Alpha-synuclein oligomers are found in exosomes

Increasing evidence suggests that αsyn can be released by neurons and neuronal like cells [17, 31–33] although extracellular αsyn and its pathological relevance are still hotly debated in the field. Recent work from our own group and the elegant study from Desplats et al. suggest that αsyn can be transferred from cell to cell and thus may provide an explanation for the spread of αsyn pathology in PD patients [16, 17]. However, little is known about the mechanism of αsyn secretion.

Recently, secretion of αsyn in association with membrane vesicles, identified as exosomes based on their composition and biophysical properties, has been described [23, 24]. However, the specific αsyn species (monomers vs pathogenic oligomers) secreted with exosomes and the location of αsyn remains to be determined.

To investigate whether oligomeric species of αsyn are present in the exosome enriched fractions we employed a bioluminescent protein-fragment complementation assay [25, 26, 30]. In this strategy, αsyn was fused to non-bioluminescent amino-terminal (S1) or carboxy-terminal fragments (S2) of Gaussia princeps luciferase [28] that can reconstitute when brought together by αsyn-αsyn interactions [25], thus providing a readout of αsyn oligomerization (Additional file 1: Figure S1A). The same principle of protein complementation with fluorescent venus YFP was used generating the fluorescent protein-fragment complementation pair V1S or SV2 whereby N-terminal half of Venus YFP is fused to the N-terminus of αsyn (V1S) and C-terminal half of Venus YFP is fused to the C-terminus of αsyn (SV2) [25] (Additional file 1: Figure S1B).

Human H4 neuroglioma cells were co-transfected with S1 and S2 that reconstitute luciferase activity upon αsyn oligomerization. Exosomes were isolated from conditioned media (CM) of H4 cells using an established subcellular fractionation methodology [34, 35] and the exosomal pellet was analyzed for luciferase activity that is indicative of αsyn oligomers. Interestingly, we witnessed a large increase in luciferase activity in the exosomal fraction derived from H4 cells transfected with S1 and S2 compared to exosomes from mock transfected cells (Figure 1A), suggesting that αsyn, and specifically αsyn oligomers are present in the exosomal fraction. To exclude the possibility that N- or C-terminal fragments of human Gaussia Luciferase can interfere with protein sorting in exosomes, our results were verified in exosomes isolated from human H4 cells transfected with untagged wild-type (wt) αsyn using a human αsyn ELISA. Significant levels of αsyn were present in the exosomal fraction from wt αsyn and S1/S2 transfected H4 cells compared to exosomes derived from empty vector (mock) transfected cells (Figure 1B).

We extended these observations to primary cortical neurons where αsyn oligomers were also found in the exosomal fraction isolated from primary neurons co-transduced with adeno-associated virus (AAV) encoding S1 (AAV-S1) and S2 (AAV-S2) as demonstrated by a significant increase in luciferase activity compared to exosomes isolated from naive neurons (Figure 1C). In accord with the experiments performed in H4 cells, we also confirmed the presence of αsyn in exosomes derived from primary neurons infected with a variety of different AAV constructs encoding either αsyn-ires-GFP, AAV-S1 and AAV-S2 or αsyn-venusYFP fluorescent protein-fragment complementation pair (AAV8-V1S or AAV8-SV2) (Figure 1D) using an αsyn ELISA. Taken together, our data provide evidence that αsyn oligomers are present in the exosomal fractions from both neurons and non-neuronal cells.

Characterization of exosomes

To confirm the presence of exosomes, fractions from both primary neurons and H4 cells were subjected to SDS-PAGE and immunoblotting. All exosomal fractions were found to be immunopositive for the exosome-specific proteins alix and flotillin, whereas the ‘exosome-free’ supernatant was immuno-negative for alix and flotillin (Figure 2A).

Moreover, exosome-enriched fractions isolated from CM of H4 cells transfected with the αsyn complementation pair S1 and S2 were also analyzed using electron microscopy and demonstrated the distinctive vesicular morphological structures characteristic of exosomes (Figure 2B). Immuno-electron microscopy with an antibody against the exosomal marker CD63 [36], confirmed characteristic exosomal vesicles typically 60–100 nm in size in exosome enriched fractions from CM of primary neurons co-transduced with AAV expressing the αsyn complementation pair V1S or SV2 (Figure 2C).

