In vitro studies
Synthesis of ZGM1
(2,3-Bis (methoxymethoxy)phenyl)-5,7-dimethoxychroman-4-one (0.2 mmol) was dissolved in 2 M HCl (10 mL), and then the mixture was stirred while refluxing for 2 h. The concentrated mixture was treated with cold water and extracted with ethyl acetate. The ethyl acetate layer was washed with brine, dried over anhydrous magnesium sulfate, and concentrated in vacuo. ZGM1 was finally produced from the residue that was purified on a silica gel column.
Preparation of Aβ aggregates
Synthetic Aβ42 peptides (Abcam, ab120301) or synthetic Aβ40 peptides (Abcam, ab120479) were dissolved in DMSO to obtain a 5 mM stock solution. Each stock solution was then diluted with D-PBS. ZGM1 was dissolved in DMSO to obtain a 0.1 M stock solution and then diluted with D-PBS to obtain the working concentration. Aβ was incubated with ZGM1 at 37 °C for 12–120 h at specific proportions. Congo red, which has been reported to inhibit Aβ aggregation, was used as a control.
A total of 3 μl of each sample was incubated for 0 h, 12 h, and 24 h with 3 μl Nu-PAGE™ LDS Sample Buffer (Invitrogen) and 6 μl ddH2O. The mixture was electrophoresed in a Nu-PAGE 4%~ 12% Bis-Tris premade gel (Invitrogen NP0321BOX) at 200 V for 30 min, and the protein was transferred to a 0.2 μm PVDF membrane. The 6E10 antibody was used for Western blotting to detect Aβ aggregation. Another 2 μl of the sample was incubated for 24–120 h or was not incubated and was spotted on an NC membrane (Sangon) for dot blotting analysis to detect Aβ oligomers.
Thioflavin T fluorescence
Thioflavin T (ThT, Sigma) was dissolved in DMSO to obtain a 0.1 M stock solution. The ThT, Aβ, and ZGM1 stock solutions were diluted with D-PBS. Aβ (30 μM) was centrifuged at 17,000×g for 20 min, and then the supernatant was retained for subsequent experiments. These reagents were mixed at a ratio of 1:1:1 so that the final concentration of Aβ was 10 μM. Then, the mixtures were added to a black-walled 96-well plate and incubated at 37 °C, and the fluorescence signals were detected at 0 h, 28 h, 50 h, 72 h, 98 h, 118 h, and 166 h. The excitation wavelength was 440 nm, and the emission wavelength was 476.5 nm.
Transmission electron microscopy
The edge of the copper mesh was clamped with tweezers, and 6 μl of the incubated sample was added to the center of the front side of the copper mesh and allowed to remain for 90 s. The sample was gently removed with absorbent paper, and a drop of uranyl acetate was added to the front of the copper mesh and immediately removed. The processed was repeated. After the third drop of uranyl acetate was added, it was allowed to remain on the mesh for 30 s before being removed. The copper mesh was dried and put into the storage box for observation. The images were obtained by transmission electron microscopy (FEI Tecnai Spirit with iCorr D1319, Tsinghua University).
Aβ42 linked to a 5-carboxyfluorescein tag at the N-terminus (5’FAM-Aβ42, Chinese Peptide) was dissolved in DMSO to obtain a 5 mM stock solution. Each stock solution was diluted with D-PBS to obtain a concentration of 400 nM and centrifuged at 17,000×g for 20 min at 4 °C, and then the supernatant was retained. The ZGM1 stock solution was diluted to a concentration of 2 mM with D-PBS. ZGM1 was titrated at a 1:1 dilution 16 times beginning at 2 mM. 5’FAM-Aβ was added to each tube and mixed; the final concentration of 5’FAM-Aβ was 200 nM, and the highest concentration of ZGM1 was 1 mM. A capillary tube (NanoTemper, MO-K002) was inserted into each tube to allow the sample to enter the capillary. The capillary was placed in each sample well in order of the ZGM1 concentration (from low to high) and was detected using microscale thermophoresis (MST, NanoTemper, Monolith NT.115).
