Astrocyte clues such as secreted factors (secretome) and cell–cell contact signals are essential for proper development, maintenance, and functioning of individual neurons, as well as for the wiring of the central nervous system (CNS) by controlling axonal guidance and dendritic complexity [1
Several studies have addressed the molecular mechanisms mediating the role of the astrocyte-derived secretome on dendritic morphology. To name some of these factors, it has been shown that astrocytes release phosphatidic acid to promote dendritic complexity in cultured hippocampal neurons as well as apolipoprotein E-complexed cholesterol and hevin to increase synaptogenesis in cultured retinal ganglion cells [4
]. Very recently, it has also been shown that astrocyte-derived small extracellular vesicles (sEVs) act as regulators of cell functions and signaling in CNS cells, especially between astrocytes and neurons [7
]. The most studied types of sEVs, also known as exosomes, are vesicles of 30–120 nm in diameter with an endocytic origin. These nanosized sEVs are released to the extracellular space from multivesicular bodies (MVBs) after their fusion with the plasma membrane [10
]. It has been claimed that sEVs’ molecular cargo could modify the physiology of recipient cells through the transfer of active micro RNAs (miRNAs) [10
]. For instance, mature miRNAs, i.e., small non-coding RNAs, 20–22 nucleotides long, recognize specific sequences located mainly at the 3` untranslated region of mRNA transcripts and, thus, they can determine either their translational quiescence or downregulation. The mechanisms involved in regulating miRNA loading in sEVs as well as the roles of miRNAs contained in astrocyte-derived sEVs remain mostly unexplored [14
We have shown that the astrocyte’s specific glycolytic enzyme Aldolase C (Aldo C) is present in sEVs and that its content in the vesicles is regulated in vivo and in vitro: Astrocyte-derived sEVs contain Aldo C and its levels increase in rat cerebrospinal fluid and in serum sEVs after exposure of animals to stress induced by movement restriction [17
]. In addition, we have recently shown that Aldo C expressed in brain astrocytes can be collected in sEVs isolated from rat blood serum, supporting the capacity of astrocyte-derived sEVs to cross biological barriers and to serve as regulators of intercellular signaling [19
]. However, it is not known whether neurons, which are in close proximity to astrocytes, can incorporate the derived sEVs containing Aldo C nor whether they could impact neuronal function.
Here, we showed that cultured astrocytes that express GFP-tagged Aldo C (Aldo C-GFP) transfer the derived sEVs, carrying the recombinant protein to developing hippocampal neurons, impacting their dendritic complexity. Using bioinformatics combined with biochemical and molecular approaches, we postulated and then confirmed that the content of miRNA-26a-5p is regulated in Aldo C-GFP-electroporated astrocytes and their sEVs. Finally, we showed that the miRNA-26a-5p carried by Aldo C-GFP-containing sEVs (Aldo C-GFP sEVs) actively regulates the expression of some neuronal proteins that are relevant in morphogenesis and the regulation of dendritic complexity in a manner dependent on the activity of miRNA-26a-5p.
2. Materials and Methods
2.1. Animal Procedures
For all described protocols, pregnant Sprague Dawley rats were used at E18 following ethical guidelines approved by the Universidad de Los Andes Bioethical Committee associated to Fondecyt project number: 1140,108 (Protocol # 09112013) and in accordance with the National Institute of Health’s Guide for the Care and Use of Laboratory Animals. All techniques were performed with all efforts to minimize animal suffering.