Localization of αsyn oligomers in the extracellular space

Cytosolic proteins can be secreted from cells via at least two distinct pathways which include exocytosis and fusion of multi-vesicular bodies with the plasma membrane to release exosomes. Defining the localization of αsyn in the extracellular space will provide insight into the mechanisms and pathways involved in αsyn release. To examine the localization of αsyn oligomers in the extracellular space we first digested exosome-enriched fractions containing αsyn S1/S2 oligomers with 0.25% trypsin. Interestingly, trypsin digestion significantly reduced luciferase activity in the exosome fraction by 62% (Figure 3A) suggesting the presence of αsyn oligomers either on the external surface of the exosomes or outside of exosomes. However, trypsin treatment did not eliminate luciferase activity to background levels completely, indicating that αsyn oligomers must exist in the lumen of the exosomes that are insensitive to the trypsin treatment. To further clarify, we cotreated exosome fractions with trypsin and the detergent saponin (0.1%). The presence of saponin results in exosome-membrane permeabilization. Notably, we saw a complete elimination of luciferase activity in the presence of trypsin and saponin (Figure 3A). Treatment with saponin alone slightly increased luciferase activity compared to untreated control exosomes, although it was not a significant increase. This could be due to enhanced substrate availability to lumenal αsyn oligomers. The same experimental paradigm was tested on the "exosome-free" supernatant fraction. As expected, trypsin eliminated all luciferase activity from "free" αsyn oligomers in the supernatant fraction in the presence or absence of saponin (Figure 3B). These data confirm the absence of exosomes from the supernatant fraction and verify that the experimental paradigm is sufficient to digest all available αsyn oligomers.

To confirm our results on the localization of αsyn oligomers inside/outside exosomes we examined samples prepared under the same experimental conditions using dot blot immunoblotting. Probing with Syn-1 antibody showed that exsosome free αsyn oligomers (sup) were completely digested by trypsin independent of saponin treatment (lane 1, sup, Figure 3C). In contrast, Syn-1 signal was not completely eliminated when exosome fractions were treated with trypsin (lane 2, exo, Figure 3C). Only the combination of trypsin and saponin resulted in a complete digestion of αsyn oligomers and a consequent abolishment of αsyn immunostaining in exosome fractions. Probing with an antibody against the exosomal marker CD63, which is known to be located solely on the outside of exosomes, shows reactivity only in the exosome fractions not treated with trypsin and no reactivity at all in supernatant-associated αsyn oligomers. Dot blots were also performed on fractions prepared from CM of cells transfected with wt untagged αsyn. As expected, trypsin treatment resulted in a reduction in Syn-1 signal in exosome associated αsyn oligomers but only the combination of trypsin and saponin resulted in a complete digestion and abolishment of Syn-1 signal. Together, the data indicate that αsyn oligomers are located on the inside and outside of exosomes.

Exosome-associated αsyn oligomers are more prone to internalization than exosome-free αsyn oligomers

It has been reported that recombinant αsyn or αsyn oligomers can be internalized by cells and result in various cellular effects [18–20, 33, 67]. Furthermore, we and others have shown that cell produced αsyn oligomers can be secreted and taken up by proliferating cells and primary neurons [17, 23]. To investigate if exosomes are required for the internalization of αsyn oligomers, we exposed naive H4 cells to exosome-associated αsyn oligomers or exosome-free supernatant containing αsyn oligomers derived from S1/S2 transfected H4 cells for 3 days. Concurrently, exosomes or exosome-free supernatant from ”mock” transfected cells were added to naïve H4 cells. Interestingly, we found that exosome associated αsyn oligomers are more prone to being taken up than exosome free asyn oligomers (Figure 4A). To control for the variable amounts of αsyn in each exosome or supernatant preparation added to cells, the luciferase signal detected in the recipient cells was normalized back to the initial luciferase counts added to the naïve cells. Data analysed in this way revealed a 2.4 fold increase in uptake of exosome-associated αsyn oligomers compared to exosome-free αsyn oligomers (Figure 4B).

