Animals
Pregnant Sprague Dawley rats were acquired at day 10 after crossings in the Animal Care Facility of Pontificia Universidad Católica de Chile. The progeny was used at postnatal day one. Rats were housed in individual cages under a 12:12 light:dark cycle under ad libitum access to food and water.
The care of the animals used in this study to perform cell cultures was in accordance with the recommendations of the National Institute of Health Guide for the Care and Use of Laboratory Animals and the ARRIVE Guidelines. The protocol was approved by the Universidad de los Andes Bioethical Committee in the frame of the Fondecyt Project 1140108 and (# CEC202039). In total, the progeny of n = 15 female rats were used. This study was not pre-registered. No blinding and sample size calculations were performed.
Antibodies
The antibodies used in this study are listed in Table 1.
Table 1
List of primary and secondary antibodies used for Western blot and immunofluorescence.
Primary antibody | | Secondary antibody |
Antibody | Company | Dilution used (WB or IF) | Manufacturer proof of validation | References | | Antibody | Company | Dilution |
anti-ALDOA | Santa Cruz | 1/1000 (WB) | (C-10) sc-390733 | [59] | | Goat anti-mouse | Licor | 1/5000 |
anti-ALIX | Santa Cruz | 1/500 (WB) | (1A12): sc-53540 | [60] | | Goat anti-mouse | Licor | 1/5000 |
anti-CD63 | Santa Cruz | 1/500 (WB) | (H-193): sc-15363 | [61] | | Goat anti-rabbit | Licor | 1/5000 |
anti-EF-2 | Santa Cruz | 1/1000 (WB) | (C-9): sc-166415 | [62] | | Goat anti-mouse | Licor | 1/5000 |
anti-FLOTILLIN | BD Transduction Labs | 1/500 (WB) | Monoclonal (18/Flotillin-1) | [63] | | Goat anti-mouse | Licor | 1/5000 |
anti-GFAP | Thermo Fisher | 1/500 (IF) | Glial fibrillary acidic protein PA1-10004 | [64] | | Goat anti-mouse Alexa Fluor 488 | ThermoFisher | 1/800 |
anti-GM130 | BD Transduction Labs | 1/1000 (WB) | Monoclonal (35/GM130) | [65] | | Goat anti-mouse | Licor | 1/5000 |
anti-MAP2 | Millipore | 1/1000 (IF) | Bovine MAP2 (AP20) MAB378 | [66] | | Goat anti-mouse Alexa Fluor 488 | ThermoFisher | 1/1000 |
anti-SUMO-1 | Santa Cruz | 1/500 (WB) | (D-11): sc-5308 | [67] | | Goat anti-mouse | Licor | 1/5000 |
anti-SUMO-1 | Cell Signaling | 1/300 (IF) | 2A12 Mouse mAb #5718 | [68] | | Donkey anti-mouse Alexa Fluor 555 | ThermoFisher | 1/800 |
anti-SUMO-2/3 | Donated by Dr. c, University of Dundee, UK | 1/500 (WB) | Not applicable | [69] | | Donkey anti-sheep | ThermoFisher | 1/5000 |
Cell culture
Astrocytes were obtained from the telencephalon of Sprague Dawley rats at postnatal day one as previously described [21]. For this, P1 rats were anesthetized under CO2 for 3 minutes between 10:00 to 11:00 AM and decapitated. Astrocytes were maintained in DMEM (#12100046, ThermoFisher, Massachusetts, USA) containing 10% Fetal bovine serum (#26140079, ThermoFisher, Massachusetts, USA), with 100 units/ml of penicillin and 100 µg/ml of streptomycin (#15140122, ThermoFisher, Massachusetts, USA) and incubated at 37°C, with 5% CO2 and 95% humidity. On days 4 and 8 in vitro (DIV), the total media volume was changed. After 15 DIV, the astrocytes were re-plated to decrease microglia and seeded at a confluence of 70–80%. For pharmacological treatments, the culture medium was completely changed to sEV free culture medium (i.e., FBS was depleted from EVs by ultracentrifugation before addition). The following compounds were added 1 hour later: 1 µM corticosterone (CORT #27840, Sigma, Missouri, USA), or 30 µM 2′,3′,4′-trihydroxy-flavone,2-(2,3,4-Trihydroxyphenyl)-4H-1-Benzopyran-4-one (2-D08, #1052, Sigma, Missouri, USA), an inhibitor of the SUMO-conjugating enzyme UBC9, or DMSO as a control, (#85190, ThermoFisher, Massachusetts, USA). The addition of these compounds was repeated 24 and 48 hours later, and the conditioned medium was collected 72 hours after the first addition. The medium was collected to isolate sEVs, and the cells were trypsinized (Trypsin, #15090046, ThermoFisher Massachusetts, USA), resuspended in phosphate-buffered saline (PBS #14190, ThermoFisher, Massachusetts, USA). An aliquot was counted using a Neubauer chamber, and the remaining cells were homogenized and stored at -80°C until further use.
