Leech central nervous system structure and isolation
Ten leeches were anesthetized in ethanol 10% at room temperature (RT) for 15 min, the CNS were dissected out in a sterile Ringer solution (115 mM NaCl, 1.8 mM CaCl2, 4 mM KCl, 10 mM Tris maleate, pH 7.4) under a laminar flow hood. After isolation of CNS, the samples were placed in 3 successive baths of antibiotics (100 UI/ml penicillin, 100 μg/ml streptomycin and 100 μg/ml gentamycin) for 15 min and later incubated in complete medium, made of Leibovitz L-15 medium (Invitrogen, Carlsbad CA, USA) complemented with 2 mM l-glutamine, 100 UI/ml penicillin, 100 μg/ml streptomycin, 100 μg/ml gentamycin, 0.6% glucose, 10 mM Hepes and 10% exosome-depleted FBS Media Supplement (SBI System Bioscience, Palo Alto CA, USA).
Neuron and Microglial cell preparation
The whole CNS were placed in 35 mm Petri dishes with 500 μl of complete medium. Ganglia and connectives were carefully decapsulated by removing the collagen layer enveloping the nerve cord. The nerve cells, neurons (10–70 μm) and microglial cells (5 μm), were mechanically collected by gentle scraping and dissociated through filters of different size. The cell debris were eliminated in a 100 μm pluriStrainer filter (Dominique Dutscher, Brumath, France). Microglia were selected through a 6 μm pluriStrainer filter and the neurons were collected in the upper part of this filter. In order to eliminate cell debris, complete medium containing microglial cells or neurons were centrifuged at 1200×g for 10 min at RT. Regarding the preparation of conditioned medium, the pellet of microglial cells corresponding to 10 nerve cords, was resuspended in 500 μL of complete medium, and plated in 4-well petri dishes. After 15 min of incubation, the enriched microglial cells or neurons were centrifuged at 1200×g for 10 min at RT. All the cell cultures (neurons and microglia) were maintained at 18 °C in atmospheric conditions.
Primary embryonic neuronal culture
Rat primary embryonic cortical neurons (primary neurons) were prepared from 17 to 18-day-old Wistar rat embryos as follows. The brain and meninges were removed. The cortex was dissected out and mechanically dissociated in culture medium by trituration with a polished Pasteur pipette. Once dissociated and after blue trypan counting, cells were plated in 6-well plate (800,000 cells/well) or 8-well Labtek plate (50,000 cells/well) (Sarstedt, Nümbrecht, Germany) coated with poly-d-lysine (0.5 mg/ml) and laminin (10 μg/ml). For dissociation, plating, and maintenance, we used Neurobasal medium supplemented with 2% B27 and containing 200 mM glutamine and 1% antibiotic–antimycotic agent (Invitrogen, Carlsbad CA, USA).
Preliminary EV isolation by ultracentrifugation (UC)
The supernatants of conditioned medium from leech microglial culture were transferred into canonical tubes and centrifuged at 1200g for 10 min at RT to pellet the cells. The resulting supernatants were transferred into new tubes and centrifuged at 1200g for 30 min at RT to eliminate the apoptotic bodies. In order to pellet the EVs, the supernatants from the second centrifugation were transferred into 10.4 ml polycarbonate bottle with Cap Assembly tubes (Beckman Coulter, Brea CA, USA). The tubes were filled with PBS to a final volume of 9 ml and samples were ultracentrifuged at 100,000×g for 90 min at 4 °C (70.1 Ti rotor, k-factor 36, Beckman Coulter, Brea, CA, USA). The supernatants were carefully removed and the UC pellets were resuspended in 200 µl of 0.20 μm filtered PBS (Invitrogen, Carlsbad CA, USA).
EV isolation by UC coupled to Optiprep™ Density Gradient (ODG)
The UC pellets were subjected to a further purification step by Optiprep™ Density Gradient. Briefly, the pellets were loaded at the bottom of gradient prepared by diluting a stock solution of Optiprep™ (60% w/v iodixanol; Sigma Aldrich, Saint Louis MO, USA) as previously described [62]. The gradient was prepared by carefully deposite 2 mL of Optiprep™ solutions: 40%, 20%, 10% and 5% in a 14 ml polyallomer Beckman coulter tubes. The samples were ultracentrifuged at 100,000×g for 16 h at 4 °C (SW 40 Ti rotor, k-factor 137, Beckman Coulter, Brea, CA, USA). The ODG fractions of 1 ml were carefully harvested from the top to the bottom and resupended in 8 ml of PBS for 90 min of centrifugation at 100,000×g at 4 °C (70.1 Ti rotor, k-factor 36, Beckman Coulter, Brea, CA, USA). After the supernatant removal, the pellets were resuspend in 100 μl of filtered PBS.
