Animals
Three-week-old male ICR mice were purchased from Sankyo Labo Service Co. (Toyama, Japan). Mice were fed an ERGO-free diet (Basal Diet 5755, PMI Nutrition International) to prepare ERGO-deficient mice, following a previously reported protocol 19, whereas mice fed a normal diet (PicoLab® Rodent Diet 5053, PMI Nutrition International) were used as controls. Mice were kept in a room under pathogen-free conditions at a controlled temperature (21–25°C) with a 12 h dark/light cycle and fed food and water ad libitum. To evaluate the effect of ERGO on cognitive decline, ERGO-deficient and normal mice at the age of 9 weeks were orally administered ERGO dissolved in sterile distilled water at 0, 2, or 20 mg/kg on days 0, 2, 4, 7, 9, and 11, followed by behavioral testing. To examine the effect of ANA-12, an inhibitor of TrkB, ERGO-deficient mice at the age of 6 weeks were orally administered ERGO (50 mg/kg) on days 0, 2, 4, 7, 9, and 11, and the mice were administered intraperitoneally with ANA-12 (0.5 mg/kg) or vehicle (1% DMSO) daily or 30 min before each administration of ERGO.
Behavioral tests
NORT and SRT were performed to assess learning and memory abilities according to the method described by Nakamichi et al. (19), with minor modifications. Fourteen days after the first administration of ERGO, NORT was performed. Each mouse was placed in an acrylic chamber (45 cm × 45 cm × 45 cm) without any objects and allowed to explore for 10 min. The following day, the mouse was placed in the same chamber with two identical objects located on a diagonal line and allowed to explore the chamber for five minutes. The time spent exploring each object was then recorded. Eight hours later, one of the objects was replaced with a novel object of a different shape at the same location in the chamber. Each mouse was allowed to explore the chamber for five minutes. The exploration time for each object was recorded. The DI, an indicator of learning and memory ability, was calculated as: [(novel object exploration time/total exploration time) − (familiar object exploration time/total exploration time)] × 100.
The SRT was conducted on the day following NORT. Each mouse was placed in an acrylic chamber (45 cm × 45 cm × 45 cm) without any objects and allowed to explore for 10 min. The next day, the mouse was placed in the same chamber with two identical objects located on a diagonal line and was allowed to explore the chamber for nine minutes. The time spent exploring each object was then recorded. One hour later, one of the objects was moved, and each mouse was allowed to explore the chamber for five minutes. The exploration time for each object was recorded. The DI was calculated as: [(moved object exploration time/total exploration time) − (unmoved object exploration time/total exploration time)] × 100.
In the experiment evaluating the effect of ANA-12, 8 h of retention time in the NORT was increased to 24 h, and 9 min of exploration time in the SRT was reduced to 5 min, without any change in other experimental conditions.
Immunohistochemistry
After the behavioral tests, each mouse was anesthetized with isoflurane and subjected to transcardial perfusion with 5 mL PBS and 20 mL 4% paraformaldehyde in phosphate buffer. Each brain sample was isolated and incubated with 4% paraformaldehyde in phosphate buffer at 4°C for 24 h. After washing with PBS, each brain was sectioned with a Neo-LinearSlicer (DOSAKA EM, Kyoto, Japan) at a thickness of 100 µm. The sections were incubated for 30 min in 0.1% Tween 20 in PBS (PBST) containing 3% bovine serum albumin (BSA) at room temperature. They were incubated with PBST containing goat anti-doublecortin (Dcx) antibody (1:500 dilution; sc-8066, goat, Santa Cruz; Dallas, TX, USA) and mouse anti-NeuN antibody (1:500 dilution; MAB377, mouse, Merck Millipore; Burlington, MA) overnight at room temperature, followed by washing with PBST and probing with anti-goat Alexa555 (1:500 dilution; A21432, donkey, Sigma-Aldrich; St. Louis, MO) and anti-mouse Alexa488 (1:500 dilution; A21202, donkey, Thermo Fisher Scientific; Waltham, MA) overnight at room temperature. The sections were rinsed again with PBS, treated with a mounting medium containing 2-(4-aminophenyl)-1H-indole-6-carboxamidine, and observed under an LSM710 confocal laser-scanning microscope (Carl Zeiss, Jena, Germany). The newborn neuron marker Dcx and the neuronal nuclei marker NeuN were colored red and green, respectively. Quantification was performed by measuring the immunoreactive area of Dcx or NeuN in double immunocytochemical analysis.
