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Prions and exosomes: From PrPc trafficking to PrPsc propagation
Blood Cells, Molecules, and Diseases 35 (2005) 143 – 148 www.elsevier.com/locate/ybcmd
Prions and exosomes: From PrPc trafficking to PrPsc propagation Isabel Porto-Carreiro a,*, Benoıˆt Fe´vrier a, Sophie Paquet b, Didier Vilette b, Grac¸a Raposo a a
b
Institut Curie, CNRS UMR 144, 75248 Paris, France Institut National de la Recherche Agronomique, Unite´ de Virologie Immunologie Mole´culaires, 78350 Jouy-en-Josas, France Submitted 20 June 2005 Available online 15 August 2005 (Communicated by M. Lichtman, M.D., 21 June 2005)
Abstract Exosomes are membrane vesicles released into the extracellular environment upon exocytic fusion of multivesicular endosomes with the cell surface. Exosome secretion can be used by cells to eject molecules targeted to intraluminal vesicles of multivesicular bodies, but particular cell types may exploit exosomes as intercellular communication devices for transfer of proteins and lipids among cells. The glycosylphosphatyidylinositol-linked prion protein (PrP) in both its normal (PrPc) and scrappie (PrPsc) conformation is associated with exosomes. Targeting of exosomes containing the normal cellular PrP could confer susceptibility of cells that do not express PrP to prion multiplication. Furthermore, exosomes bearing proteinase-K resistant PrPsc are infectious, suggesting a model in which exosomes secreted by infected cells could serve as vehicles for propagation of prions. Thus, cells may exploit the nature of endosome-derived exosomes to communicate with each other in normal and pathological situations, providing for a novel route of cell-to-cell communication and therefore of pathogen transmission. These findings open the possibility that methods to interfere with trafficking of such unconventional pathogens could be envisioned from insights on the mechanisms involved in exosome formation, secretion and targeting. D 2005 Elsevier Inc. All rights reserved. Keywords: Exosomes; Prions; Multivesicular bodies; ESCRT machinery; Glycosylphosphatidylinositol-linked proteins; Transmission
Introduction Endosomal multivesicular bodies (MVBs) in certain cell types fuse with the cell surface in an exocytic manner. During this process, the small 50– 90 nm vesicles contained in their lumen are released into the extracellular environment and are then called exosomes. Exosomes were shown to be secreted from hematopoietic (reticulocytes, B lymphocytes, dendritic cells, mast cells, T cells, platelets) and non-hematopoietic cells (intestinal epithelial cells, melanoma and mesothelioma cells) (reviewed in [1,2]). Exosomes released from these cells harbor functional molecules and can be targeted to other cells in which these molecules can elicit function. Exosomes released from B lymphocytes, dendritic cells (DCs), mast cells, tumor cells and intestinal epithelial cells can be targeted to T cells, bone-marrow-derived and splenic dendritic cells and can induce immunomodulatory functions for exosome* Corresponding author. E-mail address: [email protected] (I. Porto-Carreiro). 1079-9796/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bcmd.2005.06.013
associated antigens [3– 11]. Furthermore, vesicles with the hallmarks of exosomes are present, in vivo, at the surface of follicular dendritic cells (FDCs) in germinal centers [12], in malignant effusions [13], in broncho alveolar lavage [14], in urine [15] and in serum [16,17]. These and other studies support the idea that exosomes, in addition to their role in eradication of ‘‘unwanted’’ molecules [18,19], could provide for acellular vehicles to transfer molecules among cells in normal and pathological states [1,2,20]. Recent findings revealed an unexpected role for exosomes in the vehiculation of prions [21]. Prion diseases are invariably fatal neurodegenerative disorders that affect both humans and animals. They are associated with the conversion of the cellular prion protein (PrPc) into the scrappie PrP (PrPsc), an abnormal conformational state that tends to form amyloid deposits in brain tissue and that is thought to be infectious [22,23]. In the infectious forms of prion diseases, such as Kuru and variant Creutzfeldt – Jakob disease in humans, scrappie in sheep and bovine spongiform encephalopathy in cattle, the infectious agent
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enters the host through the gastrointestinal tract and then replicates in peripheral nerves and usually in lymphoid tissue before invading the central nervous system (CNS) [24,25]. There is increasing evidence that phagocytic mononuclear cells, including bone marrow, spleen-derived and follicular dendritic cells (FDCs), accumulate infectious prions and may play a key role in the transfer of prions from the gastrointestinal tract to the brain and therefore in the onset of disease [26,27]. However, the cellular mechanisms by which infectious prions are transferred from cell to cell are far from being fully unraveled. In agreement with previous studies, our observations provided evidence for a noncellular form of prions [28] corresponding to exosomal membranes bearing PrPsc which are infectious in vitro and in vivo [21]. These observations lead to the hypothesis that exosomes may constitute a potential vehicle for prions [21,29]. Here, we highlight the emerging link between the mode of propagation of this unconventional infectious agent and its intracellular trafficking through the endosomal system. Finally, we further discuss how insights from MVB biogenesis could contribute to design new approaches that interfere with PrP trafficking and secretion.