EV isolation by UC coupled to Size-exclusion chromatography (SEC)
The UC pellets were subjected to a size-exclusion chromatography (SEC) isolation. SEC were performed using a home-made column with a 0.7 cm internal diameter and a 26 cm height. Briefly, the glass column was washed with water and ethanol. Subsequently, a 60 μm filter (pluriselect, Leipzig, Germany) was placed at the bottom of the column which was stacked with sepharose 2B (Sigma Aldrich, Saint Louis MO, USA) to create a 19 cm height stationary phase. Then, 50 ml of PBS were loaded to rinse and equalize the phase. The resuspended UC pellet was loaded at the top of the stationary phase. The eluates were collected in 20 sequential fractions of 250 μl. For each fraction, the number of particles was determined by NTA. After analysis, SEC fractions were pooled (P) in three samples: P1-EV- (F1-F4 SEC fractions), P2-EV+ (F5–F7 SEC fractions) and P3-EV‒ (F8–F20 SEC fractions). Each sample was conserved at ‒ 20 °C for further analyses.
Nanoparticle tracking analysis (NTA)
NTA was performed using a NanoSight NS300 instrument and an automated syringe pump (Malvern Panalytical Ltd, UK). The script was adapted as follows: samples were diluted (1:100) in filtered PBS and loaded using an automated syringe pump. The infusion rate was initially fixed to 1000 for sample loading and chamber filling and then decreased to 25 for video recording. A delay of 15 s was applied to stabilize the flow before acquisition. Video captions of 60 s were performed in triplicate for each sample with a camera level setting at 13 and a screen gain at 3. The NTA 3.2 software was used to process the recorded movies with a camera level setting at 13 and a detection threshold at 3. PBS used for EV recovery was used for negative controls. As a control for ODG experiments, 200 µl of PBS were loaded at the bottom of the tube that was then processed exactly in the same conditions as the EV-containing samples. As a control for SEC experiments, 250 μl of PBS were collected before the loading of the sample on the column.
Transmission electron microscopy (TEM)
The observation of EVs by TEM was performed as previously described [63]. Briefly, isolated EVs were resuspended in 30 μl of 2% paraformaldehyde (PFA) in PBS. Then, 3 × 10 μl of sample were deposited on Formvar-carbon-coated copper grids. The adsorption was performed for 3 × 20 min in a wet environment and the grids were transferred into a drop of 1% glutaraldehyde in PBS for 5 min at RT. After several rinsing steps with ultrapure water, samples were contrasted for 10 min on ice with a mixture of 4% uranyl acetate and 2% methylcellulose (1:9, v/v). The excess of mixture was removed using Whatman filter paper. After drying, the samples were observed under a JEOL JEM-2100 TEM at 200 kV. The acquisitions were made with Gatan Orius SC200D camera.
RNase A treatment of EVs positive fractions
The EV positive fractions (both ODG and SEC isolation methods) were treated with RNase A solution (0.1 mg/ml) (Sigma Aldrich, Saint Louis MO, USA) for 90 min at 37 °C. Then they were transferred into 10.4 ml polycarbonate bottle with Cap Assembly tubes (Beckman Coulter, Brea CA, USA), filled with PBS to a final volume of 9 ml and ultracentrifuged at 100,000×g for 90 min (70.1 Ti rotor, k-factor 36, Beckman Coulter, Brea CA, USA) to eliminate the RNase A. The EV pellets were resuspended in 200 μl of PBS for further analyses or directly lysed in TRIzol® reagent for RNA extraction.
Total RNA extraction and processing from microglia EVs
The EV samples (from UC, UC-ODG or UC-SEC procedures) were mixed in 300 μl of TRIzol® reagent (ThermoFisher Scientific, Waltham MA, USA) and incubated 5 min at RT. Then, 3 μl of cel-mir-39 spike in kit (Norgen, Thorold ON, Canada) was added to the mixture as normalizer for quantitative PCR. RNA were extracted with Direct-zol™ RNA Miniprep according to manufacturer’s protocol (Zymo Research Corp, Irvine CA, USA). The extracted RNAs were analyzed with a Nanospectrophotometer MultiSkan GO (ThermoFisher Scientific, Waltham MA, USA) to evaluate their quantity and quality.