To obtain the stable expression of transgenic proteins in astrocytes, we used a system consisting of a donor and helper plasmid (piggyBac), as published elsewhere [20
]. All reading frames were under the control of the ubiquitous and strong cytomegalovirus early enhancer/chicken β actin promoter (CAG) previously described in [21
]. To construct the donor, plasmid rat gene sequence coding for Aldo C or GFP proteins were cloned into pPBCAG_eGFP plasmids using the restriction sites of EcoRI and AgeI. pPBCAG-Pbase plasmids were used as helpers. Both backbone constructs were kindly donated by LoTurco [20
2.3. Primary Antibodies and Dilutions
The primary antibodies dilutions in this study were used at 1:1000, except when indicated. They were: MAP2 A/B (MAB5622, Millipore, Billerica, MA, United States); MAP2 A/B (MAB378, Millipore, Billerica, MA, United States); Aldo C (sc-12065, Santa Cruz Biotechnology, Dallas, TX, United States); TSG101 (Ab83, Abcam, Cambridge, MA, United States); Flotilin-1 (610821, BD, Franklin Lakes, NJ, United States); CD63 (sc-15363, Santa Cruz Biotechnology, Dallas, TX, United States); GM130 (Ab52649, Abcam, Cambridge, MA, United States); GFAP ( G2032-28B-PE, US Biological, Salem, MA, United States); GFP (Ab6673, Abcam, Cambridge, MA, United States); GFP (MAB3580, Millipore, Billerica, MA, United States) (From our lab. This antibody detects a faint band over 63 kDa in astrocyte homogenates); Alix (sc-53540, Santa Cruz Biotechnology, Dallas, TX, United States); and β-actin (A5441, Sigma-Aldrich, St Louis, MO, United States).
2.4. Secondary Antibodies and Dilutions
The following secondary antibodies were used at 1:1000 dilutions in immunofluorescence and 1:5000 dilutions for Western blots: Alexa Fluor® 488 donkey anti mouse IgG (H+L) (a21202, Thermo Fisher Scientific, Waltham, MA, United States); Alexa Fluor® 555 goat anti rabbit IgG (H+L) (a21429, Thermo Fisher Scientific, Waltham, MA, United States); Alexa Fluor® 488 goat anti rabbit IgG (H+L) (a11034, Thermo Fisher Scientific, Waltham, MA, United States); anti-mouse rabbit anti-IgG horseradish peroxidase conjugated antibody (# 31430, Thermo Fisher Scientific, Waltham, MA, United States); anti-rabbit IgG horseradish peroxidase conjugated antibody (# 31460, Thermo Fisher Scientific, Waltham, MA, United States); and anti goat IgG horseradish peroxidase conjugated antibody (# 31402,Thermo Fisher Scientific, Waltham, MA, United States).
2.5. Immunofluorescence (IF)
Cultured neurons and astrocytes were fixed with 100% w/v methanol at −20 °C for 5 min, further permeabilized with 0.2% w/v Triton X-100 in phosphate-buffered saline (PBS) for 5 min and blocked with 10% w/v BSA in PBS for 10 min. Then, the cells were incubated overnight at 4 °C with the corresponding primary antibody diluted in 10% w/v BSA in PBS. Then, cells were washed 3 times with PBS (5 min each) and incubated at room temperature for 1 h with the corresponding secondary antibody coupled to a fluorescent dye. Subsequently, the cells were washed 3 times (for 5 min) and incubated with 300 nM 4ʹ, 6-diamidino-2-phenylindole (DAPI) in PBS for 3 min. Finally, the cells were mounted using the fluorescence-mounting medium (DAKO, Hamburg, Germany). The samples were analyzed in a NIKON TE-2000U epifluorescence microscope (Nikon Instruments Inc, Melville, NY, United States) equipped with a DS-2MBWC camera (2.0 monochromatic CCD megapixels). In addition, confocal microscopy was performed in an Olympus FluoView FV1000 device (Olympus, Shinjuku, Tokyo, Japan)with a UPLSAPO 60×/1.35 objective. Some samples were analyzed under Leica SP8 confocal microscope (Leica, Wetzlar, Germany).
2.6. Western Blot
For protein extraction, cells were washed twice with cold PBS and lysed with cold RIPA buffer (50 mM Tris-HCl (pH 7.4),150 mM NaCl, 0.25% deoxycholic acid, 1% NP-40, 1% SDS, and 1 mM EDTA). Protein concentration was measured using the bicinchonic acid method (BCA), according to the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, United States).
