4.2. Enzyme Activity Assay
Protein lysates were obtained by resuspending fibroblast pellets in an ice-cold lysis buffer (CelLytic, Sigma-Aldrich, St. Louis, MO, USA) without phosphatase and protease inhibitors. After centrifugation, the supernatant was collected, and protein concentration was measured using a bicinchoninic acid protein (BCA) assay (Sigma-Aldrich, St. Louis, MO, USA).
GCase enzymatic activity in fibroblast cell lines was assessed in 5 μg of protein and determined by fluorometry using 4-methylumbelliferone as fluorochrome, as described before [44
]. Fluorescence (excitation: 355 nm; emission: 460 nm) was measured using a microplate reader (CLARIOStar Plus, BMG LABTECH, Ortenberg, Germany) and results were reported as nmol of substrate/hour/mg protein.
Cathepsin D activity was assessed on fibroblast and SH-SY5Y lysates (1 μg of protein) with a fluorimetric assay kit (BioVision, Milpitas, CA, USA). Briefly, cell pellets were resuspended in 100 ul cathepsin D lysis buffer, incubated for 10 min on ice, and centrifuged at maximum speed for 5′. Fifty μL of samples were loaded together with negative controls in 96-well plates and incubated with a reaction mix (reaction buffer and substrate) at 37 °C for 1 h. Fluorescence (excitation: 328 nm; emission: 460 nm) was measured using a microplate reader (CLARIOStar Plus, BMG LABTECH, Ortenberg, Germany) and results were expressed as relative fluorescence unit.
4.3. EV Isolation and Quantification
EVs were isolated from conditioned media (CM) of fibroblast cell lines as described below. When cells were subconfluent (≈80%), the culture medium was removed, and cells were washed with PBS. New complete medium with EV-depleted bovine serum was added to the cells and incubated for 24 h. Following incubation, the CM was collected and centrifuged at 300 g × 10′, 2000 g × 20′ (Mikro 220R equipped with 1195-A rotor, Hettich, Tuttlingen, Germany) at 4 °C to eliminate dead cells and cellular debris/large apoptotic bodies, respectively. The CM was then centrifuged at 20,000 g × 60′, at 4 °C, to collect medium EVs (mEVs). A pellet containing mEVs was resuspended in 20 mM HEPES and underwent additional centrifugation. The supernatant was filtered through 13 mm sterile syringe filters with a 0.2 mm Supor (polyethersulfone ) membrane (Pall Corporation, Port Washington, NY, USA) and then centrifuged at 100,000 g for 60 min at 4 °C (Optima Max–XP equipped with TLA55 rotor, Beckman Coulter, Brea, CA, USA) to collect small EVs (sEVs). As for mEVs, the pellet containing sEVs underwent a further ultracentrifugation step in 20 mM HEPES. After this procedure, the pellet of vesicles was resuspended in 20 µL of 20 mM HEPES and used immediately for quantification experiments and SH-SY5Y treatment or processed for characterization experiments as detailed below.
Isolated EVs were analyzed by nanoparticle tracking analysis (NTA) to determine the particle number (for quantification and SH-SY5Y studies) and size distribution (for characterization experiments). A Nanosight NS300 instrument equipped with a 488 nm laser (Malvern Panalytical Ltd, Malvern, United Kingdom) was calibrated with polystyrene latex microbeads at 100, 200, and 400 nm prior to NTA. Resuspended vesicles were diluted with 20 mM HEPES to achieve between 20 and 100 objects per frame. Each sample was measured in quintuplicate at camera setting 15 with an acquisition time of 30 s and a detection threshold setting of 7. At least 200 completed tracks were analyzed per video. For each of the fibroblast cell lines, the analysis was repeated three times using different EV preparations. The NTA analytical software version 3.4 was used for capturing and analyzing the data. Data were expressed as the ratio between vesicle concentration/number of cells per flask at the time of isolation.
4.6. Western Blot Analysis
Protein lysates were obtained by resuspending fibroblast, SH-SY5Y cell or EV pellets in ice-cold lysis buffer (CelLytic, Sigma-Aldrich, St. Louis, MO, USA) containing protease and phosphatase inhibitors (protease inhibitor cocktail, 1:100; phosphatase inhibitor cocktail 1 and 2, 1:100, Sigma-Aldrich, St. Louis, MO, USA). The samples were kept on ice and placed on a shaker for 30 min, then centrifuged (16,000 g for 20 min at 4 °C). After centrifugation, the supernatant was collected, and protein concentration was measured by BCA assay (Sigma-Aldrich, St. Louis, MO, USA). Protein samples (20–30 μg) were resolved by electrophoretic run on a Mini-Protean® TGX™ gel (AnyKD, Biorad, Hercules, CA, USA) and transferred to nitrocellulose membranes (Trans-Blot® Turbo™ Transfer Pack, Biorad) through the Trans-Blot Turbo Transfer System (Biorad, Hercules, CA, USA). Membranes were blocked with a solution containing 5% BSA, 0.05% Tween-20 (Sigma) in PBS for 1 h and probed with the following primary antibodies: (a) anti-calnexin (1:2000, Proteintech, Manchester, United Kingdom), anti-flotillin (1:500, Proteintech, Manchester, United Kingdom), anti-TSG101 (1:500, Proteintech, Manchester, United Kingdom), and anti-GAPDH (1:2500, GeneTex, Irvine, CA, USA) for sEV characterization; and (b) anti-α-synuclein (1:2000, BD Biosciences, San Jose, CA, USA), anti-phospho-α-synuclein (S129) (1:500, Abcam, Cambridge, United Kingdom), and anti-β-actin (1:10,000, Santa Cruz, Dallas, TX, USA) for the evaluation of α-synuclein expression in SH-SY5Y cells. After washing, membranes were incubated with secondary antibodies (1:5000, Bio-Rad, Hercules, CA, USA) for 1 h at room temperature (RT) and visualized with enhanced chemiluminescence reagents (Biorad, Hercules, CA, USA) by using Azure600 (Azure Biosystems, Dublin, CA, USA).
Fluorescent near-infrared Odyssey® scanner and software (Li-Cor Biosciences, Lincoln, NE, USA) were used for the detection of cathepsin D and autophagy-related protein. Membranes were blocked with the Odyssey blocking buffer and incubated overnight with the following primary antibodies: anti-cathepsin D (1:500, Santa Cruz, Dallas, TX, USA), anti-p62 (1:2000, Sigma-Aldrich, St. Louis, MO, USA), anti-beclin 1 (1:500, Cell Signaling Technology, Inc., Danvers, MA, USA), anti-LC3B (1:1000, Cell Signaling Technology, Inc., Danvers, MA, USA), and anti-β-actin (1:10,000, Santa Cruz, Dallas, TX, USA). As secondary antibodies, IRDye® 700 and IRDye® 800 (1:10,000) (Li-Cor Biosciences, Lincoln, NE, USA) were used.
The signal obtained from each protein was normalized with the corresponding signal obtained for the housekeeping protein. Data were expressed as a percentage compared to control samples in each membrane.