Primary culture of cortical neurons
Mice at 17–18 days of pregnancy were sacrificed. The abdominal cavity was carefully opened, and the embryos were removed; the whole brain was also removed and placed in DMEM/F12 (1:1) medium. The olfactory bulb and brain stem were removed, and the vascular membrane was peeled off. The remaining tissue was crushed with a yellow pipet tip, transferred into a 15 mL centrifuge tube containing 0.05% Trypsin (Gibco, 25300054), placed on ice for 15 min, and then incubated at 37 °C for 10–15 min for digestion. Most of the supernatant was aspirated. Then, 50 μL DNase I (Thermo, EN0523) was added, and the tissue was digested at 37 °C for 3 min. A total of 10 mL of DMEM/F12 (1:1) medium containing 10% FBS was added to terminate digestion. The mixture was mixed 20 times by aspiration and then centrifuged at 1200 rpm for 4 min, and the supernatant was discarded. The cells were resuspended gently in 3 ml of neurobasal (Gibco, 21103049) medium, and then a cell sieve (40 μm, Falcon, 352340) was used to filter the sample. The cells were diluted in adherent medium (89.75% neurobasal medium + 10% FBS + 0.25% GlutaMAX) and then inoculated into 96-well plates at a density of 40,000 cells per well. The growth medium (97.75% neurobasal medium + 2% B27 + 0.25% GlutaMAX) was prewarmed for fluid exchange after 4 h.
SH-SY5Y cells were grown in 1:1 DMEM/F12 medium (Thermo, 11330032) supplemented with 10% fetal bovine serum (Biowest), 0.5% MEM non-essential amino acids 100× solution (NEAA, Thermo), and 0.5% sodium pyruvate 100× solution (SP, Thermo) at 37 °C with 5% CO2 in a humidified atmosphere.
The experimental groups were treated with ZGM1, Aβ, or both, while the control groups were treated with an equal amount of DMSO; each gradient used three replicates. After 48 h, the medium was changed to a medium containing 5% CCK-8 reagent (BBI, E606335), and the incubation was continued for 1 h in the dark. Then, the absorbance was measured at a wavelength of 450 nm using a microplate reader (Polarstar Omega, BMG Labtech).
Mouse feeding and intragastric administration
All animal experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 1978) and Regulations for the Administration of Affairs Concerning Experimental Animals (China, Revised in 2011). APP/PS1 mice and 129/C57BL/6 wild type mice were purchased from the Model Animal Research Center of Nanjing University and were raised in the same environment with sufficient food and water and a light to dark cycle of 12:12 h.
Four-month-old APP/PS1 TG mice (male) were divided into three groups: Group T, L-Z, and H-Z, and 4-month-old WT (male) comprised the W group. The dry ZGM1 powder was resuspended in 0.1% sodium carboxymethylcellulose (SCC, Solarbio). The drug was delivered into the stomach by a gavage needle, and the dosage was determined by the body weight. The dosage of the L-D group was 40 mg/kg per day, and the dosage of the H-D group was 120 mg/kg per day. Groups W and T were fed an equal amount of 0.1% SCC as controls for 8 weeks. The mice were administered the drug daily except for the weekends.
ZGM1 tissue abundance analysis
The mice were orally administered the drug at a dosage of 40 or 120 mg/kg. The mice were sacrificed by perfusion, and the brain tissue was obtained. The brain tissue, an equal mass of glass beads (Sigma, G8772), and 4 volumes of methanol prechilled at − 80 °C were added to the homogenate tubes, and the mixture was homogenized (6000 g) for 30 s and then cooled for 1 min on ice, which was repeated 6 times. The homogenate was placed at − 80 °C for 2 h. The homogenate was centrifuged at 15000×g for 15 min at 4 °C, and 200 μL of the supernatant was added to a new 1.5 mL EP tube. The 0 h mouse blood sample was used as a blank. ZGM1 powder was dissolved to generate a standard curve for the absolute quantification of ZGM1 in the samples. All samples and standards were tested by the Metabolomics Facility at the Technology Center for Protein Science, Tsinghua University.
The data analyses, including the recordings of all behavioral responses, were transcribed manually into a computer-acceptable format by researchers blinded to the group assignments.
