2.1 Materials
GW9662 (HY-16578, purity = 99.83%) and Mdivi-1 (HY-16578, purity = 99.73%) were supplied by MedChem Express (Shanghai, China). Bovine serum albumin (BSA), Triton X-100, isoflurane and paraformaldehyde were purchased from Sigma–Aldrich (MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin, Lipofectamine 6000 reagent, RIPA buffer, phosphate-buffered saline (PBS), saline, and phospho-Parkin (Ser65) polyclonal antibody (PA5-114616) were obtained from Invitrogen (CA, USA). Anti-Aβ (ab201060), anti-PPARγ (ab178860), anti-PINK1 (ab23707), anti-LC3B (ab192890), anti-SQSTM1/p62 (ab109012), and anti-beta tubulin (ab6046) antibodies, as well as Alexa Fluor® 647-conjugated goat anti-rabbit IgG H&L (ab150083), HRP-conjugated goat anti-rabbit IgG H&L (ab6721), and HRP-conjugated goat anti-mouse IgG H&L (ab6789), were all purchased from Abcam (CA, USA). The bicinchoninic acid (BCA) kit and enhanced chemiluminescence (ECL) kit were purchased from Pierce Biotechnology (IL, USA). The 18-Fluoro-6-deoxyglucose (18F-FDG) was obtained from Union Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology.
2.2 Synthesis of ligustrazine piperazine derivative
Ten mmol of 2-chloromethyl-3,5,6-trimethylpyrazine hydrochloride and 10 mmol of N-monosubstituted piperazine were added to 70 mL of toluene, followed by addition of 40 mmol of NaHCO3 and a catalytic amount of NaI. The mixture was heated and refluxed for 10 h. TLC showed that the reaction was complete. After filtering, the filter cake was washed 3 times with a small amount of toluene, combined with filtrate, vacuum dtilled to obtain oil, fast column separated to produce a light yellow powder, and recrystallized with n-hexane to obtain white crystal 1-benzhydryl-3-((3,5-dimethylpyrazin-2-yl)methyl)hexahydropyrimidine (LPD).
2.3 Cell culture
PC12 cells were obtained from the Cell Resource Center of the Institute of Basic Medical Sciences, Peking Union Medical College and Chinese Academy of Medical Sciences (Beijing, China). PC12 cells were maintained in DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37°C and 5% CO2 in a humidified incubator. Cell viability was determined using a Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan), according to the manufacturer’s instructions.
2.4 Dual-luciferase reporter assay
To determine whether LPD is a novel PPARγ agonist, the PPRE-TK-luc vector (1 µg) and PPARγ expression plasmid (1 µg) were cotransfected with 20 ng of pRL-TK (Promega Corp., Madison) into PC12 cells using Lipofectamine 6000 when the cells reached 80% confluence. To construct the PPARγ expression plasmid, human PPARγ (NCBI reference sequence: NM_001354666) was PCR amplified and then fused with the GV230 vector (Shanghai Genechem Technology Co., Ltd., Shanghai, China). To construct the PPRE luciferase reporter plasmid, human PERM1 (NCBI reference sequence: NM_001291366-promoter) was PCR amplified and subcloned into the GV238 luciferase reporter vector (Shanghai Genechem Technology Co., Ltd., Shanghai, China). pRL-TK was used to adjust for transfection efficiency. After 48 h of transfection, PC12 cells were treated with various concentrations of LPD (5, 10, 20, and 40 µM) or rosiglitazone (40 µM) or cotreated with LPD (20 µM) and GW9662 (10 µM) for 12 h. Luciferase activity was measured using a dual-luciferase reporter assay system (Promega, WI, USA) with a Glomax 20/20 luminometer (Turner Designs, CA, USA). The luciferase activity was normalized to Renilla luciferase activity.
