Materials
Genipin was purchased from MedChemExpress (Monmouth Junction, New Jersey, USA). Tau-R3 was obtained from ChinaPeptides (Shanghai, China). Heparin sodium salt was obtained from Aladdin (Shanghai, China). Thioflavin T (ThT) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco's modified Eagle medium (DMEM), F12-DMEM, opti-MEM, neurobasal medium, B27 supplement, streptomycin, penicillin, L-glutamine and phosphate buffer solution (PBS) were purchased from Gibco (Grand Island, NY, USA). Fetal bovine serum (FBS) was supplied from Biological Industries (Kibbutz Beit Haemek, Israel). The cell counting kit (CCK)-8 and bicinchoninic acid (BCA) protein assay kit were provided by Beyotime (Jiangsu, China). Protease and phosphatase inhibitors were obtained from Bimake (Shanghai, China). The following antibodies were used in this study: anti-Tau, anti-phospho-T231, anti-phospho-S396, anti-phospho-S404, anti-CDK5, anti-phospho-GSK-3β (Tyr216), anti-LC3, anti-amyloid precursor protein (APP), anti-Aβ, anti-BACE1, and anti-β-actin (Abcam, Cambridge, UK); anti-p62, anti-Beclin-1, anti-SIRT1, anti-LKB1, anti-phospho-LKB1, anti-AMPK, anti-phospho-AMPK, anti-mTOR, anti-phospho-mTOR, anti-p70S6K, anti-phospho-p70S6K, anti-PERK, anti-phospho-PERK, anti-eIF2a, and anti-phospho-eIF2a (Cell Signaling Technology, Beverly, MA, USA).
ThT fluorescence assay
ThT fluorescence assay was performed according to the method of our previous study[19]. Tau-R3 was prepared freshly and mixed with heparin (16 μM heparin and 20 μM ThT in 50 mM PBS). Genipin or dimethyl sulfoxide (DMSO) was then added to the mixture. After mixing, the samples were immediately incubated at 37°C and analyzed with a microplate reader (Fluoroskan Ascent FL, Thermo Scientific, USA) at different time points. The ThT fluorescence of the samples was measured with an excitation wavelength of 440 nm and an emission wavelength of 485 nm.
Transmission electron microscopy (TEM)
The procedure of TEM was performed based on our previous study with some modifications[19]. Tau-R3 (20 μM) and heparin (16 μM) were incubated with or without genipin (20 μM) at 37°C for 24 h. Then, one drop of the sample was deposited on copper grids (230 mesh, 5 μm aperture, Beijing Zhongjingkeyi Technology Co., China) and allowed to dry at 25°C. After rinsing twice with water, the grids were dyed with 5 μL 1% uranyl acetate. Then, the excess solution was removed using filter paper, and the grids were dried at room temperature (RT). The grids were analyzed using a JEM-1230 transmission electron microscope (JEOL, Tokyo, Japan).
Molecular docking
Molecular docking studies between genipin and the human Tau protein were examined with Sybyl-X 2.0 software. The three-dimensional (3D) coordinate of the Tau protein (PDB ID: 5O3L) was downloaded from the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank (PDB) (http://www.rcsb.org/pdb/home/home.do). The 2D structure of genipin was drawn using ChemBioDraw Ultra 14.0 software and then optimized by ChemBio3D Ultra 14.0 software with the MM2 method to obtain the 3D structure. The Surflex-Dock program was used to identify the potential interaction modes between genipin and the human Tau protein. Visualization of the docked conformation was performed by Chimera molecular graphics software (http://www.cgl.ucsf.edu/chimera/) and Ligplot (http://www.ebi.ac.uk/thornton-srv/software/LigPlus/).
