2.1 Materials
NGR1 (Purity ≥ 98.0%; HY-N0615), pioglitazone (Purity = 99.66%; HY-13956), GW9662 (Purity = 99.83%; HY-16578), SBE-β-CD (Purity ≥ 98.0%; HY-17031), and cytarabine (Purity = 99.43%; HY-13605) were all acquired by MedChem Express (Shanghai, China). Bovine serum albumin (BSA), dimethylsulfoxide (DMSO), Triton X-100, paraformaldehyde and D-Glucose were obtained from Sigma-Aldrich (MO, USA). Phosphate-buffered saline (PBS), Dulbecco’s modified Eagle’s medium (DMEM), neurobasal medium, B-27, GlutaMAXTM-I, L-Glutamate, fetal bovine serum (FBS), penicillin, streptomycin, isoflurane, saline, radioimmunoprecipitation assay (RIPA) buffer, human enzyme-linked immunosorbent assay (ELISA) kits for Aβ42 (KHB3544), and DAPI (D1306) were supplied from Invitrogen (CA, USA). cOmplete™ protease inhibitor cocktails were purchased from Roche (IN, USA). The antibodies against glial fibrillary acidic protein (GFAP, ab279290), microtubule-associated protein 2 (MAP2, ab32454), beta amyloid (ab11132), Iba-1 (ab178846), neuronal nuclei (NeuN, ab104224), GLUT4 (ab33780), β-tubulin (ab179513), PPARγ (ab178860), IRS-1 (ab52167), p-IRS-1 (Ser616, ab4776), Akt (ab8805), p-Akt (Ser473, ab81283), Alexa Fluor® 594-conjugated goat anti-rabbit IgG (ab150080), Alexa Fluor® 488-conjugated goat anti-mouse IgG (ab150113), HRP-linked goat anti-rabbit IgG (ab6721), HRP-linked goat anti-mouse IgG (ab6789), glucose uptake colorimetric assay kits (ab136955), and mouse ELISA kits for tumor necrosis factor-alpha (TNF-α, ab208348), interleukin-1β (IL-1β, ab197742), and insulin (ab277390) were all obtained from Abcam (CA, USA). The control siRNA (sc-37007) and PPARγ siRNA (sc-156077) were purchased from Santa Cruz Biotechnology (CA, USA). 18-fluoro-2-deoxyglucose (18F-FDG) were obtained from the first afflicted hospital of Jinan University.
2.2 Dual-luciferase reporter assay
HT22 cells (the mouse hippocampal neuronal cell line) were procured from Department of cell resource center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College (Beijing, China). Maintained in DMEM supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (100 µg/mL), HT22 cells were grown in a humidified incubator at 37°C and 5% CO2. For experiments, HT22 cells at passages 3–5 were transiently co-transfected with the peroxisome proliferator response element (PPRE) luciferase reporter plasmid (100 ng), the PPARγ expression plasmid (100 ng), and the Renilla luciferase plasmid (pRL-TK, 50 ng) using the Gene Pulser Xcell Electroporation System (Bio-rad laboratories Inc, USA), following the protocol from our previous study(Z. Li et al., 2022). NGR1, pioglitazone and GW9662 stock solution (1 mM) was prepared in DMSO and then diluted with DMEM (with DMSO at a concentration below 0.1%). After 48 h of transfection, HT22 cells were incubated for 24 h with pioglitazone (40 µM), or varying concentrations of NGR1 (2.5, 5, 10, and 20 µM), or co-treated with GW9662 (10 µM) and NGR1 (10 µM). Luciferase activity was detected on a multi-mode microplate reader (BioTek, USA) using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA). Luciferase activity was normalized to that of Renilla luciferase.
