The present study is reported in accordance with ARRIVE guidelines.
Animals and treatment
Four-week-old female 3×Tg-AD mice harboring three mutant genes (APPswe, PS1 M146V, and tau P301L) 17 were purchased from the Jackson Laboratory, Bar Harbor, ME, USA. The mice were housed individually at a temperature of 24°C + 2°C at 40%–50% relative humidity and a 12 h light-dark cycle with ad libitum access to sterilized water. Starting at 1 month of age, the mice were divided into two groups: the Control group was fed with a Control diet (AIN-76 diet) 19 (n = 9), and the Mucuna group was fed with a diet containing Mucuna bean powder (Mucuna diet, n = 9) for 13 months. The Mucuna bean powder used for preparing the Mucuna diet was processed as follows: Mucuna beans (whose chemical composition is shown in the Table S1), were of a gift from a farmer who had been cultivating this bean in Kumamoto prefecture in Japan. The written consent of the beans to be used for this research has been obtained. The use of the Mucuna beans in the present study complies applicable with international, national and /or institutional guidelines. The Mucuna beans were soaked in distilled water for 24 h and then autoclaved at 121°C for 40 min, lyophilized, and milled (New Power Mill, Osaka Chemical Co., Ltd., Japan). The Mucuna bean powder contained 2.88% L-DOPA. The daily intake of L-DOPA derived from Mucuna bean powder was adjusted to 2 mg/kg body weight/day per mouse throughout the feeding period. The Mucuna bean powder content in the Mucuna diet varied from 0.028% to 0.091% in accordance with body weight. Body weight was monitored weekly. At the age of 14 months, mice were sacrificed under anesthesia with 200mg/kg body weight of sodium pentobarbital delivered intraperitoneally, and the brain of each mouse was quickly harvested and sagittally bisected. The left hemisphere was fixed in 4% paraformaldehyde overnight for histological studies. The right hemisphere was dissected to isolate the striatum, hippocampus, and cerebral cortex and was stored at −80°C for biochemical analyses.
All experiments reported herein were approved by the Animal Care Advisory Committee of Kagawa Nutrition University and were performed in accordance with the relevant guidelines and regulations (Permit Number: 18-2).
Behavioral test: Y-maze test
The Y-maze apparatus consisted of three arms (38 cm long, 12.5 cm wide, and 12.5 cm deep; Sanki Kagaku Kogei, Japan). Each mouse, at the age of 12 months, was placed at the center of the maze and allowed to freely explore the maze for 5 min. An entry into an arm was considered complete when all four limbs were within the arm. An alteration was defined as three consecutive entries into three different arms (A, B, C or B, C, A, etc.) 20. The percentage alteration score was calculated as follows: the total alteration number/(total number of entries − 2) × 100.
Preparation of recombinant Tau protein
Recombinant human tau protein (2N4R) cDNA in pET vectors was expressed in BL21 (DE3) Escherichia coli cells and purified as previously reported 13. After E. coli expressing tau was sonicated and boiled, recombinant tau proteins in the heat-stable fraction were purified using ion-exchange chromatography (Cellufine Phosphate; JNC Corp), ammonium sulfate fractionation, gel filtration chromatography (NAP10 column; GE Healthcare), and reverse-phase HPLC (COSMOSIL-R Waters; Nacalai Tesque Inc.). After freeze-drying, recombinant tau proteins were dissolved in Milli-Q water and stored at −80°C.
Thioflavin T assay
Mucuna bean extracts for the Thioflavin T (ThT) assay were prepared as follows: Mucuna beans were incubated in distilled water at 100°C for 4 h and boiled for 1 h. Boiled Mucuna beans were homogenized in 0.4 M phosphate buffer (pH = 4.0) and centrifuged (9,000 rpm, 10 min, 4°C). The supernatant was filtered through 0.45 μm filters and subjected to a ThT assay. The ThT assay for Aβ was performed as previously reported 21. Mucuna bean extracts (extracted from 0.5 mg or 5 mg dried bean powder containing 10 μM L-DOPA or 100 μM L-DOPA), Aβ1-42 (10 μM, PEPTIDE INSTITUTE, INC, Osaka, Japan), and ThT (10 μM) were mixed in PBS and incubated at 37°C. The ThT fluorescence intensity was recorded with an Ex/Em = 450 nm/492 nm using a microplate-reader fluorometer (Corona Electric, Japan). The ThT assay for tau was also performed as previously reported 13. Recombinant wild-type 2N4R tau (10 μM), Mucuna bean extracts (extracted from 0.5 mg or 5 mg dried bean powder containing 10 μM L-DOPA or 100 μM L-DOPA), and ThT (10 μM) were mixed in HEPES buffer (10 mM HEPES, pH = 7.4; 100 mM NaCl) and incubated with heparin (0.06 mg/ml; Acros Organics) at 37°C. The ThT fluorescence intensity was recorded every hour with an Ex/Em = 420 nm/500 nm using a multiplate-reader fluorometer (1420 ARVO MX, Perkin Elmer). All samples were tested in triplicate.
