Int. J. Biol. Sci. 2023, Vol. 19 721 Ivyspring International Publisher International Journal of Biological Sciences 2023; 19(3): 721-743. doi: 10.7150/ijbs.79063 Research Paper Extracellular vesicles, the emerging mirrors of brain physiopathology Amanda Cano1,2,3,4, Miren Ettcheto2,5, Mireia Bernuz6,7, Raquel Puerta1, Ester Esteban de Antonio1, Elena Sánchez-López2,3,4,8, Eliana B. Souto9,10, Antonio Camins2,5, Mercè Martí6, María Isabel Pividori6,7, Mercè Boada1,2, Agustín Ruiz1,2 1. Ace Alzheimer Center Barcelona – International University of Catalunya (UIC), Barcelona, Spain 2. Biomedical Research Networking Centre in Neurodegenerative Diseases (CIBERNED), Madrid, Spain. 3. Institute of Nanoscience and Nanotechnology (IN2UB), Barcelona, Spain. 4. Department of Pharmacy, Pharmaceutical Technology and Physical Chemistry, Faculty of Pharmacy and Food Sciences, University of Barcelona, Spain. 5. Department of Pharmacology, Toxicology and Therapeutic Chemistry, Faculty of Pharmacy and Food Sciences, University of Barcelona, Spain. 6. Biosensing and Bioanalysis Group, Institut de Biotecnologia i de Biomedicina (IBB-UAB), Mòdul B Parc de Recerca UAB, Campus Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain. 7. Grup de Sensors i Biosensors, Departament de Química, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain. 8. Unit of Synthesis and Biomedical Applications of Peptides, IQAC-CSIC, 08034 Barcelona, Spain. 9. Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Porto, Porto, Portugal. 10. REQUIMTE/UCIBIO, Faculty of Pharmacy, University of Porto, Porto, Portugal.  Corresponding author: Amanda Cano, Ace Alzheimer Center Barcelona. C/ Marquès de Sentmenat, 57, 08029, Barcelona, Spain. E-mail address: acano@fundacioace.org © The author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/). See http://ivyspring.com/terms for full terms and conditions. Received: 2022.11.01; Accepted: 2022.11.01; Published: 2023.01.01 Abstract Extracellular vesicles are secreted by a wide variety of cells, and their primary functions include intercellular communication, immune responses, human reproduction, and synaptic plasticity. Their molecular cargo reflects the physiological processes that their cells of origin are undergoing. Thus, many studies have suggested that extracellular vesicles could be a promising biomarker tool for many diseases, mainly due to their biological relevance and easy accessibility to a broad range of body fluids. Moreover, since their biological composition leads them to cross the blood-brain barrier bidirectionally, growing evidence points to extracellular vesicles as emerging mirrors of brain diseases processes. In this regard, this review explores the biogenesis and biological functions of extracellular vesicles, their role in different physiological and pathological processes, their potential in clinical practice, and the recent outstanding studies about the role of exosomes in major human brain diseases, such as Alzheimer’s disease (AD), Parkinson’s disease (PD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), or brain tumors. Key words: Exosomes; extracellular vesicles; Alzheimer’s disease; Parkinson’s disease; glioblastoma; multiple sclerosis, amyotrophic lateral sclerosis; neurodegenerative diseases 1. Introduction Intercellular communication involves various EVs encompass three major subcategories: molecules such as RNA, amino acids, lipids, apoptotic bodies, microvesicles, and exosomes. The metabolites, and proteins, which are commonly largest of the EVs are the apoptotic bodies and packaged into extracellular vesicles (EVs) (1). EVs are microvesicles. These EVs directly originate from the membrane-enclosed structures surrounded by a invagination of the plasma membrane in an endocytic phospholipid bilayer membrane secreted by different process. Exosomes, the smallest EVs, are located in cells, whose constituents reflect their cells of origin multivesicular bodies (MVBs) formed intracellularly and their physiological and pathological processes (1). by multiple invaginations of the late endocytic Several reports have highlighted that these EVs acts membrane (2). like messengers in intercellular communication, A growing number of studies have suggested conveying information to neighboring or remote cells. that EVs could be a promising tool as new biomarkers https://www.ijbs.com Int. J. Biol. Sci. 2023, Vol. 19 722 for many diseases, mainly due to their biological may also fuse with ESEs pre-formed by the relevance and easy accessibility to a broad range of endoplasmic reticulum (ER), Golgi network (GN), and body fluids (3). Moreover, it has been reported that mitochondria. Likewise, the ESEs could also fuse with cells suffering from any pathological process, such as the ER and TGN, which could explain how the cancer cells, release more EVs than healthy cells (4,5). endocytic cargo reaches these organelles and vice Thus, the appearance of EVs with specific cargoes and versa. Then, ESEs evolve into late endosomes (LSEs), surface markers of a wide variety of pathological where the exchange between the components of the processes has been described in different human endosome and those present in the cytosol is diseases such as AD (6), cancers (7), cardiovascular significantly increased. The second invagination of diseases (8), sarcoidosis (9), or prion diseases (10), LSEs leads to the generation of intraluminal vesicles among others. (ILVs). In this step, proteins (originally belonging to In this review, we explore the biological the cell surface) could be distinctly distributed among functions of EVs, their role in different physiological ILVs. Moreover, depending on the invagination and pathological processes, their potential in clinical volume, ILVs of different sizes and variable content practice, and the recent findings about the role of EVs could be found (11). in major human brain diseases. Encapsulating several ILVs (future EVs) generates MVBs, also known as multivesicular 2. Biogenesis and main hallmarks of EVs endosomes (2). Then, MVBs can follow several routes: EVs biogenesis is initiated by plasma membrane (i) MVBs can fuse with autophagosomes, whose invagination of the origin cell. The first invagination content will undergo degradation in the lysosomes; forms a structure that includes cell-surface proteins (ii) MVBs can directly fuse with lysosomes for and soluble molecules associated with the extracel- degradation, and the cells could recycle the lular matrix, such as ions, metabolites, peptides, or degradation products; (iii) MVBs that escape this lipids (Figure 1) (11). This leads to the formation of a trajectory are transported to the plasma membrane new early endosome (ESE). The primary endosome through the microtubule network and the Figure 1. Biogenesis pathway of extracellular vesicles. (1) Endocytosis. Invagination of plasma membrane, involving extracellular constituents together with cell surface proteins. The resulting endosome membrane presents with outside-in plasma membrane orientation. (2) Formation of the endosome. This budding process leads to the formation of early sort endosomes (ESEs) or possible fusion of the bud with endosomes pre-formed by the endoplasmic reticulum (ER), Golgi network (GN) and mitochondria. Then, ESEs give rise to late sort endosome (LSEs). (3) Formation of the multivesicular body. Second invagination in the LSE leads to the generation of intraluminar vesicles (ILVs), which in turn give rise to multivesicular bodies (MVBs) with defined cargo of ILVs (future exosomes). (4) MVBs fusion. The MVB can fuse either with lysosomes or autophagosomes to be degraded. (5) Exocytosis. MVBs can be transported to the plasma membrane through the cytoskeletal and microtubule network and dock on the luminal side of the plasma membrane. Exocytosis involves the release of the extracellular vesicles, like exosomes, microvesicles and mitovesicles (extracellular vesicles of mitochondrial origin) with a similar lipid bilayer orientation as the plasma membrane. https://www.ijbs.com Int. J. Biol. Sci. 2023, Vol. 19 723 cytoskeleton (2). On the luminal side of the plasma autocrine, or endocrine activity (18,19). However, it is membrane, MVB-docking proteins facilitate