Total RNA extraction from leech microglia and neurons
Total RNAs were extracted from microglia and neurons corresponding to ten leech nerve chains. The cell pellets of the microglia or neurons were resuspended in 1 ml of TRIzol® (Thermo Fisher Scientific, Waltham MA, USA) to be processed according to the manufacturer’s instructions. The total RNA pellet were resuspended in 20 μl of DEPC-treated water (Thermo Fisher Scientific, Waltham MA, USA). After their quantification and a quality analysis at 260 nm using a Multiskan Go spectrophotometer (Thermo Fisher Scientific, Waltham MA, USA) the total RNAs were treated with RQ1-DNase1 in 10 × RQ1-DNase buffer for 30 min at 37 °C (Promega, Madison, WI, USA) to prevent any contamination by genomic DNA. The quality of total RNAs was finally analysed in a 1% agarose gel electrophoresis.
RNA Seq analysis
The CNS isolation and microglial cell preparation were performed from 60 adult leeches as presented above. In this experiment, the microglia-derived EVs were isolated from the UC procedure as described above. Following the RNA extraction, the quantification and quality controls previously described, 300 ng of RNA extract were fragmented using RNAse III reaction and used to prepare a representative cDNA library according to the manufacturer’s instructions (Ion Total RNA-Seq Kit v2, Life Technologies). The library was diluted at 9 pM before a strand-specific RNA sequencing on the Ion Personal Genome Machine™ system (Ion Torrent chip 318, Ion Torrent Systems, Inc., Life Technologies). Raw fastQ files obtained from RNA sequencing were trimmed and aligned using the web-based platform Galaxy (https://usegalaxy.org/), a custom interface for the online use of bioinformatic tools for manipulating nucleotide sequences [64]. Preprocessed reads were aligned using BWA (Burrows-Wheeler Aligner) on the complete collection of known microRNA precursors (all species) retrieved from miRbase [31]. Reads with a corresponding extended sequence identity to any known microRNA were counted and ranked according to the number of copies. Putative microRNAs having at least 50 reads were selected and then validated as described below.
Reverse transcription of total RNAs
The total RNAs were reverse transcribed according to the NCode™ miRNA First-Strand synthesis kit protocol (Invitrogen, Life Technologies, Carlsbad, CA, USA) and used 1 μg of cellular RNA extracts and 500 ng extracellular vesicle RNA extracts. For any sample, the polyadenylation reaction was necessarily performed before the first-strand cDNA synthesis. In order to validate the nature of miRNA, the same amount of RNA extracts were reverse transcribed without the poly (A) tail grafting and were used as negative controls. The reaction mixes were stored at − 20 °C for subsequent PCR studies.
Gene expression analysis
The cDNAs were amplified by PCR with GoTaq® DNA Polymerase (Promega, Madison WI, USA) according to manufacturer’s instructions. The reactions were carried out with a Biorad T100 thermocycler (BioRad, Hercules CA, USA) with the following amplification conditions: 3 min at 95 °C, 50 cycles of: 30 s at 94 °C, 20 s at 51 °C and 30 s at 72 °C; and a final step at 72 °C for 5 min. The PCR products were loaded on a non-denaturing 12% polyacrylamide gel and migrated in 1X TBE buffer for 15 min at 50 V and then 45 min at 100 V. The gels were revealed after a 10 min incubation in a TBE-SYBR Gold Nucleic Acid Gel Stain 1X solution (Molecular probes, Invitrogen). The image captures of the gels were performed under a UV camera.
Quantitative gene expression analysis
Real-time quantitative PCR (qPCR) reactions were performed using Platinum® SYBR® Green qPCR SuperMix-UDG kit (Thermo Fisher Scientific, Waltham MA, USA) on a CFX96™ Real-Time PCR Detection System instrument (Biorad, Hercules CA, USA) with the following program: 2 min at 50 °C, 1 min at 95 °C, and 50 cycles of: 15 s at 95 °C, 15 s at 51 °C, 20 s at 60 °C. Data were analyzed with the CFX Manager software (Biorad, Hercules CA, USA). The relative gene expression of the different miRNAs of interest were standardized using the miRNA cel-mir-39 spike-in control and were calculated using the 2−∆∆Ct method [65].
Subcloning and sequencing
PCR products were extracted with NucleoSpin Gel and PCR clean-up kit (Macherey–Nagel, KG, Düren, Germany) according to the manufacturer’s instructions. Extracted PCR products were ligated into the pGEM T-easy vector (Promega, Madison WI, USA) and cloned into JM109 cells according to the manufacturer’s instructions. Finally, products were sequenced using BigDye Terminator v3.0 polymerization kit before detection on Genetic Analyzer (Applied Biosystems, Foster City CA, USA).
