Chemicals and antibodies

The FDA-approved oncology drug set II was obtained from the open chemical repository of the National Cancer Institute/National Institutes of Health Developmental Therapeutics Program (NCI/NIH DTP) [27]. 1, 3 bis (2-chloroethyl)-1-nitrosourea (BCNU) or carmustine (cat# C0400), thioflavin S (cat #T1892), ethylene glycol tetraacetic acid (EGTA) (cat # E4378), paraformaldehyde (PFA) (cat # P6148), dimethyl sulfoxide (DMSO) (cat # 472301) and glutaraldehyde (cat # G-7776) were all purchased from Sigma-Aldrich (St. Louis, MO, USA). EZ-Link Sulfo-NHS-LC-biotin was from Pierce (Rockford, IL, USA). The monoclonal antibody Ab9 used for immunoprecipitation of Aβ was purified from supernatants of the hybridoma generated in mice by Biomatik Corporation (Ontario, ON, Canada). The polyclonal antibody CT15 (against C-terminal 15 residues of APP) and the polyclonal antibody, 63G (against mid region of APP) have been described previously [28, 29]. The monoclonal antibody 6E10 (cat # SIG-39300, recognizing 1- 17 of Aβ sequence) was obtained from Covance Research (Denver, CO, USA). Polyclonal anti-sAPPβ-WT antibody (cat # 18957) was purchased from IBL Co. Ltd (Gunma, Japan). Monoclonal anti-Iba1-AIF1 antibody (cat # MABN92) was purchased from Millipore (Billerica, MA, USA). Polyclonal anti-TGF beta 1 antibody (cat # NBP1-67698) was purchased from Novus Biologicals (Littleton, CO, USA). Mouse monoclonal antibody against beta-actin (cat # A00702) was purchased from Genscript USA Inc. (Piscataway, NJ, USA). Secondary antibodies, such as peroxidase-conjugated AffiniPure goat anti-mouse (Code # 115-035-146) and ant-rabbit (code # 111-035-144) immunoglobulin Gs (IgGs), were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). Anti-mouse IgG and anti-rabbit IgG-agarose beads were from American Qualex International (San Clemente, CA, USA). ADAM10 and 17 enzymes, and the substrate for inhibition assays were purchased from R&D Systems (Minneapolis, MN, USA; cat#: 936-AD-020, 930-ADB-010, ES010, respectively).

Quantitation of Aβ, CTFs and sAPPs in 7WD10 cells

The methods for the generation and characterization of CHO cells stably expressing APP751wt (7WD10 cells) for the secretion of Aβ into the conditioned medium (CM) have been described previously [28, 29]. To immunoprecipitate Aβ, 7WD10 cells were grown in six-well plates and were treated with BCNU at final concentrations of 0, 0.5, 1.0, 5.0, 10.0 and 20.0 μM in duplicate wells. After 48 hours, the CM was collected, centrifuged to remove cell debris and immunoprecipitated overnight using a monoclonal Ab9 antibody (recognizes 1 - 16 amino acids of Aβ) to pull-down total Aβ. After SDS-PAGE electrophoresis using NuPAGE 4% to 12% bis-tris gels, total Aβ was detected by immunoblotting using a mixture of 6E10/82E1 antibodies which reliably detects total Aβ as described previously [28, 29]. The CM was also immunoblotted to detect sAPPα (6E10), sAPPβ (anti-sAPPβ-wt rabbit IgG from IBL America Ltd) and sAPPtotal (63G) using the indicated antibodies. To detect APP holoprotein and CTFs (CT15 antibody raised against the last 15 amino acids of APP), the cells were lysed using lysis buffer (1% Nonidet P-40) with complete protease inhibitor mix (Sigma) and equal amounts of proteins were loaded into each well and subjected to SDS-PAGE electrophoresis. Following transfer onto polyvinylidene difluoride (PVDF) membranes, they were blocked with 5% milk in Tris-buffered saline with Tween (TBS-T) and incubated overnight with primary antibodies followed by one to four hours of incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies, such as monoclonal mouse anti-goat IgG light chain or monoclonal mouse anti-rabbit IgG light chain. The protein signals were detected using Super Signal West Pico Chemiluminescent substrate (Pierce). Quantitation of Western blot signals was done using Java-based ImageJ software available freely from NIH.