The prion protein and its trafficking The glycosylphosphatidylinositol (GPI)-anchored prion protein is ubiquitously expressed, although in higher levels, in neurons, some non-neuronal tissues and in cells from the immune system [22]. Its expression could be associated with a number of cell functions, including copper and/or zinc ion transport or metabolism; protection from oxidative stress; cellular signalling; membrane excitability and synaptic transmission; apoptosis and neurite outgrowth [30,31]. PrPc is synthesized in the endoplasmic reticulum, travels through the Golgi apparatus, before being transferred to the plasma membrane [32]. After synthesis, PrPc processing includes cleavage of the amino (N)-terminal signal peptide, addition of two N-linked oligosaccharide chains, formation of a disulfide bond and the insertion of its GPI anchor, responsible for its association with lipid-rafts at the plasma membrane. Once at the cell surface, PrPc can be constitutively internalized either by clathrin-coated vesicles or caveolae [32,33]. Studies on Chinese hamster ovary (CHO) cells which express caveolin-1 revealed that PrPc is clustered in caveolar-structures at the cell surface as well as in interconnected chains of endocytic caveolae inside the cytoplasm [34]. The choice of internalization pathway is not fully understood and may depend upon the cell type under study and the lipid microenvironment of the plasma membrane where PrPc is inserted. Furthermore, as proposed, PrP endocytosis via clathrin coated pits could be due to its association with a still unidentified accessory protein carrying signals for adaptor and clathrin recruitment [33]. Once internalized by either mechanism, PrPc has been
shown to transit through late endosomes and lysosomes [34,35], consistent with the steady state localization of a fraction of PrPc to endocytic MVBs in neurons in situ, in brain and in non-neuronal cell systems [21,34,36].
The infectious prion protein Based on the protein only hypothesis, the infectious agent is thought to be the PrPsc which arises by a conformational conversion of PrPc [22]. The transconformation implies a switch from a form rich in a-helices to another form where h-sheets predominate. The rate of this conversion can be dramatically increased by the presence of preformed PrPsc as a ‘‘seed’’. The seed acts as the infectious agent and recruits PrPc molecules into an oligomer that has all the characteristics of an amyloid fibril. Prion can then replicate the altered amyloid conformer through the generation of new protein seeds that can then be transmitted to other cells to nucleate further polymerization and thus propagate the infectious prion form [37]. Several intracellular locations have been proposed as potential sites for conversion of PrPc to PrPsc. Conversion could take place in the endoplasmic reticulum, in lipid-rafts at the cell surface, inside an endocytic organelle or even in the cytosol in the absence of membranes [32,36,38]. It is important to note, however, that the location where normal PrP is actually converted into PrPsc may not reflect the sites of PrPsc accumulation in infected cells. The characterization of the exact site of accumulation of PrPsc has been hampered by the lack of antibodies that recognize the specific conformation of PrPsc using electron microscopy. Nevertheless, visualization of PrPsc after denaturation by guanidinium treatment prior to antibody detection revealed late endosomes and lysosomes of infected neuronal cells as possible sites of accumulation [39 –42].