Cortical neurons from Sprague Dawley rat embryo brain (E18) were dissociated from the cerebral cortex, and 20,000–40,000 cells were seeded on coverslips coated with Poly-D-Lysine (#4174, Sigma Missouri, USA) using 35 mm plates. Cortical neurons were maintained in vitro for 21 days in neurobasal medium supplemented (#21103049, ThermoFisher, Massachusetts, USA) with B27 (#17504044, ThermoFisher Massachusetts, USA) and 100 units/mL of penicillin and 100 µg/mL of streptomycin (#15140122, ThermoFisher, Massachusetts, USA) incubated at 37°C, with 5% CO2 and 95% humidity.
HeLa cells were maintained in DMEM (#12100046, ThermoFisher, Massachusetts, USA) containing 10% Fetal bovine serum (#26140079, ThermoFisher Massachusetts, USA) with 100 units/ml of penicillin, 100 µg/ml of streptomycin (#15140122, ThermoFisher, Massachusetts, USA, and 200 µg/ml of G418 (#11811023, ThermoFisher, Massachusetts, USA) for maintaining stable cell lines, and were incubated at 37°C, with 5% CO2 and 95% humidity. SUMO-1 HeLa and SUMO-2 HeLa stable cell lines were generously donated by Dr. Ronald T. Hay, University of Dundee, UK [22].
Transfections
Astrocytes were transfected by using Lipofectamine 2000 in accordance with the manufacture’s instruction. Briefly, Transfections were performed after reaching 60% confluency using a 3:1 ratio of DNA: Lipofectamine (#11668030, ThermoFisher, Massachusetts, USA), in Opti-MEM (#31985062, ThermoFisher, Massachusetts, USA). pCDNA3.1 HIS-SUMO-1 and HIS-SUMO-2 plasmids were generously donated by Dr. Ronald T. Hay, University of Dundee, UK [22]. We used GFP plasmid as a control. After 6 h incubation, the medium was exchanged with sEV free culture medium (DMEM supplemented with 100 units/ml of penicillin, 100 µg/ml of streptomycin and 10% sEV depleted FBS). After 24h post transfection the efficiency of transfection was determined by GFP expression by fluorescent microscopy.
Astrocyte immunostaining and image acquisition
Astrocytes were fixed on coverslips with 4% paraformaldehyde (#158127, Sigma, Missouri, USA) in PBS for 10 minutes, washed with PBS and permeabilized with 0.2% Triton X-100. Then, astrocytes were incubated with blocking solution (10% BSA in PBS) for one hour followed by overnight incubation with anti-GFAP from ThermoFisher, Massachusetts, USA or anti-SUMO-1 from Cell Signaling, Massachusetts, USA, in blocking solution. Finally, astrocytes were incubated with the corresponding secondary antibodies in blocking solution, washed with PBS and mounted with a mounting medium with DAPI (#104139, Abcam, Cambridge, UK). The images were acquired with a Leica TCS SP8 laser scanning confocal microscope using LAS X 3.5.2.18963 software at 20x magnification, Zoom: 1, laser lines: 405, 488 and 552nM.
