Cellular Aβ assay
SK-N-BE(2) human neuroblastoma cells were maintained in RPMI1640 medium supplemented with 10% fetal bovine serum and penicillin/streptomycin. Cells were cultured in 96-well plates overnight and then treated with each drug at various concentrations in 0.5% dimethylsulfoxide for 6 h. Aβ1-42 levels in the media were measured using the 384-well plate-based sandwich-ELISA system constructed with 44A3 anti-Aβ42 monoclonal antibody (IBL, Gunma, Japan), biotin-labeled 82E1 anti-Aβ N-terminal monoclonal antibody (IBL), streptavidin-horseradish peroxidase conjugate (Invitrogen, Carlsbad, CA, USA), and TMB as the chromogen. Aβ1-40 levels in the media were measured using an ELISA kit (IBL). Cell viability was evaluated using the CellTiter-Glo Luminescent Cell Viability Assay (Promega, Fitchburg, WI, USA).
Tg2576 mouse brains at age 6, 10, 14, and 18 months were immersion-fixed in 4% paraformaldehyde/phosphate buffer for 2 days at 4°C, then placed in 16% sucrose/phosphate buffer for 2 days at 4°C. The brains were frozen and cut into 20-μm-thick coronal sections with a cryostat and then incubated with a mixture of anti-Aβ42 and anti-Aβ40 polyclonal antibodies (diluted 1 : 1000; IBL) on free-floating overnight at room temperature. Aβ immunoreactivity was visualized using the Vector Elite horseradish-peroxidase ABC kit (Vector Laboratories, Burlingame, CA, USA) with 3,3′-diaminobenzidine as the chromogen.
Spatial working memory in mice was evaluated by recording spontaneous alternation behavior in the Y-maze task as previously described (Mitani et al. 2012). Briefly, each drug was orally administered once daily for 8 days. Three hours after final administration, each animal was placed at the end of one arm of the apparatus and allowed to freely explore for 8 min. The alternation rate was defined as entries into all three arms on consecutive occasions using the following formula:
Alternation rate (%) = Number of alternations/(Number of total arm entries−2) × 100
Data were eliminated in cases where the number of total arm entries was less than 10.
Quantitation of hippocampal Aβ
Tris-buffered saline (TBS)-soluble Aβ in the hippocampus was quantified via ELISA as previously described (Mitani et al. 2012). Briefly, the hippocampus was isolated immediately after the Y-maze test followed by storage at −80°C. Frozen samples were homogenized in an ultrasonic homogenizer with 10-fold volume of TBS (Tris 25 mM, NaCl 137 mM, KCl 2.68 mM; pH 7.4) containing Complete Protease Inhibitor Cocktail (Roche Diagnostics, Mannheim, Germany) followed by ultracentrifugation at 100 000 g and 4°C for 1 h. Levels of Aβ1-42 and Aβ1-40 in the supernatant were measured using ELISA kits (IBL). To quantify insoluble Aβ, aliquots of the homogenate were solubilized with final 5 M guanidine hydrochloride for 1 h at 23 ± 3°C and applied to ELISA.
Stable isotope-labeling study
Newly synthesized Aβ in the Tg2576 mouse brain was measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS) as previously described (Bateman et al. 2006; Castellano et al. 2011) with some modifications. Mice, aged 10 and 18 months, were injected intraperitoneally with 300 mg/kg 13C6-labeled leucine (Cambridge Isotope Laboratories, Andover, MA, USA) dissolved in saline at 20 mg/mL followed by the oral administration of 10 mg/kg GSM-2 or vehicle. Brains were harvested 2 h after injection and hemisected, including removal of the olfactory bulb and cerebellum, followed by snap freezing with liquid nitrogen and storage at −80°C. Frozen hemispheres were weighed and homogenized in a teflon homogenizer with 1 mL TBS containing 1% Triton X-100 and Halt protease inhibitor cocktail (Thermo Scientific, Rockford, IL, USA) on ice followed by ultracentrifugation at 100 000 g and 4°C for 1 h. Small aliquots of supernatant were subjected to Aβ x-42 ELISA (Wako, Osaka, Japan).
The remaining supernatant was incubated with 5 μg of 44A3 anti-Aβ42 monoclonal antibody and Protein G-Sepharose 4 fast flow beads (GE Healthcare, Uppsala, Sweden) for 16 h at 4°C, then separated using spin columns. Small aliquots of the solution separated from the beads were subjected to Aβ x-40 ELISA (Wako), and the remaining solution was immunoprecipitated with 5 μg of anti-Aβ40 polyclonal antibody (IBL). All beads that trapped either Aβ42 or Aβ40 were washed with TBS and 25 mM ammonium bicarbonate. Aβ was eluted with 1,1,1,3,3,3-hexafluoroisopropanol, then dried and re-suspended with 25 μL of 20% acetonitrile in 25 mM ammonium bicarbonate followed by digestion with 1 μg Sequencing Grade Modified Trypsin (Promega) for 18 h at 37°C.
Samples were then subjected to an API 2000 electrospray ionization-tandem mass spectrometer (Applied Biosystems, Carlsbad, CA, USA) automatically tuned with a synthetic Aβ17-28 peptide, LVFFAEDVGSNK (Peptide Institute, Osaka, Japan), and coupled to an Agilent 1100 high-performance liquid chromatography system (Agilent Technologies, Santa Clara, CA, USA). Samples were injected into (10–20 μL each) and separated using a CAPCELL PAK C18, 3 μm particle, 2.0 × 35 mm column (Shiseido, Tokyo, Japan) at a flow rate of 0.5 mL/min and with a gradient mixture of solvents A (5% acetonitrile and 0.1% formic acid in water) and B (5% water and 0.1% formic acid in acetonitrile), using the following program: 10% B for 1 min, 10% to 30% B in 0.1 min, 30% B for 2 min, 30 to 90% B in 0.1 min, and 90% B for 2.5 min.
Aβ17-28 tryptic peptide and its 13C6-leucine-labeled form were monitored with multiple reaction monitoring (MRM) of m/z ([M + 2H]2+) 663.5 213.3 and 666.5 219.4, respectively. The 13C6-labeling rate was calculated from the peak height of each peptide form, since the peak height of the synthetic peptide was proportional to its amount in the range 2–10 000 fmol (r2 = 0.999), which covered all sample measurements. Newly synthesized Aβ amount was calculated using the following formula:
Newly synthesized Aβ amount = ELISA measured Aβ amount × 13C6-labeling rate.
Data are presented as mean ± SEM or individual plots, and all statistical analyses were conducted using SAS software (SAS Institute, Cary, NC, USA). The Dunnett's multiple comparison test was used for the concurrent studies of GSM-2 and the enantiomer. The unpaired Student's t-test was used for the study of only GSM-2 or comparison between Tg2576 group and wild-type group. For all tests, a value of p < 0.05 was considered statistically significant.