Proteins were separated using sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) under fully denaturing conditions. Electrophoresis was performed at 70 V for 45 min, finishing at 100 V in linear 12% p/v acrylamide gels. The transfer of proteins from the gel to a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA, United States ) was performed using a constant current of 350 mA for 90 min. Then, membranes were blocked with 5% w/v skim milk in PBS for 1 h at room temperature under constant agitation. Membranes were washed 3 times for 5 min with PBS to remove the excess milk and incubated at 4 °C with the corresponding antibody diluted in PBS with constant shaking overnight. Membranes were then washed 3 times with 0.1% w/v Tween in PBS for 10 min and incubated with the corresponding secondary antibody in a 1:5000 dilution with 0.1% w/v Tween in PBS and 5% p/v skim milk for 1 h at room temperature. Membranes were washed 2 times with 0.1% w/v Tween in PBS for 10 min and once with PBS. Finally, membranes were incubated for 1 min with the chemiluminescent reagent (Amersham Bioscience, Piscataway, NJ, United States) and then exposed to the film (Hyperfilm, ECL, Amersham Bioscience, Piscataway, NJ, United States). Bands were quantified by densitometry using the Adobe Photoshop 7.0 software (Adobe Inc., San José, CA, United States).
2.7. RNA Extraction
Isolated sEVs and cell cultures were processed with the miRNeasy Plus Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. The starting material was quantified as the total amount of proteins: 400 μg for cells and 10 μg for sEVs were used for each experimental condition. The samples were quantified using a microvolume spectrophotometer Nanodrop 2000 (Thermo Fisher Scientific, Waltham, MA, United States). The concentration was determined with the absorbance at 260 nm (A260), while the purity was estimated by measuring the absorbance ratio 260/280.
2.8. Reverse Transcription Quantitative PCR
The reverse transcription to obtain the cDNAs was done with the TaqMan®
MicroRNA Assays (Roche, Basilea, Switzerland) commercial kit, according to the manufacturer’s instructions. For each experimental condition in all the experiments of this publication, 100 ng of total RNA were mixed with primers of miR-26a-5p and miR-26a-3p or U6 (a component of the splicing machinery), plus the Mixing buffer for retro-transcription. The reaction was performed in a thermocycler with the following temperature program: 30 min at 16 °C; 30 min at 42 °C; and 5 min at 85 °C. The reaction was stopped at 4 °C to then perform a reverse transcription quantitative PCR (RT-qPCR) according to the manufacturer’s instructions. Briefly, specific Taqman®
primers were mixed together with the cDNAs, plus a solution composed of RNAase free water and Taqman universal master mix II. Each one of those mixes was submitted to the following temperature cycle program: an initial step to activate DNA polymerase for 10 min at 95 °C, followed by 40 cycles of 15 s at 95 °C; and 60 s at 60 °C to allow amplification. Once the amplification cycles were completed, we obtained the value of cycle threshold (Ct). The U6-corrected fold change of miRNA content was calculated using the procedure described in [22
2.9. In Utero Electroporation
In utero electroporation was performed to stably express proteins in astrocytes that can be maintained in culture in excellent conditions for at least 3 weeks. Electroporation of rat cerebral cortices was done in E18-E19 embryos as previously described [23
]. The fetuses were exposed through a 4 cm incision over the linea alba of the muscular layer. Once exposed, the fetuses were constantly moisturized with a saline solution (0.9% w
NaCl in distilled water) plus antibiotics, using a dilution of 50 U/mL penicillin and 10,000 μg/mL streptomycin at 37 °C. Previously, we prepared glass capillaries with a 100–150 μm inner radius using a P97 pipette puller (Sutter Instruments, Novato, CA, United States) filled with 25 μL of a solution with the following composition: 0.75 μg/μL Fast Green dye (Sigma-Aldrich, St Louis, MO, United States); 1 μg/μL pPBCAG_Pbase plasmid, and 1 μg/μL pPBCAG_Aldo C-GFP or pPBCAG_ GFP plasmids, all diluted in distilled water. Then, 1–2 μL of this solution was injected into the left lateral ventricle of each embryo by means of a peak-pressure pump PV830 ( World Precision Instruments, Sarasota, FL, United States). An electric pulse of 60–70 Volts was given by a capacitor of 500 μF previously charged with a 250 V power source. The discharge was done through copper alloy plates (1 × 0.5 cm) and arranged over the brain with the positive electrode facing the left hemisphere. Fetuses were allowed to grow in utero until day 21 of gestation to perform pure astrocyte cultures. For this, the brain was observed under a stereo microscope with the help of a fluorescence adapter (NightSea, Lexington, MA, United States) to select tissue with transgene expression from the electroporated left telencephalon.
