J Neural Transm (2010) 117:1–4 DOI 10.1007/s00702-009-0288-8
BASIC NEUROSCIENCES, GENETICS AND IMMUNOLOGY - RAPID COMMUNICATION
Astrocytes and Glioblastoma cells release exosomes carrying mtDNA Michele Guescini Æ Susanna Genedani Æ Vilberto Stocchi Æ Luigi Francesco Agnati
Received: 20 July 2009 / Accepted: 29 July 2009 / Published online: 13 August 2009 Ó Springer-Verlag 2009
Abstract Cells can exchange information not only by means of chemical and/or electrical signals, but also via microvesicles released into the intercellular space. The present paper, for the first time, provides evidence that Glioblastoma and Astrocyte cells release microvesicles, which carry mitochondrial DNA (mtDNA). These microvesicles have been characterised as exosomes in view of the presence of some protein markers of exosomes, such as Tsg101, CD9 and Alix. Thus, the important finding has been obtained that bonafide exosomes, constitutively released by Glioblastoma cells and Astrocytes, can carry mtDNA, which can be, therefore, transferred between cells. This datum may help the understanding of some diseases due to mitochondrial alterations. Keywords Glioblastoma cells Astrocytes Mitochondrial DNA Exosomes Intercellular communication
Introduction Evidence has been provided that small vesicles are released either constitutively and/or upon stimulation by many cell
M. Guescini V. Stocchi Department of Biomolecular Sciences, University of Urbino ‘Carlo Bo’, Urbino, Italy S. Genedani L. F. Agnati (&) Department Biomedical Sciences, University of Modena and Reggio Emilia, Via Campi 287, 41100 Modena, Italy e-mail:
[email protected] L. F. Agnati IRCCS Lido, Venezia, Italy
types and can function as vehicles for intercellular communication. These microvesicles can carry signals either in their limiting membrane and/or in their interior lumen (Cocucci et al. 2009). Thus, cells can exchange more complex information than it was previously thought, when the communication between cells was considered to occur only by chemical signals released by the source-cell and decoded by the target-cell upon receptor binding. Two modes of microvesicle release have been characterised: the exocytosis of multivesicular bodies (MVBs) and the direct budding from the plasma membrane of small vesicles. Meldolesi and coauthors (Cocucci et al. 2009) suggest to keep the term of exosomes for the MVB-derived microvesicles, while vesicles directly budded from the plasma membrane are referred by the different term, the ‘shedding vesicles’. Thus, MVBs are cytoplamic organelles, which contain intra-luminal vesicles (ILVs) and one possible fate of MVBs is their exocytic fusion with the plasma membrane leading to the release of the ILVs (i.e. of exosomes) into the extracellular milieu (van Niel et al. 2006). Exosomes are classically defined as vesicles with a diameter of 40–100 nm that originate in MVBs of the endosomal system with an emerging role as intercellular signalling devices (Barral and von Herrath 2005). As mentioned above, exosomes are released both constitutively and in a regulated manner (Lakkaraju and Rodriguez-Boulan 2008). Exosomes express certain marker proteins (e.g., Alix and Tsg101, which are involved in endosomal–lysosomal sorting) but lack the proteins of the MVB-limiting membrane (Lakkaraju and Rodriguez-Boulan 2008). The present paper analyses exosomes released by Glioblastoma and Astrocyte cell cultures, which have been characterised by means of their specific marker proteins Alix, Tsg101 and CD9 (Fevrier and Raposo 2004; Simons
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and Raposo 2009) and for the first time it has been demonstrated that exosomes carry mitochondrial DNA (mtDNA).
