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Exosomes as a potential novel therapeutic tools against neurodegenerative diseases
Accepted Manuscript Title: Exosomes as a potential novel therapeutic tools against neurodegenerative diseases Author: Akvil˙e Jarmalaviˇci¯ ut˙e Augustas Pivori¯ unas PII: DOI: Reference:
S1043-6618(16)30021-4 http://dx.doi.org/doi:10.1016/j.phrs.2016.02.002 YPHRS 3061
To appear in:
Pharmacological Research
Received date: Revised date: Accepted date:
11-1-2016 1-2-2016 1-2-2016
Please cite this article as: Jarmalaviˇci¯ ut˙e Akvil˙e, Pivori¯ unas Augustas.Exosomes as a potential novel therapeutic tools against neurodegenerative diseases.Pharmacological Research http://dx.doi.org/10.1016/j.phrs.2016.02.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Exosomes as a potential novel therapeutic tools against neurodegenerative diseases
Akvilė Jarmalavičiūtė1, Augustas Pivoriūnas1* 1
Department of Stem Cell Biology, State Research Institute Centre for Innovative Medicine,
LT-08406, Vilnius, Lithuania.
* Corresponding author: Augustas Pivoriūnas, Department of Stem Cell Biology, State Research Institute Centre for Innovative Medicine, Santariškių 5, LT-08406 Vilnius, Lithuania; Tel: +370 5 2628413; Fax: +370 5 2123073; E-mail: [email protected]
Graphical abstract: Multimodal neuroprotective effects of exosomes. Exosomes may exert pleiotropic effects in CNS including support of oligodendritic precursor cells, protection of different types of neurons and also suppression of neuroinflammatory responses by microglial cells.
Abstract Exosomes are extracellular vesicles that can transfer biological information over long distances affecting normal and pathological processes throughout organism. It is known that very often composition and therapeutic properties of exosomes depends on cell type and its physiological state. Thus, depending on tissue of origin and physiological context exosomes may act as promoters, or suppressors of pathological processes in CNS. From the therapeutic perspective, the most promising cellular sources of exosomes are mesenchymal stem cells, dendritic cells and inducible pluripotent stem cells. In this review, we will summarize the current state of knowledge on the molecular mechanisms underlying neuroprotective actions of exosomes derived from these cells. New therapies for the neurodegenerative disorders are often halted by the inability of drugs to cross blood–brain barrier. In this respect exosomes have a critical advantage, because they can cross blood–brain barrier. Despite the great importance, surprisingly little is known about mechanistic details of this process. Therefore we will discuss some recent findings that may explain mechanisms of exosomal entry into the brain. Keywords: Exosomes; Neurodegeneration; Neuroprotection; Blood-brain barrier.
1. Introduction
The development of new potential therapies against neurodegenerative diseases represents a crucial prerequisite for improving and extending quality of life among older people. These challenges require novel approaches, because current treatments are often symptomatic and do not stop or slow underlying neurodegenerative processes. Stem cell-based therapeutic strategies for neurological disorders have attracted a great deal of interest during the last decade, but clinically useful therapies are still not available for most patients [1]. The main reason for this is insufficient understanding about the process of engraftment and subsequent integration into targeted brain circuits of transplanted human cells [2]. In this regard, use of therapeutic factors produced by stem cells instead of classical transplantation have several advantages. First, it does not require direct use of cells, thereby avoiding the limitations and risks associated to cell transplantation. Second, this approach allows more precise control over the entire process during cellular therapy product preparation and therefore is more suitable for the large scale clinical manufacturing. The study of intercellular communication via extracellular vesicles (EVs) represent one of the most rapidly emerging fields of modern cell biology. Recently, several excellent comprehensive reviews have been published [3-5], thus, here we will give only brief introduction into the topic. According
to the origin and size EVs could be classified as exosomes, microvesicles and apoptotic bodies. Exosomes are 30-100 nm in diameter and originate from endosomal compartments, known as multi-vesicular bodies (MVBs), while microvesicles are larger particles (50-1000 nm) produced by budding from the plasma membrane. Apoptotic bodies are generated from cells undergoing programmed cell death, they are 800-5000 nm in diameter and contain extensive amounts of phosphatidylserine, DNA, histones and ribosomal RNA. It is now widely accepted that both exosomes and microvesicles play important roles in intercellular communication via the transfer of membrane receptors, proteins, lipids, RNA and miRNA between cells. Thus, EVs can transfer biological information over long distances affecting normal and pathological processes throughout organism. In this review we will focus mainly on the neuroprotective properties of exosomes. Importantly, the composition of exosomes depends on cell type and its physiological state. Accordingly, exosomes derived from diseased tissues may be important for the maintenance and spread of pathological processes. Indeed, recent studies demonstrated that exosomes are involved in the propagation of several neurodegenerative diseases [6,7]. Another striking example comes from the recent finding that tumour-secreted exosomes specifically promote organotropic metastasis and prepare pre-metastatic niches in target tissues [8]. Thus, targeting formation, release, or uptake of disease-promoting exosomes represent promising therapeutic strategy. On the other hand, exosomes derived from healthy cells may have a comparable therapeutic potential as cells themselves. From this perspective most promising cellular sources are human mesenchymal stem cells (MSCs), dendritic cells (DCs) and human inducible pluripotent stem cells (iPS). In this review, we will summarize the current state of knowledge on the molecular mechanisms underlying neuroprotective actions of exosomes derived from these cells. The inability of most drugs to cross the blood–brain barrier (BBB) represent major problem of modern neuropharmacology. In this respect exosomes have a crucial advantage, because they can cross BBB. Moreover, exosomes might be engineered to target neurons or even specific neuronal populations [9]. These new developments open possibilities for intravenous, or even intranasal delivery methods avoiding the need for neurosurgical intervention. Despite the apparent ability of exosomes to cross BBB, surprisingly little is known about the mechanistic details of this process. However, several very recent studies coming from the cancer biology field have shed new light on the potential mechanisms by which exosomes may enter into the CNS. We will discuss these findings and also propose hypothetical model on the mechanism by which exosomes can cross BBB.