2.3 Primary cultures of mouse hippocampal neurons
Primary mouse hippocampal neurons were isolated from C57BL/6J mouse embryos (day 17). Briefly, hippocampal tissues were dissected and then incubated at 37°C for 10 min with papain (10 mg/mL). After washing with DMEM supplemented with 10% FBS, the cells were seeded at a density of 1 × 105 cells/mL in poly-L-lysine-precoated (4.5 µg/cm2) 6-well plates and grown in a humidified incubator at 37°C and 5% CO2. The cells were cultured in complete neurobasal™ medium containing 20% FBS, streptomycin (100 µg/mL), and penicillin (100 U/mL). After 4 h of incubation, the culture medium was replaced with serum-free neurobasal medium containing GlutaMAXTM-I (0.5 mM), L-Glutamate (25 µM), and 2% B-27. After 48 h of incubation, the cells were cultured in serum-free neurobasal medium supplemented with cytarabine (5 µM), GlutaMAXTM-I (0.5 mM), L-Glutamate (25 µM), and 2% B-27. On day 7, primary mouse hippocampal neurons were detected by immunofluorescence staining with mouse anti-GFAP and rabbit anti-MAP2 antibodies.
2.4 Assay of neuronal glucose uptake
To assess the impact of NGR1 on glucose uptake in neurons, primary mouse hippocampal neurons underwent a 24 h treatment with varying concentrations of NGR1 (2.5, 5, 10, and 20 µM), pioglitazone (40 µM), or co-treatment with GW9662 (10 µM) and NGR1 (10 µM), or NGR1 (10 µM) for different durations (3, 6, 12, and 24 h). NGR1, pioglitazone, and GW9662 were dissolved in DMSO (1 mM) and then diluted in DMEM (with DMSO at a concentration below 0.1%). To investigate if NGR1 increases glucose uptake in a PPARγ-dependent manner, primary mouse hippocampal neurons were transiently transfected with PPARγ siRNA or an equivalent concentration of control siRNA. Transfection efficiency was confirmed through western blotting using the anti-PPARγ antibody. After 48 h of transfection, primary mouse hippocampal neurons transfected with PPARγ siRNA or control siRNA were incubated for 24 h with vehicle or NGR1 (10 µM). The 2-deoxyglucose (2-DG) uptake in transfected primary mouse hippocampal neurons was measured using glucose uptake colorimetric assay kits, following the manufacturer’s instructions.
2.5 Visualization of neuronal GLUT4 membrane translocation
Primary mouse hippocampal neurons were transfected with lenti-GLUT4-GFP as detailed in our previous study(Meng et al., 2023). To assess whether NGR1 induces membrane translocation of GLUT4, the transfected primary mouse hippocampal neurons were incubated for 24 h with NGR1 (10 µM) alone or co-treated with GW9662 (10 µM) and NGR1 (10 µM). GLUT4 membrane translocation was captured using a Carl Zeiss LSM800 confocal microscope (Göttingen, Germany).
2.6 Animal ethics
All animal protocols were approved by the Committee on the Ethics of Animal Experiments of Shenzhen Second People’s Hospital. The animal experiments adhered to the Regulations of Experimental Animal Administration. The animals were kept in a specific pathogen-free barrier facility, maintaining standard conditions with a temperature of 22 ± 1°C, humidity at 50 ± 10%, and a 12 h light-12 h dark cycle. Food and water were provided ad libitum. Double-blinded principles were employed throughout the animal procedures.
2.7 Animal treatments
APP/PS1xdb/db mice were generated by crossbreeding db/m mice with APPswe/PS1dE9 (APP/PS1) mice, as outlined in our previous study(Meng et al., 2023). Twenty 3-month-old male db/m mice (20 ± 2 g) were procured from Carvens Laboratory Animal Co., Ltd. (Changzhou, China). Additionally, twenty 3-month-old female APP/PS1 mice (20 ± 2 g) were obtained from Beijing HFK Bioscience Co. Ltd. (Beijing, China). All mice shared a C57BL/6 background. Genotyping was conducted using polymerase chain reaction and DNA sequencing of three genes (APPswe, PS1dE9, and Leprdb) following the genotyping protocol database of the Jackson Laboratory (Protocol 33897, 23050, and 21995). A blood glucose level ≥ 16 mmol/L diagnosed diabetes in APP/PS1xdb/db mice.