APP turn-over and surface biotinylation experiments

Near confluent 7WD10 cells in duplicate wells were treated with BCNU at 10.0 mM concentration and after 24 hours, washed two times with cold PBS and incubated with 2.0 mg/ml sulfo-NHS-LC-biotin in PBS, pH 8.0 under ultra-low shaking on ice in the cold room. After an hour of incubation, cells were washed three times in PBS and lysates were prepared using 1% Nonidet P-40 lysis buffer containing complete protease mix as described above. Biotinylated proteins were pulled-down by immunoprecipitation with anti-biotin antibody plus anti-mouse agarose beads. The samples were subjected to SDS-PAGE and APP was detected with CT15 antibody. To assess the effect of BCNU on APP stability, cycloheximide experiments were done essentially as previously described in our published papers [28, 29]. Briefly, 7WD10 cells were incubated with cycloheximide at a concentration of 100 mg/ml in PBS at 37°C in a CO 2 incubator. Treated and untreated cells were harvested and lysed at 0, 15, 30, 60, and 120 minutes. The lysates were processed to detect APP with CT15 antibody as described above.

Measurement of activities of secretase enzymes

Enzyme activities were measured using partially purified enzymes from mouse brain homogenates using commercially available kits for BACE (cat # 565785, EMD Millipore, a division of Merck KGaA, Darmstadt, Germany) and γ-secretase (cat # FP003, R&D Systems) according to the manufacturer's instructions. The fluorogenic substrate used for BACE was glu-val-lys-met-asp-ala-glu-phe-lys, and for γ-secretase was NMA-GGVVIATVK (DNP)-DRDRDR-NH2. The procedure briefly includes membrane isolations from the mouse brains at 4°C by homogenization using an extraction buffer (20 mM HEPES, pH 7.5; 50 mM KCl and 2 mM EGTA). Lysates were centrifuged at 800 g for 10 minutes to remove nuclei and large cell debris in the pellet and the supernatants collected. The pellets were re-homogenized and more supernatants were collected in the same way. The resulting supernatants were pooled and centrifuged at 100,000 g for 1 hour at 4°C. The resulting membrane pellet was washed once in extraction buffer and suspended in the same buffer plus 10% glycerol and flash-frozen in liquid nitrogen and stored at -80°C until used. The total protein concentrations in membrane preparations were determined using the BCA method (Pierce). Membranes were resuspended at 0.5 mg/ml concentrations in resuspension buffer and solubilized at 4°C for one hour with end-over-end rotation. Following centrifugation at 100,000 g for one hour, the supernatants were collected.

The assay mixture consisted of 50 ul of partially purified enzyme preparation, 48 ul of 2 times reaction buffer (20 mM HEPES, pH, 7.0; 150 mM of KCl; 2 mM EGTA; 1% (W/V) CHAPSO) and 2 ul of BACE or γ-secretase substrate. The mixture was incubated for two hours in the dark and fluorescence was read at 320/420 nm for BACE and 355/440 nm for γ-secretase. Some samples also included BACE or γ-secretase specific inhibitors to confirm the specificity of enzyme activity. One negative control without lysate and another without substrate were included. The positive control included was 2.0 ul of recombinant BACE enzyme. The enzyme activity was calculated per mg protein and was expressed as percentage change in BCNU-treated samples from untreated controls.

ADAM10 and 17 inhibition assays followed the same general protocol: 5 μL of 3x enzyme solution (3 and 30 nM) in assay buffer (10 mM HEPES, 0.001% Brij-35, pH 7.5) were added to solid bottom white 384 low volume plates (Nunc cat# 264706). Next, 5 μL of test compounds or pharmacological controls were added to corresponding wells. After 30 minutes incubation at room temperature (RT) the reactions were started by addition of 5 μL of 3x solutions of substrate (30 μM). Fluorescence was measured every 30 minutes for 2 hours using the multimode microplate reader Synergy H4 (Biotek Instruments, Winooski, VT, USA) using λ excitation = 324 nm and λ emission = 405 nm. Rates of hydrolysis were obtained from plots of fluorescence versus time, and inhibition was calculated using rates obtained from wells containing substrate only (100% inhibition) and substrate with enzyme (0% inhibition). The IC 50 value of the pharmacological control ((N-hydroxy-1-(4-methoxyphenyl)sulfonyl-4-(4-biphenylcarbonyl)piperazine-2-carboxamide, Calbiochem cat#: 444252) was also calculated to ascertain the assay robustness.

Cytotoxicity assays

As oncology drugs are generally cytotoxic, we wanted to identify the minimal concentrations of BCNU necessary for cytotoxicity. Neuro-2A (N2a) cells were incubated with BCNU at final concentrations of 0, 0.1, 1.0, 5.0, 10.0, 20.0, 80.0 and 240.0 uM for 24 hours. To determine cell viability, first we used the calorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) metabolic activity assay using a cell growth determination kit (cat # CGD-1, Sigma Aldrich) according to the manufacturer's instructions. Briefly, the supernatant was removed, cells were washed two times with PBS and 20 μl of MTT solution (5 mg/ml in PBS) plus 100 μl of medium were added. Following four hours of incubation at 37°C, the resulting formazan crystals were dissolved in 100 μl of DMSO and the absorbance was read at 570 nm within an hour using the Smart Spec Plus spectrophotometer (Bio-Rad). Cells treated with medium only served as controls.

