Mouse embryonic fibroblasts and N2A neuroblastomas were maintained with DMEM (Gibco, Carlsbad, CA), 1% Penicillin/Streptomycin (Gibco, Carlsbad, CA), and 10% FBS (Gibco, Carlsbad, CA). SHSY5Y neuroblastomas were maintained with DMEM/F12 (Gibco, Carlsbad, CA), 10% FBS, 1% NEAA (Gibco, Carlsbad, CA), and 1% Penicillin/Streptomycin.
Protein extracts were prepared when cells were 85–95% confluent using M-PER protein extraction reagent (Pierce, Rockford, IL) with complete mini protease inhibitor cocktail tablets (Roche, Indianapolis, IN). Protein concentrations were determined by the Bradford method. Equal amounts of protein (12–20 μg) were separated by SDS/PAGE on a 4–12% or 10% Bis/Tris or 3–8% Tris-Acetate gels (Invitrogen, Carlsbad, CA), transferred to PDVF membranes, blocked for 1 hour in 5% (vol/vol) nonfat milk in Tris-buffered saline (pH 7.5) supplemented with 0.2% Tween20, and incubated overnight at 4°C with the appropriate primary antibody. Antibodies used in this study include LC3 (MBL Int, Woburn, MA), mTOR (Sigma, St. Louis, MO), p-mTOR (S2448, Cell Signaling, Danvers, MA), beclin1 (Santa Cruz Biotechnology, Santa Cruz, CA and Novus, Littleton, CO), UVRAG (Abgent, San Diego, CA), p150 (Abnova, Taipei City, Taiwan), Vps34 (Invitrogen, Carlsbad, CA), Atg12 (Novus, Littleton, CO), LAMP2a (Invitrogen, Carlsbad, CA), Ubiquitin (DAKO, Carpinteria, CA), CT20 (Calbiochem, Gibbstown, NJ), and Actin (Sigma, St. Louis, MO). Membranes were washed 5x and then incubated with HRP-conjugated secondary antibodies at room temperature. Quantitative densiometric analyses were performed on digitized images of immunoblots with ImageJ (National Institutes of Health). The background from each blot was subtracted from the raw data for each protein band and was then normalized to the protein loading control, β-actin.
EFGP-LC3 and LysotrackerRed quantification
EGFP-LC3 (Addgene- Plasmid 11546, Cambridge, MA) was transiently transfected using Lipofectamine2000 (Invitrogen, Carlsbad, CA). At the time of media replacement, complete medium alone, with Rapamycin (Calbiochem, Gibbstown, NJ, 0.2 μM in DMSO), or DMSO was used. LysotrackerRed (75nM, Invitrogen, Carlsbad, CA) was loaded into cells for 30 minutes prior to fixation. After treatment, cells were fixed using 4% paraformaldehyde. The Leica DM2500 (Leica Microsystems, Bannockburn) confocal microscope on was used to obtain the images.
Quantifications for EGFP-LC3 and LysotrackerRed were performed using manual counts of puncta per cell on ImageJ (NIH). The identity of the cell type was blinded to the experimenter and the same puncta criteria were used throughout. Cells expressing high levels of EGFP-LC3 were excluded to avoid counting aggregates of GFP and not autophagosomes.
Transient transfections of siRNA into SHSY5Y neuroblastomas were performed using the Amaxa nucleofector system (Kit V, Lonza, Basel, Switzerland). TwoμM of each siRNA (scramble-5′-AAATGTGTGTACGTCTCCTCC-3′, PSEN1- validated Mission siRNA Sigma-Aldrich SASI_Hs01_00043630, and PSEN2- validated Mission siRNA Sigma-Aldrich SASI_Hs01_00033516, St. Louis, MO) was used and knockdown was allowed to proceed for 40 hrs where after protein extracts were made for immunoblotting. For esiRNA, 2μM of each MISSION esiRNA (esiRNA EGFP Cat #:EHUEGFP, esiRNA PSEN1 Cat#: EHU073361, esiRNA PSEN1 Cat#: EHU070541, Sigma-Aldrich, St. Louis, MO) was used and knockdown was allowed to proceed for 48 hours after which protein extracts were made for immunoblotting.