Cell culture
The human embryonic kidney 293 (HEK293)/Tau cells were obtained from HEK293 cells (Shanghai Cell Bank of the Chinese Academy of Sciences, Shanghai, China) stably expressing cDNA of the longest human Tau (Tau441) and cultured in DMEM. The SH-SY5Y/Tau cells were obtained from human neuroblastoma cells (SH-SY5Y, Shanghai Cell Bank of the Chinese Academy of Sciences, Shanghai, China) stably expressing Tau441 cDNA and propagated in DMEM mixed with nutrient F12 (25:18, v/v) supplemented with 1% nonessential amino acids, 1% L-glutamine and 1% sodium pyruvate. Mouse neuroblastoma N2a cells (Shanghai Cell Bank of the Chinese Academy of Sciences, Shanghai, China) overexpressing the Swedish mutant APP (N2a/SweAPP cells) were grown in 50% opti-MEM and 40% DMEM. In addition, these cells were grown with 10% FBS, 100 μg/mL streptomycin and 100 IU/mL penicillin and incubated in a humidified 5% CO2 atmosphere at 37°C.
Primary neuron culture
Primary neuron culture referred to our previous study[20]. Briefly, the hippocampi of newborn 3×Tg-AD mice (JAX order number 3591206, Bar Harbor, ME, USA) were obtained and then were cut into pieces using a scalpel and digested by papain (2 mg/mL) at 37°C for 30 min. The suspension was filtered and centrifuged at 1,000 rpm for 5 min. The primary neuronal cells were obtained and seeded in poly-L-lysine (0.1 mg/mL)-coated culture flasks, and were grown in neurobasal medium with 2% B27 supplement, 1% L-glutamine, penicillin and streptomycin at 37°C in 5% CO2.
Cell viability assay
Cell viability was evaluated using a CCK-8 assay. In brief, HEK293/Tau cells, SH-SY5Y/Tau cells or N2a/SweAPP cells in 96-well plates (1×105 cells/well) were pretreated with various concentrations of genipin (0, 5, 10, 20, 30 and 40 μM) for 24 h. Then, the cells were incubated with CCK-8 solution approximately 2 h after removing the culture supernatant. Finally, the absorption was measured by a microplate reader (BioTek, Vermont, USA) at 450 nm.
Western blot analysis
After treatment with genipin (0, 10, 20 and 40 μM) for 24 h, total protein was collected from HEK293/Tau cells, SH-SY5Y/Tau cells or N2a/SweAPP cells using lysis buffer containing 1% protease and phosphatase inhibitor. Then, the protein concentration was quantitated by a protein assay kit, and the protein was separated by SDS-PAGE. The protein was blotted onto nitrocellulose (NC) membranes (Merch/Millipore, Schwalbach, Germany) and blocked with 5% bovine serum albumin. After hybridization with primary antibodies (1:1000) at 4°C overnight, the NC membranes were washed with PBS three times and hybridized with secondary antibodies (1:5000) at RT for 1 h. Subsequently, the immunoreactive protein was analyzed using an electroluminescence kit (Thermo Fisher Scientific, Waltham, MA, USA).
Immunofluorescence staining
After 40 μM genipin treatment for 24 h, HEK293/Tau cells or the primary neuronal cells of 3×Tg-AD mice were fixed with methanol for 10 min on ice. Then, the cells were incubated with 10% goat serum and 0.1% Triton X-100 at RT for 1 h. After incubation with primary antibodies at 4°C overnight, the cells were rinsed with PBS three times and treated with Alexa Fluor-conjugated secondary antibody at RT for 1 h. Then, the secondary antibody was removed, and the cells were rinsed with PBS three times. After the cell nuclei were dyed with DAPI (Invitrogen, Carlsbad, CA) at RT in a dark room, micrographs of cells were visualized using a confocal microscopy (Carl Zeiss, Thornwood, NY, USA).
Statistical analysis
The data are expressed as the mean ± standard deviation (SD) and were processed by GraphPad Prism 6.0. Significant differences were analyzed using a two-tailed Student’s t-test. When P values < 0.05, differences were considered to reach statistical significance. * p < 0.05, ** p < 0.01, *** p < 0.001.