2. Identifying the best cellular source 2.1. Mesenchymal stem cells According to the traditional definition, MSCs have the potential to differentiate into mesenchymal tissues including osteoblasts, adipocytes and chondocytes [10]. MSC-like cells with similar characteristics have been isolated from virtually all vascularized organs and tissues [11]. These cells can be relatively easy isolated and expanded in vitro, therefore they became very popular among many researchers. However, a lot of controversies and misconceptions regarding the true nature and functions of MSCs still exist in the field, even the use of term „mesenchymal stem cell“ has been challenged repeatedly [12]. There are several key points to consider about MSCs. We think, that these considerations also should be taken into account during evaluation of potential therapeutic properties of MSC-secreted exosomes. First, it is essential to make a clear distinction between in vitro cultures of MSCs and their in vivo counterparts. The vast majority of information about MSCs comes from the in vitro models which not necessarily correctly reflect situation in vivo [13]. Second, MSC cultures comprise functionally different heterogeneous subpopulations. For example, only a relatively small fraction of freshly isolated adherent cells from bone marrow (BM) are colony forming unit-fibroblasts (CFU-Fs) and only a part of clonal progenitors derived from these individual CFU-Fs formed bone when transplanted in vivo [14]. This assumes, that cellular heterogeneity of MSCs is also reflected in cargo content and functional properties of secreted exosomes. Third, MSC-like cells isolated from different tissues are not equivalent and display distinct tissue-specific differentiation capacities. For instance, ectopic transplantation of BM-derived MSCs produced heterotopic bone/marrow organ structures, whereas dental pulp-derived MSCs gave dentin and pulp tissue [15]. Thus, MSC-like cells from different tissues demonstrate distinct functional traits, implying they also produce exosomes with different properties. Here we will summarize current data about neuroprotective properties of exosomes produced by MSCs derived from different tissues. For a long time it was thought, that MSCs isolated from dental pulp, also known as dental pulp stem cells (DPSCs), or stem cells derived from the dental pulp of human exfoliated deciduous teeth (SHEDs) originate from the cranial neural crest cells which are precursors of both neural and skeletal tissues [16]. However, recent study demonstrated that DPSCs originate from peripheral nerve-associated glia [17]. Thus, in contrast to MSC-like cells from other mesodermal tissues (bone marrow, adipose tissue), DPSCs could be particularly suitable for the induction of neural differentiation and also for the neuroregenerative therapies. Indeed, several studies have demonstrated, that DPSCs can be differentiated into neurons in vitro [18-20]. Using human DPSCs and SHEDs for the transplantation into the completely transected adult rat spinal cord [21], the
transplantation resulted in marked recovery of hind limb locomotor functions. Importantly, transplantation of BM-MSCs or skin-derived fibroblasts gave substantially less recovery of the locomotor function. Three major mechanisms of neuroregeneration were identified and one of them was associated with paracrine mechanisms [21]. The importance of paracrine DPSC signalling for the neuroregeneration has been known for relatively long time. For example, DPSCs produced neurotrophic factors, interacted with trigeminal neurons in vitro, and rescued motoneurons after spinal cord injury [22]. The same group showed, that paracrine factors produced by DPSCs protected dopaminergic neurons against 6-hydroxy-dopamine (6-OHDA) in vitro [23]. Recent study demonstrated that dopaminergic neuron-like cells could be efficiently induced from SHEDs and that these cells had therapeutic benefits in a 6-OHDA-induced Parkinsonian rat model [24]. Importantly, paracrine effects of differentiated SHEDs contributed to neuroprotection against 6-OHDA-induced neurodegeneration and to nigrostriatal tract restoration [24]. These studies demonstrated that DPSCs and SHEDs have unique neurogenic properties which could be potentially exploited for therapeutic use. We asked, whether exosomes and microvesicles derived from SHEDs display neuroprotective properties during 6-OHDA-induced oxidative stress in human dopaminergic neurons. We found that exosomes, derived from the microcarrier cultures of SHEDs suppressed 6-OHDA-induced apoptosis in dopaminergic neurons [25]. Importantly, exosomes derived from SHEDs grown under standard conditions did not suppress apoptosis indicating that culture conditions had a profound influence on functional properties of the exosomes. Our preliminary data from comparative proteomic and RNAomic analyses (unpublished) revealed that bioreactor culture significantly affected cargo content of exosomes. Future studies will identify unique proteins and (or) microRNAs responsible for the neuroprotective properties of exosomes. What is a possible mechanism for neuroprotective action of SHED-derived exosomes ? Since 6-OHDA triggers apoptosis mostly through production of reactive oxygen species (ROS) in mitochondria [26], we speculate that exosomes may reduce sensitivity of dopaminergic neurons to the 6-OHDA-induced oxidative stress. Indeed, several recent studies using exosomes derived from MSCs, or other types of cells, demonstrated that they may provide protection against oxidative stress. For example, human umbilical cord (UC) MSC-derived exosomes reduced cisplatin-mediated renal oxidative stress and apoptosis in vivo [27]. Another study demonstrated, that chaperone αB crystallin is secreted within exosomes by human retinal pigment epithelium and provide protection from oxidative stress in adjacent retinal cells [28]. Exosomes secreted by mouse macrophages and ex vivo loaded with potent antioxidant catalase significantly increased viability of 6-OHDA-pre-treated PC12 cells [29]. Importantly, the same study demonstrated that catalase-loaded and unloaded exosomes decreased ROS levels in activated macrophages, suggesting they may act via similar
mechanisms in microglial cells during neuroinflammation. Furthermore, in vivo experiments confirmed that catalase-loaded exosomes reduced microgliosis and increased survival of dopaminergic neurons in 6-OHDA-treated mice [29]. Thus, protection against oxidative stress may represent one of the major neuroprotective mechanisms of exosomes. Adipose tissue (AT) represents an abundant and easily accessible source of MSC-like cells, therefore it is suitable for large scale clinical production of exosomes. Several studies addressed neurotherapeutic effects of exosomes derived from AT-MSCs. Interestingly, AT-MSCs secrete functional neprilysin-bound exosomes [30]. Neprilysin (also known as CD10) is a type II membrane associated metalloendopeptidase important for the the proteolysis of β-amyloid peptide (Aβ), its activity and expression is decreased during Alzheimer’s disease (AD) [31]. AT-MSCs-derived exosomes demonstrated neprilysin-specific enzyme activity and contributed to decrease of both secreted and intracellular Aβ levels in N2a neuroblastoma cells genetically modified to overproduce human Aβ. Importantly, exosomes from AT-MSCs expressed significantly higher levels of neprilysin than BM-MSCs, highlighting the differences between functional properties of exosomes derived from different tissues [30]. In this regard, it would be interesting to investigate in vivo therapeutic potential of exosomes derived from AT-MSCs using animal models of AD. Another recent study demonstrated that exosomes and microvesicles derived from the murine AT-MSCs rescued human neuroblastoma cells SH-SY5Y and primary murine hippocampal neurons exposed to oxidative damage with H2O2 [32]. Interestingly, both exosomes and microvesicles demonstrated an inverse dose-dependent effect on cell viability. Furthermore, both exosomes and microvesicles derived from murine AT-MSCs increased the process of remyelination and activated nestin-positive oligodendroglial progenitors in cerebellar slice cultures demyelinated with lysophosphatidylcholine [32]. Very recently, the same group presented evidence for neuroprotective effects of exosomes derived from murine AT-MSCs using in vitro model of amyotrophic lateral sclerosis [33]. For this purpose they used motoneuron-like cell line NSC-34 overexpressing different mutants of human superoxide dismutase 1 (SOD1) and exposed to oxidative stress with H2O2. Exosomes were able to protect NSC-34 cells from oxidative damage showing potential for future therapeutic applications in motor neuron disease [33]. To date, BM represents the most common source of MSCs. Several studies demonstrated therapeutic potential of exosomes derived from BM-MSCs in different clinical settings. For example, MSC-derived exosomes have been successfully used for treatment of therapy-refractory graft versus host disease [34]. Another study demonstrated that human MSCs and MSC-derived exosomes very similarly improved post-stroke neuroregeneration in C57BL6 mice [35]. Interestingly, exosomes also modulated peripheral post-stroke immune responses by attenuating
post ischemic immunosuppression [35]. Exosomes derived from rat BM-MSCs promoted functional recovery and neurovascular plasticity after stroke [36] and traumatic brain injury [37] in rats. One mechanism contributing to promotion of brain remodeling during stroke recovery may be related to exosomal miR-133b. It has been demonstrated, that administration of MSCs restored downregulated expression of miR-133b in ischemic brain tissues. Furthermore, ischemic conditions increased miR133b levels in MSC-secreted exosomes and promoted neuron outgrowth in vitro via downregulated expression of RhoA protein, which is known suppressor of neurite outgrowth [38]. Interestingly, miR-133b is specifically expressed in midbrain dopaminergic neurons and is deficient in midbrain tissue from patients with Parkinson's disease [39]. This can be exploited for future therapeutic strategies using MSC-derived exosomes. Human UC tissue represents another source of MSCs and several recent studies demonstrated therapeutic potential of UC-MSC-derived exosomes in several experimental models [27,40]. However, there are no data in literature about neuroprotective properties of UC-MSCs. In conclusion, MSCs represent the most promising source of exosomes for the neurotherapeutic applications. However, there are issues to be resolved. First, systematic and comprehensive studies are required to compare proteomic and RNAomic profiles of exosomes produced by MSC-like cells derived from different tissues and grown under standard conditions. In our opinion, these should be free of xenogeneic supplements (for example, medium supplemented with platelet-rich plasma (PRP), or serum-free medium of known formulation [41]). In addition, MSCs polarized into proinflammatory and anti-inflammatory phenotypes [42] should be included into these studies. Second, we need systematic comparison of therapeutic properties of exosomes produced by MSCs derived from different tissues. At the first stage these studies could employ in vitro experimental models of neurodegeneration (for example, ReNcell VM immortalized neural stem cells differentiated into human dopaminergic neurons) for functional testing of different exosomal preparations, then extended by using in vivo experimental models. We believe that such an approach will facilitate selection for optimal source of exosomes targeting specific neurodegenerative diseases.