Protein extraction and mass spectrometry analysis from neurons
Total protein extraction
Rat primary neurons were prepared as described above. After a 7 day culture, the cells were exposed to 106, 107 EVs/well or SEC negative fractions (P3-EV‒). Each condition was done in triplicate. After a 48 h exposure, cells were washed with ice-cold PBS and then lysed with RIPA buffer for total protein extraction (150 mM NaCl, 50 mM Trizma base, 1 mM PMSF, 5 mM EGTA, 2 mM EDTA, 100 mM Sodium Fluoride, 10 mM Sodium Pyrophosphate, 1X protease inhibitors and 1% NP40) for 5 min on ice. The lysate was sonicated twice 10 s with a probe sonicator (500 W, 20 kHz). The cell debris were pelleted by centrifugation at 20,000×g for 10 min at 4 °C, and the supernatants containing proteins were collected for subsequent analysis.
Filter-aided sample preparation (FASP)
Each total protein extract was used for FASP analysis. The FASP procedure used Amicon® Ultra-0.5 30 kDa Centrifugal Filter Devices (Millipore, Burlington, VT USA) as previously described [66] before adding trypsin (Promega, Madison WI, USA) for protein digestion (20 μg/ml in 50 mM NH4HCO3). The samples were incubated with trypsin overnight at 37 °C. The peptide digests were collected by centrifugation, and the filter device was rinsed with 100 μl of 0.5 M NaCl. Next, 5% TFA was added to the digests, and the peptides were desalted with a Millipore® ZipTips C18 device (Millipore, Burlington, VT USA). The solution was then dried and solubilized in water/0.1% formic acid/2% ACN before the nLC-MS/MS analysis. The experiments were done in triplicate.
Protein extraction and mass spectrometry analysis from microglia EVs
Total protein extraction
The SEC fractions were pooled with Amicon® Ultra-0.5 50 kDa Centrifugal Filter Devices (Millipore, Burlington, VT USA) and organized in three samples: P1-EV‒ (fractions 1–4), P2-EV+ (fractions 5–7) and P3-EV‒ (fractions 8–20). Concentrated samples were lysed with RIPA buffer for total protein extraction.
In-gel digestion of EV proteins
The EV Proteins were loaded onto a 4% polyacrylamide gel for separation using a TGS solution (25 mM Tris, 192 mM Glycine and 0.1% SDS) as running buffer. An electrophoresis was performed at 70 V for 30 min to stack the proteins in the stacking gel. In order to fix proteins, the gel was stained with InstantBlue™ Coomassie protein staining solution (Expedeon, Cambridgeshire, UK) for 20 min. Each gel lane was excised and cut into small pieces of 1 mm3. The strips of gel were washed with a succession of solutions: 300 μl of ultrapure water for 15 min, 300 μl of ACN for 15 min, 300 μl of 100 mM NH4HCO3 (pH 8) for 15 min, 300 μl of NH4HCO3/ACN (1:1) for 15 min, then 300 μl of ACN for 5 min. The pieces were dried under vacuum for 5 min. The reduction of cysteines was performed using 50 μl of a solution of 10 mM DTT in 100 mM NH4HCO3 (pH 8) and incubated at 56 °C for 1 h. The alkylation of the cysteines was carried out using 50 μl of 50 mM IAA in 100 mM NH4HCO3 (pH 8) at RT in the dark for 30 min. Gel pieces were washed with 300 μl of 100 mM NH4HCO3 (pH 8) for 15 min, 300 μl of 20 mM NH4HCO3 (pH 8) / ACN (1: 1) for 15 min and 300 μl of ACN during 5 min. The pieces were dried under vacuum for 5 min and subjected to enzymatic digestion using a solution of trypsin (12.5 μg/ml) in 20 mM NH4HCO3 (pH 8) overnight at 37 °C. The peptides were then extracted using the following incubations: in 50 μl of ACN allowing the retraction of the gel band and the exit of the peptides; in 50 μl of 1% TFA in order to inhibit the action of the trypsin remaining in the tube; and finally in 150 μl of 100% ACN in order to ensure the complete release of the peptides. The supernatants were transferred to a new tube, dried, and then resuspended in 20 μl of a 0.1% TFA solution for a desalting step as previously described. The sample was finally dried and solubilized in water/0.1% formic acid/2% ACN before the nLC-MS/MS analysis. The experiments were done in triplicate.