Dissemination of prions, a role for exosomes? In most natural infections, prions are introduced peripherally and have to find their way to the CNS to produce the disease. A number of cellular players have been identified. For instance, it is thought that prions are transferred from FDCs to peripheral nerve endings within lymphoid organs. A model requiring cell-to-cell contact for intercellular prion transfer would necessitate additional cell types to bridge the gap between FDCs and neurons. Alternatively, prions could be transmitted to the nerves by cell-free short-range diffusion mechanisms. So far, the molecular and cellular mechanisms by which prion-infected cells contaminate their neighbors remain unclear. Work from Kanu and colleagues suggested that cell-to-cell contacts can promote the infection of target cells adjacent to infected cells [43]. Other studies, in contrast, reported on the presence of prion infectivity in the cell culture medium of a prion-
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infected neuronal cell line [28]. Although the physical nature of the associated prion infectivity remained uncertain, this observation supports the hypothesis that prion transfer could also occur by a means other than direct cell contact. We found that both PrPsc and PrPc are present in cell culture supernatants in a secreted exosome-associated form. As observed by electron microscopy, PrP pelleted from the cell culture supernatants was associated with membrane vesicles similar in size and morphology to previously described exosomes. Moreover, the PrP detected by Western blot floated at an equilibrium density of ¨1.14 g/ml in sucrose density gradients, consistent with its association with membrane vesicles. Western blot and mass spectroscopy analyses of the purified vesicles showed that they also contained proteins previously shown to be present in exosomes from other cells [21]. Interestingly, exosomal membranes bearing PrPsc were infectious in vivo after
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intracerebral injection in mice. All the exosome inoculated mice died as a consequence of acute typical neurological disorders. In addition, exosome-associated PrPsc released by the infected cells elicited conversion of endogenous PrPc to PrPsc when incubated with uninfected recipient cells [21]. We recently visualized prion dissemination in cell cultures infected with ovine prions. Our results indicate that transmission of infection is an efficient active biological process that proceeds mainly step by step through the infection of adjacent cells (Paquet et al., submitted to publication). Thus, release of exosome-associated PrPsc by infected cells could provide, in addition to cell – cell contact, an acellular mechanism underlying the spread of prions. These findings allow us to propose a model where exosomes from infected cells could transfer prion to distant cells and also to cells in close contact to the infected cells. Consistent with the observations that cellular PrP can be transferred between
Fig. 1. The ESCRT machinery involved in protein sorting at the limiting membrane of endosomes to ILV. (A) Distribution of ESCRT complexes at the limiting membrane of MVB. (B) Molecular components of each ESCRT complex. The heteromeric protein complexes – ESCRT-0, ESCRT-I, ESCRT-II and ESCRT-III – are thought to be involved in protein sorting to the intraluminal vesicles of multivesicular bodies (MVB). The current model implies that ubiquitinated cargo proteins are first recognized by the ubiquitin-binding proteins of the Hrs complex (Hrs, STAM and Eps15, called ESCRT-0). Hrs recruits ESCRT-I by interacting with one of its protein—Tsg101, which is also a ubiquitin-binding protein. Tsg101 then recruits ESCRT-III via ESCRT-II or AIP1/Alix, and these complexes function together to position cargo proteins into the inward-budding vesicles of the MVB. ESCRT-III interacts with the AAA-ATPase Vps4, which dissociates the ESCRT machinery and releases the class E proteins for further rounds of sorting.
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cells in a regulated manner [44], targeting of exosomes containing the normal PrP could also confer susceptibility to prion multiplication in cells that do not express PrP. Exosome delivery could also be relevant to the transconformation process. First, the raft-like nature of exosomal membranes [45] may be a favorable environment for transconformation and amyloid fiber formation [46]. Second, it has been reported that conversion of PrPc into PrPsc is greatly facilitated when the cellular and the transconformed PrP proteins are in contiguous rather than in facing membranes [47]. Exchange of membranes can result to insertion of incoming PrPsc into the raft domains of recipient cells [47] and exosomes bearing PrPsc released from infected cells could undergo fusion with the plasma membrane of non-infected cells. Since conversion has been proposed to occur at a low pH [48,49], perhaps a more likely scenario would involve internalization of exosomes and fusion with endosomal limiting membranes, similar to the process of back fusion of the internal vesicles of MVBs reported to occur in DCs [20].