2.2. Dendritic cells DCs represent another promising source of exosomes suitable for neurotherapeutic applications. DC-derived exosomes participate in antigen presentation and also as regulators of innate and adaptive immunity. They can carry MHC I and (or) II-peptide complexes that can be captured and presented by other DCs to activate naive T cells, alternatively, these exosomes can be directly recognized by primed effector T cells [43]. Importantly, maturation status of DCs determines production and functional properties of exosomes. For example, immature DCs produced greater
amounts of exosomes, but these were less potent inducers of antigen-specific effector immune responses. By contrast, exosomes from mature DCs carry MHC II and co-stimulatory molecules and can directly activate T cells [44]. Another study demonstrated, that DCs at different stages of maturation release exosomes with different miRNA content [45]. Interestingly, 139 miRNAs were detected in both types of exosomes, 5 only in immature vesicles and 58 exclusively in mature exosomes [45]. DCs also can capture exosomes derived from tumour cells and then activate populations of tumour-specific cytotoxic T lymphocytes [46]. These findings have led to the development of novel cancer immunotherapy approach currently being tested in numerous clinical trials [47]. A recent study demonstrated, that DC cultures stimulated with low-level IFNγ released exosomes that increased myelination, reduced oxidative stress and improved remyelination following acute lysolecithin-induced demyelination in hippocampal slice cultures [48]. Furthermore, nasal administration of exosomes derived from IFNγ-stimulated DCs increased CNS myelination in vivo [48]. IFNγ-stimulated DCs produced exosomes containing a high levels of miRNAs known to be involved in oligodendrocyte differentiation and anti-inflammatory pathways [48]. Among them, miR-219 plays an essential role during differentiation of oligodendritic precursor cells and improvement of remyelination [49]. Further functional studies confirmed downregulation of several miR-219 targets in slice cultures [48]. Interestingly, the same group demonstrated that exosomes derived from serums of young, or environmentally enriched old rats contained high levels of miR-219 [50]. Moreover, nasal delivery of the exosomes isolated from serums of young rats enhanced remyelination in old rats, demonstrating that DC-derived exosomes may be a useful therapy for remyelination during multiple sclerosis or other dysmyelinating disorders [50]. Thus, despite the fact that only very few studies are available so far, DCs have a great potential as a therapeutic source of exosomes for neurodegenerative diseases. Again, comprehensive proteomic and RNAomic profiling and functional evaluation of exosomes using standardized conditions is needed to ensure reproducibility and therapeutic efficacy.
2.3. Human inducible pluripotent stem cells Human inducible pluripotent stem cells (iPS) as a source of exosomes for neurotherapeutic applications have several important advantages over DCs and MSCs. First of all, current technologies enable efficient in vitro generation of different types of human neurons and glial cells and use them as producers of exosomes targeting specific pathological processes in the brain. These cells can be cultivated in vitro for a long periods of time allowing collection of large quantities of exosomes. Another important advantage is that iPS cells can be derived from patient tissues minimizing risks of immune rejection. Two recent studies demonstrated potential of iPS-derived
exosomes for the treatment of ischemic diseases [51,52]. Undoubtedly, future research will address neuroprotective properties of iPS-derived exosomes.
3. Exosomes as suppressors of neuroinflammation Increasing evidence suggests that chronic neuroinflammation has a causal role in pathogenesis of neurodegenerative diseases, therefore, new therapies directed against neuroinflammatory processes may be beneficial [53]. Several recent reports demonstrated, that exosomes display antineuroinflammatory properties in CNS. For instance, exosome-encapsulated curcumin inhibited LPS-induced brain inflammation and myelin oligodendrocyte glycoprotein induced autoimmune responses in experimental autoimmune encephalitis model [54]. Importantly, intranasally injected exosomes were rapidly transported to the brains of mice and were taken up by microglial cells. Moreover, exosomes significantly reduced numbers of activated inflammatory microglial cells [54]. Interestingly, this study also demonstrate that exosomes were taken up by both activated and resting microglial cells. The pathophysiological importance of this finding remains to be established. As we have already mentioned, exosomes derived from DCs suppressed neuroinflammatory responses in vitro [48] and in vivo [29]. Thus, there is a growing body of evidence that exosomes may exert pleiotropic effects in CNS including support of oligodendritic precursor cells, protection of different types of neurons and also suppression of neuroinflammatory responses by microglial cells.