a docking well known that EVs play a vital role in cellular process between the MVB and the lipid bilayer. intercommunication (20). In this process, both EV Finally, exocytosis results in the release of EVs (Figure uptake and secretion pathways coexist, resulting in a 1). mixed production of EVs (newly synthesized or EVs can be divided into different types based on recycled) over time (2,20). This horizontal transfer of their size and origin: exosomes are 30–150 nm in size material mediated by EVs exchange causes various and derived from intracellular vesicles (multivesi- phenotypic and molecular alterations in the receptor cular bodies, a form of late endosome); microvesicles cells of the different organs and tissues. Likewise, it directly bud off the plasma membrane and are usually has also been reported that the “turnover rate” of between 100 and 1000 nm; mitovesicles, EVs with internalized EVs may vary depending on the mitochondrial content and similar size and route than metabolic state of the recipient cell (2). Thanks to the exosomes; while apoptotic bodies are generated from wide range of molecules that EVs can carry, this dying cells and are generally between 100 and 5000 cellular communication strategy leads to much more nm (12). complex events than the signaling mediated by a EVs frequently contain apoptosis-linked gene 2 single ligand and receptor. Several mechanisms have interacting protein X (ALIX), endosomal sorting been described by which bioactive molecules complexes required for transport proteins (ESCRT), transported by EVs affect target cells: (i) epigenetic Ras-related protein GTPase Rab, soluble N-ethylma- reprogramming of recipient cells through delivery of leimide–sensitive factor attachment protein receptor functional proteins, lipids, ions, and RNAs; (ii) (SNARE), tumor susceptibility gene 101 (TSG101), transfer of activated receptors to recipient cells; and syntenin-1, syndecan-1, ceramides, phospholipids, (iii) direct stimulation of target cells through sphingomyelinases, and tetraspanins (13). In addition, surface-bound ligands (11). CD9, CD81, CD63 or flotillin are commonly used as Different mechanisms are associated with EV surface biomarkers for EV tracking. However, it is uptake (Figure 2). For example, it has been described remarkable that these molecules are not only present that the uptake of EVs in human pancreatic cancer in exosomes surface and they are also found, for cells is caused by macropinocytosis through example, in multinucleated giant cells (14). Likewise, oncogenic signals induced by the expression of EV cargo can contain DNA, RNA, metabolites, amino mutant KRAS (21,22). In human melanoma cells, this acids, different types of cell surface proteins, and uptake is carried out through the fusion of the EV intracellular proteins (15). with the plasma membrane (23). It is currently The heterogeneity of EVs is large and is mainly unknown why this variation exists and whether the determined by four factors (2,16): (i) EVs’ size, which uptake of EVs results in a different functionality from could be due to uneven invagination of the MVB’s that of the EV constituents themselves (24). Tracing limiting membrane, affecting the different amounts of the intercellular exchange of EVs is also a great EVs content; (ii) e EVs’ content that may involve challenge today. Several in vivo studies explored extracellular matrix, membrane, cytosolic and nuclear different genetic strategies in animal models. Under proteins, lipids, metabolites, and nucleic acids physiological conditions, these studies demonstrated (noncoding RNA species and DNA). The inherent that EVs do not usually deliver mRNA to a recipient biology conditions this cargo and the cell. In contrast, this delivery, promoted by the microenvironment of the cells of origin; (iii) EVs’ activation and expansion of EV-producing immune origin: Different cells and organs use EVs as a tool for cells, was increased in mouse models of acute and intercellular communication. Heterogeneity can be chronic inflammation (25). Likewise, therapeutic found in the tissue of origin, giving them different interventions can also influence EV uptake. In this properties such as uptake by specific cell types or regard, Parolini et al. showed that the inhibition of tropism toward certain organs; (iv) functional impact: proton pumps led to an inhibition of EV uptake. The The effects of EVs on target cells can be different due acidification of the cellular pH increased the EV to the diversity of cell surface receptor expression. release and uptake in melanoma cell lines (26). This can result in EVs inducing cell survival, EVs have also been shown to be involved in apoptosis, or immunomodulatory functions (17). maintaining and improving synaptic plasticity and neurotransmission in the adult brain, which is crucial 3. Physiological functions of EVs for cognitive function (27). It has been shown that EVs The biological functions of EVs are complex and modulate synaptic activity in rapid and progressive diverse. Some of their cellular pathways are not yet manners and have some similarities with synaptic completely understood, as well as their paracrine, vesicles in biogenesis, content, and function Together https://www.ijbs.com Int. J. Biol. Sci. 2023, Vol. 19 724 Figure 2. Physiological pathways of extracellular vesicles’ biological uptake. with neurons, glial cells, such as astrocytes and (31,32). The protection of neurons by astrocyte- microglia, are mediated by neural synaptic plasticity derived EVs is dependent on astrocyte-derived EV processes (28). Active zones on the presynaptic PrP transport into neurons, a physiologically plasma membrane are responsible for depositing important receptor protein that protects against synaptic vesicles into the cytoplasm of the nerve oxidative stress in the CNS (33). Other studies indicate terminal. This synaptic vesicle exocytosis is typical of that oligodendrocyte- and microglia-derived EVs synaptic neurotransmission (27). However, signal transfer several enzymes to neurons, thus transduction of neurons through the secretion of EVs contributing to different energy metabolism pathways can also induce a wide range of neurobiological (34,35). All these processes comprise normal brain functions, including synaptic plasticity (29). In this function. regard, Bahrini et al. demonstrated that PC12 The role of EVs in innate immune responses and cell-derived EVs could stimulate synaptic pruning by their homeostasis has been extensively studied (36– enhancing the complement component 3 level in 41). Several mechanisms have been described by microglial MG6 cells. Furthermore, it has also been which EVs contribute to this physiological function: described that neuronal-derived EVs might affect (i) induction of signaling pathways by ligands present synaptic plasticity by managing the number of AMPA on the surface of EVs; (ii) transfer and presentation of receptors for glutamate transmission (30). Addition- antigenic peptides; (iii) gene-expression promoted by ally, increased secretion of EVs derived from cortical EV miRNA cargo; and (iv) the delivery of DNA- neurons, which carry neurotransmitter receptors, led inducing cyclic GMP-AMP synthase stimulators of to enhanced glutamatergic activity (30). interferon genes in recipient cells, in which sensing of Numerous mechanisms of intercellular commu- cytosolic DNA triggers the expression of a type I IFN nication are orchestrated by neuron-glia homeostasis response and inflammatory gene expression (2). EVs balance. EVs are involved in the physiological secreted by antigen-presenting cells (APCs) carry interactions between all the cells that compose the p-MHC-II and costimulatory signals. This directly neurovascular unit. In addition, EVs are present in presents the peptide antigen to specific T cells to neuronal tissue’s intrinsic development and induce their activation. However, T-cell stimulation protection processes (27). Evidence has indicated that by APCs is more effective than EV stimulation (42– communication between glia and neurons through 44). The role of EVs in antigen presentation has also EVs is required for neuronal growth and cell survival been described in the context