To reproduce and confirm these results, we also measured cell viability using an in vitro toxicology assay kit based on secretion of lactic dehydrogenase (LDH) (cat # TOX7, Sigma Aldrich). The assay is based on reduction of nicotinamide adenine dinucleotide (NAD) by LDH enzyme into NADH which converts tetrazolium dye to a colored compound that can be quantitated spectrophotometrically. The procedure briefly is as follows: after 24 hours incubation of cells with BCNU at different concentrations, 50 μl of LDH assay lysis solution was added to each well and further incubated at 37°C for 45 minutes. The assay mixture was freshly prepared by adding equal volumes of substrate, dye and cofactor; 50 μl of LDH assay mixture was added to each of the 50 μl aliquots of the test medium. The plates were sealed with aluminum foil to protect from light and incubated for 30 minutes at 37°C. The reaction was terminated by adding 10 μl of 1 N HCl and the absorbance was measured at a wavelength of 490 nm.

Staining of amyloid plaques

APdE9 mice that overexpress both APP with Swedish mutation and PS1 with ΔE9 deletion were used as a robust mouse model of Alzheimer's disease. All animal procedures were carried out strictly following the National Institutes of Health's 'Guide for the Care and Use of Animals' and using the animal protocol as approved (protocol # TPI-03-11) by the Torrey Pines Institute's Animal Care and Use Committee (IACUC). BCNU, dissolved initially in DMSO and further diluted in saline, was administered to mice daily by intraperitoneal injections at 0.5 mg/kg body weight starting from four months of age until six months of age for 60 days. Age and genotype-matched control mice received vehicle injections for the same period of time. Following the treatment period, mice were anesthetized by isoflurane and perfused using a mixture of 4% PFA and 0.02% glutaraldehyde in PBS. After 72 hours, the brains were dehydrated using sucrose gradient. Brains were frozen in optimal cutting temperature (OCT) solution and coronal sections of 16 μM thickness were cut by cryostat at -19°C to 21°C and transferred to superfrost slides. The slides were rinsed twice with distilled water for five minutes. The slides were then immersed in 1% thioflavin S solution prepared in 50% ethanol for five minutes and then differentiated in 70% ethanol for five minutes, rinsed again twice in water for five minutes and cover-slipped with Sure mount (EMS) mounting media and held at 4°C until they were imaged. Images were captured using a Zeiss Examiner D1 microscope. All images were acquired at the same exposure and were automatically aligned using the stitching tool in the Axiovision LE software. Once acquired, all images were opened in ImageJ and were normalized; the threshold was set for each image using the histogram mean at the same standard deviation. Each image was adjusted to the threshold and set to scale in pixels. The parameters measured include the area, integrated density, perimeter, and feret's diameter for each plaque. To help eliminate background the particle size pixel was set at 30-infinity pixel. To quantify plaques, the brain level of cut sections was fixed for all mice at the region of the motor cortex and hippocampus corresponding to the starting section at interaural 2.34 mm and Bregma -1.46 mm of 'The Mouse Brain Atlas' by George Paxinos and Keith Franklin. A fixed thickness of 16 μM coronal sections at regular intervals was maintained in all animals. The amyloid plaques were quantified from throughout the sections from five sections per mouse and mean values were generated for each mouse. Pictures were montaged and, for quantification by image J software, the color images were converted in to HSV format and 8-bit channels. Plaques were quantified in an unbiased manner by an investigator blind to the treatment nature of the samples. Plaque burden was calculated as the area occupied by the plaques divided by the total brain region area. The data are expressed as percent change in means from the controls.

Quantitation of Aβ40 levels in the brain by ELISA

Aβ40 levels in the brain extracts were determined by sandwich ELISA. Briefly, the wet mass of the brain was weighed and homogenized thoroughly in cold 1% CHAPSO/PBS with protease inhibitors. The homogenate was ultra-centrifuged at 100,000 g for 60 minutes. The samples were further diluted to 40-fold and stored on ice until use. The Aβ standard (Bachem) was dissolved in hexafluoroisopropanol at 1 mg/ml, sonicated and dried under nitrogen. The dried Aβ40 was resuspended in DMSO, separated into aliquots and frozen at -80°C. The rest of the protocol is exactly as described previously from our laboratory [28]. The quantity of Aβ40 in each sample was measured in quadruplicate. Total protein concentrations were determined using the BCA assay (Pierce).