4. Exosomes can cross blood-brain barrier: how do they do it ? The BBB represents dynamic physiological barrier between the brain and circulating blood that regulates local brain microenvironment necessary for proper neuronal function and is formed by the continuous layer of specialized brain capillary endothelial cells. These endothelial cells are surrounded by basement membrane, pericytes and astrocytes forming neurovascular unit (NVU). Astrocytes play an essential role in differentiation and support of brain capillary endothelial cells, while pericytes are important integrators of endothelial and astrocyte functions at the NVU. Studies with pericyte-deficient mouse mutants revealed increased permeability of BBB, which occurred through increased transcytosis in endothelial cells [55]. The BBB restricts diffusion of hydrophilic or most large (> 400 Da) molecules into the CNS [56]. It is now established that four fundamental molecular properties of endothelial cells contribute to BBB integrity and functions. First, tight junctions (TJ) between endothelial cells prevent paracellular flux of small hydrophilic molecules and ions. Second, brain endothelial cells display low rates of transcytosis. Third, selective influx and efflux transporters allow uptake of nutrients and molecules from blood and elimination of toxins from the CNS. Finally, endothelial cells express low levels of leukocyte adhesion molecules,
limiting entry of immune cells into the brain parenchyma and creating immunoprivileged environment [57]. What could be the mechanism of exosomal entry into the CNS ? In principle, there are two possible ways of entry. First, it has been proposed that exosomes may be internalized by endothelial cells, undergo transcytosis, then released again to be re-internalized by other recipient cells [58]. Thus, according to this model, exosomes can cross tissue by ,,jumping‘‘ from one cell to another operating in a similar manner to some viruses. However, currently there is no experimental evidence to support this model. Alternatively, exosomes may enter into CNS via intercellular junctions of endothelial cells. Despite the current lack of direct experimental proof that exosomes can enter into the CNS paracellulary, numerous recent studies demonstrate, that exosomes increase permeability of vascular barriers throughout the organism. Hence, recent study demonstrated, that metastatic breast cancer-secreted exosomes destroys vascular endothelial barriers to promote metastasis [59]. Exosome-associated miR-105 significantly downregulated expression of ZO-1, a central molecular component of TJs, destroying barrier function in endothelial monolayers [59]. In vivo studies demonstrated that exosomes with high content of miR-105 significantly increased miR105 levels in lung and brain, accompanied by reduced ZO-1 expression and also enhanced vascular permeability in lungs, liver and brain. Furthermore, in vivo treatment with the anti-miR-105 compound reduced the volume of primary tumours, suppressed distant metastases to the lung and brain and restored vascular integrity in tumour-bearing animals [59]. More recently, it has been demonstrated, that breast cancer-secreted exosomes can trigger breakdown of BBB by yet another mechanism [60]. Exosomal miR-181c downregulated expression of 3-phosphoinositide-dependent protein kinase-1 (PDPK1), leading to decreased levels of phosphorylated cofilin and abnormal polymerization of actin in brain endothelial cells [60]. These findings demonstrate, that cancersecreted exosomes may employ different mechanisms to increase permeability of BBB and other vascular barriers. Most likely, other miRNAs and yet unidentified proteins and possibly lipids are also important mediators of exosome-dependent breakdown of BBB. Very recently another important study revealed mechanism by which cancer exosomes direct metastatic organotropism and prepare the pre-metastatic niches in target tissues [8]. This report demonstrated, that distinct expression patterns of exosomal integrins were associated with organotropic metastasis. Strikingly, treatment with exosomes from lung-tropic cancer cells redirected the metastasis of bone-tropic tumour cells [8]. Importantly, exosomes promoted vascular leakiness, before exosome uptake by specific lung cells suggesting that exosomes first permeabilize vessels, allowing for exosome entry into the target tissue. Several other pathologies have also been associated with exosome-dependent increase of vascular permeability. Recent findings demonstrated, that exosomes from patients with
cardiovascular disease or from endothelial cells exposed to stressful conditions can disrupt endothelial TJs. For instance, plasma-derived exosomes from obese children with obstructive sleep apnea altered endothelial TJs [61]. In addition, some viruses are using highly sophisticated mechanisms to trigger breakdown of BBB. It has been demonstrated, that human immunodeficiency virus type 1 (HIV-1)-infected microglia and astrocytes produced viral proteins including gp120, Tat, and Nef affecting the integrity of the BBB [62]. Interestingly, Nef-transfected, or HIV-infected microglia released Nef protein in exosomes. Studies using in vitro model of BBB revealed, that these Nef-positive microglial exosomes significantly reduced expression of the TJ protein ZO-1, suggesting a mechanism for exosomal Nef-mediated neuropathogenesis [63]. Of note, exosomes
and HIV-1 display very similar biophysical and molecular properties. In addition, HIV-1 and exosomes utilize the same biogenesis pathway [64]. These similarities led to the Trojan exosome hypothesis which states that retroviruses exploit preexisting pathways of exosome biogenesis and uptake [65]. All these examples demonstrate, that exosomes secreted by diseased cells can affect vascular permeability and also in some cases integrity of BBB. But what about exosomes derived from healthy cells ? Several studies convincingly demonstrated, that exosomes from different types of cells can effectively cross BBB [9,66]. However, it is presently unclear, whether they employ similar mechanisms as exosomes from diseased tissues. Interestingly, our in vivo imaging experiments revealed, that exosomes derived from human dental pulp stem cells (DPSCs) increased vascular permeability at the intitial phases of carrageenan-induced acute inflammation in BALB/c mice [67]. Despite of that, exosomes reduced the carrageenan-induced edema to similar levels seen with the positive control (prednisolone), showing potent anti-inflammatory properties [67]. Therefore, it is possible, that as in the case of cancer exosomes, the first step of entry into the tissue might be related with permeabilization of vascular endothelial barriers. Accordingly, we suggest that exosomes cross BBB via two-stage process (Fig. 1). During the first stage exosomes are internalized by brain capillary endothelial cells and release specific cargo components affecting integrity of intercellular junctions. Thus, this ,,first wave‘‘ of exosomes increase intercellular permeability of endothelial cells allowing a massive entry of exosomes into the CNS during the second stage of the process. If correct, this model might be helpful for the establishment of new and more effective protocols of exosome administration. For example, initial pretreatment with exosomes may be followed by a ,,window‘‘ allowing maximal permeabilization of endothelial barriers of BBB and then by the next ,,therapeutic‘‘ dose of exosomes.
It should be noted, however, that various neurological disorders are associated with increased permeability of BBB, therefore more experiments are needed to evaluate and understand possible adverse effects related to therapeutic administration of exosomes.
5. Conclusions and future perspectives Accumulating evidence suggests that exosomes derived from different types of cells can be successfully used for the treatment of various neurodegenerative disorders. However, there are several problems that need to be solved before their widespread use in the clinical setting. First of all, investigators often use different methods of isolation, characterization and quantification of exosomes in their studies. For example, very often researchers quantify exosomes using standard protein quantification methods, others perform nanoparticle tracking analysis, or use empirical activity units reflecting yield of exosomes prepared from supernatants of cells grown under strictly defined conditions. Thus, it is increasingly difficult to compare and contrast study outcomes hindering progress in the field. It is also important to perform a comprehensive proteomic and RNAomic profiling and functional evaluation of exosomes using standardized conditions to ensure reproducibility and therapeutic efficacy. Finally, exosomes are complex systems containing a great number of molecular constituents raising multiple safety and predictability issues. Therefore, it seems likely, that future studies will focus on engineering of exosome mimetic delivery systems targeting specific cells and containing only desired therapeutic molecules.
Acknowledgements This work was supported by National Research Programme ,,Healthy ageing’’(Grant Nr. SEN15090) from Research Council of Lithuania.
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Figure legends Figure 1. The proposed mechanism by which exosomes cross blood-brain barrier. During the first stage exosomes are internalized by brain capillary endothelial cells, release specific cargo components and affect integrity of intercellular junctions via different mechanisms. A- Exosomal miR-105 downregulates expression of tight junction protein ZO1 [59]; B-Exosomal miR-181c induces abnormal polymerization of actin in brain endothelial cells [60]. Increased intercellular permeability of endothelial cells allows a massive entry of exosomes into the CNS during the second phase of the process.