of bacterial infection (2). https://www.ijbs.com Int. J. Biol. Sci. 2023, Vol. 19 725 EVs could enhance antibacterial immune responses gradient centrifugation, size exclusion chromato- by promoting bacterial antigen presentation from graphy, filtration, and polymer-based precipitation macrophage-derived EVs, which subsequently are the most popular. influences the adaptive immune response (45). Differential centrifugation remains the most Likewise, Patil et al. also demonstrated the opsoni- commonly used technique for physically EVs zation role of EVs in mediated phagocytosis of dead isolation. The main objective of this method is to cells in the injured heart (46). The nucleic acids’ EV eliminate particulate interferents, including dead cells cargo, mainly DNA and miRNAs, has also been and debris, by successive centrifugation steps. An identified to regulate innate and adaptive immune updated protocol with minor changes in responses. EVs DNA has been shown to play a role in centrifugation rate and times was recently proposed cancer progression, and miRNA transfection may also (53). In brief, four successive centrifugations are regulate the immune response by influencing gene necessary to separate: 1) cells at 300 g; 2) dead cells expression and signaling pathways in recipient cells and large cell debris at 2,000 g; 3) large and (2). medium-sized EVs (or apoptotic bodies and EVs are present in breast milk, amniotic fluid, microvesicles) at 10,000 g; and 4) small EVs (or and semen, suggesting that EVs possess putative exosomes) at 100,000 g. Differential centrifugation is a functions in human reproduction, pregnancy, and well-established and moderately time-consuming embryonic development (2). Seminal plasma EVs method that can be used with little or no sample have been implicated in sperm maturation (47). The pre-treatment. In addition, this approach is generally miRNA signature of EVs has been found to play a less expensive than density-gradient centrifugation vital role in the expression of interleukins involved in and can provide high yields because EVs s are genitalia-resident immunity and the prevention of concentrated in the residual pellet, which contains infections in the placenta (48). Moreover, miRNAs fewer contaminants (54). In contrast, differential and the protein cargo of plasma EVs dynamically ultracentrifugation methods require costly equipment evolve during pregnancy (49). EV-derived miRNAs in and can contribute to mechanical damage and low breast milk also seem to promote postnatal health and purity due to the co-existence of other non-EV vesicles growth (50). and protein aggregates. Finally, another biological function of natural Size-exclusion chromatography (SEC) is a EVs is wound healing. EVs derived from bone technique for separating particles based on their size. marrow mesenchymal stem cells have been shown to The interaction of the analysed particles with activate several signaling pathways in the wound heterosporous polymeric beads results in hydrody- healing process, such as STAT3, ERK, and Akt (51). namic radius separation (55). Compared to individual Likewise, they are also implicated in the expression of proteins, protein aggregates, and other contaminants, many wound healing-related growth factors, such as the relatively larger size of EVs leads to high EV nerve growth factor, insulin-like growth factor 1, purity even from complex biologic matrices. stromal cell-derived factor 1, or hepatocyte growth Sepharose-based SEC columns are the most factor (51). In addition, several studies have pointed commonly used for EV isolation. For example, out the crucial role of EVs in controlling the Sepharose CL-2B (Sigma-Aldrich Inc., St. Louis, MO, inflammation associated with wound healing. The USA) can separate vesicles of 70 nm in diameter or release of EVs by mesenchymal stem cells creates a larger. Although long elution times can occur, the suitable microenvironment through the transfer of EV mild conditions and reagent compatibility minimally miRNA. This micro RNA could regulate the affect EVs’ biological activity, contributing to expression of important immuno-modulators that maintaining the integrity of the proteins and enzymes fine-tune, for example, the TLR-induced NFκB present in the EVs. The resulting high purity of these signaling pathways and their downstream targets, preparations enables their use for high-demanding thus reducing the inflammatory process surrounding “omics” downstream applications. The main short- wounds without associated toxic effects (52). coming is related to the fact that the EVs are not preconcentrated by this strategy. It is important to 4. Isolation techniques note that commercial pre-packed Sepharose columns The purification of EVs is mandatory for most are specifically designed for EV isolation. These downstream analysis techniques. However, the include Exopure™ (BioVision, Milpitas, CA, USA), separation is challenging due to the EVs’ unique PURE-EVs Columns (HansaBioMed, Tallinn, Estonia), structure, complexity, and low concentration in and qEV Exosome Isolation (Izon Science, complex biologic matrices. Among the different Christchurch, Canterbury, New Zealand). methods for EV isolation, differential and density As an alternative, microfiltration and ultrafiltra- https://www.ijbs.com Int. J. Biol. Sci. 2023, Vol. 19 726 tion membranes can also be used for EVs isolation Moreover, there are commercially available kits for and, in some instances, in combination with separating EVs based on this principle, such as the ultracentrifugation and SEC. This approach is based MagCapture exosome isolation kit (Wako Life on pore size filtration that allows particles with Sciences, Richmond, VA, USA). Likewise, several EV specific sizes or molecular weights (MW) to pass receptors have been used for IMS, including the most through while retaining larger-sized particles. Some common tetraspanins CD9, CD63, and CD81 (60,61). commercial membranes are available in various pore The main advantage of the IMS is the ability to isolate sizes (800-100 nm). These include ExoMirTM (Bioo EVs by a specific immunological reaction, separating Scientific Corporation, Austin, TX, USA), Durapore® an EV subpopulation expressing a biomarker. Membrane Filter (Merck Chemicals & Life Science Moreover, IMS is a very gentle approach that ensures S.A., Darmstadt, Hessen, Germany), Vivaspin® (Sar- both EV integrity and purity. It is easy to carry out by torius Lab Instruments GmbH & Co. KG, Goettingen, one-step incubation with the sample to form Lower Saxony, Germany), and the exoEasy Maxi Kit EV-magnetic particle complexes and does not require (Qiagen, Hilden, North Rhine-Westphalia, Germany). benchtop instrumentation. However, a remarkable Polymer-based precipitation relies on precipi- disadvantage is that IMS is significantly more tation using polymers such as PEG (56). The principle expensive than SEC, polymer-based precipitation, and of polymer-based precipitation is to use a reagent that filtration. binds to water molecules, thereby forcing the Finally, although EVs are commonly described less-soluble components, such as EVs, to sediment, to be isolated from body fluids and culture medium, thus allowing collection by short, relatively low-speed there are also been described several methods to centrifugation or filtration. Some commercial kits extract them from different tissues, including the based on this approach include ExoQuick™ (System brain. Most common method is based on a sucrose Biosciences, Mountain View, CA, USA), Exo-spin™ gradient ultracentrifugation of brain homogenates (Cell Guidance Systems, Babraham, Cambridge, UK), (62,63). Briefly, post-mortem tissues (previously ExoPrep (HansaBioMed, Tallinn, Estonia), Exosome stored at −80°C) are sliced lengthways on ice using a Purification Kit (Norgen Biotek, Thorold, Ontario, razor blade or cryostat (for human and mouse brains Canada), miRCURY Exosome Isolation Kit (Exiqon, respectively) to generate wide sections of brain. The Vedbaek, Denmark), and Total Exosome Isolation slices are homogenized and immersed in collagenase Reagent (Life Technologies Corporation, Carlsbad, to dissociate the tissue. After a few cycles of CA, USA). Although