L-DOPA analyses
The L-DOPA content of Mucuna bean extracts or powder was analyzed using a reversed-phase column WAKOSIL Ⅱ 5C18 RS (Φ4.6 × 250mm). This column was placed in a JASCO CO-2067 Plus HPLC column oven and analysis was conducted at 40℃. The mobile phase was phosphate buffer (pH = 2) / methanol (90:10) with a flow rate of 1 mL/min. The eluate was monitored at 200 nm.
Tissue extraction for biochemical studies
Tissue extraction was performed as previously described 13. Frozen brain tissues (striatum, hippocampus, and cerebral cortex) were homogenized in 7.5 volumes of TBS buffer containing 50 mM Tris (pH = 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, protease inhibitors, and phosphatase inhibitors. The homogenates were centrifuged (23,000 rpm, 15 min, 4˚C) in a TLA-55 rotor (Beckman Coulter) and separated into the supernatant (TBS-soluble fraction) and pellet. The pellets were resuspended in 0.32 M sucrose containing 10 mM Tris (pH = 7.4), 0.8 M NaCl, and 1 mM EGTA and were then centrifuged (23,000 rpm, 15 min, 4°C) in a TLA-55 rotor. Supernatants were collected and treated with 1% Sarkosyl for 1 h at 37°C. They were then centrifuged (60,000 rpm, 1 h, 4°C) in a TLA-110 rotor and separated into the supernatant and pellet (Sarkosyl-insoluble fraction). Samples from TBS-soluble and Sarkosyl-insoluble fractions were dissolved in Laemmli SB including 2-mercaptoethanol and were then boiled for 5 min.
Immunoblotting analysis
Immunoblotting analysis was performed as previously described 22. The protein concentration of the TBS-soluble and Sarkosyl-insoluble fractions was determined using a DC protein assay kit (Bio-Rad, Hercules, CA, USA). Fifty-microgram protein from the TBS-soluble fraction was applied to NOVEX 10%–20% Tricine gel (Thermo Fisher). The separated proteins were transferred to nitrocellulose membranes (Millipore, Billerica, MA, USA) and were subjected to heat treatment (boiling in PBS, 15 min). After blocking for 60 min at room temperature with 2.5% skim milk in PBS containing 0.05% Tween 20, the membranes were incubated for 40 h at 4°C with primary antibody (6E10, 1:1000, BioLegend). After washing the membrane with PBS containing 0.05% Tween 20, the blots were incubated with horseradish peroxidase-linked secondary antibodies at room temperature. Immune complexes were visualized by Western Lightning Ultra (Perkin Elmer).
Thirty-microgram protein from the Sarkosyl-insoluble fraction was applied to NOVEX 5%–20% e-Pagel (ATTO). The separated proteins were transferred to nitrocellulose membranes (Millipore). After blocking for 1 h at room temperature with 2.5% skim milk in PBS containing 0.05% Tween 20, the membranes were incubated overnight at 4°C with primary antibody (AT8, 1:1000, Thermo Fisher Scientific). After washing the membrane with PBS containing 0.05% Tween 20, the blots were incubated with horseradish peroxidase-linked secondary antibodies (anti-mouse IgG, GE Healthcare) at room temperature. Immune complexes were visualized by Western Lightning Ultra (Perkin Elmer). The results were analyzed by densitometry (NIH Image J-Fiji, v-1.45 software) and expressed as percentage of Control values.
Immunohistochemistry
Immunohistochemical analysis was performed as previously described 22. The left hemisphere was fixed in 4% paraformaldehyde overnight. Paraffin-embedded sections (sectioned in the coronal plate at 4 μm thick) were deparaffinized. For antigen retrieval, the sections for Aβ were treated with proteinase K (DAKO, S3004) for 10 min and then treated with 90% formic acid for 5 min. The sections for phosphorylated tau were treated with Target Retrieval Solution (pH 9.0; DAKO, S2367) at 120°C for 10 min. Then, both sections were immersed in 0.3% hydrogen peroxide in methanol for 10 min. After treating with 5% normal rabbit serum for 10 min, the sections were incubated overnight at 4°C with primary antibodies that were pre-mixed with secondary antibodies (anti-mouse IgG2b, goat Fab) at room temperature for 20 min. Primary antibodies for Aβ and phosphorylated tau were 4G8 (1:1000, BioLegend) and AT8 (1:1000, Thermo Fisher Scientific), respectively. The secondary antibodies were visualized using a HistoFine kit (Nichirei, Japan), followed by a 3,3′-diaminobenzidine reaction. Finally, the sections were counterstained with hematoxylin and observed using an Olympus BX63 microscope equipped with an Olympus DP73 digital camera. Consecutive sections were incubated in the absence of primary antibodies to ensure the specificity of staining.
Quantification of the 4G8-positive Aβ area and the AT8-positive tau area in the brain was performed using Image J-Fiji (v-1.45 NIH) after adjusting for the threshold, and the results were expressed as percentage of Control values.
Statistical analysis
Values are expressed as means ± SD (or SEM). Differences were analyzed using Student’s t-test, Welch’s t-test, or one-way analysis of variance followed by Tukey’s multiple comparisons test. Statistical significance was considered when p < 0.05.