Clinical study
A randomized, placebo-controlled, double-blind, parallel-group clinical study (placebo group, n = 23; group treated with ERGO-containing tablets, n = 25), including healthy volunteers and subjects with MCI, was conducted for 12 weeks, as previously described 21. Subjects were divided into two groups based on their age and MMSE scores. Participants in the ERGO-treated group were orally administered four food extract tablets containing 5 mg ERGO daily with water, whereas those in the placebo group were administered ERGO-free tablets in the same manner as the ERGO-treated group. Neither tablet could be distinguished by its appearance or smell. All participants were instructed to refrain from eating foods containing ERGO, such as mushrooms, during the study period. Serum collection and cognitive function tests were performed using Cognitrax at weeks 0, 4, 8, and 12.
Measurement of ERGO and its metabolites
Hippocampal samples were weighed and homogenized using a Precellys homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France) in 12 volumes of methanol containing L-(+)-ergothioneine-d9 (ERGO-d9; Toronto Research Chemicals Inc., North York, ON) as an internal standard, whereas plasma and serum samples were mixed with nine volumes of methanol containing ERGO-d9. The mixtures were centrifuged (21,500 × g, 10 min, 4°C) twice and subjected to liquid chromatography-mass spectrometry/mass spectrometry (LCMS-8040; Shimadzu, Kyoto, Japan) equipped with a Luna 3.0 µm HILIC column (200 Å, 150 × 2.0 mm; Phenomenex, Torrance, CA) to quantify ERGO, S-methyl-ERGO, and hercynine. Chromatography was performed using step-gradient elution (flow rate, 0.4 mL/min) as follows: The mobile phases were (A) 10 mM ammonium acetate/0.1% formic acid/80% water/20% acetonitrile and (B) 10 mM ammonium acetate/0.1% formic acid/5% water/95% acetonitrile. 0–0.5 min: 1% A/ 99% B; 0.5–1.9 min: 1% A/ 99% B to 20% A/ 80% B; 1.9–4.5 min: 20% A/ 80% B; 4.5–5.5 min: 20% A/ 80% B to 60% A/ 40% B; 5.5–6.5 min: 60% A/ 40% B; 6.5–6.7 min: 60% A/ 40% B to 1% A/ 99% B; 6.7–7.2 min: 1% A/ 99% B. Each compound was measured in the ESI positive mode (ERGO: 230.3 > 127.0, S-methyl-ERGO: 244.1 > 140.95, hercynine: 198.0 > 95.05, ERGO-d9: 239.2 > 127.0). The area under the serum concentration curve (AUC) of ERGO subtracted from the baseline level of ERGO at week 0 (ΔAUC) was calculated using the following equation:
$${\Delta }\text{A}\text{U}\text{C}={\sum }_{n=1}^{3}2\left({y}_{n-1}{+y}_{n}{-2y}_{0}\right)$$
where \({y}_{0}\), \({y}_{1}\), \({y}_{2}\), and \({y}_{3}\) represent the serum ERGO concentrations at weeks 0, 4, 8, and 12, respectively.
Isolation of EVs from serum
One milliliter of serum was first centrifuged at 1,200 × g for 20 min, and the supernatant was then centrifuged at 17,000 × g for 20 min. The collected supernatant was passed through a 0.22 µm filter, followed by ultracentrifugation at 100,000 × g for 90 min (TLS-55 Rotor, Optima MAX-TL, Beckman Coulter, Brea CA). The pellets were resuspended in 50 µL of phosphate-buffered saline (PBS) and used for each experiment.
Nanoparticle analysis
The concentration and size distribution of EVs were measured with tunable resistive pulse sensing by qNano (Izon Science, UK) using an NP100 nanopore (Izon Science, UK), according to the method described by Lane et al45 with minor modifications. All measurements were calibrated with 100 nm polystyrene beads (CPC100; Izon Science, UK). Data analysis was carried out using Izon Control Suite software v3.1 (Izon Science, UK).