From ILV formation to exosome secretion The finding that exosomes may constitute a ‘‘bubbleride’’ for prions open the possibility that methods to interfere with prion trafficking and propagation could be designed from insights on the mechanisms involved in
exosome formation. Exosomes correspond to the ILVs of MVBs, and the molecular requirements for ILV generation constitute new targets to potentially regulate exosome formation [2,50]. The sorting of cargo into ILVs from MVBs is a tightly regulated process that depends on the function of a complex composed by at least 18 proteins. The ESCRT (Endosomal Sorting Complex Required for Transport) sorting machinery was firstly described in the yeast Saccharomyces cerevisiae as the class E ‘‘Vacuolar Protein Sorting’’ (Vps) proteins (reviewed in [51]). The ESCRT machinery is constituted by a group of cytosolic wellconserved proteins from yeast to mammals which are transiently recruited to the cytosolic side of the endosomal membrane for sorting of selected cargo to ILVs and for ILV formation. The ESCRT machinery is organized into four heteromeric protein complexes – ESCRT-0, ESCRT-I, ESCRT-II and ESCRT-III – that act in a sequence. Ubiquitinated cargo proteins are first recognized by the ubiquitin-binding proteins of the Hrs complex (Hrs, STAM and Eps15), known as ESCRT-0. Hrs recruits Tsg101 and its complex (ESCRT-I), which can also bind to ubiquitinated proteins. Tsg101 then recruits ESCRT-III via ESCRT-II or AIP1 (also known as Alix), and these complexes function together to sequester cargo proteins into the inward-budding vesicles of the MVB. ESCRT-III interacts with the AAAATPase Vps4, which is required for recycling of the ESCRT machinery (Fig. 1). Targeting the components of the ESCRT machinery, either by overexpression of dominant-negative
Fig. 2. The DN Vps4 overexpression in Rov cells retains PrPc at the limiting membrane of endosomes. (A) Immunolabeling with anti-PrP antibodies in Rov control cells revealed PrP at the cell surface as well as in punctuate structures inside the cells. (B – D) Rov cells that overexpress the DN Vps4 mutant tagged with the virus protein V5 were double labeled with an anti-PrP antibody and an anti-V5-FITC antibody. Expression of the DN Vps4 promoted an enlargement of perinuclear endosomes (C). PrPc accumulated at the limiting membrane of these endosomes (B), coinciding with Vps4 localization, as seen in the merged image (D).
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constructs or the use of small interfering RNA technology, could thus be used to interfere with protein sorting to exosomes and exosome formation (Fig. 1) [2]. It is not known however if the sorting of PrP into ILVs is facilitated by the ESCRT machinery as PrP is GPI-anchored, and it is not supposed to be monoubiquitinated prior to its association to the endosomal membrane. Some ILV cargo, such as Sna3p and Cvt17p in yeast [52], appear to bypass the requirement for direct ubiquitination and viruses such as human immunodeficiency virus (HIV)-1, and equine infectious anemia virus EIAV subvert the ILV pathway for budding by directly targeting downstream components such as ESCRT-I or Alix [53]. In mammalian cells, the delta opioid receptor (DOR), a G-protein-coupled receptor, is an example of non-ubiquitinated cargo that requires some components of the ESCRT machinery. Lysosomal degradation of DOR was significantly inhibited by the dominant-negative overexpression of Vps4, the AAA-family ATPase that participates at the late step of the ESCRT machinery process [54]. Sorting of DOR at the endosomal level appears also to be dependent on Hrs function. On the other hand, siRNA-mediated knockdown of Tsg101, a protein of the complex that functions at an intermediate step, does not affect the sorting of DOR, suggesting that the non-ubiquitinated receptor does utilize some components of the ESCRT machinery but not all. With the goal of interfering with PrP sorting at the level of MVBs and by consequence with its secretion via exosomes, we are currently investigating the requirements for components of the ESCRT machinery on PrPc sorting into ILVs. In Rov cells [55], overexpression of a dominantnegative (DN) Vps4 promoted an enlargement of endosomes and PrPc accumulated at the limiting membrane of these organelles (Fig. 2). Interestingly, in MOV cells [56], overexpression of Hrs disturbed intracellular lipid distribution as probed with the tracer phosphatidylethanolaminerhodamine and promoted accumulation of PrPc in caveolae and caveosomes (our unpublished observations). Thus, alteration of the function of the ESCRT machinery components appears to modify the intracellular trafficking of the GPI-linked PrPc and in particular its transfer to late endocytic MVBs. These observations will give us the opportunity to further analyze the implication of the endocytic pathway in PrPc transconformation and the role of exosomes in the trafficking of prions.
Acknowledgments This paper is based on a presentation at a Focused Workshop on ‘‘Exosomes: Biological Significance’’ sponsored by The Leukemia and Lymphoma Society in Montreal (19 –20 May 2005). Thanks to Rose Johnstone for her energy and motivation in organizing the Workshop. We thank Hubert Laude (INRA, Jouy-en-Josas, France) for stimulating discussions. Work in the author’s laboratories is supported
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by CNRS, Institut Curie, INRA and Ministe` re de la Recherche (GIS, infections a` prions). We thank Paul Bates, Univ. of Pennsylvania for providing the VPS4 constructs.