NanoLC-HR-MS/MS
Samples were separated by online reversed-phase chromatography using a Thermo Scientific Proxeon EASY-nLC 1000 system equipped with a pre-column (Acclaim Pepmap, 75 µm ID × 2 cm, Thermo Scientific, Waltham, MA, USA) and a C18 packed-tip column (Acclaim PepMap, 75 µm ID × 50 cm, Thermo Scientific, Waltham MA, USA). Peptides were separated using a gradient of ACN (5–35% for 120 min) at a flow rate of 300 nL/min. The LC eluent was electrosprayed directly from the analytical column and a voltage of 1.7 kV was applied via the liquid junction of the nanospray source. The chromatography system was coupled to a Thermo Scientific Q-exactive mass spectrometer programmed to acquire in a data-dependent mode Top 10 most intense ion method. The survey scans were done at a resolving power of 70,000 FWHM (m/z 400), in positive mode and using an AGC target of 3e6. Default charge state was set at 2, unassigned and 1 charge states were rejected and dynamic exclusion was enabled for 25 s. The scan range was set to 300–1600 m/z. For ddMS2, the scan range was between 200 and 2000 m/z, 1 microscan was acquired at 17,500 FWHM and an isolation window of 4.0 m/z was used.
MS data analysis
All the MS data were processed with the MaxQuant (version 1.5.8.3) software using the Andromeda search engine. The proteins were identified by searching MS and MS/MS data against Rattus norvegicus database or homemade H. medicinalis database described in detail [24]. Trypsin specificity was used for the digestion mode with N-terminal acetylation and methionine oxidation selected as the variable. Carbamidomethylation of cysteines was set as a fixed modification, with up to two missed cleavages. For MS spectra, an initial mass accuracy of 6 ppm was selected, with a minimum of 2 peptides and at least 1 unique peptide per protein, and the MS/MS tolerance was set to 20 ppm for HCD data. For identification, the FDR at the peptide spectrum matches (PSMs) and protein level was set to 0.01. A label-free quantification of proteins was performed using the MaxLFQ algorithm integrated into MaxQuant with the default parameters. The analysis of the proteins identified was performed using Perseus (version 1.6.2.3) software. The file containing the information from identification was used with hits to the reverse database, and proteins only identified with modified peptides and potential contaminants were removed. Then, the LFQ intensity was logarithmized (log2[x]). Categorical annotation of rows was used to define different groups after pooling replicates. Multiple-sample tests were performed using ANOVA test with a p-value of 5% and preserving grouping in randomization. The visual heatmap representations of significant proteins were obtained using hierarchical clustering analysis. The normalization was achieved using a Z-score with a matrix access by rows. For the statistical analysis, only proteins presenting as significant by the ANOVA test were used. Hierarchical clustering depending on protein extract were first performed using the Euclidean parameter for distance calculation and average option for linkage in row. An integrated Venn diagram analysis was performed using “Draw Venn diagram”, a web-based tool for the analysis of complex data sets. The analysis of gene ontology, cellular components and biological processes were performed with FunRich 3.0 analysis tool.
Prediction of mRNA targets
Predicted mRNA targets were extracted from two independent programs miRDB (https://mirdb.org) [35] and TargetScan (https://www.targetscan.org/vert_72/) [36]. Only common results between the two programs were considered.
Neurite outgrowth assay
The rat primary neurons were prepared as described above and put in 8-well LabTek culture chambers at a concentration of 50,000 cells/well. After a 3 day culture, the cells were exposed to 105, 106 and 107 EVs/well (from P2-EV+ sample) or to P3-EV- as negative control. Each condition was performed in triplicate. After a 48 h exposure, cells were fixed with 4% PFA for 20 min. After 3 washes with PBS, cells were stained with rhodamine-conjugated phalloidin (Santa Cruz, Dallas TX, USA) for 30 min at 4 °C to evaluate neurite length. After 3 washes with PBS, the nuclei were stained with diluted Hoechst 33342 (1:10,000) (Euromedex, Souffelweyersheim, France) for 30 min at RT. Finally, after a last PBS washing, cells were mounted on a slide with Dako Fluorescent Mounting Medium (Agilent, Santa Clara CA, USA) and kept in the dark before acquisition. The analyses were conducted using a Zeiss Axiovert 200 M with a 63 × 1.4 numerical aperture oil immersion objective. The neurite length was measured with NeuriteTracer ImageJ software program [67]. For all assays, the significance was calculated by one-way ANOVA followed by Tukey post hoc test [19].