Recombinant oligomers as well as physiologically secreted αsyn oligomers can cause cell death when applied to culture medium of different cell lines and primary neurons [18–20, 23, 33, 67]. To determine if exosome-associated αsyn oligomers confer more cytotoxicity compared to exosome-free αsyn oligomers, we applied exosome-enriched fractions or exosome-free fractions derived from S1/S2 or MOCK transfected H4 cells to naïve proliferating H4 cells and found an increase in Caspase 3/7 activation conferred by exosome associated αsyn oligomers (Figure 4C). To ensure the same amount of αsyn oligomers in each fraction, the level of Caspase 3/7 activation was normalized to the amount of αsyn oligomers prior to the addition to naïve cells. Interestingly, a significant 1.5-fold increase in Caspase3/7 activation and resulting apotosis induction from exosome-associated αsyn oligomers compared to exosome-free αsyn oligomers was detected (Figure 4D).

In accordance with our data for human H4 cells we confirmed that exosome-associated αsyn oligomers could also be taken-up by naive primary neurons (Figure 4E) and induce apoptosis as characterized by an increase in caspase3/7 activity (Figure 4F). Unfortunately, due to high levels of non-specific background bioluminescence from B-27 supplement in our neuronal cell culture medium, we were unable to assess the internalization of exosome-free αsyn oligomers by primary neurons.

Exosomes need to be intact to be internalized

Because our data suggest that exosome-associated αsyn may be preferentially taken up by neighboring cells, we next asked whether exosomes need to be intact for uptake to occur. To explore this question, we labeled purified exosome-enriched fractions derived from S1/S2 transfected H4 cells with the membrane dye DiD [68]. To delineate the morphology of H4 cells or primary neurons, we transfected cells with venus-YFP prior to exosome addition resulting in a subpopulation of H4 cells or primary neurons that could be identified via green fluorescence. As expected when labeled exosomes were exogenously added to H4 cells or primary neurons in culture, we observed a rapid uptake of labeled exosomes into the cytosol of cells. To investigate whether membrane integrity is important for uptake, exosomes were subjected to sonication which is known to disrupt lipid bilayer integrity [69–71]. Sonication of exosomes prevented the uptake of exosomes by recipient cells (Figure 5A). These results were confirmed by measuring luciferase activity inside naive cells following incubation with intact or sonicated exosome fractions from syn-luc transfected H4 cells. Where exosomes were sonicated, significantly less luciferase activity was detected inside the naive cells (Figure 5B) although sonication itself had no effect on luciferase activity of exosomal fractions. This uptake was not unique to proliferating cells as we were also able to confirm the uptake of labeled exosomes into primary cortical neurons (Figure 5C). These results demonstrate that intact exosomes promote exosomal uptake.

To confirm that the labeled microvesicles being observed by confocal microscopy were actually exosomes, we immunostained cells with the exosomal marker flotillin and found that the flotillin immunoreactivity colocalized with the DiD labeled exosomes (Figure 5D).

Autophagy regulates αsyn secretion

Exosomes are derived from multivesicular bodies (MVBs), which are endocytic organelles generated by membrane invagination [72–74]. Proteins that are designated for lysosomal degradation are sequestered by MVBs, however, an alternative destination of MVBs is their exocytic fusion with the plasma membrane leading to the release of intraluminal vesicles (ILVs; i.e., exosome) into the extracellular environment. Interestingly, induction of autophagy can markedly increase the interaction of MVBs and autophagosomes and thereby block exosome secretion [75].

We detected a higher luciferase activity extracellularly (Figure 6A) which may suggest that more oligomers are secreted. However, it’s also possible that coelentrazine is more sensitive to extracellular αsyn oligomers although it is cell permeable. Nevertheless, given that we detected αsyn oligomers in the exosomal fraction of cells and observed that αsyn oligomer secretion could be modulated by autophagic activity (Figure 6A) we asked whether autophagy would be a release pathway for exosome-associated αsyn oligomers.