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Prions and exosomes: From PrPc trafficking to PrPsc propagation Isabel Porto-Carreiro a,*, Benoıˆt Fe´vrier a, Sophie Paquet b, Didier Vilette b, Grac¸a Raposo a a
b
Institut Curie, CNRS UMR 144, 75248 Paris, France Institut National de la Recherche Agronomique, Unite´ de Virologie Immunologie Mole´culaires, 78350 Jouy-en-Josas, France Submitted 20 June 2005 Available online 15 August 2005 (Communicated by M. Lichtman, M.D., 21 June 2005)
Abstract Exosomes are membrane vesicles released into the extracellular environment upon exocytic fusion of multivesicular endosomes with the cell surface. Exosome secretion can be used by cells to eject molecules targeted to intraluminal vesicles of multivesicular bodies, but particular cell types may exploit exosomes as intercellular communication devices for transfer of proteins and lipids among cells. The glycosylphosphatyidylinositol-linked prion protein (PrP) in both its normal (PrPc) and scrappie (PrPsc) conformation is associated with exosomes. Targeting of exosomes containing the normal cellular PrP could confer susceptibility of cells that do not express PrP to prion multiplication. Furthermore, exosomes bearing proteinase-K resistant PrPsc are infectious, suggesting a model in which exosomes secreted by infected cells could serve as vehicles for propagation of prions. Thus, cells may exploit the nature of endosome-derived exosomes to communicate with each other in normal and pathological situations, providing for a novel route of cell-to-cell communication and therefore of pathogen transmission. These findings open the possibility that methods to interfere with trafficking of such unconventional pathogens could be envisioned from insights on the mechanisms involved in exosome formation, secretion and targeting. D 2005 Elsevier Inc. All rights reserved. Keywords: Exosomes; Prions; Multivesicular bodies; ESCRT machinery; Glycosylphosphatidylinositol-linked proteins; Transmission
Introduction Endosomal multivesicular bodies (MVBs) in certain cell types fuse with the cell surface in an exocytic manner. During this process, the small 50– 90 nm vesicles contained in their lumen are released into the extracellular environment and are then called exosomes. Exosomes were shown to be secreted from hematopoietic (reticulocytes, B lymphocytes, dendritic cells, mast cells, T cells, platelets) and non-hematopoietic cells (intestinal epithelial cells, melanoma and mesothelioma cells) (reviewed in [1,2]). Exosomes released from these cells harbor functional molecules and can be targeted to other cells in which these molecules can elicit function. Exosomes released from B lymphocytes, dendritic cells (DCs), mast cells, tumor cells and intestinal epithelial cells can be targeted to T cells, bone-marrow-derived and splenic dendritic cells and can induce immunomodulatory functions for exosome* Corresponding author. E-mail address: [email protected] (I. Porto-Carreiro). 1079-9796/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bcmd.2005.06.013
associated antigens [3– 11]. Furthermore, vesicles with the hallmarks of exosomes are present, in vivo, at the surface of follicular dendritic cells (FDCs) in germinal centers [12], in malignant effusions [13], in broncho alveolar lavage [14], in urine [15] and in serum [16,17]. These and other studies support the idea that exosomes, in addition to their role in eradication of ‘‘unwanted’’ molecules [18,19], could provide for acellular vehicles to transfer molecules among cells in normal and pathological states [1,2,20]. Recent findings revealed an unexpected role for exosomes in the vehiculation of prions [21]. Prion diseases are invariably fatal neurodegenerative disorders that affect both humans and animals. They are associated with the conversion of the cellular prion protein (PrPc) into the scrappie PrP (PrPsc), an abnormal conformational state that tends to form amyloid deposits in brain tissue and that is thought to be infectious [22,23]. In the infectious forms of prion diseases, such as Kuru and variant Creutzfeldt – Jakob disease in humans, scrappie in sheep and bovine spongiform encephalopathy in cattle, the infectious agent
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enters the host through the gastrointestinal tract and then replicates in peripheral nerves and usually in lymphoid tissue before invading the central nervous system (CNS) [24,25]. There is increasing evidence that phagocytic mononuclear cells, including bone marrow, spleen-derived and follicular dendritic cells (FDCs), accumulate infectious prions and may play a key role in the transfer of prions from the gastrointestinal tract to the brain and therefore in the onset of disease [26,27]. However, the cellular mechanisms by which infectious prions are transferred from cell to cell are far from being fully unraveled. In agreement with previous studies, our observations provided evidence for a noncellular form of prions [28] corresponding to exosomal membranes bearing PrPsc which are infectious in vitro and in vivo [21]. These observations lead to the hypothesis that exosomes may constitute a potential vehicle for prions [21,29]. Here, we highlight the emerging link between the mode of propagation of this unconventional infectious agent and its intracellular trafficking through the endosomal system. Finally, we further discuss how insights from MVB biogenesis could contribute to design new approaches that interfere with PrP trafficking and secretion.