Iba1 I mmunohistochemistry

Brain sections (18 um) from saline- and BCNU-treated mice were washed two times with PBS 1X for five minutes. Antigen retrieval was carried out by immersing slides in 10 mM citric acid (pH 6.0) for 10 minutes at 90°C. Sections were washed three times with PBS 1X for five minutes and incubated in blocking solution (10% normal goat serum, 1% BSA, 0.1% Triton X-100 in PBS 1X) for one hour at RT. The sections were incubated overnight with anti-Iba1/ALF1 mouse monoclonal antibody (Millipore) in blocking solution (1:200) at 4°C. After washing three times in PBS 1X for 5 minutes, the sections were incubated with Alexa Fluor® 568 goat anti-mouse IgG (Invitrogen) in blocking solution (1:500) at RT for two hours in the dark. Finally, slides were washed three times with PBS for five minutes, covered with mounting medium for fluorescence with 4',6-diamidino-2-phenylindole (DAPI) (Vector Laboratories) and sealed with nail clear. Sections were visualized in a fluorescence microscope (Axio Examiner D1) and a confocal microscope (Nikon 90i, scan head-C1 SHS, Melles Griot laser system). Microglia were counted in a defined area of the motor cortex and hippocampus (CA3) using the Image-Pro Plus (Media Cybernetics) software package. Positive cells were defined as those whose nuclei and processes were evidently stained for Iba1 and whose nuclei were co-localized with DAPI.

Quantitation of BCNU drug levels in the brain

Two sets of three mice each were injected with BCNU at 4 mg/kg body weight and euthanized after either 5 or 20 minutes. The brains were rapidly removed, frozen and weighed before being homogenized in 250 μL of Dulbecco's PBS (dPBS), immediately followed by 750 μL of acetonitrile. The samples were spun for 10 minutes at 10,000 rpm at 4°C. The supernatant was removed and the samples were dried for two hours. The pellets were left at 4°C overnight, and on the following day samples were reconstituted in 30% acetonitrile, vortexed, and spun at 13,000 rpm for five minutes at 4°C. To assess stability of BCNU in the blood, blood samples were collected from several mice after anesthesia with isoflurane and 200 μL of blood was mixed with 8 μL of EDTA (final concentration 1.5 mg/mL) and about 100 μg of BCNU in 4 μl. The mixture was allowed to stand in a 37°C water bath for 0, 5, 10, 15 and 30 minutes. After the chase time, 600 μL of 100% acetonitrile was added to the sample. The samples were spun at 3,000 g for five minutes and the supernatant was collected. Each sample was made in triplicate, with the exception of the 0 minute blood which was done in duplicate. The blood samples were dried for approximately three hours. The pellet was reconstituted using 100 μL of 30% acetonitrile, vortexed and spun at 13,000 g for five minutes. The samples were transferred to shelf pack vials and run on the UV mass spectrometer. Similarly, to assess BCNU stability in the brain homogenates, three mice were anesthetized with isoflurane, perfused with dPBS and their brains collected. Brains were homogenized in 250 μL of dPBS and 200 μL of brain homogenate was incubated with 100 μg of BCNU for 1, 15, or 30 minutes at 37°C. After the chase time, 750 μL of 100% acetonitrile was added to the sample. The rest of the procedure was similar to the samples prepared for blood. All analyses were performed in triplicate.

The samples were then run on the UV mass spectrometer. The liquid chromatography system (Shimadzu, Kyoto, Japan) consisted of a LC20AD binary solvent delivery pump, a DGU-20A 5 degasser, CTO-20A column oven and a SPD-M20A photodiode array detector. Chromatographic separation was carried out on a C-18 reverse phase column (Luna 50 μ, 100 Å, 50 × 4.6 mm) fitted with a C-18 reverse phase guard cartridge (Phenomenex, 4 × 3.00 mm) and BCNU was eluted using a gradient of solvents A (0.1% formic acid in water) and B (0.1% formic acid in acetonitrile) at 0.5 mL/minute flow rate. The gradient was 5% to 65% B over 20 minutes, 65% to 95% B over one minute, and kept for two minutes and restored to 5% B in one minute followed by re-equilibration for five minutes. The peak for BCNU was monitored at 280 nm by injecting 5 μL of the sample.

Statistical analysis

The signal intensities of immunoblots of the samples treated with BCNU in CHO cells as well as plaque burden in the mouse brain were quantified using publicly available Java-based ImageJ software developed at the National Institutes of Health. All data were analyzed by Student's t test using Instat3 software (GraphPad Software, San Diego, CA, USA). We used a two-tailed P value assuming populations may have different standard errors. The data for dose-response experiments were analyzed by analysis of variance (ANOVA) followed by either Dunnett or Bonferroni multiple comparison tests. The data were considered significant only if the P < 0.05, * indicates P < 0.05, **, P < 0.01 and ***, P < 0.001.