S1043-6618(16)30021-4 http://dx.doi.org/doi:10.1016/j.phrs.2016.02.002 YPHRS 3061
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Pharmacological Research
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11-1-2016 1-2-2016 1-2-2016
Please cite this article as: Jarmalaviˇci¯ ut˙e Akvil˙e, Pivori¯ unas Augustas.Exosomes as a potential novel therapeutic tools against neurodegenerative diseases.Pharmacological Research http://dx.doi.org/10.1016/j.phrs.2016.02.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Exosomes as a potential novel therapeutic tools against neurodegenerative diseases
Akvilė Jarmalavičiūtė1, Augustas Pivoriūnas1* 1
Department of Stem Cell Biology, State Research Institute Centre for Innovative Medicine,
LT-08406, Vilnius, Lithuania.
* Corresponding author: Augustas Pivoriūnas, Department of Stem Cell Biology, State Research Institute Centre for Innovative Medicine, Santariškių 5, LT-08406 Vilnius, Lithuania; Tel: +370 5 2628413; Fax: +370 5 2123073; E-mail: [email protected]
Graphical abstract: Multimodal neuroprotective effects of exosomes. Exosomes may exert pleiotropic effects in CNS including support of oligodendritic precursor cells, protection of different types of neurons and also suppression of neuroinflammatory responses by microglial cells.
Abstract Exosomes are extracellular vesicles that can transfer biological information over long distances affecting normal and pathological processes throughout organism. It is known that very often composition and therapeutic properties of exosomes depends on cell type and its physiological state. Thus, depending on tissue of origin and physiological context exosomes may act as promoters, or suppressors of pathological processes in CNS. From the therapeutic perspective, the most promising cellular sources of exosomes are mesenchymal stem cells, dendritic cells and inducible pluripotent stem cells. In this review, we will summarize the current state of knowledge on the molecular mechanisms underlying neuroprotective actions of exosomes derived from these cells. New therapies for the neurodegenerative disorders are often halted by the inability of drugs to cross blood–brain barrier. In this respect exosomes have a critical advantage, because they can cross blood–brain barrier. Despite the great importance, surprisingly little is known about mechanistic details of this process. Therefore we will discuss some recent findings that may explain mechanisms of exosomal entry into the brain. Keywords: Exosomes; Neurodegeneration; Neuroprotection; Blood-brain barrier.
1. Introduction
The development of new potential therapies against neurodegenerative diseases represents a crucial prerequisite for improving and extending quality of life among older people. These challenges require novel approaches, because current treatments are often symptomatic and do not stop or slow underlying neurodegenerative processes. Stem cell-based therapeutic strategies for neurological disorders have attracted a great deal of interest during the last decade, but clinically useful therapies are still not available for most patients [1]. The main reason for this is insufficient understanding about the process of engraftment and subsequent integration into targeted brain circuits of transplanted human cells [2]. In this regard, use of therapeutic factors produced by stem cells instead of classical transplantation have several advantages. First, it does not require direct use of cells, thereby avoiding the limitations and risks associated to cell transplantation. Second, this approach allows more precise control over the entire process during cellular therapy product preparation and therefore is more suitable for the large scale clinical manufacturing. The study of intercellular communication via extracellular vesicles (EVs) represent one of the most rapidly emerging fields of modern cell biology. Recently, several excellent comprehensive reviews have been published [3-5], thus, here we will give only brief introduction into the topic. According
to the origin and size EVs could be classified as exosomes, microvesicles and apoptotic bodies. Exosomes are 30-100 nm in diameter and originate from endosomal compartments, known as multi-vesicular bodies (MVBs), while microvesicles are larger particles (50-1000 nm) produced by budding from the plasma membrane. Apoptotic bodies are generated from cells undergoing programmed cell death, they are 800-5000 nm in diameter and contain extensive amounts of phosphatidylserine, DNA, histones and ribosomal RNA. It is now widely accepted that both exosomes and microvesicles play important roles in intercellular communication via the transfer of membrane receptors, proteins, lipids, RNA and miRNA between cells. Thus, EVs can transfer biological information over long distances affecting normal and pathological processes throughout organism. In this review we will focus mainly on the neuroprotective properties of exosomes. Importantly, the composition of exosomes depends on cell type and its physiological state. Accordingly, exosomes derived from diseased tissues may be important for the maintenance and spread of pathological processes. Indeed, recent studies demonstrated that exosomes are involved in the propagation of several neurodegenerative diseases [6,7]. Another striking example comes from the recent finding that tumour-secreted exosomes specifically promote organotropic metastasis and prepare pre-metastatic niches in target tissues [8]. Thus, targeting formation, release, or uptake of disease-promoting exosomes represent promising therapeutic strategy. On the other hand, exosomes derived from healthy cells may have a comparable therapeutic potential as cells themselves. From this perspective most promising cellular sources are human mesenchymal stem cells (MSCs), dendritic cells (DCs) and human inducible pluripotent stem cells (iPS). In this review, we will summarize the current state of knowledge on the molecular mechanisms underlying neuroprotective actions of exosomes derived from these cells. The inability of most drugs to cross the blood–brain barrier (BBB) represent major problem of modern neuropharmacology. In this respect exosomes have a crucial advantage, because they can cross BBB. Moreover, exosomes might be engineered to target neurons or even specific neuronal populations [9]. These new developments open possibilities for intravenous, or even intranasal delivery methods avoiding the need for neurosurgical intervention. Despite the apparent ability of exosomes to cross BBB, surprisingly little is known about the mechanistic details of this process. However, several very recent studies coming from the cancer biology field have shed new light on the potential mechanisms by which exosomes may enter into the CNS. We will discuss these findings and also propose hypothetical model on the mechanism by which exosomes can cross BBB.
