 min. The behavior was recorded by video, and the movement distance, percentage of resting time in the zone and trajectory were calculated for each mouse by use of Smart v3.0 software with the Panlab Harvard apparatus (Cornellà, Barcelona, Spain). The floor and internal walls were cleaned with ethanol between each trial.
The nest-building test was performed as described . Each mouse was housed in single cages containing two pieces of cotton (5 × 5 cm). The presence and quality of nests built were recorded by the nesting score, measured on a 5-point scale: 1 = cotton not noticeably touched, 2 = cotton partially torn up, 3 = mostly shredded cotton but often no identifiable nest location, 4 = a markedly nesting site but flat nest, and 5 = a (near) perfect nest. Nesting score was recorded manually at 72-h intervals.
The Y-maze test was performed as described . The Y-maze apparatus consists of three arms of channels made of stainless steel joined in the middle to form a “Y” shape. The mice were placed into one of the arms (start arm) and allowed to explore the maze with only one of the arms closed for 10 min (training trial). After 3 h, mice were placed back in the start arm of the Y maze. Then, mice were allowed to explore all three arms freely for 5 min (test trial). The number of entries into each arm, the distance of movement and the first choice of entry were assessed in video recordings.
Morris water maze (MWM)
MWM was performed as described . A large circular tank (0.8 m diameter, 0.4 m depth) was filled with water (25 ± 1 °C, 20 cm depth), and the escape platform (8 × 4 cm) was submerged 1 cm below the surface. The training section was monitored by a video system. The escape latency and trajectory of swimming were recorded for each mouse. The hidden platform was located at the center of one of the four quadrants in the tank. The location of the platform was fixed throughout the testing. Mice had to navigate using extra-maze cues that were placed on the walls of the maze. From days 1 to 4, mice went through three trials with an inter-trial interval of 5 min. The mouse was placed into the tank facing the side wall randomly at one of four start locations and allowed to swim until it found the platform or for a maximum of 120 sec. Mice that failed to find the platform within 120 sec were guided toward the platform. The animal then remained on the platform for 20 sec before being removed from the pool. The day after the hidden platform training, a probe trial was conducted to determine whether the mouse used a spatial strategy to find the platform. On day 5, the platform was removed from the pool and the mouse was allowed to swim freely for 120 sec. The proportion of time spent in each quadrant of the pool and the number of times the mouse crossed the former position of the hidden platform were recorded.
Results are presented as mean ± SEM. Data from cell studies were evaluated by non-parametric tests. Mann-Whitney U test was used to compare 2 independent groups. Kruskal-Wallis followed by Bonferroni post-hoc analyses was used to account for multiple testing. Data from animal studies were evaluated by parametric tests. Two-way ANOVA followed by LSD test was used for multiple comparisons. The reagent effect and genotypic effect were two independent factors for this analysis. SPSS v20.0 (SPSS Inc, Chicago, IL) was used for analysis. Differences were considered statistically significant at P < 0.05.