Materials and methods Microvesicle isolation Human glioblastoma cells (U87MG) and primary astrocyte cells (prepared using cortices obtained from neonatal rat) were cultured at 37°C with 5% CO2 in DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 10% heat-inactivated FBS, penicillin (100 U/mL, Invitrogen) and streptomycin (100 mg/mL, Invitrogen). Conditioned medium from 5 9 107 cells was collected after 48 h. Microvesicles were purified by differential centrifugation. Glioblastoma- or astrocyte-conditioned medium was centrifuged for 15 min at 1,000g to eliminate cell contamination. Supernatants were further centrifuged for 20 min at 12,000g and subsequently for 20 min at 18,000–20,000g. The resulting supernatants were filtered through a 0.22 lm filter and then microvesicles were pelleted by ultracentrifugation at 110,000g for 70 min. The microvesicle pellets were washed in 13 mL PBS, pelleted again and resuspended in PBS. For further experiments, the isolated exosomes from U87MG cells were purified by immobilization onto magnetic beads. Briefly, 25 lL Dynabeads, precoated with goat antimouse immunoglobulin G (Invitrogen) was incubated for 1 h with 30 lL of an anti-CD9 monoclonal antibody (clone MM2/57, Chemicon International Inc., Temecula, CA, USA). Subsequently, beads were incubated by rotation with 30 lg exosomes for 3 h at 4°C. After washing four times, beads and bound exosomes were resuspended in PBS for further experiments. Western blotting analysis For electrophoresis, samples containing 50 lg of protein were mixed with Laemmli sample buffer (1:1 ratio) and loaded onto 12% SDS-PAGE gels. Subsequently, proteins were blotted to a nitrocellulose membrane (GE Healthcare). Primary antibodies used were Alix (1:1,000 dilution, clone sc-49268 Santa Cruz), Tsg101 (1:2,000 dilution, clone 4A10 Abcam) and CD9 (1:500 dilution, clone MM2/57 Chemicon). Primary antibodies were incubated overnight at 4°C followed by washing and the application of secondary HRPconjugated antibody (Pierce). Immune complexes were visualised using the Supersignal Dura reagent (Pierce). DNA isolation and quantification Genomic DNA was isolated from purified exosomes using the Qiamp Mini kit (Qiagen) according to the manufacturer’s
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instructions. Thereafter, nuclear and mitochondrial DNA (nDNA and mtDNA) content from exosome preparations were measured by real-time PCR. To confirm that the DNA is confined inside the exosomes, U87MG exosomes were treated with DNaseI (Ambion) for 30 min at 37°C before DNA purification. The following specific primers for human b actin (ACTB, ACTB-F: 50 -TGA CTG GCC CGC TAC CTC TT-30 and ACTB-R: 50 -CGG CAG AAG AGA GAA CCA GTG A-30 ), human mitochondrial encoded NADH dehydrogenase subuniut 1 (HmMT-ND1, MT-ND1-F: 50 -ACG CCA TAAAAC TCT TCA CCA AAG-30 and HmMTND1-R: 50 -TAGTAG AAG AGC GAT GGT GAG AGC TA-30 ) and rat mitochondrial encoded NADH dehydrogenase subuniut 1 genes (RnMT-ND1, MT-ND1-F: 50 -ACT CCC TAT TCG GAG CCC TA-30 and RnMT-ND1-R: 50 CAG GCG GGG ATT AAT AGT CA-30 ) were used in the quantitative amplification. Real-time PCR amplifications were conducted using LightCyclerÒ 480 SYBR Green I Master (Roche) according to the manufacturer’s instructions, with 500 nM primers and a variable amount of DNA standard in a 20 lL final reaction volume. Thermocycling was conducted using a LightCyclerÒ 480 (Roche) initiated by a 10 min incubation at 95°C, followed by 40 cycles (95°C for 5 s, 60°C for 5 s, 72°C for 10 s) with a single fluorescent reading taken at the end of each cycle. Each reaction was conducted in triplicate. All the runs were completed with a melt curve analysis to confirm the specificity of amplification and the lack of primer dimers. Cp (second derivative method) values were determined by the LightCyclerÒ 480 software version 1.2.