Isolation of brain-derived EVs via immunoprecipitation
Serum EVs were isolated via size exclusion chromatography according to the method described by Böing et al. (46), with minor modifications. Briefly, the tip of a 10 mL plastic syringe (Terumo, Tokyo, Japan) was stuffed with nylon stocking (DAISO, Tokyo, Japan), and the syringe was stacked with 5 mL of washed Sepharose CL-2B (GE Healthcare, Uppsala, Sweden). After equilibration with PBS, 1 mL of serum was loaded onto the column and eluted with PBS. The eluate was collected into 25 sequential fractions (0.5 mL). The number and size of particles were determined using qNano for each fraction, and particles were detected in fractions 6–12. These fractions (3.5 mL) were combined and concentrated to approximately 200 µL using Amicon Ultra-15 (Merck Millipore, Burlington, MA, USA). To isolate brain-derived EVs, an antibody against the mature neuronal marker SNAP25 (1:25 dilution; sc-390644, mouse, Santa Cruz; Dallas, TX, USA) was first incubated with Protein G Sepharose 4 Fast Flow (Cytiva, Marlborough, MA, USA) in PBS containing 0.01% Triton-X 100 and phosphatase inhibitors (10 mM sodium fluoride, 10 mM β-glycerophosphate disodium salt hydrate, 10 mM sodium pyrophosphate decahydrate, and 1 mM sodium orthovanadate) at 4°C for 2 h, followed by blocking with PBS containing 10% BSA and phosphatase inhibitors. The beads and EV samples were then mixed and incubated under rotation at 4°C for 2 h. The samples were centrifuged at 15,000 × g for 2 min at 4°C, and the supernatant was discarded. The pellets were washed twice with PBS containing 0.01% Triton-X 100 and phosphatase inhibitors, followed by western blotting as described below.
Western blotting
Western blotting was performed according to the method described by Ishimoto et al. (28), with minor modifications. Briefly, isolated hippocampal dentate gyrus and EVs were homogenized using a tip sonicator (TOMY SEIKO Co. Ltd., Tokyo, Japan) with 20 mM Tris–HCl buffer (pH 7.5) containing 1 mM EDTA, 1 mM EGTA, protease inhibitors (0.1 mM 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, 1 µg/mL leupeptin, 1 µg/mL antipain, and 0.5 mM benzamidine hydrochloride hydrate), and phosphatase inhibitors (10 mM sodium fluoride, 10 mM β-glycerophosphate disodium salt hydrate, 10 mM sodium pyrophosphate decahydrate, and 1 mM sodium orthovanadate), followed by protein concentration measurements with a Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific, Waltham, MA). The homogenates were added at a volume ratio of 1:4 to 10 mM Tris–HCl buffer (pH 6.8) containing 10% glycerol, 2% sodium dodecyl sulfate, 0.01% bromophenol blue, and 5% 2-mercaptoethanol, and the mixtures were incubated on a shaker at room temperature for 1 h, followed by incubation at 95°C for 5 min. Each aliquot of 10 µg of protein from the hippocampal dentate gyrus was loaded onto a 10% polyacrylamide gel for electrophoresis at a constant current of 40 mA/plate for 90 min at room temperature using a PAGE system (Sima Biotech, Chiba, Japan). For EV samples, each aliquot of 20 µg of protein was loaded on a 12.5% polyacrylamide gel for electrophoresis at a constant current of 21 mA/plate for 30 min at room temperature using a compact-slab size PAGE system (ATTO, Tokyo, Japan), followed by blotting to a polyvinylidene fluoride membrane pretreated with 100% methanol. For blocking, BSA or skim milk dissolved in 20 mM Tris–HCl buffer (pH 7.5) containing 137 mM NaCl and 0.1% Tween 20 were used depending on the antibodies (2% BSA: p-TrkB, TrkB, NT-5, Flag, and β-actin; 1% skim milk: SNAP25; 3% skim milk: CD63). The membranes were probed with antibodies against TrkB (1:1,000 dilution; 4603S, rabbit, Cell