sEV isolation
Cell cultures were grown for 72 hours in an sEV free culture medium (DMEM supplemented with 100 units/ml of penicillin, 100 µg/ml of streptomycin and 10% sEV depleted FBS via depletion of vesicles after ultracentrifugation for 2 hours at 100,000 g). The conditioned media was harvested to isolate sEVs by serial centrifugations as previously described [21, 23]. After 30 minutes at 2,000 g, the supernatant was recovered and centrifuged for 45 minutes at 10,000 g. Then, the supernatant was centrifuged for 2 hours at 100,000 g, and the new supernatant was eliminated. The sEV enriched pellet was washed once in PBS (#14190, ThermoFisher, Massachusetts, USA), centrifuged for 2 hours at 100,000 g and re-suspended in PBS to store at -80°C until use. Alternatively, and as indicated in each case, the Total Exosome Isolation kit (#4478359, ThermoFisher, Massachusetts, USA), was used following the provider’s instructions. The sEV pellet was resuspended in PBS and stored at -80°C until use. For characterization of astrocyte sEVs, iodixanol density gradients were performed. For this, astrocyte sEVs (1mg) were resuspended in 1 mM EDTA and 36% iodixanol/PBS pH 7.4. The solution was placed at the bottom of the tube and covered by solutions of descending concentrations of iodixanol in PBS (30%, 24%, 18% and 12%) to centrifuge at 163,000 g for 24 hours in a TH-641 Swinging Bucket rotor. Fractions (1ml) were collected to measure their density and assess their protein profile.
Nanoparticle tracking analysis
The nanoparticle tracking analysis (Nanosight NS300, Malvern Instruments, Malvern, UK) was used to determine particle concentration and size distribution. Samples were diluted 5 or 10 times in PBS to obtain ≥ 80 particles per field for analysis, and three videos were recorded for 30 seconds each.
Western blots
Cells were lysed in RIPA buffer (150 mM NaCl, 25 mM Tris-HCL pH 7.4, 0.5% NP-40, 0.5% sodium deoxycholate, 20 mM NEM (N-Ethylmaleimide (NEM, #34115, Sigma, Missouri, USA). Protein from the homogenates and sEVs (sEV resuspended in PBS containing 0.1% SDS) was quantified using the bicinchoninic acid method [24]. The samples were boiled with loading buffer and 20 mM NEM for 5 minutes at 100°C to prevent de-SUMOylation. As depicted, each lane was loaded with either the same amount of total protein or the same number of vesicles. Proteins were separated by polyacrylamide gel electrophoresis under denaturing conditions (SDS-PAGE). Gels were stained with Coomassie dye or transferred to nitrocellulose membranes to proceed with standard Western blot procedures. Briefly, membranes were incubated with blocking solution (10% non-fat milk in PBS) for one hour followed by overnight incubation with anti-ALDOA, anti-ALIX, anti-EF-2, anti-SUMO-1 and anti-CD63 from Santa Cruz, Texas, USA. Anti-FLOTILLIN, and anti-GM130 from BD Transduction Labs, New Jersey, USA). Anti-SUMO-2/3 (was generously donated by Dr. Ronald T. Hay, University of Dundee, UK [22]). Then, membranes were washed in PBS-T (PBS containing 0.05% Tween20) and incubated 1 hour with the corresponding secondary antibodies. Finally, membranes were washed in PBS-T and reveal with chemiluminescence kit (#32106, ThermoFisher, Massachusetts, USA).
Proteomics
sEV proteins were separated using SDS-PAGE. Each lane was divided into 8 sections to perform in-gel digestion. Liquid chromatography followed by tandem-mass spectrometry (MS/MS) of the sample fractions was performed on a hybrid dual-pressure linear ion trap/orbitrap mass spectrometer (LTQ Orbitrap Velos Pro, Thermo Scientific) equipped with an EASY-nLC Ultra HPLC (Thermo Scientific). Peptide samples were dissolved in 10 µL of 2% acetonitrile/0.1% trifluoric acid and fractionated on a 75-µm i.d., 25-cm PepMap C18-column, packed with 2 µm of resin (Dionex, Germany). Separation was achieved by applying a gradient of 2–35% acetonitrile in 0.1% formic acid over 150 minutes at a flow rate of 300 nL/min. The LTQ Orbitrap Velos Pro MS was exclusively used for CID fragmentation when acquiring MS/MS spectra, which consisted of an orbitrap full mass spectrometry (MS) scan followed by up to 15 LTQ MS/MS experiments (TOP15) on the most abundant ions detected in the full MS scan. The essential MS settings were as follows: full MS (resolution, 60,000; mass to charge ratio range, 400–2000); MS/MS (Linear Trap; minimum signal threshold, 500; isolation width, 2 Da; dynamic exclusion time setting, 30 seconds; and singly charged ions were excluded from the selection). Normalized collision energy was set to 35%, and activation time was set to 10 milliseconds. Raw data processing and protein identification were performed by Peaks Studio 8.0 (Bioinformatics solutions Inc.). The false discovery rate was set to < 1%. The bio-informatic analysis was done by DAVID Bioinformatics Resources 6.8. pValue was represented as -Log10.