Following guidelines for assays monitoring autophagy [76] we measured the levels of LC3.II (ratio with ß-actin) and p62 (ratio with α-tubulin) in S1/S2 transfected H4 cells treated with rapamycin, bafilomycin A1 and DMSO. Levels of LC3.II and p62 significantly increased when H4 cells were treated with bafilomycin A1 compared to DMSO controls, indicating an abundance of autophagosomes due to reduced downstream fusion with lysosomes. The opposite effect was observed when treated with rapamycin (Figure 6 B, C). We speculated that an increased pool of autophagosomes could be the basis for an increased exosomes secretion. Exosomes were isolated from conditioned media of S1/S2 transfected H4 cells treated with bafilomycin A1, rapamycin or DMSO, and luciferase activity was monitored. A significant increase in luciferase activity in the exosomal fraction from cells that were treated with bafilomycin A1 compared to DMSO control (Figure 6D) was observed which suggests that inhibition of the fusion of the autophagosome with the lysosome by bafilomycin A1 provides an increased pool autophagosomes which enhances exosomal release. Likewise, treatment with the autophagy enhancer rapamycin showed a decreased αsyn oligomer signal in the exosomal fraction as measured by luciferase activity compared with DMSO treatment (Figure 6D), suggesting that enhanced lysosomal activity results in effective αsyn oligomer degradation and less secretion. We extended these findings to primary neurons and observed an increase in the αsyn oligomer signal in the exosomal fraction from neurons treated with bafilomycin A1 and a decrease in the αsyn oligomer signal with rapamycin treatment in the exosomal fraction compared to DMSO control (Figure 6E), although these effects did not reach statistical significance (p = 0.17), probably due to the fact that the yield of exosomes are significantly less from primary neuronal preparations resulting in barely detectable luciferase and an decrease in the signal/noise ratio. Together, these experiments indicate autophagy can be a specific release pathway for secretion of αsyn oligomers.

Plasmid generation

Fusion constructs αsyn-hGLuc1 (S1), αsyn-hGLuc2 (S2) and Venus1-αsyn (V1S), αsyn-Venus2 (SV2) were generated by subcloning αsyn into Not1/Cla1 sites of humanized Gaussia Luciferase and VenusYFP constructs provided by Dr. Stephen Michnick of University of Montreal [25, 28].

AAV vectors construction and production

The viral vectors rAAV-CBA-WPRE, rAAV-CBA-IRES-EGFP and rAAV-CBA-SYNUCLEIN-IRES-EGFP were described previously [29]. rAAV-CBA-SYNUCLEIN-LUC1-WPRE (AAV-S1) and rAAV-CBA-SYNUCLEIN-LUC2-WPRE (AAV-S2) were constructed as follows: αsyn -hGLuc1 (S1) and αsyn -hGLuc2 (S2) were subcloned into Not1/Nhe1 sites of AAV-CBA-WPRE vector. rAAV-CBA- VENUS1-SYNUCLEIN-WPRE (AAV-V1S) and rAAV-CBA-SYNUCLEIN-VENUS2-WPRE (AAV-SV2) were constructed as follows: the fragments Venus1-Synuclein and Synuclein-Venus2 was inserted into the EcoRV and NheI sites of the pAAV-CBA-WPRE vector. Recombinant adeno-associated type 2/8 was generated by tripartite transfection (AAV-rep/cap expression plasmid, adenovirus miniplasmid and AAV vector plasmid) into 293A cells and purified by iodixanol gradient followed by Q sepharose column chromatography (Harvard Gene Therapy Initiative, Harvard Medical School). The purified virus was dialyzed against PBS, concentrated by Amicon spin column, and tittered by dot blot hybridization. Final titers for virus were for AAV-S1 1.5E13 gc/ml, AAV-S2 1.3E13 gc/ml, V1S 8.3E12 gc/ml and SV2 8.7E12 gc/ml.

Human αsynuclein ELISA

Alpha synuclein concentration was quantified using human αsyn specific ELISA (#KHB0061, Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Absorbance is read at 450 nm. The absorbance is directly proportional to the concentration of αsyn present in the original specimen. αSyn concentration was determined by plotting sample absorbances against standards using Graph Pad Prism fitting software (four parameter algorithm).

Cell culture and transfections

Unless otherwise stated, human H4 neuroglioma cells (HTB-148 - ATCC, Manassas, VA, USA) were maintained in OPTI-MEM medium supplemented with 10% fetal bovine serum (both from Invitrogen) and incubated at 37°C. Cells were plated 24 hours prior to transfection, growing to 80–90% confluency prior to transfection. Transfection was performed using Superfect (Qiagen, Chatsworth, CA, USA) using equimolar ratios of plasmids according to the manufacturer’s instructions. Conditioned media was collected 48 hours post-transfection and centrifuged for 5 min at 3000 g to eliminate floating cells before being used.