Stable knockdown of beclin1 in wild-type mouse embryonic fibroblasts were performed using shRNA lentiviral delivery. Lentiviral shRNA beclin1 constructs were purchased from Open Biosystems. shRNAs were cotransfected into 293FT cells together with packaging plasmids by following the manufacturer’s protocol (Invitrogen, Carlsbad, CA ViraPowerTM Lentiviral Expression Systems kit). Mouse embryonic fibroblasts were passaged and plated in a 6-well plate and allowed to adhere for 24 hrs. Cells were subjected to lentiviral infection in the presence of polybrene overnight, and media was replaced the following day. After 24 hrs, cells were selected by treating with media containing 1.5 μg/ml puromycin. After 3–4 passages, cells were considered to have stable knockdown of beclin1 as confirmed by immunoblot.
Pulse-chase for long-lived proteins and long-lived protein proteolysis
Two protocol were used to assess the breakdown of long-lived proteins. Both were adapted from (Bauvy et al., 2009
First, to confirm that methionine treatment would not cause autophagy inhibition, cells were incubated with 10mM L-methionine (Sigma, St. Louis, MO) for 10hrs and then protein was extracted for immunoblot analysis. In the first analysis, cells were incubated with media containing 2μCi/mL of L-METHIONINE, [35S] (MP Biomed, Solon, OH) for 18 hours. Pulse samples were then lysed using M-PER protein extraction reagent (Pierce, Rockford, IL) containing a protease inhibitor cocktail (complete-mini, Roche, Indianapolis, IN). Cells for chase analysis were then washed with PBS and replaced with media containing 10mM L-methionine for one hr. Media was then removed and replaced again with media containing 10mM L-methionine for 4 hrs. The media was then removed and cells were lysed using M-PER protein extraction reagent (Pierce, Rockford, IL) containing a protease inhibitor cocktail (Complete-mini, Roche, Indianapolis, IN). Protein concentrations were then determined by the Bradford method. Equal amounts of protein (20 μg) were separated by SDS/PAGE on a 4–12% Bis/Tris gel (Invitrogen, Carlsbad, CA). The gels were then fixed for one hour and then incubated in Amplify solution (Amersham Biosciences, Piscataway, NJ) for 30 minutes. The gels were then dried using a slab gel dryer (Savant, SGD-5040, Thermo Scientific, Billerica, MA) and exposed to film at room temperature. Quantitative densiometric analyses were performed on digitized images of film using ImageJ (NIH).
For the second analysis, control and PSDKO fibroblasts were pulsed with 0.2μCi/ml L-valine-[H3] (MP Biomed, Solon, OH) for 48 hours. Cells were then washed extensively and media was replaced with 10mM valine for one hour. Once more, cells were washed extensively and media was replaced with 10mM valine and 0.2μM rapamycin when applicable. Subsequent time points of media and cells were collected at 4, 8, 12, and 24 hours. Rapamycin treated cells were taken at 12 hours. The media was precipitated with tricholoacetic acid (TCA) and the acid soluble radioactivity was measured. The cells were washed with cold 10% TCA with 10mM valine and then lysed with sodium hydroxide and the radioactivity was measured. Proteolysis is presented as the percentage of initial acid-precipitatable radioactivity transformed to acid-soluble radioactivity.
For immunocytochemical analysis of ubiquitin, cells were fixed on glass slides with 4% paraformaldehyde. The cells were then permeablelized and blocked using 10% NGS with Triton X100 in PBS. The cells were then incubated overnight at 4°C in the primary antibody solution (Ubiquitin 1:200, DAKO, Carpinteria, CA). After washing with PBS, the cells were incubated in the secondary antibody conjugated to Alexa-488 (Molecular Probes, Invitrogen, Carlsbad, CA). After washing with PBS, the slides were then coverslipped and allowed to dry for 48 hours before confocal microscopy was performed on the Leica DM2500 confocal microscope (Leica Microsystems, Bannockburn).
For fluorgenic proteasome substrate functional analysis using confocal microscopy, ZsProsensor-1 vector (Clontech, Mountain View, CA) was transiently transfected using the Amaxa nucleofector system (Lonza, Kit MEF 1, Basel, Switzerland). Twenty-four hours after transfection, cells were fixed using 4% paraformaldehyde and coverslipped. After 48 hrs, confocal microscopy was performed on the Leica DM2500 confocal microscope (Leica Microsystems, Bannockburn).