2.10. Cell Cultures and Isolation of sEVs
Hippocampal neurons were obtained from embryonic Sprague Dawley rats (E18) as previously described [24
]. Primary astrocyte cultures (90% of GFAP-positive cells) were obtained following established procedures with slight modifications [25
]. In order to clearly identify fetuses electroporated with different plasmids, we started astrocyte cultures at E21. When cultured astrocytes reached 70–90% confluence, culture media was replaced by a sEV free medium for 72 h. Subsequently, the medium was collected, and successive centrifugations were performed: 30 min at 2000× g
to eliminate cells and debris; 40 min at 10,000× g
to eliminate microvesicles; and 2 h at 100,000× g
to obtain the sEV enriched fraction in the pellet. Finally, this pellet was washed by resuspension in phosphate buffer saline (PBS) at pH = 7.4 and centrifuged again for 2 h at 100,000× g
to obtain the 100 K pellet fraction. The resulting pellet is enriched in sEVs [26
2.11. Transwell Astrocyte-Neuron Co-Culture
In order to obtain astrocytes expressing Aldo C-GFP or GFP, in-utero electroporation was performed as described above and astrocyte primary cultures were performed. After 15 days in vitro (DIV), the Aldo C-GFP or GFP-electroporated astrocytes were treated with trypsin (Sigma-Aldrich, St Louis, MO, United States) for 1 min at 37 °C, and, finally, the cells were seeded in a polycarbonate Transwell system of 0.4 µm pores (Corning Costar Co., Cambridge, MA, United States ) to reach 70% confluency. The Aldo-GFP or GFP astrocytes were transferred 24 h later to 24 plate wells to be co-cultured with 3 DIV hippocampal neurons. Three days later, both astrocytes and neurons were fixed using 4% PFA, and samples were submitted to IF.
2.12. Nanoparticle Tracking Analysis (NTA)
The sEVs were analyzed with the NanoSight LM-10 device (Malvern Instruments, Malvern, UK) equipped with a green laser, as described in [19
2.13. Sucrose Flotation Assay
To perform the assay, 300–400 µg of sEVs were resuspended in 1 mL of 2.5 M sucrose with 50mM HEPES buffer at pH = 7.2, diluted in deuterated water, and loaded at the bottom of a 13 mL ultracentrifuge tube. A continuous linear gradient was made from 2 M and 0.5 M sucrose solutions prepared with 50 mM HEPES buffer at pH = 7.2, diluted in deuterated water, and added over exosomes. The gradient was centrifuged for 17 h at 200,000× g and stopped with the free braking mode of the ultracentrifuge. Then, 1 mL fractions were collected from the top of the tube with the sucrose gradient. Each of these fractions was resuspended in 12 mL of 50 mM HEPES at pH = 7.4, and then centrifuged at 200,000× g for 2 h in order to collect sEVS in the precipitate. Each pellet was resuspended in 30 μL of loading buffer and boiled under denaturant conditions and fully loaded on a 12% w/v acrylamide gel with SDS for analysis by Western blot.