Gaussia luciferase protein-fragment complementation assay

Fusion constructs αsyn hGLuc1 (S1) and αsyn -hGLuc2 (S2) were generated as described previously [25]. S1 and S2 were transfected into H4 cells in a 96-well plate format as described above. 48 h after transfection, culture media was transferred to a new 96 well plate (Costar, Corning, NY, USA). Cells were washed with PBS and replaced with serum- and phenol-red free media. Luciferase activity from protein complementation was measured for conditioned media and live cells in an automated plate reader at 480 nm following the injection of the cell permeable substrate, coelenterazine (20 μM) (Prolume Ltd, Pinetop, AZ) with a signal integration time of 2 seconds.

Primary cortical cell culture

Primary cortical neurons were prepared from cerebral cortices of E14-16 mouse embryos. Cortices were dissected from embryonic brain and the meninges were removed. Cortices were dissociated by titruation at RT and cells were resuspended in Neurobasal (NB) (Gibco) medium supplemented with 10% fetal bovine serum, 2 mM Glutamax, 100 U/mL penicillin, and 100 μg/mL streptomycin and plated at a density of 3.84 x 10⁴ cells/well on 96w plates (Corning, NY, USA), 60 mm dishes at a density of 3.6 x 10⁶ cells/dish (Costar, Corning, NY, USA) coated with 20μg/mL poly-D-lysine(Sigma-Aldrich, St. Louis, MO, USA). After 2 h medium was changed into NB/B-27 [NB medium containing 2% (v/v) B-27 supplement], 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM/L glutamine. Cells were maintained at 37°C in 5% CO₂ in a humidified incubator. Medium was changed every third day. Neurons were grown for 4–5 days in vitro (DIV) before infected with AAV8-S1/AAV8-S2, AAV8-V1S/AAV8-SV2, AAV8-syn-ires-GFP or AAV8-GFP. Infections were carried out as followed: 6 μl rAAV2/8 expressing eGFP (1.3E13gc/ml) per 60 mm dish, 6μl rAAV2/8 expressing αsyn(wt)-ires-GFP (1.1E13gc/ml) per 60 mm dish and 3 μl rAAV2/8 expressing V1S (8.3E12gc/ml) together with 3 μl rAAV2/8 expressing SV2 (8.7E12gc/ml) per 60 mm dish, as well as 3 μl rAAV2/8 expressing S1 (1.5E13gc/ml) together with 3 μl rAAV2/8 expressing S2 (1.3E13gc/ml) per 60 mm dish.

Exosome isolation

Exosomes from Human H4 cells and primary neurons were prepared as described earlier [34, 35] with minor modifications. Briefly, conditioned medium was collected and spun for 5 min at 500xg to remove floating cells. The supernatants were then sequentially centrifuged at 300xg (10 min) and 2x 200xg (10 min) at 4°C each. Then supernatants were filtered through a 0.45 μm (Whatmann, Florham Park, NJ) and then 0.22 μm (Millipore, Carrigtowhill, Cork, Ireland) filter, and centrifuged for 30 minutes at 10,000xg (2X) at 4°C. After ultracentrifugation at 100,000xg for 70 min at 4°C, exosomal pellet was then resuspended in 1xPBS for Western Blotting, electron microscopy or luciferase assay or resuspended culture medium for cell treatments.

Exosome depleted medium was prepared as described above, except after ultracentrifigation at 100,000xg for 70 min at 4°C, exosome free supernatant was filtered through a 0.22 μm filter before used in cell culture.

Digestion of exosomes

Exosome associated or exosome free αsyn oligomers were digested by addition of 0.25% trypsin (Invitrogen, Carlsbad, CA, USA) and/or 0.1% saponin and incubated for 20 min at 37°C. After complete digestion samples were analyzed in luciferase assay or Dot blot approach.