2. Identifying the best cellular source 2.1. Mesenchymal stem cells According to the traditional definition, MSCs have the potential to differentiate into mesenchymal tissues including osteoblasts, adipocytes and chondocytes [10]. MSC-like cells with similar characteristics have been isolated from virtually all vascularized organs and tissues [11]. These cells can be relatively easy isolated and expanded in vitro, therefore they became very popular among many researchers. However, a lot of controversies and misconceptions regarding the true nature and functions of MSCs still exist in the field, even the use of term „mesenchymal stem cell“ has been challenged repeatedly [12]. There are several key points to consider about MSCs. We think, that these considerations also should be taken into account during evaluation of potential therapeutic properties of MSC-secreted exosomes. First, it is essential to make a clear distinction between in vitro cultures of MSCs and their in vivo counterparts. The vast majority of information about MSCs comes from the in vitro models which not necessarily correctly reflect situation in vivo [13]. Second, MSC cultures comprise functionally different heterogeneous subpopulations. For example, only a relatively small fraction of freshly isolated adherent cells from bone marrow (BM) are colony forming unit-fibroblasts (CFU-Fs) and only a part of clonal progenitors derived from these individual CFU-Fs formed bone when transplanted in vivo [14]. This assumes, that cellular heterogeneity of MSCs is also reflected in cargo content and functional properties of secreted exosomes. Third, MSC-like cells isolated from different tissues are not equivalent and display distinct tissue-specific differentiation capacities. For instance, ectopic transplantation of BM-derived MSCs produced heterotopic bone/marrow organ structures, whereas dental pulp-derived MSCs gave dentin and pulp tissue [15]. Thus, MSC-like cells from different tissues demonstrate distinct functional traits, implying they also produce exosomes with different properties. Here we will summarize current data about neuroprotective properties of exosomes produced by MSCs derived from different tissues. For a long time it was thought, that MSCs isolated from dental pulp, also known as dental pulp stem cells (DPSCs), or stem cells derived from the dental pulp of human exfoliated deciduous teeth (SHEDs) originate from the cranial neural crest cells which are precursors of both neural and skeletal tissues [16]. However, recent study demonstrated that DPSCs originate from peripheral nerve-associated glia [17]. Thus, in contrast to MSC-like cells from other mesodermal tissues (bone marrow, adipose tissue), DPSCs could be particularly suitable for the induction of neural differentiation and also for the neuroregenerative therapies. Indeed, several studies have demonstrated, that DPSCs can be differentiated into neurons in vitro [18-20]. Using human DPSCs and SHEDs for the transplantation into the completely transected adult rat spinal cord [21], the
transplantation resulted in marked recovery of hind limb locomotor functions. Importantly, transplantation of BM-MSCs or skin-derived fibroblasts gave substantially less recovery of the locomotor function. Three major mechanisms of neuroregeneration were identified and one of them was associated with paracrine mechanisms [21]. The importance of paracrine DPSC signalling for the neuroregeneration has been known for relatively long time. For example, DPSCs produced neurotrophic factors, interacted with trigeminal neurons in vitro, and rescued motoneurons after spinal cord injury [22]. The same group showed, that paracrine factors produced by DPSCs protected dopaminergic neurons against 6-hydroxy-dopamine (6-OHDA) in vitro [23]. Recent study demonstrated that dopaminergic neuron-like cells could be efficiently induced from SHEDs and that these cells had therapeutic benefits in a 6-OHDA-induced Parkinsonian rat model [24]. Importantly, paracrine effects of differentiated SHEDs contributed to neuroprotection against 6-OHDA-induced neurodegeneration and to nigrostriatal tract restoration [24]. These studies demonstrated that DPSCs and SHEDs have unique neurogenic properties which could be potentially exploited for therapeutic use. We asked, whether exosomes and microvesicles derived from SHEDs display neuroprotective properties during 6-OHDA-induced oxidative stress in human dopaminergic neurons. We found that exosomes, derived from the microcarrier cultures of SHEDs suppressed 6-OHDA-induced apoptosis in dopaminergic neurons [25]. Importantly, exosomes derived from SHEDs grown under standard conditions did not suppress apoptosis indicating that culture conditions had a profound influence on functional properties of the exosomes. Our preliminary data from comparative proteomic and RNAomic analyses (unpublished) revealed that bioreactor culture significantly affected cargo content of exosomes. Future studies will identify unique proteins and (or) microRNAs responsible for the neuroprotective properties of exosomes. What is a possible mechanism for neuroprotective action of SHED-derived exosomes ? Since 6-OHDA triggers apoptosis mostly through production of reactive oxygen species (ROS) in mitochondria [26], we speculate that exosomes may reduce sensitivity of dopaminergic neurons to the 6-OHDA-induced oxidative stress. Indeed, several recent studies using exosomes derived from MSCs, or other types of cells, demonstrated that they may provide protection against oxidative stress. For example, human umbilical cord (UC) MSC-derived exosomes reduced cisplatin-mediated renal oxidative stress and apoptosis in vivo [27]. Another study demonstrated, that chaperone αB crystallin is secreted within exosomes by human retinal pigment epithelium and provide protection from oxidative stress in adjacent retinal cells [28]. Exosomes secreted by mouse macrophages and ex vivo loaded with potent antioxidant catalase significantly increased viability of 6-OHDA-pre-treated PC12 cells [29]. Importantly, the same study demonstrated that catalase-loaded and unloaded exosomes decreased ROS levels in activated macrophages, suggesting they may act via similar
mechanisms in microglial cells during neuroinflammation. Furthermore, in vivo experiments confirmed that catalase-loaded exosomes reduced microgliosis and increased survival of dopaminergic neurons in 6-OHDA-treated mice [29]. Thus, protection against oxidative stress may represent one of the major neuroprotective mechanisms of exosomes. Adipose tissue (AT) represents an abundant and easily accessible source of MSC-like cells, therefore it is suitable for large scale clinical production of exosomes. Several studies addressed neurotherapeutic effects of exosomes derived from AT-MSCs. Interestingly, AT-MSCs secrete functional neprilysin-bound exosomes [30]. Neprilysin (also known as CD10) is a type II membrane associated metalloendopeptidase important for the the proteolysis of β-amyloid peptide (Aβ), its activity and expression is decreased during Alzheimer’s disease (AD) [31]. AT-MSCs-derived exosomes demonstrated neprilysin-specific enzyme activity and contributed to decrease of both secreted and intracellular Aβ levels in N2a neuroblastoma cells genetically modified to overproduce human Aβ. Importantly, exosomes from AT-MSCs expressed significantly higher levels of neprilysin than BM-MSCs, highlighting the differences between functional properties of exosomes derived from different tissues [30]. In this regard, it would be interesting to investigate in vivo therapeutic potential of exosomes derived from AT-MSCs using animal models of AD. Another recent study demonstrated that exosomes and microvesicles derived from the murine AT-MSCs rescued human neuroblastoma cells SH-SY5Y and primary murine hippocampal neurons exposed to oxidative damage with H2O2 [32]. Interestingly, both exosomes and microvesicles demonstrated an inverse dose-dependent effect on cell viability. Furthermore, both exosomes and microvesicles derived from murine AT-MSCs increased the process of remyelination and activated nestin-positive oligodendroglial progenitors in cerebellar slice cultures demyelinated with lysophosphatidylcholine [32]. Very recently, the same group presented evidence for neuroprotective effects of exosomes derived from murine AT-MSCs using in vitro model of amyotrophic lateral sclerosis [33]. For this purpose they used motoneuron-like cell line NSC-34 overexpressing different mutants of human superoxide dismutase 1 (SOD1) and exposed to oxidative stress with H2O2. Exosomes were able to protect NSC-34 cells from oxidative damage showing potential for future therapeutic applications in motor neuron disease [33]. To date, BM represents the most common source of MSCs. Several studies demonstrated therapeutic potential of exosomes derived from BM-MSCs in different clinical settings. For example, MSC-derived exosomes have been successfully used for treatment of therapy-refractory graft versus host disease [34]. Another study demonstrated that human MSCs and MSC-derived exosomes very similarly improved post-stroke neuroregeneration in C57BL6 mice [35]. Interestingly, exosomes also modulated peripheral post-stroke immune responses by attenuating
post ischemic immunosuppression [35]. Exosomes derived from rat BM-MSCs promoted functional recovery and neurovascular plasticity after stroke [36] and traumatic brain injury [37] in rats. One mechanism contributing to promotion of brain remodeling during stroke recovery may be related to exosomal miR-133b. It has been demonstrated, that administration of MSCs restored downregulated expression of miR-133b in ischemic brain tissues. Furthermore, ischemic conditions increased miR133b levels in MSC-secreted exosomes and promoted neuron outgrowth in vitro via downregulated expression of RhoA protein, which is known suppressor of neurite outgrowth [38]. Interestingly, miR-133b is specifically expressed in midbrain dopaminergic neurons and is deficient in midbrain tissue from patients with Parkinson's disease [39]. This can be exploited for future therapeutic strategies using MSC-derived exosomes. Human UC tissue represents another source of MSCs and several recent studies demonstrated therapeutic potential of UC-MSC-derived exosomes in several experimental models [27,40]. However, there are no data in literature about neuroprotective properties of UC-MSCs. In conclusion, MSCs represent the most promising source of exosomes for the neurotherapeutic applications. However, there are issues to be resolved. First, systematic and comprehensive studies are required to compare proteomic and RNAomic profiles of exosomes produced by MSC-like cells derived from different tissues and grown under standard conditions. In our opinion, these should be free of xenogeneic supplements (for example, medium supplemented with platelet-rich plasma (PRP), or serum-free medium of known formulation [41]). In addition, MSCs polarized into proinflammatory and anti-inflammatory phenotypes [42] should be included into these studies. Second, we need systematic comparison of therapeutic properties of exosomes produced by MSCs derived from different tissues. At the first stage these studies could employ in vitro experimental models of neurodegeneration (for example, ReNcell VM immortalized neural stem cells differentiated into human dopaminergic neurons) for functional testing of different exosomal preparations, then extended by using in vivo experimental models. We believe that such an approach will facilitate selection for optimal source of exosomes targeting specific neurodegenerative diseases.
