Animals All procedures were carried out in accordance with the National Institutes of Health Guidelines for the Care and Use of the Laboratory Animals and were approved by the Animals Advisory Committee at Jinan University. C57BL/6 mice were obtained from Guangdong Provincial Zoological Experimental Centre. Heterozygous 5XFAD mice on a B6SJL background were obtained from the Jackson Laboratory (stock no.006554), these 5XFAD transgenic mice overexpress mutant human amyloid beta precursor protein 695 (APP) with the Swedish (K670N, M671L), Florida (I716V), and London (V717I) Familial Alzheimer's Disease (FAD) mutations along with human presenilin 1 (PS1) harboring two FAD mutations, M146L and L286V. mGluR5 knockdown mice (B6.129-Grm5tm1Rod/J) (mGluR5+/−) (stock no.003558) were purchased from the Jackson Laboratory and maintained on a C57BL/6J genetic background. For the purposes of the present study, we crossed 5XFAD mice with B6.129-Grm5tm1Rod/J mice to obtain 5XFAD/mGluR5+/− double-transgenic mice along with 5XFAD, mGluR5+/−, and wild-type (C57BL/6) littermates.
2- and 6-month-old mice were housed on a 12/12 h light/dark cycle with free access to food and water, at 20 ~ 24°C. The mice were divided into four groups: WT group (n = 15), mGluR5+/− group (n = 15), 5XFAD group (n = 15), and 5XFAD/ mGluR5+/− group (n = 15), half male and half female.
Aβ42 drugs To prepare oligomeric Aβ42, the Amyloid β Protein Fragment 1–42 (A9810, Sigma) was first suspended in 100% 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP,52517, Sigma) at a concentration of 1 mM. The Aβ42-HFIP solution was then incubated in polypropylene vials to complete solubilization at room temperature (RT) for 2 hours. HFIP was allowed to evaporate under a slight stream of nitrogen until a clear peptide film was observed at the bottom of the vials and stored at -80℃ until use. The film was re-suspended by adding DMSO (D2650, Sigma) at a concentration of 5 mM and sonicated at room temperature for 10 min. The solution was then diluted to a final concentration of 100 µM in ice-cold cell culture medium (phenol red-free Ham's F-12; BioSource), immediately vortexing for 30 s and incubated at 4℃ for 24 h. Aβ (1–42) oligomers were formed by fibrils.
Cultured neural stem cell and EdU staining Primary hippocampal neural stem cell cultures were prepared from new-born mice. Hippocampi were dissected and minced to 1 mm2 in high glucose medium. Then tissue was mechanically triturated using a 10 mL pipette, filtered through a 40 µm cell strainer (Corning 352340), pelleted at 1000 × g for 5 minutes and washed twice with high glucose medium, finally were plated with appropriate densities in culture medium (DMEM/F12, 20 ng/mL EGF, 20 ng/mL bFGF, 2% B27 compound). Cultures were kept at 37℃ in a 5% v/v CO2 humidified incubator. Thereafter, one third to half of the medium was replaced twice a week. For EdU staining, neural stem cells (NSCs) were cultured in confocal dishes, and the cells were treated with different concentrations of Aβ42 oligomers until cell density reached over 70% of the dish area. The EdU (KeyGEN BioTECH, KFluor488 Click-iT EdU, KGA331-100) operating solution (10µM) was preheated at 37℃ and added to cell culture medium for 24hr. After completion of EdU labeling cells, the medium was removed and 2 mL fixture (4% PFA) was added and fixed for 15 min at room temperature. Then the NSCs were washed three times with PBS for 10 min and later were incubated with 0.3% Triton X-100 in PBS for 15 min at room temperature. Finally, click reaction solution was added to the cells and incubated for 30 min at room temperature. Fluorescence images were acquired with a confocal laser microscope (Leica TCS SP8 MP) using the sequential scanning mode.
Apoptosis assay Cell apoptosis was identified using an Annexin V-EGFP Apoptosis Detection Kit (Cat: KGA108, Jiangsu, China). NSCs were uniformly inoculated in 6-well plates coated with PLL. Waiting for 6 hours, the cells were tightly to the cell plate, treated with different concentrations of Aβ42 (0, 1 pM, 10 pM, 100 pM, 1 nM, 10 nM, 100 nM) and cultured for 24 h. The cells were digested with EDTA-free trypsin, centrifuged at 1000 rpm for 5min, collected and rinsed 3 times with PBS. Single suspension cells were prepared by adding 500 µL Bingding Buffer and 5 µL Annexin V-EGFP (Cat: KGA108) later, then were added 5 µL Propidium Iodide, reacting at room temperature under dark conditions for 5 ~ 15 min. Flow cytometry was performed within 1 h.