Results We previously demonstrated the release of exosomes in the culture medium by glioblastoma (U87MG) and astrocyte cells. The exosomes were collected by differential centrifugation as previously described in Skog et al. (2008). The presence of exosomes was demonstrated by western blot analysis for Alix, Tsg101 and CD9 (Fig. 1a). Since some proteomic profiles reported that released microvesicles can contain mitochondrial proteins (Mears et al. 2004; Staubach et al. 2009), we further analysed exosomes for the presence of mtDNA. DNA was isolated from purified exosomes and quantified by real-time PCR. This analysis revealed the presence of mtDNA (Fig. 1b) but not of nDNA (data not shown) in the exosomal pellet of both glioblastoma cells and astrocytes. To confirm that mtDNA is confined inside the exosomes and its presence was not the result of simultaneous pelleting during ultracentrifugation steps, DNaseI treatment of U87MG exosomes was performed after resuspension in PBS. The results showed a
Astrocytes and Glioblastoma cells release exosomes
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Fig. 1 a Western blot characterisation of glioblastoma and astrocyte exosomes. Exosomes (Exo), supernatant (Sup) and whole cellular lysate proteins (Lys) from glioblastoma and astrocyte cells were separated on SDS-PAGE and electroblotted to nitrocellulose membrane. Blots were probed with antibodies against: Alix, Tsg101 and CD9. Molecular mass markers are shown at left. b Mitochondrial DNA quantification from glioblastoma- and astrocyte-conditioned medium. mtDNA was isolated from exosome pellets and quantified by real-time PCR using specific primers for the mitochondrially encoded gene NADH dehydrogenase subunit 1 (MT-ND1). Ultracentrifugation pellet obtained from DMEM medium added of 10% heatinactivated FBS was used as negative control. c Mitochondrial DNA
quantification after DNaseI treatment. Intact U87MG exosomes treated with DNaseI show a significant decrease in mtDNA quantity respect to control exosomes although about 10% of mtDNA was protected from degradation. Exogenous DNA (Exg DNA) added to exosomes was completely degraded by DNase treatment. d Exosomes purification using magnetic beads. mtDNA was quantified from U87MG exosome fraction after purification with magnetic beads. CD9 antibodies were used in the binding of exosomes to the beads. This figure shows that most of the pelletted mtDNA is free (SUPERNATANT) and only about 5% of the released mtDNA is associated with exosomes (BEADS ? Anti CD9)
significant difference in mtDNA quantity between DNasetreated and control exosomes (Fig. 1c). This demonstrated that a marked portion of released mtDNA is probably free, however the DNase-resistant mtDNA, about 10% of the total, may be enveloped in the interior of microvescicles. Importantly, exogenous DNA added to exosomes was completely degraded by the same treatment. In order to investigate if mtDNA is associated to the exosomal fraction, exosomes were further purified by magnetic beads coated with a CD9 antibody. CD9 is a tetraspanin protein abundant on the exosome surface (Jansen et al. 2009). Indeed, mtDNA was further quantified from bead–exosome
complexes. This approach showed that only the 5% of mtDNA is confined within exosomal vesicles (Fig. 1d).
Discussion It has been shown that several messages can be sent via exosomes and hence dispatched into the extra-cellular space within protective containers diffusing until their targets. The view that exosomes, as well as shedding vesicles (Cocucci et al. 2009) are safe vesicular carriers for targeted intercellular communication, is largely supported
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by experimental data (Simons and Raposo 2009). As a matter of fact, it has been shown that intrinsic features of exosomes (and of shedding microvesicles) allow their specific interaction with the appropriate target cells and fulfil their task of ‘roamers’ moving from the source-cell to the target-cell(s) (Belting and Wittrup 2008; Mallegol et al. 2007; Schorey and Bhatnagar 2008; Simons and Raposo 2009). Thus, Raposo states that the most distinctive function of exosomes is to specifically interact with target-cells and hence they play an important role in cell–cell communication (Simons and Raposo 2009). In this context, it is interesting to cite the Smalheiser’s (2007) proposal that exosomal transfer of proteins and RNAs, especially from the post-synaptic to the pre-synaptic terminal, can play a role in synaptic plasticity. The present findings demonstrate that some of the microvesicles released by glioblastoma and astrocyte cells are exosomes as shown by the presence of the specific protein markers Alix and Tsg101 (Raiborg et al. 2003). Therefore, our data suggest that exosomes can work as vesicular carriers of mtDNA and this finding can be of an enormous physiological and pathological importance. This proposal is also supported by the discovery of some mitochondrial proteins within the exosomes released by melanoma and human breast carcinoma cells (Mears et al. 2004; Staubach et al. 2009). Previous papers have shown that mitochondria can migrate from one cell to neighbouring cells via Tunneling NanoTubes (TNTs) (Gerdes et al. 2007, Rustom et al. 2004). In view of the demonstration of a pathogenic role of altered mitochondria in Alzheimer’s disease (AD) (Schapira 2006), the finding that altered (rotenone-poisoned) mitochondria can migrate via TNTs (Agnati et al. 2009) has a substantial interest. The present paper shows that migration of mtDNA can take place also via exosomes and hence microvesicles can represent an alternative pathway, through which altered mtDNA can enter into other cells, favouring the diffusion of various pathologies. In agreement with the possible role of the microvesicle transfer of altered mtDNA in AD, recent evidence has also pointed out the pathogenic Ab peptides can be secreted from the cells in association with exosomes and exosomal proteins have been found to accumulate in the plaques of AD patient brains, suggesting a role in the pathogenesis of AD (Rajendran et al. 2006).
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