Cell culture
The primary murine cortex neuronal cells were prepared from embryonic 18-day ICR mice. Briefly, the brain was dissected in Hanks Buffered Saline Solution without Mg2+ and Ca2+ (HBSS: GIBCO, Grand Island, NY, USA), and digested in 0.25% trypsin containing DNAse I (2000 Units/mg) (GIBCO, Carlsbad, CA, USA) for 20 min at 37 °C. The obtained cell suspension was diluted in DMEM containing 25 mM glucose and 10% fetal bovine serum (FBS), and cultured in tissue culture flasks coated with 50 µg/ml poly-D-lysine at a density of 3–4 × 105 cells/cm2. Human neuroblastoma cell line SK-N-SH was obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). SK-N-SH cells were cultured in Minimum Essential Medium (Hyclone Laboratories, Logan, UT, USA) containing 10% FBS (GIBCO, Grand Island, NY, USA) and gentamycin (0.1 mg/mL) in a humidified incubator at 37 °C with 5% CO2. SK-N-SH (human neuroblastoma cell line, passage no.14) was acquired from the American Type Culture Collection (ATCC, Rockville, MD, USA). SK-N-SH cells were cultured in Minimum Essential Medium (Hyclone Laboratories, Logan, USA) with 10% fetal bovine serum (GIBCO, NY, USA) and gentamycin (0.1 mg/mL) at 37 °C and 5% CO2.
PRP (106-126) Treatment
Synthetic prion peptides PrP (106–126) (sequence, Lys-Thr-Asn-Met-Lys-His-Met-Ala-Gly-Ala-Ala-Ala-Ala-Gly-Ala-Val-Val-Gly-Gly-Leu-Gly) were synthesized by Peptron (Seoul, Korea). The peptides were dissolved in sterile dimethyl sulfoxide (DMSO) at a concentration of 10 mM (stock) and stored at -20 °C.
Annexin V Assay
Cells in the logarithmic phase were collected and cultured in 24-well plate at 4 × 104 cells/well. Cell survival was evaluated using an annexin V Assay kit (Santa Cruz Biotechnology, CA, USA) following to the manufacturer's procedure. The fluorescence was determined at 488-nm excitation and 525/30 emission using a Guava EasyCyte HT System (Millipore, Bedford, MA, USA).
Lactate Dehydrogenase Assay
Cytotoxicity was measured by evaluating the levels of lactate dehydrogenase in the cell culture supernatant using the lactate dehydrogenase (LDH) detection kit (Takara Bio, Inc., Tokyo, Japan) following the manufacturer’s protocol. LDH activity was detected by measuring absorbance at 490 nm using a microplate reader (Spectra Max M2, Molecular Devices, Sunnyvale, CA, USA).
BacMam Transduction
GFP-tagged wild-type or mutant LC3B was overexpressed in neuronal cells via viral transduction using Premo Autophagy Sensor LC3B-GFP (BacMam 2.0) kit (Life Technologies, P36235). Briefly, LC3B-FP or LC3B (G120A)-FP viral particles (MOI = 30) were added to the growth medium and autophagosomes dynamics were visualized using fluorescence microscopy. The mutant LC3B (G120A)-FP was used as negative control.
Immunocytochemistry
Cells in the logarithmic phase were collected and cultured in 1% gelatin-coated cover-slit (12 mm; Nalge Nunc International, Naperville, IL) in 24-well plate at 4 × 104 cells/well. After treatment, fixed with 4% PFA in Phosphate Buffered Saline (PBS; 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, 0.24 g KH2PO4 in 1L distilled water to desired pH 7.4) for 20 minutes at room temperature (RT). Cells were washed in sterilized Tris-Buffered Saline (TBS; 2.423 g Tris, 8.006 g NaCl in 1L distilled water to desired pH 7.6) with 0.1% Tween 20 (TBST) for 10 minutes, then blocked for 15 minutes in TBST with 5% FBS, and then incubated for 3 h at RT with the primary antibodies (anti-phospho-AMPKα diluted 1:100; #2535, Cell Signaling Technology) diluted with in TBST with 5% FBS. Alexa Fluor 488-labeled donkey anti-rabbit IgG antibody (Molecular Probes, A21206) diluted 1:1000 was employed to visualize channel expression using fluorescence microscopy (Nikon Eclipse 80i). Image was evaluated using the NIS-Elements F ver4.60 Imaging software.