The prion protein and its trafficking The glycosylphosphatidylinositol (GPI)-anchored prion protein is ubiquitously expressed, although in higher levels, in neurons, some non-neuronal tissues and in cells from the immune system [22]. Its expression could be associated with a number of cell functions, including copper and/or zinc ion transport or metabolism; protection from oxidative stress; cellular signalling; membrane excitability and synaptic transmission; apoptosis and neurite outgrowth [30,31]. PrPc is synthesized in the endoplasmic reticulum, travels through the Golgi apparatus, before being transferred to the plasma membrane [32]. After synthesis, PrPc processing includes cleavage of the amino (N)-terminal signal peptide, addition of two N-linked oligosaccharide chains, formation of a disulfide bond and the insertion of its GPI anchor, responsible for its association with lipid-rafts at the plasma membrane. Once at the cell surface, PrPc can be constitutively internalized either by clathrin-coated vesicles or caveolae [32,33]. Studies on Chinese hamster ovary (CHO) cells which express caveolin-1 revealed that PrPc is clustered in caveolar-structures at the cell surface as well as in interconnected chains of endocytic caveolae inside the cytoplasm [34]. The choice of internalization pathway is not fully understood and may depend upon the cell type under study and the lipid microenvironment of the plasma membrane where PrPc is inserted. Furthermore, as proposed, PrP endocytosis via clathrin coated pits could be due to its association with a still unidentified accessory protein carrying signals for adaptor and clathrin recruitment [33]. Once internalized by either mechanism, PrPc has been
shown to transit through late endosomes and lysosomes [34,35], consistent with the steady state localization of a fraction of PrPc to endocytic MVBs in neurons in situ, in brain and in non-neuronal cell systems [21,34,36].
The infectious prion protein Based on the protein only hypothesis, the infectious agent is thought to be the PrPsc which arises by a conformational conversion of PrPc [22]. The transconformation implies a switch from a form rich in a-helices to another form where h-sheets predominate. The rate of this conversion can be dramatically increased by the presence of preformed PrPsc as a ‘‘seed’’. The seed acts as the infectious agent and recruits PrPc molecules into an oligomer that has all the characteristics of an amyloid fibril. Prion can then replicate the altered amyloid conformer through the generation of new protein seeds that can then be transmitted to other cells to nucleate further polymerization and thus propagate the infectious prion form [37]. Several intracellular locations have been proposed as potential sites for conversion of PrPc to PrPsc. Conversion could take place in the endoplasmic reticulum, in lipid-rafts at the cell surface, inside an endocytic organelle or even in the cytosol in the absence of membranes [32,36,38]. It is important to note, however, that the location where normal PrP is actually converted into PrPsc may not reflect the sites of PrPsc accumulation in infected cells. The characterization of the exact site of accumulation of PrPsc has been hampered by the lack of antibodies that recognize the specific conformation of PrPsc using electron microscopy. Nevertheless, visualization of PrPsc after denaturation by guanidinium treatment prior to antibody detection revealed late endosomes and lysosomes of infected neuronal cells as possible sites of accumulation [39 –42].