For dose-response experiments, sixty 4-month-old male APP/PS1xdb/db mice were randomly assigned to six groups: vehicle, pioglitazone (10 mg/kg/day), and different doses of NGR1 (10, 20, 40, and 80 mg/kg/day), with each group consisting of 10 mice. Additionally, 20 wild-type (WT) littermates were randomly divided into two groups: vehicle and NGR1 (80 mg/kg/day). Pioglitazone and NGR1 were solubilized in DMSO and then diluted with saline containing 20% SBE-β-CD. The mice received intragastric administration of NGR1, pioglitazone, or an equivalent volume of vehicle for consecutive 16 weeks.
To investigate the mechanisms underlying the protective effects of NGR1, sixty 4-month-old male APP/PS1xdb/db mice were randomly divided into three groups: vehicle, NGR1 (40 mg/kg/d), and GW9662 (5 mg/kg/d) + NGR1 (40 mg/kg/d), with each group consisting of 20 mice. Twenty WT littermates were used as controls. NGR1, pioglitazone, and GW9662 were solubilized in DMSO and then diluted with saline containing 20% SBE-β-CD. The mice received intragastric administration of NGR1, pioglitazone, GW9662 + NGR1, or an equivalent volume of vehicle for consecutive 16 weeks.
The body weights of mice were measured weekly throughout the study. The volumes of vehicle, pioglitazone, NGR1, and GW9662 were adjusted according to mouse body weight. The levels of fasting blood glucose were detected every 4 weeks using an Accu-Chek Performa glucometer (Roche, Mannheim, Germany). After 16 weeks of treatment, ten mice were randomly selected from each group for 18F-FDG micropositron emission tomography (microPET) scans, Morris water maze test, novel object recognition test, open-field test, and glucose tolerance test (GTT). The remaining mice (n = 10) from each group were subjected to nest construction tests and insulin tolerance test (ITT). Neurobehavioral tests were performed under constant light and environmental conditions. Drug administration continued until the end of the behavioral experiment.
2.8 Tissue processing
All mice underwent an overnight fast and were anesthetized with isoflurane (4% for induction and 2% for maintenance). From each group, 10 mice were randomly chosen, and blood was collected via cardiac puncture. Serum separation was achieved by centrifugation at 1,000 × g for 10 min at 4°C and stored at − 80°C. Fourteen mice were randomly selected from each group and transcardially perfused with cold PBS containing a protease inhibitor cocktail. The brains were promptly removed, and the hippocampus tissues were carefully dissected, frozen in liquid nitrogen, and stored at − 80°C. ELISA utilized the hippocampal tissues of 11 mice, while the remaining hippocampal tissues (n = 3 per group) were allocated for western blot analysis. For immunofluorescence staining, the remaining mice (n = 6) in each group were transcardially perfused with cold PBS, followed by 4% paraformaldehyde, and their brains were isolated.
2.9 Novel object recognition test
The short-term object recognition memory of the mice was evaluated using the novel object recognition test. The apparatus comprised a 40 × 40 × 40 cm3 transparent plastic chamber and plastic objects. Mouse behavior was recorded using a video tracking system and analyzed with SMART 3.0 software (RWD Life Science, Shenzhen, China). During the habituation phase, mice (n = 10 per group) were placed in the center of an empty chamber and allowed to explore freely for 10 min/day over 3 days. In the familiar phase, mice were placed in a chamber with two identical objects and allowed to explore for 10 min. After a 6 h retention interval, the testing trial began. One familiar object was substituted with a novel object, and the mouse freely explored for 10 min. The apparatus was cleaned with 75% alcohol at the end of each trial to eliminate odors. Exploration was defined as direct contact of the nose or front paws with the object at a distance ≤ 2 cm. The time spent exploring each object was recorded. The discrimination index was calculated as the ratio of the exploration time for the novel object to the total time spent with familiar and novel objects.
