All chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO), unless stated otherwise. All cell culture supplies, murine biotinylated 4G8 and 6E10 antibodies, human amyloid beta ELISA kit, and streptavidin Dynabeads T1 were purchased from Invitrogen (Carlsbad, CA). HCT-1026 was synthesized according to literature procedures (Bolla et al. 2005
Cell culture and treatments
The murine neuroblastoma N2a cells obtained from American Type Culture Collection (ATCC, Manassas, VA) were cultured in 1:1 DMEM and OPTI-MEM supplemented with 5% fetal bovine serum, 100U/mL penicillin, and 100U/mL streptomycin. N2a cells stably transfected with the Swedish mutant of human APP (N2a/APPsw a kind gift of Dr. Gopal Thinakaran, University of Chicago) were additionally supplemented with 200 μg/mL G418 which was omitted during all drug treatments. Cells were maintained at 37 °C and 5% CO2. RAW 264.7 mouse macrophage-likecells, provided by ATCC or Dr. J. Cook (University of Illinois at Chicago, Chicago, IL), were maintained in DMEM, supplemented with 1% penicillin-streptomycin and 10% fetal bovine serum and incubated in 5% CO2 at 37°C. For co-culture experiments, N2a/WT cells were plated in 6 well plates at a density of 10 × 104 cells/well. RAW cells were plated at a concentration of 20 × 104 cells/insert on 0.4 μm pore inserts fitted into 6 well plates. After 24 h, the N2a media was replaced with 3 mL fresh DMEM media supplemented with 0.2% FBS and the inserts containing RAW cells were transferred to the N2a 6 well plates. The co-cultured cells were treated with DMSO as vehicle control or different concentrations of flurbiprofen or HCT-1026 for 1 h prior to addition of 1 μg/mL LPS. The inserts were removed 48 h after treatment and the MTT assay was performed on N2a cells to determine cell viability. Rat primary cortical glial cultures were established from cortices of newborn rats (1–2 days old). The cells were grown in minimum essential medium (DMEM) supplemented with 10% fetal calf serum, amino acids, vitamins, D-glucose (5 mmol/L), penicillin (100 IU/mL) and streptomycin (100 μg/mL). Cells were ready to use at day 14.
Amyloid beta measurement from N2a/APPsw cell supernatant
N2a/APPsw cells were plated 24 h before the experiment at a density of 25 × 104 cell/well in a 24 well plate. Cells were washed with PBS (50mM, pH 7.4) before the addition of 500 μL of DMEM supplemented with 0.2% FBS followed by drug treatment for a period of 24 h. Conditioned media was collected, followed by the addition of NaN3 (0.01% final concentration) and a mixture of protease inhibitors (10mM phenanthroline and P2714 protease inhibitor cocktail from Sigma containing AEBSF, aprotinin, bestatin, E-64, leupeptin, and EDTA), and centrifuged for 2 min at a speed of 10,000g. Aβ1–42 levels were determined by sandwich ELISA using a human Aβ1–42 ELISA kit and following the supplied protocol. For immunoprecipitation, cells were treated as described above, but plated in a 6 well plate at a density of 100 × 104 cell/well in 2 mL media. 24 h after treatment, 1 mL of the conditioned media was collected, followed by the addition of NaN3 and protease inhibitors and spiked with Aβ1–43 at approximately 1ng/mL final concentration as an internal standard before performing immunoprecipitation. Following immunoprecipitation the samples were analyzed in a MALDI-TOF instrument for the purpose of measuring the relative abundance of Aβ peptides.
Immunoprecipitation and MALDI/TOF analysis
IP-MALDI-TOF quantification was performed as described previously in detail (Abdul-Hay, 2009
). Briefly, immunoprecipitation of Aβ was performed by the addition of a mixture of biotinylated 4G8 and 6E10 antibodies (Signet, MA) followed by the addition of T1 streptavidin coated magnabeads. Beads were washed with NH4
(10 mM, pH 8.0) and the Aβ peptides were eluted prior to addition of the matrix solution (alpha-cyano-4-hydroxycinnamic acid in 1:1 acetonitrile and water). Analysis was performed using a MALDI-TOF instrument (Applied Biosystems) in linear positive mode at 1950 shot per spectrum. For each individual experiment, peak heights were normalized to the peak height of the Aβ1–43
standard; these normalized values were then expressed relative to the DMSO vehicle control in each set of experiments.
Assay of nitrite production as a measure of endogenous iNOS activation in RAW 264.7 cells was performed as described previously in detail (Hagos et al. 2008
). Cells were plated at a concentration of 25 × 104
cells/well in a 24-well plate and incubated at 37°C for 24 h. The medium was changed, and the cells were drug treated, followed 30 min later by the addition of LPS (Sigma-Aldrich). After 24 h, 100 μL of the supernatant was removed and incubated with the Griess reagent (100 μL) for 30 min at room temperature in the dark. The absorbance was measured at 530 nm and calibrated using a standard curve constructed with sodium nitrite (0–100 μM) in culture media. Nitrite release from nitrate drugs was measured in RAW cell culture without LPS addition and subtracted from the measurements obtained in induced cells to quantify endogenous cellular nitrite production. Drug concentrations were selected to be non-toxic based upon cell viability assays (data not shown). For primary cell cultures, the Griess assay was used as described previously (Bhat et al. 2002
): after pre-incubation with drugs or vehicle for 1 h, cells were incubated for a further 48 h with LPS (1μg/mL), or LPS (1μg/mL) + IFNγ (100 U/mL); aliquots of culture supernatant were mixed with an equal volume of Griess reagent as described above.