costly, these polymer-based centrifugations in PBS medium, the supernatant is precipitation kits are user-friendly, rapid, and overlaid on a triple sucrose cushion and effective in isolating EVs in the 40-180 nm range while ultracentrifuged during 3h at 180,000 × g (average) at excluding larger EVs. Moreover, these kits have lower 4°C. Obtained fractions are separated and diluted sample volume requirements, thus making them ideal with ice-cold PBS and spun at 100,000 × g (average) at for clinical use (57). However, this technique by itself 4°C for 1 h to pellet the vesicles, and characterized to is not useful for the isolation of EV from a specific determine the nature of the vesicles. In addition, SEC cellular origin, since it is based on a non-specific is sometimes included after sucrose gradient to polymer precipitation. enhance the purity of obtained EVs fractions (64). The Immunomagnetic separation (IMS) uses main advantage of this method, compared with biologically-modified magnetic particles (MPs) that previous methods in which tissue is subjected the can specifically bind targets. MPs are powerful and tissue to filtering, blending and/or homogenization versatile separation and pre-concentration tools used (65,66), or vortexing (67), is the enhancement of in various analytical and biotechnology applications isolation enrichment, EVs purity and EV structure (58,59). MPs can be coated with antibodies to integrity preservation. EV-receptor molecules. Once captured, a magnetic field is applied to separate the EV-coated MPs from 5. Biomedical applications of EVs the biologic matrix, thus significantly reducing matrix EVs have been considered as a molecular mirror effects and contamination (60,61). Commercial super- of the cells they originate from and as potential targets paramagnetic Dynabeads® M-450 Tosylactivated for determining the state of cells in pathological (Life Technologies Corporation, Carlsbad, CA, USA) conditions (68). Furthermore, they can be found in (4.5 µm in diameter) are some of the MPs most many biological fluids such as blood, CSF, urine, commonly used to achieve this task. Other types of saliva, and milk (69), making them accessible MPs are widely available from System Biosciences, biomarkers for complex diseases. Specifically, when Pierce, Miltenyi Biotec, HansaBioMed, Aethlon focusing on neuronal diseases, the ability of EVs to Medical, and New England Peptide, among others. cross the blood-brain barrier (BBB) makes them https://www.ijbs.com Int. J. Biol. Sci. 2023, Vol. 19 727 promising diagnostic and therapeutic biomarkers (70– with high specificity, sensitivity, and simplicity (56). 72). However, there is little evidence about EVs Numerous simplified analytical methods with crossing and there is needed more evidence to clarify various readouts have been reported recently for the penetration process of EVs through the BBB. detecting EVs, including optical immunoassays (60), Also, their role in intercellular communication and electrochemical biosensors (61,81), among many opens an opportunity for their modification for others. In addition to their potential role as therapeutic use. biomarkers, EVs are designed to carry molecular cargo from one cell to another (82). The EVs could 5.1 The diagnostic capability of EVs thus be loaded with therapeutic cargo, enabling EVs are released into body fluids, allowing us to highly targeted delivery of drugs to specific types of obtain representative information in biological fluids cells while sparing all other cell types from damage from less-accessible organs, as is the case of the central (83). nervous system, during non-invasive procedures such as liquid biopsies. They reflect the cellular changes in 5.2 The therapeutic potential of EVs pathobiology, even in the early stages. Because of this, EVs are garnering considerable interest as the circulating EVs are a promising source of novel promising candidates for therapeutic purposes due to biomarkers for diagnosing and monitoring brain their molecular-specific characteristics and carrying diseases. Some proteins are found to be ubiquitously properties. In fact, EVs by themselves or as vehicles useful as EV general markers (68,73,74). On the other for the delivery of drug payload(s) are being actively hand, not only are proteins used as biomarkers but explored as therapeutic agents. They are stable, miRNA EVs expression patterns have also been biocompatible, can avoid the activation of the reported to change in different brain diseases, such as immune system, contain proteins that can prevent the AD and PD (75,76). These changes have been activation of the coagulation cascade (84,85), and have observed in different biofluids, such as CSF, serum been described to be able to cross blood vessels and plasma (77). (including the BBB) (86), although this event must be Unfortunately, realizing the potential of these deepen analyzed in further studies. All these vesicles as biomarkers will require technical characteristics confer them less toxicity and a lower improvements since the EVs are exceptionally clearance ratio compared to other therapeutic challenging to characterize with current technologies. strategies. Moreover, EVs have cell tropism, allowing EVs have a diameter of 30 to 200 nm, putting them drug delivery specificity and are suitable for the outside the sensitivity range of most cell-oriented transport of biological drugs such as proteins or sorting or analysis platforms, such as classical flow nucleic acids (as short interference RNA (siRNA) or cytometers. To date, the most common methods for micro-RNA (miRNA)) (70,83). This nucleic acid cargo targeting EVs have involved purification followed by is also able to modify protein expression for specific characterization of their cargo (78). Isolation therapeutic purposes. Besides these features, natural of the EVs is best performed with differential EVs are not enough for efficient therapeutic use, and ultracentrifugation. Purification can also be done with they must be modified appropriately. Enrichment of precipitation, size-exclusion chromatography, or EVs based on their surface ligand presentation may ultrafiltration (79). Lately, immunomagnetic separa- also enable the development of receptor-mediated tion is also used to isolate EV populations based on tissue targeting (56). Pre-secretory drug loading and the universal membrane proteins, including CD9, post-secretory drug loading are the two most CD63, or CD81 (80), although as described above, common strategies for loading therapeutic cargo. In they are not only specific of EVs’ surface (14). the pre-secretory way, the modification is performed Identification of membrane vesicles as EVs also in the cells that produce the EVs, while in the requires morphological analysis (78). EVs can only be post-secretory way, modifications are performed visualized with an electron microscope, given their directly on the EV surface or into its membrane (by small size. Nanoparticle tracking analysis (NTA) is electroporation, sonication, and other procedures) usually used to count the EVs, followed by down- (56). stream processes for specific detection, including LC-MS/MS and Western Blot for proteins and qPCR 6. The role of EVs in neurological for genetic material. The whole procedure is diseases time-consuming and requires skilled personnel, laboratory facilities, and benchtop instrumentation. 