Dissemination of prions, a role for exosomes? In most natural infections, prions are introduced peripherally and have to find their way to the CNS to produce the disease. A number of cellular players have been identified. For instance, it is thought that prions are transferred from FDCs to peripheral nerve endings within lymphoid organs. A model requiring cell-to-cell contact for intercellular prion transfer would necessitate additional cell types to bridge the gap between FDCs and neurons. Alternatively, prions could be transmitted to the nerves by cell-free short-range diffusion mechanisms. So far, the molecular and cellular mechanisms by which prion-infected cells contaminate their neighbors remain unclear. Work from Kanu and colleagues suggested that cell-to-cell contacts can promote the infection of target cells adjacent to infected cells [43]. Other studies, in contrast, reported on the presence of prion infectivity in the cell culture medium of a prion-
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infected neuronal cell line [28]. Although the physical nature of the associated prion infectivity remained uncertain, this observation supports the hypothesis that prion transfer could also occur by a means other than direct cell contact. We found that both PrPsc and PrPc are present in cell culture supernatants in a secreted exosome-associated form. As observed by electron microscopy, PrP pelleted from the cell culture supernatants was associated with membrane vesicles similar in size and morphology to previously described exosomes. Moreover, the PrP detected by Western blot floated at an equilibrium density of ¨1.14 g/ml in sucrose density gradients, consistent with its association with membrane vesicles. Western blot and mass spectroscopy analyses of the purified vesicles showed that they also contained proteins previously shown to be present in exosomes from other cells [21]. Interestingly, exosomal membranes bearing PrPsc were infectious in vivo after
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intracerebral injection in mice. All the exosome inoculated mice died as a consequence of acute typical neurological disorders. In addition, exosome-associated PrPsc released by the infected cells elicited conversion of endogenous PrPc to PrPsc when incubated with uninfected recipient cells [21]. We recently visualized prion dissemination in cell cultures infected with ovine prions. Our results indicate that transmission of infection is an efficient active biological process that proceeds mainly step by step through the infection of adjacent cells (Paquet et al., submitted to publication). Thus, release of exosome-associated PrPsc by infected cells could provide, in addition to cell – cell contact, an acellular mechanism underlying the spread of prions. These findings allow us to propose a model where exosomes from infected cells could transfer prion to distant cells and also to cells in close contact to the infected cells. Consistent with the observations that cellular PrP can be transferred between
Fig. 1. The ESCRT machinery involved in protein sorting at the limiting membrane of endosomes to ILV. (A) Distribution of ESCRT complexes at the limiting membrane of MVB. (B) Molecular components of each ESCRT complex. The heteromeric protein complexes – ESCRT-0, ESCRT-I, ESCRT-II and ESCRT-III – are thought to be involved in protein sorting to the intraluminal vesicles of multivesicular bodies (MVB). The current model implies that ubiquitinated cargo proteins are first recognized by the ubiquitin-binding proteins of the Hrs complex (Hrs, STAM and Eps15, called ESCRT-0). Hrs recruits ESCRT-I by interacting with one of its protein—Tsg101, which is also a ubiquitin-binding protein. Tsg101 then recruits ESCRT-III via ESCRT-II or AIP1/Alix, and these complexes function together to position cargo proteins into the inward-budding vesicles of the MVB. ESCRT-III interacts with the AAA-ATPase Vps4, which dissociates the ESCRT machinery and releases the class E proteins for further rounds of sorting.
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cells in a regulated manner [44], targeting of exosomes containing the normal PrP could also confer susceptibility to prion multiplication in cells that do not express PrP. Exosome delivery could also be relevant to the transconformation process. First, the raft-like nature of exosomal membranes [45] may be a favorable environment for transconformation and amyloid fiber formation [46]. Second, it has been reported that conversion of PrPc into PrPsc is greatly facilitated when the cellular and the transconformed PrP proteins are in contiguous rather than in facing membranes [47]. Exchange of membranes can result to insertion of incoming PrPsc into the raft domains of recipient cells [47] and exosomes bearing PrPsc released from infected cells could undergo fusion with the plasma membrane of non-infected cells. Since conversion has been proposed to occur at a low pH [48,49], perhaps a more likely scenario would involve internalization of exosomes and fusion with endosomal limiting membranes, similar to the process of back fusion of the internal vesicles of MVBs reported to occur in DCs [20].