2.2. Dendritic cells DCs represent another promising source of exosomes suitable for neurotherapeutic applications. DC-derived exosomes participate in antigen presentation and also as regulators of innate and adaptive immunity. They can carry MHC I and (or) II-peptide complexes that can be captured and presented by other DCs to activate naive T cells, alternatively, these exosomes can be directly recognized by primed effector T cells [43]. Importantly, maturation status of DCs determines production and functional properties of exosomes. For example, immature DCs produced greater
amounts of exosomes, but these were less potent inducers of antigen-specific effector immune responses. By contrast, exosomes from mature DCs carry MHC II and co-stimulatory molecules and can directly activate T cells [44]. Another study demonstrated, that DCs at different stages of maturation release exosomes with different miRNA content [45]. Interestingly, 139 miRNAs were detected in both types of exosomes, 5 only in immature vesicles and 58 exclusively in mature exosomes [45]. DCs also can capture exosomes derived from tumour cells and then activate populations of tumour-specific cytotoxic T lymphocytes [46]. These findings have led to the development of novel cancer immunotherapy approach currently being tested in numerous clinical trials [47]. A recent study demonstrated, that DC cultures stimulated with low-level IFNγ released exosomes that increased myelination, reduced oxidative stress and improved remyelination following acute lysolecithin-induced demyelination in hippocampal slice cultures [48]. Furthermore, nasal administration of exosomes derived from IFNγ-stimulated DCs increased CNS myelination in vivo [48]. IFNγ-stimulated DCs produced exosomes containing a high levels of miRNAs known to be involved in oligodendrocyte differentiation and anti-inflammatory pathways [48]. Among them, miR-219 plays an essential role during differentiation of oligodendritic precursor cells and improvement of remyelination [49]. Further functional studies confirmed downregulation of several miR-219 targets in slice cultures [48]. Interestingly, the same group demonstrated that exosomes derived from serums of young, or environmentally enriched old rats contained high levels of miR-219 [50]. Moreover, nasal delivery of the exosomes isolated from serums of young rats enhanced remyelination in old rats, demonstrating that DC-derived exosomes may be a useful therapy for remyelination during multiple sclerosis or other dysmyelinating disorders [50]. Thus, despite the fact that only very few studies are available so far, DCs have a great potential as a therapeutic source of exosomes for neurodegenerative diseases. Again, comprehensive proteomic and RNAomic profiling and functional evaluation of exosomes using standardized conditions is needed to ensure reproducibility and therapeutic efficacy.
2.3. Human inducible pluripotent stem cells Human inducible pluripotent stem cells (iPS) as a source of exosomes for neurotherapeutic applications have several important advantages over DCs and MSCs. First of all, current technologies enable efficient in vitro generation of different types of human neurons and glial cells and use them as producers of exosomes targeting specific pathological processes in the brain. These cells can be cultivated in vitro for a long periods of time allowing collection of large quantities of exosomes. Another important advantage is that iPS cells can be derived from patient tissues minimizing risks of immune rejection. Two recent studies demonstrated potential of iPS-derived
exosomes for the treatment of ischemic diseases [51,52]. Undoubtedly, future research will address neuroprotective properties of iPS-derived exosomes.
3. Exosomes as suppressors of neuroinflammation Increasing evidence suggests that chronic neuroinflammation has a causal role in pathogenesis of neurodegenerative diseases, therefore, new therapies directed against neuroinflammatory processes may be beneficial [53]. Several recent reports demonstrated, that exosomes display antineuroinflammatory properties in CNS. For instance, exosome-encapsulated curcumin inhibited LPS-induced brain inflammation and myelin oligodendrocyte glycoprotein induced autoimmune responses in experimental autoimmune encephalitis model [54]. Importantly, intranasally injected exosomes were rapidly transported to the brains of mice and were taken up by microglial cells. Moreover, exosomes significantly reduced numbers of activated inflammatory microglial cells [54]. Interestingly, this study also demonstrate that exosomes were taken up by both activated and resting microglial cells. The pathophysiological importance of this finding remains to be established. As we have already mentioned, exosomes derived from DCs suppressed neuroinflammatory responses in vitro [48] and in vivo [29]. Thus, there is a growing body of evidence that exosomes may exert pleiotropic effects in CNS including support of oligodendritic precursor cells, protection of different types of neurons and also suppression of neuroinflammatory responses by microglial cells.