2.5 APP695swe/PSEN1dE9-overexpressing stable cell line
We previously established stable APP695swe-transfected PC12 cells(Meng et al., 2016). To generate APP/PS1 double-overexpressing cells, human PSEN1 cDNA (NCBI reference sequence: NM_000021.4) was amplified through PCR. Mutant human presenilin 1 (PSEN1dE9) was constructed and subcloned into the GV208 vector (Shanghai Genechem Technology Co., Ltd., Shanghai, China). The PS1-dE9 plasmid was cotransfected with the framework plasmid vector pHelper 1.0 and pHelper 2.0 into HEK293T cells to produce Lenti-PSEN1dE9 (Shanghai Genechem Technology Co., Ltd., Shanghai, China). APP695swe stably transfected PC12 cells were seeded on six-well plates and allowed to grow to 80% confluency. PC12 cells stably transfected with APP695swe were infected with Lenti-PSEN1dE9. Puromycin (2 µg/mL) was added, and drug-resistant cells were collected after 2 weeks for single-cell cloning. Steadily transfected cells were maintained in puromycin at a final concentration of 1 µg/mL. Resistant clones were analyzed by laser confocal microscopy and western blotting to confirm the overexpression of APP and PSEN1.
2.6 Seahorse assay
Mitochondrial oxidative phosphorylation capacity was determined as the uncoupled oxygen consumption rate (OCR) using a Seahorse XF96 extracellular flux analyzer (Seahorse Biosciences, MA, USA). Briefly, APP/PS1 cells (5 × 104 per well) were plated in XF96 extracellular flux assay plates. After 24 h, the medium was replaced with XF Assay Medium (Seahorse Bioscience MA, USA). After the cells were incubated in a CO2−free incubator at 37°C for 30 min, basal levels were measured with no additives. For OCR detection, oligomycin, FCCP, and rotenone/antimycin A were added at final concentrations of 1 µM, 0.3 µM, and 0.1 µM, respectively. Three separate measurements were taken after each of the above reagents was added. Triplicate or quadruplicate experimental wells were examined, and the results were plotted using Seahorse software.
2.7 Animals and ethical considerations
All animal protocols were approved by the ethics committee of Shenzhen Second People's Hospital. The experiments were conducted in compliance with the Guide for the Care and Use of Laboratory Animals. All efforts were made to reduce the number of animals used and minimize animal suffering in the experiments. Six-month-old male Swedish mutant APP (APP695swe)/PS1 (PSEN1dE9) transgenic mice and age-matched male C57BL/6N wild-type (WT) mice were obtained from Beijing HFK Bioscience Co., Ltd. (Beijing, China). The animals were housed in a specific pathogen-free animal facility at a constant room temperature of 22°C ± 1°C and 50 ± 10% relative humidity with 12 h light/12 h dark cycles. Access to standard rodent chow and water was available ad libitum.
2.8 Animal treatments
After adaptation for 7 d, 60 APP/PS1 mice were randomly assigned to 6 groups for dose selection: vehicle, rosiglitazone (10 mg/kg/day), GW9662 (5 mg/kg/day), LPD (5 mg/kg/day), LPD (10 mg/kg/day), and LPD (20 mg/kg/day). Each group consisted of 10 mice. Twenty age-matched C57BL/6N WT mice were randomly assigned to vehicle and LPD (20 mg/kg/day) groups. The mice were intragastrically administered LPD, rosiglitazone, GW9662 or an equivalent volume of vehicle (saline) for 12 consecutive weeks. LPD and GW9662 were prepared in saline and stored at 4°C.
In the following experiments, 60 APP/PS1 mice were randomly assigned into 3 groups: vehicle, LPD (10 mg/kg/day), and GW9662 (5 mg/kg/day) + LPD (10 mg/kg/day). Each group consisted of 20 mice. Twenty age-matched C57BL/6N WT mice were used as controls. Each mouse was intragastrically administered either vehicle, LPD or GW9662 every day from the age of 6 months for a total period of 3 months.