Labeling of exosomes

The exosomes were labelled using DiD (Biotium, Hayward, CA USA) according to manufacturer’s instructions in a 1:200 dilution. Briefly, after the final spin in exosome purification exosomal pellet was resuspended in 1 ml DiD solution and incubated for 5 minutes. After ultracentrifugation at 100,000xg for 70 min at 4°C the exosomal pellet was washed in 1xPBS centrifuged again for 90 min at 150 000 g to remove free dye. Then the pellet is resuspended finally as exosome fraction.

Dot blot

Exosomes or exosome free supernatant was collected as described previously. 100ul of each condition was applied to nitrocellulose membrane (pore size 0.22 um, Whatman Protran,Sanford, ME, USA) placed in a Dot blot apparatus (Schleicher & Schuell Minifold-I Dot-Blot System, Whatman, Sanford, ME, USA, ) and incubated at RT for 1 h. Samples were filtered through the membrane by gentle vacuum and developed using conditions as described previously (21). Briefly, the membrane was blocked with 10% non-fat dried milk in Tris-buffered saline (TBS, Sigma-Aldrich, St. Louis, MO, USA) containing 0.01% Tween 20 (TBS-T), at room temperature for 1 h. After three washes with TBS-T, the membrane was incubated with anti- Syn-1 antibody (1:1000; BD transduction, Franklin Lakes, NJ, USA) or monoclonal anti-CD63 antibody (1:500, BD Transduction) overnight at 4 C with gentle agitation. The membranes were then washed three times for 5 min with TBS-T, incubated with horseradish peroxidase conjugated anti mouse IgG (Jackson Immuno Research Laboratories, Baltimore, PA, USA) diluted 1:2000 in 5% non-fat dried milk in Tris-buffered saline (TBS) containing 0.01% Tween 20 (TBS-T) and incubated for 1 hour at room temperature. The blots were washed three times with TBS-T and developed with Pierce ECL chemiluminescence kit from Thermo Scientific (Rockford, IL, USA).

Pharmacological treatments in vitro

H4 cells were plated into 96 well plates or 60 mm dishes (Costar, Corning, NY, USA) and transfected as described above. Transfection mix was incubated for 2 h according to manufacturer’s protocol, then media was replaced by fresh culture media containing 0.4 μg/ml rapamycin (Sigma Aldrich, St. Louis, MO) or DMSO (Sigma Aldrich, St. Louis, MO) and incubated for 48 h. 200nM Bafilomycin A1 (Merck KG, Darmstadt, Germany) was added to the culture medium 20 h before harvesting the medium. Conditioned medium was collected for aluciferase assay or exosomal isolations. To ensure that pharmacological treatments result in a true increase in the secretion ratio of αsyn oligomers and not simply a matter of more available αsyn oligomers in the cytoplasm, we calculated the ratio of secreted αsyn oligomers in the conditioned medium to intracellular αsyn oligomers.

Primary neurons were plated into 60 mm dishes (Costar, Corning, NY, USA) and infected as described above. DMSO and 0.1 μg/ml rapamycin were added to the culture medium right after infection, whereas 100nM bafilomycin A1 was added after 3 days expression and incubated for 20 h. Conditioned medium was then collected to perform exosomal isolations.

Exosomal uptake experiments

Conditioned media from naïve H4 cells or naïve primary neurons was replaced by exosome containing culture media. After 3–4 days incubation cells were washed twice with 1xPBS and then assayed for luciferase activity.

Toxicity assay

Toxicity was analyzed 3–4 days after exosome application by measuring the activity of Caspases 3 and 7 using a fluorometric substrate Z-DEVD-Rhodamine 110 (Apo-ONE homogeneous Caspase-3/7 assay #G7790, Promega, Madison, WI) according to the manufacturer’s protocol.

