Reagents and antibodies
The following reagents and antibodies used in this study were purchased commercially: TNFα from AbFrontier (Seoul, South Korea); z-IETD-fmk (caspase-8 inhibitor), mouse anti-HA antibody and rabbit anti-GM130 antibody from Abcam (Cambridge, UK); 2′,7′-dichlorofluorescin diacetate (DCFH-DA), hydrogen peroxide (H2O2), 1-methyl-4-phenylpyridinium (MPP+), propidium iodide (PI), poly-D-lysine, brefeldin A (BFA), GW4869, monensin, cycloheximide (CHX), necrostatin-1 (Nec-1), DPQ, proteinase K (PK), Dynasore, heparin, Heparinase III, mouse anti-β-actin, and mouse anti-Flag antibody from Sigma-Aldrich (Saint Louis, MO, USA); 4′,6-diamidino-2-phenylindole (DAPI), horseradish peroxidase (HRP)-conjugated anti-mouse antibody, and HRP-conjugated anti-rabbit antibody from Thermo Fisher Scientific, Inc. (Rockford, IL, USA); mouse anti-Flotillin-1 from BD Biosciences (San Jose, CA, USA); bafilomycin A1, mouse anti-Alix antibody, mouse anti-β-Galactosidase antibody (40-1a), rabbit anti-Calregulin antibody, mouse anti-CD63 antibody, mouse anti-Parkin antibody, and mouse anti-FAF1 antibody from Santa Cruz Biotechnology (Dallas, TX, USA); mouse anti-Hsc70 antibody and mouse anti-Hsp90 antibody from Enzo Life Sciences (Farmingdale, NY, USA); And zVAD-fmk (z-VAD) from Calbiochem (Darmstadt, Germany).
Cell culture and transfection
SH-SY5Y, MEF, HEK293, RAW264.7, HeLa, PANC-1, MIA PaCa-2 and MCF-7 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, WelGENE, Daegu, Korea) containing 10% fetal bovine serum (FBS, Atlas Biologicals, Fort Collins, CO, USA) and 1% antibiotic-antimycotic (Gibco BRL, Grand Island, NE, USA) unless otherwise specified. Cells were transfected with the indicated plasmids using Bio T (Morganville Scientific Inc., Morganville, NJ, USA) following the manufacturer’s protocol. Rat midbrain cultures derived from postnatal day 1 were prepared using standard procedures. Briefly, material dissected form the ventral portion of the midbrain was cleaned free of meningeal tissue, minced, and enzymatically dissociated in a mixture of papain/DNase (Sigma-Aldrich). Dissociated cells were plated onto amine-coated 6-well plates (BD Biosciences). Cells were maintained in neurobasal medium (Gibco), with B27 serum-free supplements (Gibco), 0.5 mm l-glutamine. After 5 days of culture, the cells were infected using AAV1 (adeno-associated virus 1)-hFAF1 viral vectors (MOI of 10,000 and 50,000).
The siRNA-resistant parkin construct was generated using QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA). The primers were as follows: #1; 5′-GAGCTGAGAAACGACTGGACTGTGCAGAATTGTG-3′, #2; 5′-GGGAAGGAGCTGAG AAACGATTGCACTGTGCAGA -3′.
All small interfering RNAs (siRNAs) against parkin and scrambled RNA (scRNA) were purchased from Bioneer (Daejeon, South Korea). The sequences of the siRNAs used in this study were as follows: siRNA against parkin (5′-UGAGGAAUGGGACUGU-3′). The scRNA or siRNA were transfected into SH-SY5Y cells using Lipofectamine RNAi MAX (Thermo Fisher Scientific) according to the manufacturer’s instructions.
Preparation of conditioned medium
To prepare conditioned medium (CM), cells transfected with the indicated plasmids were cultured in 60 mm diameter dishes in DMEM containing 10% FBS. After 24 h, the cells were switched to serum-free DMEM for the indicated times. Here, we used serum-free medium to avoid interference from albumin-enriched FBS. Then, the CM was collected and centrifuged at 800⨯g for 5 min and 2000⨯g for 10 min to remove cellular debris. For western blot analysis, the CM was concentrated using 50 kDa or 100 kDa cutoff Amicon Ultra filters (Millipore, Billerica, MA, USA) at 4000⨯g for 10 min or 15 min.