The body weights of the mice were monitored weekly. The volumes of LPD, GW9662, and saline were adjusted according to the body weights of the mice. Fasting blood glucose levels were measured using an Accu-Chek Performa glucometer (Roche, Mannheim, Germany), with blood drawn from the tail vein every two weeks. After completion of drug treatments, 10 mice were randomly selected from each group for the Morris water maze test, and the other mice (n = 10 per group) were subjected to microPET. All mice were sacrificed with an overdose of isoflurane anesthetic, and 14 mice were randomly selected from each group and perfused transcardially with saline using a syringe infusion pump at a 5 min/min rate for 5 min. The brain tissue was quickly removed following decapitation, and the hippocampus was then dissected and frozen for ELISA (n = 8 per group) and western blotting (n = 6 per group). The other mice (n = 6 per group) were perfused with 4% paraformaldehyde following saline perfusion, and brain tissue (n = 3 per group) was collected for immunohistochemistry and immunofluorescence. The hippocampi (n = 3 per group) were then dissected and further processed with transmission electron analysis.
2.9 Morris water maze
The Morris water maze test was performed to assess the spatial learning and memory of APP/PS1 mice, and the investigator was blinded to the groups for the behavioral assessments. Briefly, the Morris water maze apparatus consisted of a pool with a diameter of 120 cm and a height of 40 cm filled with water opaque water colored with milk powder. The water temperature was maintained at 22 ± 1°C. The pool was surrounded by a white curtain. An escape platform (20 cm in diameter) was submerged 0.5 cm under the water level and located in the center of the target quadrant. Dark posters, different in shape (one per wall), provided distant landmarks. Mouse behavior was recorded using a video camera connected to a video tracking system. The mice (n = 10 per group) were subjected to training and probe tests. The training test consisted of 5 consecutive days (4 trials per day, separated by 1 h intervals). For each trial, the mouse was placed in the water facing the wall at different start locations and was required to locate a submerged platform. The time each mouse took to reach the platform was recorded as the escape latency. If the platform was not located within 60 s, the mouse was gently guided to the platform and allowed to stay on the platform for 30 s. The probe test was performed on the sixth day. During the probe trial, the hidden platform was removed, and the mice were allowed to swim for 60 s. The percentage of time spent in the target quadrant was calculated.
2.10 Micropositron emission tomography (microPET)
Brain glucose uptake was evaluated using 18F-FDG microPET imaging as described in our previous study(Li et al., 2022). After a 6 h fast, the body weight and blood glucose level of the mice (n = 10 per group) were measured. The mice received 18F-FDG (200 ± 10 µCi) from the tail vein. The mice were anesthetized at 60 min postinjection with 2% isoflurane using a Matrix VIP 3000 calibrated vaporizer (Midmark, OH, USA) and placed on a scanning bed. PET was performed for 10 min followed by a CT scan using a TransPET Discoverist 180 system (Raycan Technology Co., Ltd, Suzhou, China). Body temperature was maintained at 37°C with a heating pad during anesthesia. PET image reconstruction was performed using the 3-dimensional ordered-subject expectation maximization method with a voxel size of 0.5×0.5×0.5 mm3. CT images were reconstructed using the FDK algorithm with a 256×256×256 matrix. Images were displayed with Carimas software (Turku PET Center, Turku, Finland). The mean standardized uptake value was calculated using the following formula: mean pixel value with the decay-corrected region of interest activity (µCi/kg).
2.11 ELISA
The levels of Aβ40 and Aβ42 were detected using the respective ELISA kits. Briefly, APP/PS1 cells (1.5 × 104 per well) were plated in 6-well plates and cultured for another 24 h. The cells were incubated with different concentrations of LPD (5, 10, 20, and 40 µM). The mice (n = 8 per group) were anesthetized and transcardially perfused with PBS. The APP/PS1 cells and the hippocampus of mice were collected and lysed in RIPA buffer containing phosphatase inhibitor and protease inhibitor cocktail. The homogenates were centrifuged at 20,000 rpm for 10 min at 4°C, and the supernatants were pooled for the analysis of soluble Aβ40 and Aβ42. To extract fibrillar and membrane-bound insoluble Aβ40 and Aβ42, the pellets were homogenized in 70% formic acid and centrifuged at 40,000 rpm for 10 min at 4°C. The supernatants were neutralized with 1 M Tris-base and analyzed for insoluble Aβ40 and Aβ42.
