Primary cultures of rat hippocampal neurons were prepared from the brains of day 18 embryonic rats. Briefly, the hippocampus was dissected in free HBSS and incubated with a 0.125% trypsin solution for 15 min at 37°C. The resulting cell suspensions were diluted in neurobasal medium (#21103-049, Gibco), supplemented with SM1 components (#05711, Stemcell), and plated onto 100 μg/mL poly-D-lysine (#P0899, Sigma-Aldrich) and 2 μg/mL laminin (#11-243-217-001, Roche)-coated plates or coverslips.
Pharmacological treatment of hippocampal neuron
Propionic acid (#402907) and bafilomycin A1 (B1793) were purchased from Sigma-Aldrich, USA, and PD98059 (#513000) was purchased from Calbiochem. PPA was dissolved in phosphate-buffered saline (PBS) for treatment (100 μmole/mL). Bafilomycin A1 (2 nmole/mL) and PD98059 (10 μmole/mL) were dissolved in dimethyl sulfoxide and stored in aliquots at −20°C until the experiments. Vehicle (PBS), bafilomycin A1 (2 nmole/mL), and PD98059 (10 μmole/mL) were simultaneously treated with PPA (100 μmole/mL). PPA treatment was denoted as DIV 18, and cells were harvested on DIV 21.
Western blotting analysis
Cultured neurons were harvested by scraping in ice-cold radio-immunoprecipitation assay buffer (#89900, Thermo Scientific) solution containing a protease inhibitor (A32963, Thermo Scientific) and phosphatase cocktail inhibitors (#5970, Cell Signaling) to avoid phosphorylation and degradation of proteins. After incubation, all lysates were centrifuged at 15,000 g at 4°C for 30 min. The supernatant was then evaluated for total protein concentration using a BCA protein assay kit (#23225, ThermoFisher). Equal amounts of protein samples were incubated with 5X SDS sample loading buffer (CBSS-9005, CHEM-BIO) at 95°C for 5 min. The samples (10 μg) were subjected to SDS-polyacrylamide gel electrophoresis on precast, 4–15% gradient mini-gels (#456-1085, Bio-rad). Following transfer to PVDF membranes (#1620177, Bio-rad), the membranes were blocked in Tris-buffered saline (#CBTB-9110, CHEM-BIO) containing 3% BSA (#9048-4-8, GENEray Biotechnology) and 0.1% Tween 20 (H5152, Promega) for 1 h. Membranes were then washed with TBST and incubated overnight at 4°C with primary antibodies against phosphorylated ERK1/2 (#4370, Cell Signaling), phosphorylated AKT (#4060, Cell Signaling), LC3A/B (#12741, Cell Signaling), p62 (ab56416, Abcam), and beclin-1 (#3495, Cell Signaling). Membranes were then probed with horse radish peroxidase-conjugated secondary antibody (1:5000) for 1 h and developed using an enhanced chemiluminescence immunoblot detection system (Fusion FX7, VILBER). Immunoblots for phosphorylated ERK1/2 and phosphorylated AKT were subsequently stripped and re-probed with anti-ERK1/2 (#4692, Cell Signaling) and anti-AKT (#4691, Cell Signaling) antibodies. Immunoblots were analyzed by densitometry using ImageJ software (National Institutes of Health). Only film exposures that were in the linear range of the ECL reaction (#32106, Thermo Scientific) were used for quantification analysis.
Cultured neurons were fixed with 1% paraformaldehyde in PBS containing 4% sucrose for 5 min at room temperature. Without washing, neurons were then permeabilized and blocked simultaneously in 100% methanol for 7 min at −20°C. Primary antibodies against LC3A/B (#12741, Cell Signaling), p62 (ab56416, Abcam), lysosomal-associated membrane protein (LAMP1) (H4A3, Santa Cruz), and ubiquitin (P4D1, Santa Cruz) were added to blocking solution containing 0.1% gelatin, 0.3% Triton X-100, 16 mM sodium phosphate, and 450 mM NaCl and incubated overnight at 4°C. After washing with PBS, coverslips were incubated with AlexaFluor488 (#4412, Cell Signaling) or AlexaFluor594 (#8890, Cell Signaling)-conjugated secondary antibodies for 1 h at room temperature and then washed extensively with PBS and distilled water. Subsequently, coverslips were mounted with mounting medium (H-1000, Vector Laboratories). The samples were imaged with a confocal laser-scanning microscope (Nikon, Japan) using a 60X oil lens and 488 nm and 594 nm emission lasers.
Confocal microscopy for dendritic spines
Cells were maintained in an incubator with 5% CO2 at 37°C. The neurons were transfected with 2 μg of GFP construct with 2 μL of lipofectamine 2000 (Invitrogen). Then, cells were stained by immunofluorescence labeling with anti-GFP antibody (A11122, Invitrogen). The samples were imaged with a confocal laser-scanning microscope (Nikon, Japan) using a 60X oil lens and 488 nm laser. Our data indicate that all spine categories were represented in pyramidal neurons. Dendritic spine densities were assessed by analyzing high-resolution digital images with ImageJ software (National Institutes of Health). Dendritic spines were counted manually using the point picker function in ImageJ particle analysis. The dendritic field was estimated to be 10 μm in length.
Detection of autophagic flux
The formation of autophagosomes and autolysosomes in hippocampal neurons of control and PPA-treated cells was detected using a Premo™ Autophagy Tandem Sensor RFP-GFP-LC3B Kit (P36239, Invitrogen), according to the manufacturer’s instructions. The RFP-GFP-LC3B sensor enables the detection of LC3B-positive, neutral pH autophagosomes in green fluorescence (GFP), and LC3B-positive acidic pH autolysosome in red fluorescence (RFP). Cells were grown on coverslips and incubated with 10 μL of BacMam reagents containing RFP-GFP-LC3B for 16 h. Cells were then washed in PBS three times. Coverslips were mounted with mounting medium (H-1000, Vector Laboratories), and fluorescent images were taken using confocal microscopy (Nikon, Japan). LC3B-positive autophagosomes (GFP and YFP) and LC3B-positive autolysosomes (RFP only) were analyzed and quantified using ImageJ software (National Institutes of Health).
Transmission electron microscopy for autophagy
Neuron cells were treated with 100 μM of PPA for 3 days and then fixed at 4°C in 2.5% glutaraldehyde (#16210, EMS) and 2% paraformaldehyde (#19210, EMS); cells were then post-fixed with 2% osmium tetroxide (#19150, Sigma-Aldrich) for 30 min at 4°C. Then, the cells were stained en bloc with 0.1 mg TCH (T1136, TCI) in 10 mL distilled water and 1% uranyl acetate (#22400, EMS) and dehydrated via a graded ethanol series. Samples were then embedded with an EMBed-812 embedding kit (#14120, EMS). Embedded samples were sectioned (60 nm) with an ultra-microtome (Leica, Germany), and the sections were then viewed on a Tecnai 20 transmission electron microscope (TEM) (ThermoFisher, USA) at 120 kV. Numbers of autophagosomes and autolysosomes per 100 μm2 were measured, as well as the size of the ultrastructure, using ImageJ software (National Institutes of Health).
All data are represented as mean ± standard error of the mean (SEM) of at least three individual experiments. Statistical analysis was performed using one-way analysis of variance, and statistical significance (p-value) was calculated with GraphPad Prism 5 (GraphPad Software). Results at the 95% confidence level were considered significant.