Confocal Microscopy
For confocal microscopy, the cells were cultured on coverslips. After treatment, the cells were fixed with 4% PFA (in PBS) for 20 min at room temperature (RT) and permeabilized using 0.3% Triton X-100 (in PBS) with 5% horse serum for 10 min. Subsequent incubations were performed in permeabilization buffer. The cells were incubated with primary antibodies for 60 min at RT, washed four times with PBS and incubated with Alexa Fluor 488, Alexa Fluor 568, and Alexa Fluor 647 conjugated secondary antibodies at a concentration of 0.3 µg/ml each for 60 min at RT. The coverslips were placed in mounting medium and imaged on a Zeiss LSM710 microscope equipped with a standard set of lasers through a 63 × oil objective. Excitation wavelengths were 488, 543, and 633 nm. Bandpass filters were set at 500–550 (Alexa Fluor 488), 560–615 nm (Cy3, Alexa Fluor 568) and 650–750 nm (Alexa Fluor 647). Image acquisition was accomplished at the 12-bit rate. Settings were optimized to ensure suitable dynamic range, low background, and appropriate signal/noise ratio.
Measurement Of Ca2+
Ions
Neuronal cells cultured on collagen-coated confocal dishes were incubated with 5 µM Fluo-4 AM (Invitrogen, Thermo Fisher Scientific) in culture media containing 1% FBS at 37 °C for 40 min. The cells were washed three times with HBSS (Hank’s Balanced Salt Solution). Intracellular calcium dynamics were visualized using a confocal microscope (Zeiss) with 488 nm excitation and 530 nm emission. For [Ca2+]i calculation, the method of Tsien et al. [35] was employed with the following equation: [Ca2+]i = Kd(F − Fmin) / (Fmax − F), where Kd is 345 nM for Fluo-4, and F is the observed fluorescence level. Each tracing was calibrated for the maximal (Fmax) by adding ionomycin (2 µM) and for the minimum intensity (Fmin) by adding EGTA (5 mM) at the end of each measurement.
Western Blot Analysis
Cells in the logarithmic phase were collected and cultured in 6-well plate at 3 × 105 cells/well. After treatments, cells were washed with PBS and lysed in lysis buffer [25 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), pH 7.4, 100 mM NaCl, 1 mM ethylene diamine tetra acetic acid (EDTA), 5 mM MgCl2, 0.1 mM dithiothreitol (DTT), and a protease inhibitor mixture]. Equal quantities of cellular proteins (more than 15 µg/µl) were electrophoretically resolved on a 10% sodium dodecyl sulfate (SDS) poly-acrylamide gel and transferred to a nitrocellulose membrane. Immuno-reactivity was detected through consecutive incubation with blocking solution using 5% skim milk and primary antibodies, followed by the corresponding horseradish peroxidase-conjugated secondary antibodies, and finally edveloped using enhanced chemi-luminescence substances i.e. west save gold detection kit (LF-QC0103, AbFrontier Inc.). The primary antibodies that anti-phospho-AMPKα diluted 1:1000 (#2535, Cell Signaling Technology), anti-AMPKα diluted 1:1000 (#2532, Cell Signaling Technology), anti-LC3B diluted 1:1000 (#4108, Cell Signaling Technology), anti-P62 diluted 1:1000 (#5114, Cell Signaling Technology) and anti-β-actin diluted 1:5000 (A5441, Sigma Aldrich) using antibody solution (1% skim milk in TBST) were used for immunoblotting. Images were inspected using a Fusion FX7 imaging system (Vilber Lourmat, Torcy Z.I. Sud, France). Densitometry of the signal bands was evaluated using the Bio-1D software (Vilber Lourmat, Marne La Vallee, France).
TEM (transmission Electron Microscopy) Analysis
Cells were fixed in 2% glutaraldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) and 2% paraformaldehyde (EMS, USA) in 0.05 M sodium cacodylate (pH 7.2; Electron Microscopy Sciences) for 2 h at 4 °C. After, the samples were fixed in 1% osmium tetroxide (Electron Microscopy Sciences) for 1 h at 4 °C, dehydrated by incubating in alcohol solutions of increasing concentration (25, 50, 70, 90 and 100%) for 5 min each and entrenched in epoxy resin (Embed 812; Electron Microscopy Sciences) for 48 h at 60 °C following the manufacturers’ instructions. Ultrathin sections (60 nm) were cut using the LKB-III ultratome (Leica Microsystems GmbH, Wetzlar, Germany) and stained using 0.5% uranyl acetate (Electron Microscopy Sciences) for 20 min and 0.1% lead citrate (Electron Microscopy Sciences) for 7 min at room temperature. Images were captured on a Hitachi H7650 electron microscope (Hitachi, Ltd., Tokyo, Japan; magnification, 10,000×) at the Center for University-Wide Research Facilities (CURF), Chonbuk National University.
Statistical analysis
Results are presented as the mean of replicas ± SEM. The Welch’s correction was employed for comparing two samples. The one-way ANOVA followed by the Tukey post-hoc test was applied for comparing multiple samples. All statistical analyses were implemented with GraphPad Prism version 5.0 software. P values such as * p < 0.05, ** p < 0.01 or *** p < 0.001 were considered statistically significant.