2.12 Transmission electron analysis
The hippocampi (n = 3 per group) were fixed in 4% paraformaldehyde and 1% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) overnight at room temperature. Following fixation, the hippocampi were treated with reduced 1% osmium tetroxide, followed by 1% tannic acid in 0.1 M sodium cacodylate buffer for 1 h. The hippocampi were then stained with 2% aqueous solution of uranyl acetate for 30 min, dehydrated in a series of graded ethanol concentrations, and processed for enface embedding in PolyBed (Polysciences). Blocks were sectioned at a 90 nm thickness, poststained with Venable’s lead citrate, and viewed with a transmission electron microscope (JEOL, Tokyo, Japan). Images were obtained on a digital camera (Advanced Microscopy Techniques, MA, USA) by observers who were blinded to the experimental groups.
2.13 Immunofluorescence
Brain tissue (n = 3 per group) were fixed in 4% paraformaldehyde, dehydrated, and embedded in paraffin followed by dehydration in graded ethanol solutions and in toluene. Coronal slices (5 µm) were cut on a slicer. Immunofluorescence was performed to detect Aβ plaques. The sections were incubated with 1% BSA containing 0.1% Triton X-100 in PBS at room temperature for 1 h and then coincubated with rabbit polyclonal anti-Aβ antibody at 4°C overnight. The sections were washed three times in PBS and incubated with goat anti-rabbit IgG H&L (Alexa Fluor® 594) at room temperature for 1 h. Cover slips were mounted in Gel Mount (Vectashield, CA, USA). The nuclei were stained with DAPI. The sections were scanned using a Pannoramic MIDI scanner (3DHISTECH, Budapest, Hungary). The percentage of Aβ plaque area in the hippocampus was quantified.
2.14 western blot analysis
Western blot analysis was conducted as described previously(Li et al., 2021). Frozen hippocampal tissue from mice or cultured cells were homogenized in cold RIPA buffer containing a protease inhibitor cocktail, phosphatase inhibitor cocktail, and phenylmethanesulfonylfluoride (Roche, IN, USA) and then centrifuged at 10000 rpm at 4°C for 10 min. The supernatants were collected, and the protein concentrations were determined using BCA kits. Equal amounts of protein were separated by electrophoresis in 10% sodium dodecyl sulfate polyacrylamide gels and transferred to PVDF membranes (Bio–Rad, CA, USA). The membranes were blocked in 5% nonfat milk powder in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h and then incubated at 4°C overnight with anti-PPARγ, anti-PINK1, anti-Parkin, anti-TOMM20, anti-TIMM23, anti-LC3B, anti-SQSTM1/p62, and anti-beta tubulin antibodies. After rinsing in TBST, the membranes were incubated with goat anti-rabbit IgG H&L (HRP) or goat anti-mouse IgG H&L (HRP) antibodies at room temperature for 1 h. The protein bands were visualized by a ChemiDoc Touch Imaging System (BIO-RAD, USA) using ECL kits and quantified by Molecular Imager Image Lab software (BIO-RAD, USA). All protein band densities were normalized relative to beta tubulin. At least three separate experiments were performed.
2.15 Statistical analysis
Data are presented as the means ± SD and were analyzed using IBM SPSS Statistics version 20 (SPSS Inc., IL, USA). The independent samples t test was used to compare data between two groups. Comparisons among three or more groups were conducted using one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test. The statistical significance of the genotype and treatment effects was assessed using two-way ANOVA followed by Tukey’s post-hoc test. P < 0.05 was considered statistically significant.