4. Exosomes can cross blood-brain barrier: how do they do it ? The BBB represents dynamic physiological barrier between the brain and circulating blood that regulates local brain microenvironment necessary for proper neuronal function and is formed by the continuous layer of specialized brain capillary endothelial cells. These endothelial cells are surrounded by basement membrane, pericytes and astrocytes forming neurovascular unit (NVU). Astrocytes play an essential role in differentiation and support of brain capillary endothelial cells, while pericytes are important integrators of endothelial and astrocyte functions at the NVU. Studies with pericyte-deficient mouse mutants revealed increased permeability of BBB, which occurred through increased transcytosis in endothelial cells [55]. The BBB restricts diffusion of hydrophilic or most large (> 400 Da) molecules into the CNS [56]. It is now established that four fundamental molecular properties of endothelial cells contribute to BBB integrity and functions. First, tight junctions (TJ) between endothelial cells prevent paracellular flux of small hydrophilic molecules and ions. Second, brain endothelial cells display low rates of transcytosis. Third, selective influx and efflux transporters allow uptake of nutrients and molecules from blood and elimination of toxins from the CNS. Finally, endothelial cells express low levels of leukocyte adhesion molecules,
limiting entry of immune cells into the brain parenchyma and creating immunoprivileged environment [57]. What could be the mechanism of exosomal entry into the CNS ? In principle, there are two possible ways of entry. First, it has been proposed that exosomes may be internalized by endothelial cells, undergo transcytosis, then released again to be re-internalized by other recipient cells [58]. Thus, according to this model, exosomes can cross tissue by ,,jumping‘‘ from one cell to another operating in a similar manner to some viruses. However, currently there is no experimental evidence to support this model. Alternatively, exosomes may enter into CNS via intercellular junctions of endothelial cells. Despite the current lack of direct experimental proof that exosomes can enter into the CNS paracellulary, numerous recent studies demonstrate, that exosomes increase permeability of vascular barriers throughout the organism. Hence, recent study demonstrated, that metastatic breast cancer-secreted exosomes destroys vascular endothelial barriers to promote metastasis [59]. Exosome-associated miR-105 significantly downregulated expression of ZO-1, a central molecular component of TJs, destroying barrier function in endothelial monolayers [59]. In vivo studies demonstrated that exosomes with high content of miR-105 significantly increased miR105 levels in lung and brain, accompanied by reduced ZO-1 expression and also enhanced vascular permeability in lungs, liver and brain. Furthermore, in vivo treatment with the anti-miR-105 compound reduced the volume of primary tumours, suppressed distant metastases to the lung and brain and restored vascular integrity in tumour-bearing animals [59]. More recently, it has been demonstrated, that breast cancer-secreted exosomes can trigger breakdown of BBB by yet another mechanism [60]. Exosomal miR-181c downregulated expression of 3-phosphoinositide-dependent protein kinase-1 (PDPK1), leading to decreased levels of phosphorylated cofilin and abnormal polymerization of actin in brain endothelial cells [60]. These findings demonstrate, that cancersecreted exosomes may employ different mechanisms to increase permeability of BBB and other vascular barriers. Most likely, other miRNAs and yet unidentified proteins and possibly lipids are also important mediators of exosome-dependent breakdown of BBB. Very recently another important study revealed mechanism by which cancer exosomes direct metastatic organotropism and prepare the pre-metastatic niches in target tissues [8]. This report demonstrated, that distinct expression patterns of exosomal integrins were associated with organotropic metastasis. Strikingly, treatment with exosomes from lung-tropic cancer cells redirected the metastasis of bone-tropic tumour cells [8]. Importantly, exosomes promoted vascular leakiness, before exosome uptake by specific lung cells suggesting that exosomes first permeabilize vessels, allowing for exosome entry into the target tissue. Several other pathologies have also been associated with exosome-dependent increase of vascular permeability. Recent findings demonstrated, that exosomes from patients with
cardiovascular disease or from endothelial cells exposed to stressful conditions can disrupt endothelial TJs. For instance, plasma-derived exosomes from obese children with obstructive sleep apnea altered endothelial TJs [61]. In addition, some viruses are using highly sophisticated mechanisms to trigger breakdown of BBB. It has been demonstrated, that human immunodeficiency virus type 1 (HIV-1)-infected microglia and astrocytes produced viral proteins including gp120, Tat, and Nef affecting the integrity of the BBB [62]. Interestingly, Nef-transfected, or HIV-infected microglia released Nef protein in exosomes. Studies using in vitro model of BBB revealed, that these Nef-positive microglial exosomes significantly reduced expression of the TJ protein ZO-1, suggesting a mechanism for exosomal Nef-mediated neuropathogenesis [63]. Of note, exosomes
and HIV-1 display very similar biophysical and molecular properties. In addition, HIV-1 and exosomes utilize the same biogenesis pathway [64]. These similarities led to the Trojan exosome hypothesis which states that retroviruses exploit preexisting pathways of exosome biogenesis and uptake [65]. All these examples demonstrate, that exosomes secreted by diseased cells can affect vascular permeability and also in some cases integrity of BBB. But what about exosomes derived from healthy cells ? Several studies convincingly demonstrated, that exosomes from different types of cells can effectively cross BBB [9,66]. However, it is presently unclear, whether they employ similar mechanisms as exosomes from diseased tissues. Interestingly, our in vivo imaging experiments revealed, that exosomes derived from human dental pulp stem cells (DPSCs) increased vascular permeability at the intitial phases of carrageenan-induced acute inflammation in BALB/c mice [67]. Despite of that, exosomes reduced the carrageenan-induced edema to similar levels seen with the positive control (prednisolone), showing potent anti-inflammatory properties [67]. Therefore, it is possible, that as in the case of cancer exosomes, the first step of entry into the tissue might be related with permeabilization of vascular endothelial barriers. Accordingly, we suggest that exosomes cross BBB via two-stage process (Fig. 1). During the first stage exosomes are internalized by brain capillary endothelial cells and release specific cargo components affecting integrity of intercellular junctions. Thus, this ,,first wave‘‘ of exosomes increase intercellular permeability of endothelial cells allowing a massive entry of exosomes into the CNS during the second stage of the process. If correct, this model might be helpful for the establishment of new and more effective protocols of exosome administration. For example, initial pretreatment with exosomes may be followed by a ,,window‘‘ allowing maximal permeabilization of endothelial barriers of BBB and then by the next ,,therapeutic‘‘ dose of exosomes.
It should be noted, however, that various neurological disorders are associated with increased permeability of BBB, therefore more experiments are needed to evaluate and understand possible adverse effects related to therapeutic administration of exosomes.
5. Conclusions and future perspectives Accumulating evidence suggests that exosomes derived from different types of cells can be successfully used for the treatment of various neurodegenerative disorders. However, there are several problems that need to be solved before their widespread use in the clinical setting. First of all, investigators often use different methods of isolation, characterization and quantification of exosomes in their studies. For example, very often researchers quantify exosomes using standard protein quantification methods, others perform nanoparticle tracking analysis, or use empirical activity units reflecting yield of exosomes prepared from supernatants of cells grown under strictly defined conditions. Thus, it is increasingly difficult to compare and contrast study outcomes hindering progress in the field. It is also important to perform a comprehensive proteomic and RNAomic profiling and functional evaluation of exosomes using standardized conditions to ensure reproducibility and therapeutic efficacy. Finally, exosomes are complex systems containing a great number of molecular constituents raising multiple safety and predictability issues. Therefore, it seems likely, that future studies will focus on engineering of exosome mimetic delivery systems targeting specific cells and containing only desired therapeutic molecules.
Acknowledgements This work was supported by National Research Programme ,,Healthy ageing’’(Grant Nr. SEN15090) from Research Council of Lithuania.
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Figure legends Figure 1. The proposed mechanism by which exosomes cross blood-brain barrier. During the first stage exosomes are internalized by brain capillary endothelial cells, release specific cargo components and affect integrity of intercellular junctions via different mechanisms. A- Exosomal miR-105 downregulates expression of tight junction protein ZO1 [59]; B-Exosomal miR-181c induces abnormal polymerization of actin in brain endothelial cells [60]. Increased intercellular permeability of endothelial cells allows a massive entry of exosomes into the CNS during the second phase of the process.