Western blot analysis
Cells were harvested, washed twice with PBS and lysed with mammalian lysis buffer [50 mM Tris-Cl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.4 mM phenylmethylsulfonylfluoride]. Then, the protein concentrations were quantified by using a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA). After quantification, samples were boiled in 6× protein sample buffer [250 mM Tris-Cl (pH 6.8), 30% glycerol, 10% SDS, 5% β-mercaptoethanol]. Then, samples were electrophoresed by SDS-PAGE and transferred to nitrocellulose membranes (GE Healthcare, Maidstone, UK). The membranes were blocked with 5% skim milk in PBS with 0.1% Tween-20 (PBST) and incubated with the indicated primary antibodies overnight. After their washing with PBST, the membranes were incubated with secondary antibodies. Immunoblot signals were measured by using chemiluminescent detection (Lab Frontier, Anyang, Korea).
CHX chase assay
SH-SY5Y cells were transiently transfected with the indicated plasmids for 24 h. After transfection, the cells were switched to serum-free DMEM containing CHX (Sigma-Aldrich, 20 μg/ml) for the indicated of times. Subsequently, the medium was concentrated with 50 kDa cutoff Amicon Ultra filters (Millipore) and analyzed by western blot analysis.
Purification of exosomes
We followed a previously described protocol with some modifications . Briefly, cells were transfected with the indicated plasmids for 24 h and incubated in DMEM containing 10% exosome-depleted FBS (System Biosciences, Palo Alto, CA, USA) for 48 h. The CM was then collected and subjected to sequential centrifugation at 800⨯g for 5 min, 2000⨯g for 10 min and 10,000⨯g for 30 min at 4 °C to remove cellular debris. Using a Beckman Coulter Optima L-90 K ultracentrifuge with a type 41Ti rotor, the supernatant was then spun down at 140,000⨯g for 70 min. The pellet was resuspended in PBS and then spun again at 140,000⨯g for 70 min at 4 °C. Finally, the pellet was resuspended in PBS or radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich).
In addition to their isolation via ultracentrifugation, we isolated exosomes using ExoQuick-TC (System Biosciences) according to the manufacturer’s instructions.
Treatment of vesicles with Na2CO3
To separate integral membrane proteins and luminal proteins, purified exosomes were treated with 100 mM Na2CO3 (pH 11) for 30 min at 4 °C as previously described . After centrifugation at 50,000⨯g for 60 min, integral proteins remained in the pellet fraction, while luminal proteins remained in the supernatant fraction. The pellet fractions were resuspended in RIPA buffer (Sigma-Aldrich) and the supernatants were collected in a separate tube for western blot analysis.
Proteinase K digestion
PK (Sigma-Aldrich) was added to the samples at a final concentration of 2 μg/ml. Then, the samples were incubated at 37 °C for 30 min and 5 mM phenylmethylsulfonyl fluoride was added to inhibit the activation of PK, followed by the addition of protein sample buffer.
Immunodepletion of CM
Fifty microliters of protein G-conjugated Dynabeads (ThermoFisher Scientific) was incubated overnight with mouse monoclonal antibody against FAF1 (final concentration of 0.5 or 1 μg/ml) before its addition to CM. The antibody-Dynabeads complex was incubated with 3 ml of CM at 4 °C overnight. After the complex was removed using a magnet, the immunodepleted CM was concentrated using a 50 kDa cutoff Amicon Ultra filter and used for western blot analysis. For flow cytometry, unconcentrated immunodepleted CM was applied to recipient cells.
To evaluate cell death, we measured PI-positive cell staining by using a Guava EasyCyte flow cytometer (Millipore). Briefly, cells were switched to serum-free DMEM or neurobasal medium for the indicated time. Additionally, to measure recipient cell death, SH-SY5Y and rat primary neuronal cells were treated with CM from donor cells for the indicated times. The cells were harvested, stained with PI (50 μg/ml), and evaluated using a Guava EasyCyte flow cytometer, following which the results were quantified using InCyte software (Millipore).
Concentrated CM treatment
SH-SY5Y cells (donor cells) were plated on 60 mm tissue culture dishes and transfected with GFP-vector or GFP-FAF1 plasmid. At 24 h after transfection, the cells were incubated in serum-free medium for 24 h. After the CM was concentrated by using 50 kDa cutoff Amicon Ultra filters, the concentrated CM was dissolved in new serum-free medium that was applied to recipient cells on poly-L-lysine-coated coverslips in 12-well plates for 24 h.