Aβ42 ELISA quantification One hippocampus from a 5XFAD mouse was homogenized in 200 µL of solution (20 mM Tris-HCl, 5 mM EDTA) (pH 7.8) and protease inhibitor cocktail (20 mM Tris-HCl, 5 mM EDTA) (pH 7.8). The mixture was then centrifuged at 400,000 × g for 20 min, and the soluble Aβ supernatant was collected. The remaining insoluble pellets were re-homogenized in 200 µL of 5 M guanidine hydrochloride (GHCl), 50 mM Tris-HCl (pH 8.0), shaken for 4 h, centrifuged at 400,000 × g for 20 min and the supernatants were collected. Total protein of Tris-soluble and GHCl-soluble supernatants were determined using Mouse Amyloid Beta 1–42 ELISA Kit (Colorimetric) (Novus Biologicals, CO, USA).
Ca 2+ Measurements Primary hippocampal neural stem cells were loaded with 5 µM Fluo-4 acetoxymethyl ester (AM) for 30 min at room temperature. Fluorescence was imaged over time using a confocal microscope (Leica TCS SP8). Fluo-4 was excited with a 488 nm argon laser, and emitted light was collected at 500 ~ 580 nm. Calcium imaging was started and the initial intracellular calcium in the cells were recorded After 1 minute, the cells were treated with 100 pM or 100 nM Aβ42Os to observe calcium signals. Movies were taken over 30 min. Images were taken every 2 s. Data were obtained and processed using Leica LAS AF software. The data was normalized to the initial calcium fluorescence levels, F/F0 (F0 is the initial level of fluorescence), to take account of variation in Fluo-4 loading between cells. The figures were conducted with OriginPro 2021.
EdU labeling 5-Ethynyl-2'-deoxyuridine (EdU; ST067; Beyotime), the thymidine analog, which incorporates into the DNA of dividing cells during the S phase, was used for mitotic labeling. In brief, EdU was dissolved in PBS to make the solution at a concentration of 10 mg/ml before injection. For analysis of cell proliferation, 8- or 25-week-old mice received 6 injections of EdU (50 mg/kg, twice daily with interval of 12 h on 3 consecutive days), and were perfused 6 h later (at age 2 month or 6 month). For analysis of early cell differentiation, 7 ~ or 24 ~ week-old mice received 6 injections of EdU (50 mg/kg, twice daily with an interval of 12 h on 3 consecutive days) and were perfused 7 d later (at age 2 month or 6 month). For analysis of cell survival and maturation, 1 ~ or 5 ~ month-old mice received 6 injections of EdU (50 mg/kg, twice daily with interval of 12 h on 3 consecutive days) and were perfused 28 d after the last injection (at age 2 month or 6 month). These time points were chosen based on the mitosis of progenitor cells (0 ~ 24 h), the differentiation and migration of newborn cells (4 ~ 10 d), and the expression of mature neuronal marker (2 ~ 4 week) in rats and mice (27).
Morris water maze (MWM) Mice were placed in a plastic circular pool filled with opaque tap water (water mixed with water-soluble with paint) and maintained at 25℃. A white plastic platform (10 cm diameter) was submerged 1 cm below the surface of the water. Visual cues were positioned around the perimeter of the pool. The pool was visually divided into four quadrants, and the hidden platform was placed in one of these quadrants, where it remained throughout the acquisition trials. Mice were trained for 7 days (four trials per day, 60 s each time) to find the platform at a fixed position from a random start point of the four equally spaced points around the pool. If the mice fail to find the platform within 60 s, they were guided to the platform and allowed to spend 30 s on the platform by the experimenter. On day 8, the platform was removed from the pool. The mice were placed into the water in the opposite quadrant of the platform, and their swimming trajectories were recorded for 60 s. The mean target quadrant occupancy of all four test periods were calculated. All trials were monitored using a computerized tracking system (Chengdu Taimeng Technology & Market Co.Ltd)
Novel Object Recognition Twenty-four hours before training, mice were placed in the empty box as open field for 10 min acclimatization and removed, while the box was cleaned with 75% ethanol. During training, two identical objects A and B were placed in the box 5 cm from the edges and 5 cm apart, and mice were returned to the box for 8 min and the ratio of both the number of visits and the time spent at each object was recorded, and then the mice were put back into the box to rest. Replace the B object in the test box with the C object. After resting for 20 minutes, the mice were put back into the box for testing, and the number and time of contact between the mice and objects B and C were recorded, and the test was terminated after 8 minutes. The number of investigations/duration of the novel object was divided by the number of investigations/duration of the control object to generate the discrimination index (28).