2.10 Morris water maze test
The spatial learning and memory of the mice were evaluated using the Morris water maze test, as outlined in our previous study(Li et al., 2021). The apparatus comprised a circular pool (120 cm in diameter and 40 cm in height) surrounded by black curtains adorned with visual cues, and a platform (20 cm in diameter). The pool was filled with water. The water was colored with non-toxic white paint (titanium dioxide), and maintained at 22 ± 1°C. The mouse behaviors were recorded using a video tracking system, analyzed with SMART 3.0. Mice (n = 10 per group) underwent acquisition training 4 times daily for 5 consecutive days. In each trial, mice, facing the wall at randomly chosen starting positions, sought the submerged escape platform (1.0 cm beneath the water surface). After climbing the platform, mice remained for 10 s. Escape latency, the time taken to find the platform, was recorded. A mouse unable to locate the platform within 60 s was guided and allowed to remain for 20 s. Probe trials on the sixth day, post-platform removal, had mice placed opposite the target quadrant, swimming for 60 s. The percentage of time spent in the target quadrant was calculated.
2.11 Open-field test
The anxiety-like behaviors of the mice were assessed using the open-field test. Mice (n = 10 per group) freely explored the open field for 10 min. Video tracking and SMART 3.0 analysis measured anxiety-like behavior by assessing the percentage of time spent in the middle area.
2.12 Nest construction test
Social behavior was evaluated through a nest construction test. Mice (n = 10 per group), housed in cages with corncob bedding for 7 days before testing, received eight pieces of paper 2 h before the dark phase. On the fourth morning, nest construction was scored on a four-point system: 1, no tearing with random dispersion; 2, no tearing with gathering in a corner; 3, moderate tearing; and 4, extensive tearing.
2.13 18F-FDG-PET scans
Cerebral glucose uptake in mice was assessed using 18F-FDG-PET scans. Mice (n = 10 per group) underwent measurements of body weight and blood glucose levels after a 6 h fast. Subsequently, mice received 18F-FDG (4–6 MBq) via the tail vein. Anesthesia was induced with 2% isoflurane using a calibrated Matrix VIP 3000 vaporizer (Midmark, OH, USA), and mice were positioned on a scanning bed 50 min post-18F-FDG injection. MicroPET was performed for 10 min, followed by CT using an IRIS PET/CT system (Inviscan SAS; Strasbourg, France). CT parameters included an 80 kV bulb voltage and 0.9 mA current. Body temperature was kept at 37°C using a heating pad, and respiratory rates were continuously monitored. PET images were reconstructed using a three-dimensional ordered-subject expectation maximization method based on the Monte Carlo exact detector model with a voxel size of 0.855 × 0.855 × 0.855 mm3, and CT images were reconstructed using the FDK algorithm with a voxel size of 0.16 × 0.16 × 0.16 mm3. Pmod software (version 4.1, Inviscan SAS, Strasbourg, France) was used for image display. PET images were aligned with a predefined mouse brain atlas template, and standardized uptake value (SUV) was calculated for semiquantitative analysis using the formula: SUV = activity concentration (Bq/cm) / (injected dose (Bq) / body weight (g)).
2.14 Glucose tolerance test (GTT) and insulin tolerance test (ITT)
Following a 6 h food deprivation, mice (n = 10 per group) underwent intraperitoneal administration of d-glucose (2 g/kg body weight) or insulin (1.5 IU/kg body weight; Novo Nordisk Pharmaceuticals Co., Ltd., Tianjin, China). Blood glucose concentrations were measured at 0, 15, 30, 45, 60, and 120 min for GTT and at 0, 30, 60, 90, and 120 min for ITT. Area under the curve (AUC) for glucose concentration in GTT and ITT was calculated.