The movement and retention information of the mice over 5 min were recorded by a camera. The next mouse experiment was performed after cleaning the field with 75% alcohol. After all experiments were completed, the total distance, the central area, and the number of crossings in the central area were analyzed.
Elevated plus maze
After placing the mice in the center of the elevated plus maze (80 cm × 80 cm), the movement and retention information of the mice over 5 min were recorded by a camera located at the top of the room. The next mouse experiment was performed after cleaning the maze with 75% alcohol. After all experiments were finished, the open arm entry and time in the open arm were statistically analyzed, and the differences between the groups were compared.
Novel object recognition
On the first day, objects A (circles) and B (squares) of the same color were placed in the open field. The mouse was free to explore in this environment for 10 min. The movement and retention information of the mice were recorded. The next mouse experiment was performed after cleaning the field with 75% alcohol. The next day, object B was replaced in the open field by a circular object C, which was slightly larger than object A. The position of object A did not change. After the mice were placed in the field, the movement and retention information of the mice were recorded over 5 min. The number of times and the timing of the sniffing of objects A and C by the mice were statistically analyzed, and the differences between the groups were compared.
Morris water maze
A platform was placed 1.5 cm below the water surface in the second quadrant of a circular water tank (80 cm diameter, 25 ± 1 °C). Titanium dioxide was added to whiten the water. The test was performed for 6 days. Mice were trained for 6 consecutive days with 3 trials per day as acquisition trials. Each trial began with placing the mouse into a different quadrant and allowing it to swim freely for 60 s. After each mouse reached the platform (or were guided to the platform if the mice were unable to locate the platform after 60 s), they were returned to their cage to dry for 20 min. The time that each mouse took to reach the platform was regarded as the escape latency. On the 7th day, the mice were rested for a day. On the 8th day, a probe test was performed without the platform for 60 s. Each mouse was placed into the opposite quadrant of the target zone, from which the platform was removed. The time in the target zone and the number of crossings that occurred in the probe were recorded.
ThS staining of Aβ plaques in brain sections
After the behavioral studies, the mice were deeply anesthetized with 7% chloral hydrate (dissolved in PBS, 0.02 mL/g, intraperitoneally) and perfused. For ThS staining, excised brains were fixed overnight in 4% paraformaldehyde at 4 °C and immersed in 30% sucrose for 24 h twice for dehydration. Brain slices were cut in the coronal plane (30 μm per section) at − 26 °C. Aβ plaques in brains were visualized using ThS staining. ThS was dissolved in 50% ethanol at 0.4 M (filtered by a 0.22-μm filter), and brain sections were stained for 8 min and then washed with 50% ethanol for 3 min, which was repeated 3 times. The slices were finally sealed with 20% glycerin (dissolved in PBS). The images of the plaques were obtained with an Olympus fluorescence microscope. The number and areas of the plaques were determined using the ImageJ program (NIH). The analyses of the plaque distributions were transcribed manually into a computer-acceptable format by researchers blinded to the group assignments.
Blotting analyses and the quantification of Aβ in brain lysates
Brain tissues were homogenized in ice-cold RIPA containing a 1* proteinase inhibitor. The homogenized tissue was ultrasonically crushed on ice for 20 min at 5% of the maximum power. The supernatant of the brain lysate was used for ELISA of Aβ40 or Aβ42 and Western blotting and dot blotting to detect biochemical changes and soluble Aβ. The concentrations of the soluble fractions of brain lysates were determined by using a BCA protein assay kit (Thermo, A53225). Protein samples (20 μg) were loaded in each lane of SDS–PAGE gels for Western blot analyses. GluR1, GluR2, PSD95, synaptotagmin, and App695 were measured. The measurements of Aβ40 and Aβ42 content were completed with an ELISA kit (Invitrogen) according to the manufacturer’s instructions with fivefold diluted soluble fraction samples. Detailed information about the antibodies used is included in the Additional files.
Graphs were obtained with GraphPad Prism 6, and statistical analyses were performed with one-way analysis of variance or two factors analysis of variance (*P < 0.05, **P < 0.01, ***P < 0.001, other comparisons were not significant).
The error bars represent the s.e.m. or s.d.
The data that support the findings of this study are available within the article, in the Additional files, and from the corresponding author upon reasonable request.