Passive Avoidance Test The escape instrument consists of two parts, a bright chamber and a dark chamber, connected by small holes with gates, allowing mice to shuttle freely when the gates open. The bottom of the darkroom is provided with electric shock device. In the absence of any stimulus environment, mice have the habit of innate darkness and enter the darkroom, but when the mice are stimulated by the bottom of the darkroom, they will quickly escape to the bright room. The experiment lasted for 3 days. On the first day, no electricity was turned on, the gate was opened, and the mice were put into the bright room with the head behind the hole. The mice were allowed to shuttle freely in the dark room for 300 s. On the day 2 and 3, the power supply was turned on and the time was set as 300 s, and the voltage was 36 V. The time when the mice entered the darkroom for the first time to receive electric shock was recorded as the escape incubation period, and the number of times of entering the darkroom to receive electric shock within 300 s was recorded as the wrong number. After 300 s, the whole experiment ended.
Tissue preparation Mice were anesthetized via deep isoflurane inhalation and perfused transcardially with 0.9% saline. Immediately after sacrifice, the brains were removed and cut along the sagittal midline. The left hemisphere was dissected into hippocampus, cerebral cortex, cerebellum, and brain stem; immediately frozen on dry ice; and then stored at − 80°C for protein analysis. Right hemibrains were immersion-fixed in 4% PFA at 4°C for 48 h and then cryoprotected in 30% sucrose in PBS. Then, 20-µm-thick sagittal sections were cut on a freezing microtome. The sections were stored in glycol anti-freeze solution (ethylene glycol, glycerol, and 0.1 M PBS in 3:3:4 ratio) at − 20°C until further processing for histological analysis. Approximately 40 serial coronal sections of the hippocampal formation were obtained for each mouse.
Immunofluorescence Immunofluorescence staining was performed on eight or seven 20 µm free-floating sections, and every 5th brain section was chosen for densitometry and quantification. All the obtained measurements per section were then calculated as the mean value for each mouse brain and used for statistical analysis. Coronal brain slices were incubated in permeable buffer (0.5% Triton-X-100 in PBS) for 20 min and then blocked in 5% bovine serum albumin (BSA) for 30 min at room temperature. Following this, the slices were incubated with primary antibodies overnight at 4℃. The slices were then washed three times with PBS-T (0.1% Tween 20 in PBS) for 10 min each time and incubated with Alexa Fluor secondary antibodies at room temperature for 1 h. If co-stained with EdU, click reaction solution was added to the brain slice and incubated for 30 min at room temperature. Fluorescence images were acquired with a confocal laser microscope (Leica TCS SP8 MP) using the sequential scanning mode.
Western blotting The tissue of hippocampal dentate gyrus were obtained and homogenized using a chilled Vibrahomogenizer (Servicebio) in 1mL of RIPA buffer. The lysate was then centrifuged, and the supernatant collected for Western blot analysis. Proteins were separated by 8% or 10% of sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to Immobilon-P PVDF membranes. The membranes were blocked with 5% skim milk-TBS for 1 hour at room temperature and then were incubated with primary antibodies at 4℃ overnight followed by HRP-conjugated secondary antibodies. Proteins bands were then visualized using the ECL detection substrate and analyzed with ImageJ software.
Statistical analysis
All data were presented as mean ± standard errors of the mean (S.E.M). The data was analyzed with Social Package of Statistical Software 22.0 (SPSS Inc., Illinois, USA). The figures were conducted with GraphPad Prism version 9.0 (GraphPad Software Inc.; La Jolla, CA, USA). The normality of the data was determined by using the Kolmogorov–Smirnov test. One-way ANOVA with Tukey–Kramer post hoc test was used to compare the variables between multiple groups. Two-way ANOVA (repeated measures) was used when two factors (genotype and month) were included in the experiment. For all other comparisons between two groups, Student's t-test was used. All animals were extremely stable in the experiments, and no animal deaths and replacements occurred.P < 0.05 was considered as statistically significant.