From ILV formation to exosome secretion The finding that exosomes may constitute a ‘‘bubbleride’’ for prions open the possibility that methods to interfere with prion trafficking and propagation could be designed from insights on the mechanisms involved in
exosome formation. Exosomes correspond to the ILVs of MVBs, and the molecular requirements for ILV generation constitute new targets to potentially regulate exosome formation [2,50]. The sorting of cargo into ILVs from MVBs is a tightly regulated process that depends on the function of a complex composed by at least 18 proteins. The ESCRT (Endosomal Sorting Complex Required for Transport) sorting machinery was firstly described in the yeast Saccharomyces cerevisiae as the class E ‘‘Vacuolar Protein Sorting’’ (Vps) proteins (reviewed in [51]). The ESCRT machinery is constituted by a group of cytosolic wellconserved proteins from yeast to mammals which are transiently recruited to the cytosolic side of the endosomal membrane for sorting of selected cargo to ILVs and for ILV formation. The ESCRT machinery is organized into four heteromeric protein complexes – ESCRT-0, ESCRT-I, ESCRT-II and ESCRT-III – that act in a sequence. Ubiquitinated cargo proteins are first recognized by the ubiquitin-binding proteins of the Hrs complex (Hrs, STAM and Eps15), known as ESCRT-0. Hrs recruits Tsg101 and its complex (ESCRT-I), which can also bind to ubiquitinated proteins. Tsg101 then recruits ESCRT-III via ESCRT-II or AIP1 (also known as Alix), and these complexes function together to sequester cargo proteins into the inward-budding vesicles of the MVB. ESCRT-III interacts with the AAAATPase Vps4, which is required for recycling of the ESCRT machinery (Fig. 1). Targeting the components of the ESCRT machinery, either by overexpression of dominant-negative
Fig. 2. The DN Vps4 overexpression in Rov cells retains PrPc at the limiting membrane of endosomes. (A) Immunolabeling with anti-PrP antibodies in Rov control cells revealed PrP at the cell surface as well as in punctuate structures inside the cells. (B – D) Rov cells that overexpress the DN Vps4 mutant tagged with the virus protein V5 were double labeled with an anti-PrP antibody and an anti-V5-FITC antibody. Expression of the DN Vps4 promoted an enlargement of perinuclear endosomes (C). PrPc accumulated at the limiting membrane of these endosomes (B), coinciding with Vps4 localization, as seen in the merged image (D).
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constructs or the use of small interfering RNA technology, could thus be used to interfere with protein sorting to exosomes and exosome formation (Fig. 1) [2]. It is not known however if the sorting of PrP into ILVs is facilitated by the ESCRT machinery as PrP is GPI-anchored, and it is not supposed to be monoubiquitinated prior to its association to the endosomal membrane. Some ILV cargo, such as Sna3p and Cvt17p in yeast [52], appear to bypass the requirement for direct ubiquitination and viruses such as human immunodeficiency virus (HIV)-1, and equine infectious anemia virus EIAV subvert the ILV pathway for budding by directly targeting downstream components such as ESCRT-I or Alix [53]. In mammalian cells, the delta opioid receptor (DOR), a G-protein-coupled receptor, is an example of non-ubiquitinated cargo that requires some components of the ESCRT machinery. Lysosomal degradation of DOR was significantly inhibited by the dominant-negative overexpression of Vps4, the AAA-family ATPase that participates at the late step of the ESCRT machinery process [54]. Sorting of DOR at the endosomal level appears also to be dependent on Hrs function. On the other hand, siRNA-mediated knockdown of Tsg101, a protein of the complex that functions at an intermediate step, does not affect the sorting of DOR, suggesting that the non-ubiquitinated receptor does utilize some components of the ESCRT machinery but not all. With the goal of interfering with PrP sorting at the level of MVBs and by consequence with its secretion via exosomes, we are currently investigating the requirements for components of the ESCRT machinery on PrPc sorting into ILVs. In Rov cells [55], overexpression of a dominantnegative (DN) Vps4 promoted an enlargement of endosomes and PrPc accumulated at the limiting membrane of these organelles (Fig. 2). Interestingly, in MOV cells [56], overexpression of Hrs disturbed intracellular lipid distribution as probed with the tracer phosphatidylethanolaminerhodamine and promoted accumulation of PrPc in caveolae and caveosomes (our unpublished observations). Thus, alteration of the function of the ESCRT machinery components appears to modify the intracellular trafficking of the GPI-linked PrPc and in particular its transfer to late endocytic MVBs. These observations will give us the opportunity to further analyze the implication of the endocytic pathway in PrPc transconformation and the role of exosomes in the trafficking of prions.
Acknowledgments This paper is based on a presentation at a Focused Workshop on ‘‘Exosomes: Biological Significance’’ sponsored by The Leukemia and Lymphoma Society in Montreal (19 –20 May 2005). Thanks to Rose Johnstone for her energy and motivation in organizing the Workshop. We thank Hubert Laude (INRA, Jouy-en-Josas, France) for stimulating discussions. Work in the author’s laboratories is supported
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by CNRS, Institut Curie, INRA and Ministe` re de la Recherche (GIS, infections a` prions). We thank Paul Bates, Univ. of Pennsylvania for providing the VPS4 constructs.
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