Western blotting

Primary cortical neurons were scraped from 60 mm dishes and washed by centrifugation and resuspension in cold PBS. The cells were resuspended in 1x PBS containing protease inhibitors (protease inhibitor cocktail tablet 1 tablet/10 mL (Roche Diagnostics) sheared by passing through a 27-gauge 1 ml syringe 4–6 times and centrifuged for 5 min at 13,000 g. Lysates or exosomal samples were resolved by electrophoresis on a 4–12 % Bis-Tris gradient gel (NuPAGE Novex Bis-Tris Gel, Invitrogen, Carlsbad, CA, USA) according to manufacturer’s instructions using NuPAGE MOPS buffer. After transfer to nitrocellulose membrane (Protran, Schleicher and Schuell, Whatman GmbH, Dassel, Germany) membranes were blocked in either 5% milk inTBS-T or Li-Cor blocking buffer (LI-COR,Lincoln, NE, USA) for 1 hour at room temperature. Membranes were then incubated with primary antibodies (mouse anti-Alix, 1:500, BD Transduction; mouse anti-flotillin: monoclonal, 1:500, BD Transduction) overnight at 4 C. After three 5–10 min TBS-T washes, membranes were incubated at room temperature for 1 hour with either IR-labeled secondary antibodies (IR800 goat anti-mouse, 1:2000, Rockland Immunochemicals, PA,USA) or HRP-conjugated secondary antibodies (1:2000). After three 5– 10 min TBS-T washes, immunoblots were analyzed using either the Odyssey Infrared imaging system (Li-Cor, Lincoln, NE,USA) or the ECL chemiluminescent detection system (Amhersham/GE HealthCare, Buckinghamshire, UK).

Cell imaging and immunofluorescence staining

All images were acquired using a 20x Plan Apochromat lens (Carl Zeiss), 25x APO-Plan NEOFLU lens (Carl Zeiss) or Zeiss 63x 1.2 NA C-APO-Plan NEOFLU water immersion lens (Carl Zeiss), mounted on the microscope described before. H4 cells or cortical neurons were washed three times with phosphate-buffered saline (PBS) following 30 min incubation in a fixation solution containing 4% paraformaldehyde in PBS. After washing, the cells were permeabilized and unspecific binding sites were blocked using 0.05% Saponin and 1% bovine serum albumin in PBS followed by another washing step. The primary rabbit antibody against flotillin (1:500, BD Transduction) was added for 1 h at RT, followed by another washing step and incubation with the secondary antibody (anti-mouse antibody labeled with Alexa-Fluor 488; Invitrogen) for 1 h at RT.

Electron microscopy

An exosome pellet from either human H4 cells or primary neurons was prepared by centrifugation as described above and then resuspended in 20 μl of cold Karnovsky’s EM fixative (2% formaldehyde and 2.5% glutaraldehyde in 0.1 M Sodium Cacodylate buffer, pH 7.4.). Ultrathin sections from LR white embedded samples were picked up from the knife with a loop, dipped in a 2:1 mixture of 2.3 M sucrose and 2% methylcellulose, and adsorbed to the surface of a formvar/carbon coated copper grid. Grids are placed on 2% gelatin in a small petri dish and stored in the fridge until immunogold labeling. This was accomplished by washing grids in PBS and then either treating with CD63 antibody (BD Transduction) followed by 10 nm gold labelled secondary antibodies (Sigma Aldrich, St. Louis, MO) or processing without immunolabelling. These exosome containing grids were then post-fixed with 2.5% glutaraldehyde, washed and contrasted with 2% methyl cellulose and 3% aqueous uranyl acetate. Samples were examined and photographed with a JEOL 1200EX electron microscope.

microRNA profiling and data analysis

For nucleic acid analysis, the entire exosome pellet is gently resuspended in 20 μl of 1xPBS. Any cellular/ribosomal RNAs that may exist in the extra-exosomal solution (53) are eliminated by adding 8 μg of RNAse T1/A (Fermentas) to the 20ul preparation and incubating for 10 minutes at 37°C. Four hundred units of SuperRase-in RNAse (Ambion) inhibitor are then added to inactivate the RNAses and the sample is held at 25°C for 10 minutes. The entire mixture is then dissolved in 60 μl of miRNA extraction buffer (Arcturus), incubated at 42°C for 30 minutes, and stored at −80°C prior further processing. In order to generate amplified sense RNA from small quantities (<1 ng) of purified miRNAs, we used the NCode miRNA Amplification System (Invitrogen) according to the manufacturer’s instructions. miRNA expression profiles were generated by adding 250 ng of this amplified miRNA to the FlexMiR miRNA assay from Luminex Corporation (Austin, Texas) and running on a Luminex FlexMAP 3D system according to the manufacturer's instructions.

Statistical analysis

Statistical analyses were carried out using the program GraphPad Prism, Version 4.0. Values in the figures are expressed as means +/− SEM.