After plating for 24 h, RAW 264.7 cells were treated with flurbiprofen and HCT-1026 at different concentrations, and 1 μg/mL LPS was added 30 min later. After a further 24 h, media were collected and immediately treated with 1 N citric acid (40 μL) and 10% butylated hydroxytoluene (5 μL). Before extraction, 20 ul of PGE2
(100 ng/mL) was added to each sample as an internal standard. Prostaglandins were extracted from cell suspensions using 2 mL of hexane/ethyl acetate (1:1 (v/v)). The extraction step was repeated twice, and the organic phases were evaporated to dryness under a stream of nitrogen at room temperature. All extraction procedures were performed under low light and low temperature conditions. Samples were reconstituted in 200 μL of methanol before liquid chromatography/tandem mass spectrometry (LC/MS) analysis as described previously (Yang et al. 2002
). LC/MS was performed using an API 3000 mass spectrometer (Applied Biosystems, Foster City, CA) equipped with HPLC (Shimadzu, Kyoto, Japan) separating on a Luna 3-μm, phenylhexyl, 2 × 150-mm analytical column (Phenomenex, Torrance, CA) using a methanol/ammonium acetate (10 mM; pH 8.5) gradient at a flow rate of 400 μL/min.
The MTT assay was performed by incubating cells in a culture media containing 0.5 mg/mL MTT solution (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) for 4 h, followed by washing with PBS and solubilization of the purple formazan crystals in DMSO. The plate was shaken on a plate rocker for 30 min then the absorbance was measured at 570 nm using 630 nm as the reference wave length on a Dynex MRX II microplate spectrophotometer.
Step-through passive avoidance task
STPA was performed on male C57BL/6 mice (Charles River’s Laboratories) of 8–10 weeks of age and weighing 22–27 g. The mice were kept under standardized laboratory conditions (temperature 21 ± 1 °C, humidity 50–60%) with free access to food and tap water in a room with a natural light–dark cycle. The avoidance experiment was divided into 3 phases: habituation, training and retention phase. The apparatus used consisted of a box divided into 2 compartments, an illuminated compartment adjacent to a dark compartment, the floor consisted of an electric grid controlled by a switch that can deliver an electric shock only to the dark compartment. A doorway was located at floor level in the center of the connecting wall; the door closed once the mouse entered the dark compartment. The habituation phase was performed with no shock, by individually placing the mice in the light compartment; the door was closed once the mouse entered the dark compartment; the mouse left for 15 s before being returned to its cage. The training phase, performed 2 h after habituation consisted of individually placing animals in the illuminated compartment, with an electric shock (0.6 mA for 2 s) delivered immediately after the mouse entered the dark compartment, accompanied by the door closure. Trials were repeated for a maximum of 5 trials or until the mouse remained in the light compartment for 300 s in one trial. The retention test was conducted 48 h after training, consisting of a single repeat of the training protocol without foot shock: The retention trial ended when the mouse entered the dark box or 300 s had elapsed. Scopolamine (1mg/kg) dissolved in physiological saline was administered by i.p. injection 30 min before the training phase and drugs dissolved in 25% DMSO were administered by i.p. injection 20 min before the training.
Radial water maze task
The effect of HCT-1026 on spatial working and reference memory was assayed in a six arm radial water maze (RWM) using a scopolamine-induced cognitive deficit in male Long-Evans rats (200–250 g, Charles River). The RWM contained six swim paths extending out of an open central area with an escape platform located at the end of one arm. For two consecutive days, mice were trained for 16 one-minute trials grouped into 4 blocks (Day 1: blocks 1 & 2; Day 2: blocks 3 & 4; and 4 trials within each block with 15 sec inter-trial interval and 5 min interblock interval) with the hidden platform. Animals were released with their heads pointed toward the wall of the water pool in one arm. Entry into an incorrect arm was scored as an error. The total number of errors made in finding the hidden platform in each trail was counted. If the animal failed to reach the platform after 60 s, it was placed onto the platform for 10 s. Scopolamine (1 mg/kg, i.p.) was administered 30 min and drug (4.5 μmol/kg) or saline 20 min before training was conducted on each day. For each group of animals (N = 8), the mean (SE) number of errors was reported.
Results were analyzed by one-way ANOVA with Dunnett’s or Tukey’s post test as appropriate, using GraphPad Prism software version 5 (San Diego, California). For the Radial Water Maze task, the between group difference (p = 0.02) was assessed using a repeated-measure generalized linear model.