6.1 EVs in Alzheimer’s disease Current gold standard methodologies have limita- Dementia was the fifth-leading cause of death in tions in isolating, detecting, and characterizing EVs 2016 (87). AD is the most common form of dementia https://www.ijbs.com Int. J. Biol. Sci. 2023, Vol. 19 728 worldwide, constituting up to 50–80% of cases (87). functionally specialized synaptic protein cargo of AD is commonly diagnosed symptomatically through plasma neuronal-derived exosomes (NDEs) of the occurrence of significant memory loss, global patients with AD dementia and frontotemporal cognitive decline, and an overt impairment of daily dementia (FTD) (94). One of the main molecular life activities. Later in the course of the disease, the changes in the early stages of AD is the altered breakdown of physical functions, such as walking, synaptic protein levels (95,96). They found that four swallowing, and general movement, ultimately leads synaptic proteins (synaptophysin, synaptopodin, to death (88). EVs have an important role in AD as synaptotagmin, and neurogranin) were significantly they carry pathogenic proteins and spread them decreased not just in AD patients but also in FTD across the brain. Moreover, they are capable of patients. Moreover, the serine 9 phosphorylation of inducing apoptosis of astrocytes by impairing synapsin 1 was reduced in AD and FTD, indicating neuronal function (89). EVs are involved in APP that the protein’s functionality is also altered. Only cleavage. Some proteases like BACE 1, PSEN 1, PSEN AD patients exhibited a significant reduction in 2, and ADAM10 are present in MVB and exosomes. GAP-43 and synapsin 1 levels. Taking these together, Furthermore, EVs can deliver mRNA that activates the authors suggest that NDE synaptic proteins may toll-like receptors, which upregulate the levels of be useful for preclinical dementia detection and proteases BACE1, PSEN1, and PSEN2 (89). A PS gene disease progression (94). The same authors found that mutation reduces Cystatin C (CysC) in EVs, which protein levels of presynaptic proteins NPTX2, decreases soluble APP, and increases Aβ1-42 NRXN2α, and their postsynaptic partners AMP4 and production (90). In this sense, Pérez-González et al. NLGN1 were significantly decreased in plasma NDEs demonstrated that EVs containing CysC showed and correlated with cognitive loss (97). Additionally, neuroprotection properties in cultured cells, as well as except for NPTX2, these proteins were found to be EVs secreted by CysC-deficient cells treated with reduced even in preclinical AD patients, and all of exogenous human CysC significantly enhanced the them declined with AD progression (97). survival of the cells (91). Other molecules Liu et al. also studied the potential of the synaptic participating in APP metabolisms, such as Grb2 or protein neurogranin (Ng) as a neurodegeneration tetraspanin 6, have also been detected in EVs (77). biomarker in plasma exosomes (98). In recent years, Likewise, and study carried out with exosomes from clinical studies have reported that CSF and blood Ng human brain tissue showed that the exosome levels are closely related to the occurrence and secretory pathway transports APP carboxyl-terminal subsequent progression of AD (99–101). Thus, the fragments from the cell into the brain extracellular authors conducted a meta-analysis including 4661 space (65). Moreover, ALIX, an EV marker, has been individuals, of which 187 were selected for exosomes found in the senile plaques of AD brains (89). analysis. Ng levels were found to increase in CSF but Astrocytes surrounding the senile plaques uptake Aβ decrease in plasma exosomes of patients with AD and peptide and release EVs with apoptotic proteins that mild cognitive impairment (MCI)-AD and to be lead to the apoptosis of astrocytes even if they have no highly associated with cognitive decline. Interest- contact with Aβ (77). However, EVs have also been ingly, the concentration of Ng in plasma exosomes of described to possess neuroprotective effects in AD. AD and MCI patients was lower than that of healthy The surface protein PrPc sequesters the Aβ oligomers, controls. Moreover, this concentration was also lower thus reducing the Aβ accumulation. Similarly, in MCI-AD patients than in those with a stable MCI microglia can remove Aβ- EVs by recognizing diagnosis (98). Nielsen et al. evaluated the protein nSMase2 and EV uptake, decreasing the extracellular cargo of plasma-derived EVs related to neurological Aβ levels (92). Microglia-derived exosomes (MDEs) and inflammatory processes in patients with AD, also contribute to spreading pathogenic tau (89). MCI, and healthy controls (102). They analyzed 182 Interestingly, mouse tauopathy models that have proteins by using the proximity extension assay depleted microglia have shown an inhibition of tau technology of Olink proteomics (103–105). Although propagation (92). The genotype has also been related no group differences in particle concentration or size to EVs in AD. A study conducted in n human were found in EVs samples, the CLEC1B/CCL11 ratio postmortem tissues and humanized mouse models for showed diagnostic capabilities between healthy apolipoprotein E showed that the apolipoprotein E4 controls and AD. Similarly, Winston et al. predicted genotype leads to downregulation of exosome conversion from MCI to dementia through the study biosynthesis and release (93). of plasma NDE protein profiles (106). In this study, Many studies are currently evaluating the the authors also found that Ng levels were potential of EVs as diagnostic and therapeutic tools in significantly lower in AD and MCI-AD compared to AD (Table 1). In that sense, Goetzl et al. explored the cognitively normal controls. In contrast, the levels of https://www.ijbs.com Int. J. Biol. Sci. 2023, Vol. 19 729 p-tauT181, p-tauS396, and Aβ1–42 in plasma NDEs were showed that mice injected with AD NDEs had more significantly higher than those in AD NDEs. In p-tau positive cells in the CA1 region of the addition, the authors proved that NDEs contribute to hippocampus than those littermates injected with AD pathology by injecting plasma NDEs from AD MCI NDEs (106). and MCI patients into wild-type mice. The results Table 1. Selected relevant studies of recent findings of EVs for the diagnosis and/or treatment of Alzheimer’s disease. Fluid of Origin Isolation method Purpose of Target of study Type of study Main Results Reference EVs cell-type of the study EVs Plasma Neurons ExoQuick®, isolation and Diagnosis Synaptic proteins Clinical NDE levels of synapsynaptophysin, synaptopodin, Goetzl et immunoprecipitation synaptotagmin-2, and neurogranin were significantly al. 2016 (L1CAM) lower in patients with FTD and AD. NDE levels of growth-associated protein 43 and synapsin 1 were reduced only in patients with AD. Plasma Neurons ExoQuick,® isolation and Diagnosis NPTX2, NRXN2α, Clinical The NDE-derived proteins were significantly decreased Goetzl et immunoprecipitation AMPA4 and in AD dementia. Diminished levels of AMPA4 and al. 2018 (L1CAM) NLGN1 NLGN1 in NDEs correlated with the extent of cognitive loss. In a preclinical period, the NDE levels of all but NPTX2 were significantly lower than those of matched controls, and levels of all proteins significantly declined with the dementia conversion. Plasma Total - Diagnosis Neurogranin Clinical Ng levels increased in CSF, but decreased in blood Liu et al. plasma exosomes of patients with AD and MCI-AD, and 2020 were highly associated with cognitive decline. Plasma Total Ultracentrifugation Diagnosis Neurology and Clinical Several proteins and their ratios could distinguish Nielsen et Inflammation cognitively affected from healthy individuals. For plasma al. 2020 proteins TGF-α | CCL20 (AUC = 0.96, 95% CI = 0.88–1.00, p = 0.001) and EVs CLEC1B | CCL11 (AUC = 0.95, 95% CI = 0.86–1.00, p = 0.001) showed diagnostic capabilities. Plasma Neurons ExoQuick®, isolation and Diagnosis Protein cargo in Clinical / Pre-Clinical: Mice injected with plasma NDEs from ADC Winston et immunoprecipitation NDEs Pre-clinical patients displayed increased p-Tau positive cells in the al. 2016 (L1CAM) (In Vivo) CA1 region of the hippocampus. Clinical: Plasma NDE levels of P-T181-tau, P-S396-tau, and Aβ1–42 were significantly higher, whereas neurogranin and the repressor element 1-silencing transcription factor were significantly lower in AD and MCI converting to AD patients. Plasma Neurons ExoQuick®, isolation and Treatment GHRH Clinical Exo-derived Aβ1-42, neurogranin, synaptophysin, Winston et immunoprecipitation synaptotagmin, and synaptopodin showed the highest al. 2018 (L1CAM) diagnostic accuracy for distinguishing between CNC and MCI patients. Plasma Astrocytes ExoQuick®, isolation and Diagnosis C1q, C4b, factor D, Clinical ADE levels of complement proteins were significantly Winston et immunoprecipitation fragment Bb, C5b, higher in patients with MCIc than those with MCIs. ADE al. 2019 (GLAST/ACSA-1) C3b, C5b-C9,CD46, levels of inhibitory CPs decay-accelerating were CD59, and type 1 