For kinetic fluorogenic proteasome activity assays, cells were homogenized in assay buffer containing 25 mM HEPES, pH 7.5, 0.5 mM EDTA, and 0.05% NP-40. Immediately prior to the kinetic readings the proteasome substrates (LLVY-AMC for the chymotrypsin-like, VGR-AMC for the trypsin-like, and LLE-AMC for the PGPH/caspase-like activity) were added at 75 nM. Readings for AMC release were taken at 37 °C every 1.5 min for 60–90 min (excitation 360 nm, emission 460 nm), on the Synergy HT using KC4 software (BioTek, Winooski, VT).
γ-Secretase Inhibitor treatments
Fibroblasts and N2A neuroblastomas were treated with γ-secretase inhibitor IX (N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-S-phenylglycine t-Butyl Ester, DAPT, 500nM, Catalog#:565784, Calbiochem, Gibbstown, NJ) and γ-secretase inhibitor X (L-685,458, (1S-Benzyl-4R-[1-(1S-carbamoyl-2-phenethylcarbamoyl)-1S-3-methylbutylcarbamoyl]-2R-hydroxy-5-phenylpentyl0carbamic Acid tert-butyl Ester, 200nM, Catalog#:565771 Calbiochem, Gibbstown, NJ) for 48 hrs. Protein extracts were then prepared using M-PER protein extraction reagent (Pierce, Rockford, IL) with a Complete-mini protease inhibitor cocktail tablets (Roche, Indianapolis, Indiana). Successful γ-secretase inhibition was determined using western blot analysis for C-terminal fragments of amyloid precursor protein using the antibody CT-20.
Aβ1-40 and Aβ1-42 were measured using a sensitive sandwich ELISA system as previously described (Koike et al., 2010
). Briefly, media was collected from N2A cells treated with vehicle or γ-secretase inhibitors after the full course of treatment. MaxiSorp immunoplates (Nunc, Rochester, NY, USA) were coated with mAB20.1 (William Van Nostrand, Stony Brook, NY) antibody at a concentration of 25 mg/ml in Coating Buffer (0.1 M NaCO3 buffer, pH 9.6), and blocked with 3% BSA. Samples were diluted at 1:5 prior to loading onto ELISA plates in duplicate. Standards of both Aβ40 and 42 were made in Antigen Capture Buffer (ACB; 20 mM NaH2PO4; 2 mM EDTA, 0.4 M NaCl; 0.5 g CHAPS; 1% BSA, pH 7.0), and loaded onto ELISA plates in duplicate and incubated overnight at 4°C. Plates were washed and then probed with either HRP-conjugated anti-Aβ 35-40 (MM32-13.1.1, for Aβ1-40) or anti-Aβ 35-42 (MM40-21.3.4, for Aβ1-42) overnight at 4°C.3,3′,5,5′-tetramethylbenzidine was used as the chromagen, and the reaction stopped by 30% O-phosphoric acid, and read at 450 nm on a Molecular Dynamics plate reader.
Presenilin rescue into PS1KO
pcDNA, wild-type presenilin-1 cDNA, and ΔTM1-2 presenilin-1 cDNA were transiently transfected using lipofectamine2000 (Invitrogen, Carlsbad, CA) into PS1KO mouse embryonic fibroblasts. Expression was allowed to proceed for 72 hours. Protein extracts were then prepared using M-PER protein extraction reagent (Pierce, Rockford, IL) with a Complete-mini protease inhibitor cocktail tablets (Roche, Indianapolis, Indiana).
Rapamycin and Lysosome Inhibitor Treatments
To induce autophagy, fibroblasts were treated with Rapamycin (0.2μM, Calbiochem, Gibbstown, NJ) for 24 hrs. To inhibit autophagy, fibroblasts were treated with the vacuolar ATPase inhibitor BafilomycinA1 (100nM for 7hrs, Sigma, St. Louis, MO) or the microtubule destabilizer Nocodazole (50μM for 2.5 hrs Sigma, St. Louis, MO).
Images were acquired on the Leica DM2500 confocal microscope. Acquisition software used was LAS_AF (Leica Microsystems, Bannockburn). Slides were prepared using fluromount mounting media (Southern Biotech, Birmingham, Alabama) and images were acquired at room temperature. Fluorochromes used are listed under the specific experiment.
Data are presented as mean + 1 SEM, with n = number of samples examined. An unpaired Student’s T-test or ANOVA was used to determine statistical significance (p < 0.05) indicated within the figure legend. Jmp8 statistical software was used.