Data availability: The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD029940. For reviewers, the account details are the following: Username:[email protected] and Password:MhY1L1qp
Incubation of neurons with sEVs
A total of 1000 vesicles were added per neuron, and this was repeated 24 hours later. After 72 hours, the cells were used for fluorescence non-canonical amino acid tagging assay (FUNCAT).
Morphological analysis
Sholl analysis was used to analyze neurons incubated with astrocyte-derived sEVs obtained in the different experimental via plugins from ImageJ software (National Institute of Health, USA). The dendritic tree was examined in 3 µm increments. The following parameters were obtained: longest dendrite length (i.e., the largest radius at which there is an intersection with a neuronal process) and the total number of intersections (i.e., the sum of all intersections with each different radius) [21]. Images were processed using ImageJ software (National Institutes of Health) and and Adobe Photoshop 2020, and assembled using Adobe Illustrator 2020.
FUNCAT assay
To visualize newly synthesized proteins in cells, the FUNCAT method was used according to Daniela Dieterich [25]. Cultures were incubated for 3 hours with methionine-free HibernateA supplemented with non-canonical amino-acid AHA (L-azidohomoalanine), which was incorporated into newly synthesized proteins instead of methionine. As a negative control, methionine was used (4 mM). After metabolic labeling neurons were washed with cold PBS-MC (1 mM MgCl2, 0.1 mM CaCl2 in PBS pH 7.4), directly fixed with 4% paraformaldehyde for 30 minutes, then washed 3 times with PBS pH 7.4 and incubated with B-block solution (10% normal horse serum, 5% sucrose, 2% BSA, 0.2% TritonX-100 in PBS pH 7.4) for 1 hour at room temperature. The cells were then washed with PBS pH 7.8 and finally incubated overnight with FUNCAT solution (0.2 mM Triazole ligand Tris [(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine), 0.5mM TCEP [Tris-(2-carboxyethyl)phosphine hydrochloride], TAMRA (red-fluorescent tetramethylrhodamine) alkyne tag and 40 µg/ml CuSO4 in PBS pH 7.8. At the next day cells were washed with PBS pH 7.8, followed by PBS pH 7.4 and incubated with anti-MAP2 from Millipore Massachusetts, USA, in B-block+ 0.2% TritonX-100 for 2 hours at room temperature, washed with PBS pH 7.4 and incubated with secondary antibody in B-block solution for 1 hour at room temperature. Finally, the cells were incubated for 10 min with 300 mM DAPI 4′,6-diamidino-2-phenylindole (D9542, Sigma Missouri, USA) in PBS pH 7.4, washed with PBS pH 7.4, and mounted using MOWIOL mounting media MOWIOL (#81381, ThermoFisher, Massachusetts, USA). Immunohistochemistry staining in combination with FUNCAT were analyzed via confocal laser scanning microscopy (CLSM). For this, an Axio Z1 microscope provided with a LSM 710 confocal system (Carl Zeiss group) was used together with its user interface ZEN 2012 SP5 (Carl Zeiss group). All the images from the FUNCAT procedure were taken in Z-stacks. The FUNCAT signal was visualized with ImageJ as a fire lookup table, to visually favor the expression range of the newly synthesized proteins through a range of colors from blue to white, where blue represents the absence of new protein synthesis.
Statistical analysis and figures
Figures were created using Adobe Illustrator.
Average values are expressed as mean ± standard error mean (SEM). Statistical significance of results was assessed using one sample Student’s t-test (when assessing whether fold change over control values in Western blots were different from 1); two-tailed Student’s t-test or one-way ANOVA followed by a posthoc Dunnetts test using GraphPad Prism version 5 for Windows, GraphPad Software, La Jolla, California USA, as indicated in each case. Differences were considered statistically significant if p < 0.05. Throughout the manuscript, independent biological replicates are defined as independently performed experiments on material derived from different animals.