significantly lower in patients with MCIc than those with complement MCIc. receptor Plasma Neurons Prognosis pS312-IRS-1 and Clinical The change from baseline in insulin signaling molecules Mustapic ExoQuick®, isolation and pY-IRS-1 in neuronal-enriched EVs in response to an experimental et al. 2019 immunoprecipitation treatment (20 IU of intranasal insulin) is associated with a (L1CAM) change in cognition, which suggests engagement of the insulin cascade in the brain. Serum Neurons Total Exosome Isolation Diagnosis miRNA-135a, -193b Clinical Both serum exosome miR-135a and miR-384 were Yang et al. kit (Invitrogen Life and 384 up-regulated while miR-193b was down-regulated in 2018 Technologies) serum of AD patients compared with that of normal controls. Exosome miR-384 was the best among the three miRNAs to discriminate AD, VaD, and PDD. Combination of miR-135a, -193b, and -384 was proved to be better than a particular one for early AD diagnosis Serum, Total Total exosome Isolation Diagnosis miRNA-193b in Preclinical ( In ABCA1 and ABCA1-Labeled Exosomes Are Detected at Liu et al. CSF and kit (Invitrogen Life ABCA1-labeled vivo and in High Levels in Neurons. ABCA1-Labeled Exosomal 2021 culture Technologies) exosomes vitro)/ miR-193b Is Abundant in Serum and CSF from AD Model medium Clinical Mice. ABCA1-Labeled Exosomal miR-193b Levels Are Elevated in the CSF and Serum of AD Patients. Saliva Total Ultracentrifugation Diagnosis Salivary exosomes Clinical Significant differences in salivary exosomes concentration Rani et al. among the groups of cognitively impaired and AD 2021 patients compared to the healthy control cohort. Ab1–42, beta amiloide peptide; ABCA1, ATP Binding Cassette Subfamily A Member 1; AD, Alzheimer’s disease; ADC, converting to AD; ADEs, astrocyt-derived exosome; AMPA4, GluA4-containing glutamate receptor; CA1, cornu ammonis 1; CPs, Levels of complement proteins; CNC; cognitively normal controls; CSF, cerebrospinal fluid; C1q, C4b, Complement Protein of classical pathway; C5b, C3b, and C5b-C9, Complement Protein of both patways; CD46,CD59, and type 1 complement receptor, Proteins of the inhibitory complement pathway; CDAT; dementia of the Alzheimer type; factor D and fragment Bb, Complement Protein of the alternative pathway; FTD, frontotemporal dementia; GHRH, Growth hormone-releasing hormone; MCI, mild cognitive impairment; MCIC, MCI converting to dementia within 3 years; MCIS, MCI remaining stable over 3 years; NDE, neuronal-derived exosomes; NG, neurogranin; NLGN1, neuroligin 1; NPTX2, neuronal pentraxin 2; NRXN2α, neurexin 2α; PDD, Parkinson’s disease with dementia; pS312-IRS-1, Insulin Receptor Substrate 1 Phospho-Ser312; P-T181-tau, phospho tau T181; P-S396-tau, phospho tau S396; pY-IRS-1, Insulin Receptor Substrate; SCD; Subjective cognitive decline; VaD, vascular dementia; https://www.ijbs.com Int. J. Biol. Sci. 2023, Vol. 19 730 In a later study, the authors also evaluated the or 40 IU. Interestingly, these changes were also progression from MCI to AD dementia by analyzing correlated with cognition scores (116). the abnormalities of complement proteins (CPs) in The study of the variabilities in the miRNA plasma astrocyte-derived exosomes (ADEs) (107). profile of the EVs is another of the most interesting Several pieces of evidence have suggested that approaches to this type of study. In this regard, Yang complement system proteins may mediate the et al. evaluated the expression levels of exosomes capacity of type A1 astrocytes to damage neurons miRNA-135a, miRNA-384, and miRNA-193b, which (108–110). Moreover, astrocytes expressing type A1 were observed to be involved in the regulation of APP markers in areas of postmortem brain tissues affected and BACE1 in AD, vascular dementia (VD), and by neuroinflammatory or neurodegenerative diseases Parkinson’s disease patients (117). They found that have high levels of complement component 3 (C3) miRNA-135a and miRNA-384 were upregulated (111). In this case, ADEs showed significantly while miRNA-193b was downregulated. The best increased levels of some proteins from both the candidate for differentiating between AD, VD, and classical and alternative complement pathways. The PD was mi-RNA-384, while the combination of all CD46, CD59, and type 1 complement receptor levels three was proved to be better than a particular one for were significantly lower in MCI converting patients early AD diagnosis (117). than in stable MCI patients (107). The authors Liu et al. also studied the diagnostic potential of theorized that the increased C3b opsonization by miRNA-135a and miRNA-193b loaded into neurons might be one of the causes of the attraction ATP-binding cassette transporter ABCA1-labeled and neuronal toxicity mediated by microglia, while exosomes in both preclinical and clinical samples of the high levels of the complex C5b-C9 might cause AD (118). ABCA1 mediates cellular cholesterol efflux direct injury to neurons. However, further studies are in the brain and participates in neuroinflammation needed to clarify which CPs could distinguish and neurodegeneration processes (119). The authors between stable and converting MCIs patients, their found that ABCA1-labeled exosomes containing contribution to neurotoxic neuroinflammation, and miRNA-135a were able to cross the BBB and were their potential as predictive biomarkers (107). increased in the CSF and serum of transgenic mice. In The same authors evaluated the biomarker addition, ABCA1-labeled exosomal content of miR- profile variations in plasma NDEs from participants 135a was significantly increased in the CSF and serum enrolled in a randomized, double-blind, placebo- of MCI and AD patients compared to those of the controlled 20-week trial of growth hormone-releasing control group, agreeing with Yang et al.’s findings hormone (GHRH) administration (112). The beneficial (118). In the study of miRNA-193b, the authors found effects of insulin growth factor 1 (IGF-1), whose that wild-type mice injected with ABCA1-exosomes release is stimulated by GHRH in physiological from the CSF of APP/PS1 mice exhibited increased conditions, on brain health are well described, as is levels of miRNA-193b in both CSF and serum. As with their reduction caused by aging, which has been miRNA-135-a, ABCA1-labeled exosomal miRNA- associated with the development of AD (113–115). 193b levels were also higher in both CSF and serum of The authors found that concentrations of Ng, MCI and AD patients compared with the control synaptophysin, synaptotagmin, and synaptopodin group (120). However, in this case, miRNA-193b were significantly decreased in NDEs of patients with appeared to be up-regulated in ABCA1-labeled MCI, whereas Aβ1-42 was significantly increased, exosomes of AD patients contradicting the results independent of GHRH treatment. On the other hand, found by Yang et al. p-tauS396 and GAP43 were not affected by cognitive Other fluids have also been studied as a source status or GHRH treatment. Thus, even though NDEs of EVs. Moreover, the concentration of EV protein biomarkers cargo showed promising results, the cargo per se has also been reported as a differential treatment with GHRH did not clarify its role in the parameter of disease progression (121). In that sense, clearing mechanisms associated with reducing AD Rani et al. developed a new method based on neuropathology in the brain (112). Similarly, Mustapic nanoparticle tracking analysis to purify salivary et al. aimed to study the neuroprotection effect of exosomes from patients with AD, MCI, and VD. insulin in the brain through the analysis of exosomes Using their method, the authors observed significant protein cargo. Thus, they measured the levels of differentiation in salivary exosomal concentrations. pS312-IRS-1 and pY-IRS-1, biomarkers of insulin Specifically, exosomes concentrations were found to resistance, in NDEs to track the response of AD and be significantly higher in AD and MCI compared to MCI patients to intranasal insulin treatment (116). controls, while they were slightly higher between AD They found differences in both biomarkers in and MCI groups (122). Moreover, pathological response to the 20 IU treatment but not for the placebo proteins (Aβ, p-tau, and