2.14. Incubation with sEVs
Isolated sEVs were resuspended in Neurobasal medium and added onto 20,000 hippocampal neurons (3 DIV) to obtain a final protein concentration of 10 ng/μL, in a total volume of 400 μL. As assessed by NTA analysis, this corresponds to a total mean number of added vesicles of 0.95 × 109 particles for Aldo C-GFP sEVs and 1.2 × 109 particles for GFP sEVs. After 72 h, neurons were fixed, stained with the corresponding antibodies, and submitted to Sholl analysis. When indicated, neurons were treated with sEVs 2 h after magnetofection. For the uptake experiments of sEVs, 200,000 hippocampal neurons were seeded on 35 mm plates or 25 mm coverslips and at 6 DIV they were incubated with 10 µg (protein content) of the corresponding sEVs at 4 °C or 37 °C for three hours. For Western blot analysis, the complete cell lysate obtained from each well/condition was loaded in each lane. For IF analysis, cells were fixed with 4% PFA. Alternatively, cells were lysed for RNA extraction and submitted to quantitative RT-PCR.
2.15. Neuronal Magnetofection
For neuronal transfections, 10 pmol of miR-26a-5p mimic (mimic 26a-5p) (Ambion®
# 4464066, Thermo Fisher Scientific, Waltham, MA, United States), miR-26a-5p inhibitor or antago (antago 26a-5p)( Ambion®
# 4464084, Thermo Fisher Scientific, Waltham, MA, United States), and miR-26a-5p mimic negative control (scrambled) (Ambion®
# 4464058, Thermo Fisher Scientific, Waltham, MA, United States ) were transfected by NeuroMag Transfection Reagent (Ozbioscience, San Diego, CA, United States) following the manufacturer’s instructions. High transfection efficiency was achieved by magnetofecting small fluorescent oligonucleotides (>90%, data not shown). Similar results were obtained in the literature [27
]. When indicated, sEVs were added 2 h after neurons were magnetofected with the respective oligos. Additionally, 200,000 neurons seeded on 35 mm plates were magnetofected with 20 pmol of the respective oligos and submitted to Western blot.
2.16. Morphological Analysis
Neurons were submitted to a Sholl analysis using the plugin of the Image J software [28
]. All concentric radiui were 3 μm from each other. The following parameters were obtained: total intersections (i.e., the sum of all intersections with each different radius); primary intersections (i.e., number of intersections with the first radius); critical distance (i.e., the radius with the maximum number of intersections); maximum number of intersections (i.e., maximum number of intersections reached by a neuron at any radius); maximum distance (i.e., the largest radius at which there is an intersection with a neuronal process).
2.17. Bioinformatic Analysis
The miRECORDS platform (http://tinyurl.com/js9jr8n
) was used to identify theoretical targets of miRNAs enriched in astrocytes by the consolidation of eleven programs with different prediction algorithms: DIANA-microT, MicroInspector, miRanda, MirTarget2, miTarget, NBmiRTar, PicTar, PITA, RNA22, and RNAhybrid and TargetScan/TargertScanS. We selected the targeted genes predicted by at least four different algorithms. The obtained list was further analyzed with the functional enrichment tool in biological processes defined by the AmiGO2 platform (http://tinyurl.com/z2pn5hb
]. To obtain a functional enrichment of the genes associated with cell functions, we normalized it by the expected value of the gene from a human reference genome. The list of predicted genes and the corresponding functional enrichment analysis for each miRNA are provided in the Supplementary Materials (Folder S1: Predicted miRNA targets and functional enrichment analysis)
2.18. Statistical Analysis
Differences between two groups were determined using the Welch’s t-test. The differences between more than two groups were evaluated with one-way ANOVA followed by Tukey’s post-hoc test. The fold changes compared to a theoretical value were evaluated with one-sample t-test or Wilcoxon one-sample signed-rank test when indicated. The differences were considered statistically significant with p < 0.05.