Signaling Technology; Danvers, MA), p-TrkB at Tyr816 (1:100 dilution; ABN1381, rabbit, Merck Millipore; Burlington, MA), NT-5 (1:250 dilution; AB1781SP, rabbit, Merck Millipore; Burlington, MA), SNAP25 (1:50 dilution; sc-390644, mouse, Santa Cruz; Dallas, TX), CD63 (1:100 dilution; sc-15363, rabbit, Santa Cruz; Dallas, TX), Flag (1:1,000 dilution; 14793S, rabbit, Cell Signaling Technology; Danvers, MA), and β-actin (1:10,000 dilution; A5441-.2ML, mouse, Sigma-Aldrich; St. Louis, MO) diluted with Can Get Signal Solution 1 (TOYOBO, Osaka, Japan), followed by probing with a secondary anti-rabbit IgG antibody conjugated with peroxidase (1:2,000 dilution; 7074, goat, Cell Signaling Technology; Danvers, MA) or anti-mouse IgG antibody conjugated with peroxidase (1:10,000 dilution; A9917-1ML, goat, Sigma-Aldrich; St. Louis, MO) diluted with Can Get Signal Solution 2. Proteins that reacted with these antibodies were detected with ECL™ detection reagents using a lumino image analyzer (LAS-4000; FUJIFILM, Tokyo, Japan). Densitometric analysis of the western blots was performed using ImageJ software.
Plasmid construction
pAAV-CMV-ZsGreen1 was purchased from Takara Bio. TdTomato was amplified from the pCSCMV:tdTomato plasmid (Addgene plasmid #30530; http://n2t.net/addgene:30530; RRID: Addgene_30530) 47. Murine TrkB-3xFlag was amplified from the murine hippocampus using the DNA polymerase KOD FX NEO with the following sense and antisense primers: 5'-tgtaatcgatgtcatgatctttataatcaccgtcatggtctttgtagtccatCGCGCCTAGGATATCCAG-3', 5'-tcacagggatgccacccgtggatccTCACTTGTCATCGTCATCCTTGTAATCGATGTCATGATCTTTATA-3'. The sequence encoding tdTomato in pAAV-CMV-tdTomato was replaced with TrkB-3xFlag using a NEBuilder HiFi DNA assembly kit (New England Biolabs).
Cell culture
Neuro2a cells were cultured according to the method described by Nakamichi et al. (24), with minor modifications. Briefly, Neuro2a cells were seeded at a density of 1.2 × 106 cells/dish on φ10 cm dishes in DMEM supplemented with 100 units/mL penicillin, 100 µg/mL streptomycin, and 10% EV-depleted FBS, which was the supernatant after ultracentrifugation at 100,000 × g for 16 h at 4°C. After 24 h, the cells were transiently transfected with pAAV-CMV-TrkB-3xFlag or pAAV-CMV-tdTomato using PEI MAX (Polysciences, Warrington, PA, USA), according to the manufacturer’s instructions, and the medium was changed to DMEM supplemented with 2% EV-depleted FBS and 20 µM retinoic acid for neuronal differentiation. At 48 h after seeding, the medium was changed to DMEM containing 100 nM of the TrkB agonist 7,8-dihydroxyflavone or vehicle. At 72 h after seeding, the cells and medium were collected. The cultures were maintained in a humidified atmosphere of 5% CO2/ 95% air at 37°C.
Statistics
Data were expressed as means ± standard deviation (SD). The statistical significance of the differences was determined using the Student's t-test or one-way or repeated measures ANOVA, followed by Tukey’s post hoc test. Correlations were determined using Pearson's correlation coefficient after the Kolmogorov-Smirnov test. Statistical analyses were performed using Prism 7 (GraphPad Software, San Diego, CA, USA) and IBM-SPSS version 25.
Study approval
Animal studies were approved by the Committee on the Ethics of Animal Experiments of the University of Kanazawa (permit number: AP-183968) to minimize animal suffering and loss of life. The clinical studies were conducted following the Declaration of Helsinki and approved by the Institutional Review Boards of Kanazawa University and the Japan Food Evidence Association. All participants provided written informed consent for the use of their samples and medical information for research purposes.