Aβ oligomers/fibrils) found https://www.ijbs.com Int. J. Biol. Sci. 2023, Vol. 19 731 in exosomes of both AD and MCI were significantly L1CAM-enriched plasma exosomes, which was higher than in control groups. significantly correlated with the disease severity. Although further studies are needed to clarify the 6.2 EVs in Parkinson’s disease physiopathology molecular mechanisms, the authors PD is characterized by a progressive loss of suggest that this increased efflux of α-synuclein into dopaminergic neurons, mainly present in the exosomes is a cleavage mechanism to reduce the substantia nigra, accompanied by the degeneration of α-synuclein central toxicity (127). About L1CAM, this dopaminergic terminals in the striatum, thus leading transmembrane protein is expressed on neurons and to movement coordination impairments and cognitive widely used to isolate neuron-derived EVs (NDEVs) decline, depression, or anxiety. It is the most common in human biofluids. However, there is much motor disorder and the second most prevalent controversy about the utility of L1CAM to track neurodegenerative disease. It is incurable, with an neuronal origin exosomes. A recent study developed unknown etiology in most cases. A systematic by Norman et al. aimed to understand why there was analysis for the Global Burden of Disease Study 2016 observed increased levels of α synuclein in NDEVs pointed out that PD was the disease with the samples isolated by capture with an anti-L1CAM fastest-growing prevalence, disability, and death antibody (129). They showed that this increase were rates, affecting 1% of the population over the age of not related to EVs of plasma or CSF, but a nonspecific 60, or 1-2 individuals over the age of 60 per 1000 binding of soluble α-synuclein (and other soluble people at any given time (123). proteins) to the anti-L1CAM antibody (129). In this EVs have been observed to contain α-synuclein, sense, further studies are needed to find a real specific the essential protein involved in the pathogenesis of marker of NDEVs and highlighting the importance of PD. Acceleration of α-synuclein accumulation (124) performing rigorous controls to exclude these false and cytotoxic effects have been associated with positives. α-synuclein EV uptake (125). EVs are involved in Zheng et al. investigated α-synuclein species, transmitting α-synuclein between cells, facilitating its oligomeric and phosphorylated α-synuclein in plasma propagation (126) and the pathogenesis severity (125). exosomes as potential PD peripheral biomarkers Interestingly, some PD genes such as LRRK 2, VPS 35 (130). The authors found that aggregated α-synuclein and PARK 9 are related to the autophagic-lysosomal and p-α-synuclein existed inside and on plasma pathway of α-synuclein clearance (125). This pathway exosomes’ membrane surfaces. In addition, the ratio is inhibited or at least dysfunctional in PD subjects, of p-α-synuclein oligomer/total p-α-synuclein was thus leading to impaired protein clearance. This shown to be moderately helpful in PD diagnosis (130). alteration increases EV release to avoid intracellular The utility of EVs is not only restricted to α-synuclein accumulation but enhances extracellular diagnosis purposes but also monitoring and treatment accumulation and the spread of PD pathophysiology of PD. In this sense, Athauda et al. performed a (126). In fact, the pathological mutation of LRRK2 proteomic assay in plasma exosomes of patients resulted in an increase in the number of EVs due to an enrolled in a randomized, placebo-controlled clinical abnormal function of MVBs. Furthermore, it has been trial to assess the influence of Exenatide treatment on reported that the levels of PARK9 and EVs are brain insulin and protein activity kinase B (Akt) positively correlated, thus suggesting that α-synuclein signaling pathways (131). Exenatide, a glucagon-like extracellular levels are modulated by PARK9 through peptide 1 agonist used in type 2 diabetes, was recently EV secretion (125). found to have beneficial effects on the motor function Several studies have evaluated the diagnostic of PD (132). In this study, the authors found that potential of EVs in PD and their role in disease NDEs of treated patients exhibited elevated spreading (Table 2). In that regard, Shi et al. evaluated expression of downstream substrates, including total the performance of blood α-synuclein as a PD Akt and phosphorylated mechanistic target of biomarker through its analysis in plasma exosomes rapamycin (mTOR), as well as augmented tyrosine (127). Previous studies reported plasma α-synuclein phosphorylation of insulin receptor substrate to be inconsistent and generally ineffective for PD compared to the placebo group (131). These results diagnosis (128). Preclinical in vivo findings showed highlighted the potential of using EVs -based that the α-synuclein could be transported from the biomarkers as monitoring objective measures for brain to the peripheral blood into exosomes. clinical trials. Furthermore, they found that the patients with PD presented a higher concentration of α-synuclein in https://www.ijbs.com Int. J. Biol. Sci. 2023, Vol. 19 732 Table 2. Selected relevant studies of recent findings of EVs for the diagnosis and/or treatment of Parkinson’s Disease. Fluid of Origin Isolation method Purpose of Target of study Type of Main Results Reference EVs cell-type the study study of EVs Plasma Neurons Centrifugation and Diagnosis α-syn Clinical / Pre-Clinical: CSF α-syn was readily transported to blood, Shi et al. immunoprecipitation Pre-clinical with a small portion being contained in exosomes. 2014 (L1CAM) (In Vivo) Clinical: The study found that in contrast to CSF α-syn concentrations, the levels of plasma exosomal α-syn were substantially higher in PD patients, suggesting an increased efflux of the protein to the peripheral blood of these patients Plasma Total 1) Total exosome Isolation Diagnosis α-syn and Clinical α-syn and p-α-syn in the plasma exosomes of PD patients Zheng et al. kit (Invitrogen Life p-α-syn showed poor solubility after Protease K treatment. 2021 Technologies) 2) Differential centrifugation Plasma Neurons Centrifugation and Monitoring NDEs Clinical Exenatide-related changes in EV biomarkers were Athauda et immunoprecipitation significantly associated with clinical improvements. al. 2019 (L1CAM) Plasma Total ExoQuick®-TC kit Treatment miRNA-137 Pre-clinical Down-regulation of exosomal miR-137 alleviates oxidative Jianj et al. and OXR1 (In vitro and stress injury in PD by up-regulating OXR1. 2019 In Vivo) Culture ASCs Ultracentrifugation and Treatment miRNA-188-3p Pre-clinical miRNA-188-3p-enriched exosome treatment suppressed Li et al. medium ExoQuick®-TC (In Vivo and autophagy and pyroptosis, whereas increased proliferation 2019 reagent In Vitro) via targeting CDK5 and NLRP3 in PD mice and MN9D cells were observed. Culture BMSCs Ultracentrifugation Treatment EXO-ASO4 Pre-clinical In Vitro: Exo-ASO4 also significantly attenuated α-syn Yang et al. medium (In Vivo and aggregation induced by pre-formed α-syn fibrils. 