2.15 ELISA
Serum insulin (n = 10 per group) and HbA1c levels (n = 10 per group) were assessed using commercial ELISA kits following the provided instructions. The homeostasis model assessment-estimated insulin resistance (HOMA-IR) index was calculated using the formula: HOMA-IR = [fasting blood glucose (mmol/L) × (fasting serum insulin (mU/L)] / 22.5. For analyzing soluble Aβ42, insulin, TNF-α, and IL-1β, hippocampal tissues (n = 11 per group) were homogenized in RIPA buffer containing a protease inhibitor cocktail. The homogenates underwent centrifugation in an ultracentrifuge at 4°C, 12,000 × g for 10 min, with supernatants collected. For analyzing insoluble Aβ42, the pellets were homogenized in 70% formic acid, underwent sonication, and were centrifuged at 4°C, 20,000 × g for 10 min. Supernatants were neutralized with 1 M Tris-base.
2.16 Immunofluorescence
Brains (n = 6 per group) were embedded in paraffin and cut into 5 µm-thick serial coronal sections. Following deparaffinization and rehydration, brain sections underwent treatment with citrate buffer for 1 h. Subsequently, brain sections were treated with 0.3% H2O2, washed in PBS, and then treated for 10 min with 70% formic acid. Further permeabilization occurred with PBS containing 0.1% Triton X-100 for 30 min. After PBS rinsing, brain sections were blocked for 1 h with PBS containing 1% bovine serum albumin. Overnight incubation at 4°C followed with mouse monoclonal anti-Iba-1 and rabbit polyclonal anti-Aβ antibodies, or mouse monoclonal anti-NeuN and rabbit polyclonal anti-GLUT4 antibodies. After rinsing with PBS, brain sections were incubated at room temperature in the dark for 1 h with Alexa Fluor® 488-conjugated goat anti-mouse IgG and Alexa Fluor® 594-conjugated goat anti-rabbit IgG. Nuclei were stained with DAPI. Slides were mounted with a gel mount (Vectashield) and scanned using a Pannoramic MIDI scanner (3DHISTECH, Budapest, Hungary). Immunofluorescence images were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
2.17 Western blotting analysis
Hippocampal tissues (n = 3 per group) were homogenized in ice-cold RIPA lysis buffer supplemented with phenylmethanesulfonyl fluoride and a protease inhibitor cocktail. The homogenates underwent centrifugation at 4°C for 30 min at 12,000 × g. Supernatants were collected, protein content determined using bicinchoninic acid kits (Pierce Biotechnology, IL, USA). Equal amounts of proteins were separated by electrophoresis on 10% sodium dodecyl sulfate-polyacrylamide gels and then transferred onto polyvinylidene fluoride membranes (Merck Millipore Ltd., Carrigtwohill, Ireland). Membranes were blocked for 1 h with 5% BSA in Tris-buffered saline containing 0.1% Tween-20 (TBST) and then incubated with antibodies against PPARγ, p-IRS-1 (Ser616), IRS-1, p-Akt1 (Ser473), Akt, GLUT4, and β-tubulin overnight at 4°C. Following rinsing with TBST, membranes were incubated for 1 h at room temperature with HRP-linked goat anti-rabbit IgG or HRP-linked goat anti-mouse IgG antibodies. Protein bands were visualized using western blotting detection kits (Advansta, CA, USA) and a ChemiDoc Touch Imaging System (Bio-Rad, USA). Band intensity was quantified using the Image Lab software (Bio-Rad, USA). The densities of protein bands were normalized to those of β-tubulin.
2.18 Statistical analysis
Data are presented as the mean ± standard deviation (SD) and were analyzed using IBM SPSS Statistics version 20 (SPSS Inc., IL, USA). One-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test was used to analyze differences among multiple groups. Two-way ANOVA followed by Tukey’s post-hoc test was used to analyze the statistical significance of the genotypes and treatment effects. Two-way ANOVA with repeated measures followed by Tukey’s post-hoc test was used to analyze the results of the Morris water maze test. P < 0.05 was considered statistically significant.