SH-SY5Y cells (donor cells) were plated on 60 mm tissue culture dishes. Donor cells were transfected with GFP-vector or GFP-FAF1 plasmids. At 24 h after transfection, the cells were collected and replated in cell culture inserts (polycarbonate membrane, 3.0 μm pore size, Corning, Kennebunk, ME, USA) at a density of 1⨯105 cells. SH-SY5Y cells (recipient cells) were plated at a density of 1⨯105 cells on poly-D-lysine-coated coverslips in 12-well plates. After 24 h, the cultures were combined such that the donor cells were in the insert and separated from recipient cells plated on a coverslip.
Cells were fixed with 4% paraformaldehyde for 15 min. The cis-Golgi were stained with GM130. After the nuclei were stained with DAPI for 10 min. The coverslips were mounted onto microscope slides using fluorescence mounting medium (Dako, Carpinteria, CA, USA) and analyzed using a Zeiss LSM 510 laser scanning confocal microscope (Carl Zeiss, Oberkochen, Germany).
For transmission electron microscopy (TEM), samples were prepared using the Exosome-TEM-easy Kit containing a Formvar-carbon-coated EM mesh 400 grid, wash buffer, and EM solution (101 Bio, Mountain View, CA, USA). The pellets from the 12 ml of CM (vector- or FAF1-transfected cells) obtained by ultracentrifugation were resuspended in 60 μl of PBS, 10 μl of which was applied to the grid. All samples were prepared following the manufacturer’s instructions. For immuno-EM, the pellets were first fixed with 40 μl of 4% paraformaldehyde and 0.2% glutaraldehyde (Sigma-Aldrich) overnight at 4 °C. Then, the fixed exosome solution was transferred to grids and subsequently treated with 0.05 M glycine for 10 min to quench free aldehyde groups. After blocking with PBS containing 1% BSA for 30 min, the grids were incubated for 1 h with the indicated antibodies (diluted 1:50 in PBS containing 0.1% BSA) at room temperature. After three washes with PBS containing 0.1% BSA, the grids were incubated for 1 h with the secondary antibody (anti-mouse IgG conjugated to 10 nm gold particles, 1:25, Sigma-Aldrich) at room temperature. Three washes to eliminate secondary antibody were followed by incubation with EM solution and a wash step. Samples were viewed under a Talos F200X transmission electron microscope (FEI, Hillsboro, OR, USA) operated at 200 kV, and images were captured with a Ceta 16 M pixel CMOS camera (FEI).
Nanoparticle tracking analysis
Following isolation by differential ultracentrifugation or ExoQuick-TC (System Biosciences), the exosome pellets were resuspended in 50 μl of PBS. Then, 10 μl of the exosome solution was diluted in PBS to a total volume of 1 ml. The samples were analyzed by nanoparticle tracking analysis using a NanoSight NS300 (Malvern Panalytical Ltd., Malvern, UK), equipped with a 405 nm laser. To accurately identify the vesicles, the detection threshold was set at 5. The number of vesicles in each sample represents the number of particles per ml of medium. Cells were counted using a Muse Count & Viability Kit (Millipore) on a Muse cell analyzer (Millipore).
Caspase-3 activity assay
SH-SY5Y cells were treated with CM from FAF1-transfected cells plus caspase-8 inhibitor or TNFα (Ab frontier) plus CHX (Sigma-Aldrich) at the indicated concentrations for the indicated times. Then, caspase-3 activity was measured by using a caspase-3 colorimetric assay kit (BioVision, Milpitas, CA, USA) in accordance with the manufacturer’s protocol. The absorbance at 450 nm was measured with the use of a VICTOR microplate reader (PerkinElmer, Norwalk, CT, USA).
We investigated the presence of a signal peptide in FAF1 using SignalP-4.1 (http://www.cbs.dtu.dk/services/SignalP-4.1/) with secretogranin-1 used as a positive control as it contains a signal peptide.
Experiments were independently carried out three times (n = 3). All the data are expressed as the mean ± standard deviation (S.D.). Statistical comparisons were performed using Student’s t-test or one-way analysis of variance (ANOVA) followed by Tukey’s HSD post hoc analysis using SPSS software (statistics version 22; IBM, Inc., Chicago, IL, USA). Statistical significance was established when the P-value was lower than 0.05.