Aβ1 42

Aβ_1-42 was bought from the Peptide Institute (Osaka, Japan). VBIT-4 was bought from ChemPartner (Chengdu, China). Ferrostatin-1, Emricasan, and Necrostatin-1 were purchased from Selleckchem (Selleck Chemicals; USA). Antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). PC12 cells and SH-SY5Y cells were obtained from the Bena Culture Collection (Beijing, China).

Aβ_1–42 (human, 1–42) was purchased from the Peptide Institute (Osaka, Japan). Asiaticoside was purchased from Sigma-Aldrich. The reference dye 5-carboxytetramethylrhodamine (TAMRA) was purchased from Olympus America Inc, whereas TAMRA-Aβ_1–42 was obtained from ANASPEC Inc. CA. Other chemicals were of analytical grade. Uranyl acetate was obtained from BDH. All experiments were carried out with the approval of an appropriate ethics committee of Shimane University compiled from the Guidelines for Animal Experimentation of the Japanese Association for Laboratory Animal Science.

Dissolved lyophilized Aβ_1–42 (PEPTIDE INSTITUTE, INC., Osaka, Japan) in 2 mM NaOH produced a peptide concentration of 0.5 mg/mL, which was then subjected to sonication by a bath-type sonicator for 1 min prior to re-lyophilizing the resulting solution. The NaOH-treated Aβ_1–42 lyophilizate was dissolved in 500 μL distilled water and sonicated for 1 min. We added 500 μL of 20 mM phosphate buffer and centrifuged the resulting solution for 10 min at 16,000 × g. The supernatant was used as the Aβ_1–42 solution. Aggregation of Aβ_1–42 was measured using the SensoLyte® Thioflavin T β-Amyloid (1–42) Aggregation Kit (ANASPEC Inc., Fremont, CA.) according to the manufacturer’s protocol. To measure the Aβ_1–42 fibril formation in the 96-well black microplates, 10 μL of 2 mM ThT and 5 μL of each monoamine solution (2 mM) were added into each well and mixed with 85 μL Aβ_1–42 solution. The final concentration of Aβ_1–42 was 45 μM and that of each monoamine concentration was 100 μM. The ThT fluorescence signal was monitored with Ex/Em = 440 nm/484 nm using Varioskan LUX (Thermo Fisher Scientific) at 37 °C at intervals of 5 min and 10 min for 0–125 min and 140–175 min, respectively.
All samples were tested in triplicate, and data were shown as relative fluorescence units by subtracting the fluorescence level of the blank from the actual value.

Aβ_1–42 and Aβ_1–40 were purchased from Peptide Institute Inc. (Osaka, Japan), and SH-SY5Y cells (human neuroblastoma; EC94030304) were obtained from the European Collection of Authenticated Cell Cultures (London, UK). Dulbecco’s Modified Eagle Medium (DMEM) Ham’s/F-12 medium and all-trans retinoic acid (ATRA) were obtained from Fuji Film Wako Pure Chemical Co., Ltd. (Osaka, Japan). Penicillin G sodium, streptomycin sulfate, amphotericin B, and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific K.K. (Waltham, MA, USA). 17β-estradiol and G-15 were procured from Cayman Chemical Company (Ann Arbor, MI, USA), and raloxifene hydrochloride was obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). The other chemicals used in this experiment were commercially available and of the purest grade. Their structures are shown below (Figure 1). Special-grade products were used for other reagents.

1.1 µM of synthetic Aβ_1–40 and Aβ_1–42 (Peptide Institute, Osaka, Japan) were incubated at 37°C for 3 days with gentle shaking. 4 µl each of Aβ_1–40 and Aβ_1–42 were blotted onto nitrocellulose paper and allowed to dry. The dot blot was then stained with biotinylated β55. β55 was visualized by secondary staining with streptavidin IRDye 700DX (Rockland Immunochemicals, Gilbertsville, PA) and imaged on an Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NB). Western blots of the synthetic Aβ reaction mixtures were also performed to identify the Aβ species present in each reaction mixture. Western blots were stained with 6E10 antibody (1∶1000), which is reactive to amino acid residues 3–8 of Aβ. 6E10 was visualized by secondary staining with anti-mouse IgG antibody conjugated to IRDye800 (Rockland Immunochemicals, Gilbertsville, PA) and imaged on an Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NB).

Aβ1-42 was obtained from the Peptide Institute (Osaka, Japan). Fibril formation of Aβ1-42 (22 μM) was monitored upon measuring the fluorescence intensity of 20 μM thioflavin T (ThT; Sigma-Aldrich, St. Louis, MO, USA) in phosphate-buffered saline (PBS). The lyophilized sample of Aβ1-42 was first dissolved in 0.1% ammonia solution to prepare the stock solution. The stock solution of Aβ1-42 was centrifuged at 53,000 rpm at 4 °C for 3h using a Himac CS120GX ultracentrifuge (Hitachi Koki Co., Ltd., Tokyo, Japan) to eliminate the preformed aggregates. Then, 90 μL of PBS containing ThT in PBS was mixed with 10 μL of the Aβ1-42 stock solution. The fluorescence measurements were carried out using a Genios plate reader (TECAN, Männedorf, Switzerland) with an excitation wavelength of 450 nm and emission wavelength of 485 nm in a polystyrene 96-microwell plate. 4R-Tau (P301L) fibrils with molecular weight 16.0 kDa were purchased from Cosmo Bio Co., Ltd. (Tokyo, Japan).

Intracerebroventricular (ICV) injection was performed as described previously with some modification [19 (link)]. Briefly, each mouse was fixed in a stereotactic frame, anesthetized with Nembutal in saline, and a 28-gauge needle was inserted unilaterally 1 mm to the right of the midline, 0.2 mm posterior to the bregma and 2.5 mm deep to the skull surface. Aβ1–42 (Peptide Institute, Osaka, Japan) dissolved in PBS was injected intracerebroventricularly at 200 pmol in 3 μl PBS at rate of 1 μl/min using a syringe pump. After the injection, the needle was held in the original location for an additional 3 minutes and then withdrawn. For vehicle control mice, 3 μl PBS was injected. The body temperature was maintained with a heat lamp throughout the procedure and recovery. After they were completely alert, mice were returned to their home room, and normal food and water were given. General locomotor activity and diet volume of mice were checked daily and not changed significantly among all groups until sample preparation. Mice were weighed before and one week after ICV injection, and immediately before each analysis, and showed no significant difference at each time point in each group. In addition, we did not observe a significant change in systolic blood pressure among all groups during reagent administration.