2021 In Vitro) In Vivo: Exo-ASO4 intracerebroventricular injection into the brains of α-syn A53T mice significantly decreased the expression of α-syn and attenuated its aggregation. Furthermore, it ameliorated the degeneration of dopaminergic neurons in these mice and showed significantly improved locomotor functions. ASCs, Adipose-derived mesenchymal stem cells; ASO(s), Antisense oligonucleotide(s); BMSCs, Bone marrow Mesechymal stem cells; CDK5, cell division protein kinase 5; CSF, cerebrospinal fluid; DA, dopaminergic; EV; extracellular vesicles; Exo, Exosomes; L1, L1 cell adhesion molecule; NLRP3, Nucleotide-binding and oligomerization domain-like receptor family pyrin domain containing 3; PD, Parkinson’s disease; ROS, reactive oxygen species; SOD, superoxide dismutase; OXR1, oxidation resistance 1; α-syn, α-sinuclein; p-α-syn, Ser129 phosphorylated α-sinuclein. Blood EVs carrying miRNAs have been lein expression in PD (138). Knockdown ASO-based highlighted to serve as potential targets for diagnosis gene strategy is a reliable and well-established and PD treatment. In that sense, Jiang et al. aimed to method for treating neurodegenerative diseases, such demonstrate in a preclinical study that exosomal as PD (139). The authors found that ASO loaded into miRNA-137 isolated from serum might have an effect exosomes showed high cellular uptake and low on PD by regulating oxidation resistance 1 (OXR1), a toxicity in primary neuronal cultures. Furthermore, in protein involved in oxidative stress-induced an α-synuclein A53T transgenic mouse PD model, neurodegeneration (133). These authors found that intracerebroventricular injection of exo-ASO signifi- the inhibition of miRNA-137 or up-regulation of cantly decreased the expression of α-synuclein and OXR1 ameliorated PD-induced oxidative stress injury. attenuated its aggregation, ameliorating the degene- Furthermore, when exosomal miRNA-137 was ration of dopaminergic neurons and improving the inhibited using miR-137 antagomir, an amelioration locomotor functions of treated mice (138). of PD-induced oxidative stress injury was observed in an in vitro model. Thus, the down-regulation of 6.3 EVs in multiple sclerosis exosomal miR-137 alleviates oxidative stress injury in Multiple sclerosis (MS) is the most common PD by upregulating OXR1 (133). inflammatory neurological disease in young adults. It Li et al. explored the therapeutic potential of is a chronic, heterogeneous, disabling autoimmune exosomes by suppressing the autophagy processes in disease characterized by demyelination, neuroinflam- the dopaminergic neurons of the substantia nigra (134). mation, oligodendropathy and neuronal degeneration In this case, exosomes serve as genetic vectors for that affects both gray and white matter (140). miRNA-188-3p, which is found to be an autophagy Although the exact etiology of MS is not clear yet, and pyroptosis suppressor by targeting CDK5 and demyelination is the main alteration found in these NLRP3 (135–137). They assessed the levels of patients. In addition, EVs have a vital role in CNS autophagy, injury, and inflammasomes in PD mouse myelination development (141). Oligodendrocytes and cell models and found that these processes were secrete EVs that slow the formation of the myelin suppressed in both models after treating them with sheath, while neuronal EVs s inhibit oligodendrocyte- miRNA-188-3p-enriched exosomes (134). Yang et al. derived EVs. Therefore, EVs contribute to the also investigated the use of exosomes as carriers of homeostasis of myelin biogenesis (142). Moreover, it antisense oligonucleotides (ASO) to reduce α-synuc- has been reported that EVs, released by mature https://www.ijbs.com Int. J. Biol. Sci. 2023, Vol. 19 733 oligodendrocytes activated with glutamate through significant scores when validated in an independent the NMDA and AMPA pathway, stimulate the group of progressive MS cases (146). transduction of myelin proteins and the promotion of In an interesting study by Mrad et al., they specific RNA (143). However, EVs have also been compared the effects of serum-derived exosomes on shown to negatively contribute to the demyelination monocyte-derived macrophages (MDMs) of healthy process. Endothelial cells, leucocytes, microglia, donors and those of patients with relapsing-remitting astrocytes, and platelets can be released in response to MS in remission and relapse (147). The main purpose proinflammatory cytokines. EVs carrying metallo- of this study was to assess whether the response proteinases and caspase 1 can induce disintegration of correlates with exosomal Epstein-Barr virus (EBV) the BBB and facilitate lymphocyte and myeloid cell protein expression. EBV has been highly associated transmigration into the CNS (144). Furthermore, with MS (148). In fact, scientific evidence has shown endothelial cells transfer integrin Mac 1 and ICAM 1 that most MS patients are infected with EBV and have receptors to monocytes through EVs, which enhances increasing titers of antibodies against the EBV nuclear their ability to cross the endothelial barrier (145). antigen EBNA1 before the onset of neurologic Collectively, they facilitate the transmigration of symptoms (149,150). The results found in this study lymphocyte cells into the brain and the onset of the revealed that the expression of latent membrane autoimmune response typical of MS (141). proteins LMP1 and 2A and EBV nuclear antigen Based on this evidence, most studies have EBNA1 was higher on exosomes derived from deepened the potential of EVs in MS (Table 3). patients with active MS compared with healthy Ebrahimkhani et al. explored the exosomal miRNA controls and stable patients. Moreover, MDMs signatures as monitoring and diagnostic tools for MS differentiated from patients with active disease were (146). When comparing the exosome-associated better secretors of CXCL10, CCL2 and CXCL9, than microRNAs in serum samples of MS patients and MDMs from healthy and stable MS groups (147). healthy controls, they found several differentially Thus, the authors suggest that EBV(+) exosomes expressed exosomal miRNAs in both relapsing- could promote a proinflammatory response in MS remitting MS and progressive MS patients compared patients by antigen-presenting cells and may to controls. In addition, nine of these miRNAs showed contribute to ongoing immune activation. Table 3. Selected relevant studies of recent findings of EVs for the diagnosis and/or treatment of Multiple Sclerosis. Fluid of Origin Isolation method Purpose of Target of study Type of study Main Results Reference EVs cell-type of the study EVs Serum Total Size exclusion Diagnosis & miRNAs Clinical A group of nine miRNAs (miR-15b-5p, miR-23a-3p, Ebrahimkhani chromatography + qEV monitoring miR-223-3p, miR-374a-5p, miR-30b-5p, miR-433-3p, et al. 2017 size exclusion columns miR-485-3p, miR-342-3p, miR-432-5p) distinguished relapsing-remitting from progressive disease. Serum Total Total exosome Isolation Diagnosis Macrophages Clinical / Serum-derived exosomes of patients with MS Mrad et al. 2021 kit (Invitrogen Life Pre-clinical (In expressed higher levels of EBV proteins than HC. Technologies) vitro) Incubation with EBV(+) exosomes promoted MDMs activation and pro-inflammatory cytokines secretion. Culture BMSCs Ultracentrifugation Treatment Neuroinflammation Pre-clinical (In Exosomes from BMSCs significantly decreased Li et al. 2019 medium Vivo) neural behavioural scores, neuroinflammation, and demyelination. In addition, exosomes increased the levels of IL-10 and TGF-β, whereas TNF-α and IL-12 levels decreased significantly. Culture AMSCs Exocib® exosome Treatment Immunomodulation Pre-clinical (In Intranasal administration of MSC-SEV to EAE mice Fathollari et al. medium isolation kit Vivo) was more effective than the administration of MSC 2021 alone to reduce clinical scores and histological lesions of the CNS tissue. Culture MSCs ExoQuick®-TC kit Treatment Myelin Pre-clinical (In In Vitro: The aptamer-exosome promoted the Hosseini medium Vivo and In proliferation of OLN93 cell line. Shamili et al. Vitro) In vivo: The aptamer-exosome produced a robust 2019 suppression of inflammatory response as well as lowered demyelination lesion region in CNS, resulting in reduced severity of the disease in a C57BL/6 mice model. Culture Dendritic ExoQuick® Kit Treatment INF-γ Pre-clinical (In Nasally administered IFNγ-DC-Exos increased CNS Pusic et al. 2014 medium cells vivo) myelination in vivo. AMSCs, Abdominal adipose tissue MSC; BMSCs, Bone marrow Mesechymal stem cells; CNS; central nervous system; EAE; Experimental autoimmune encephalomyelitis; EBV, Epstein-Barr virus; INF-γ, Interferon gamma; IFNγ-DC-Exos, low-level interferon gamma released exosomes; MDMs, monocyte-derived macrophages; MSCs, Mesechymal stem cells; SEV, Small extracellular vesicle. https://www.ijbs.com Int. J. Biol. Sci. 2023, Vol. 19 734 Several authors have studied the therapeutic 6.4 EVs in amyotrophic lateral sclerosis potential of EVs in MS against the demyelination ALS is a fatal motor neuron degenerative disease process by targeting the neuroinflammation cascade. with high socioeconomic impact, affecting In that sense, Li et al. explored the use of exosomes approximately 300,000 patients worldwide and secreted by bone marrow mesenchymal stem cells expected to increase to 400,000 by 2040 (158,159). The (BMSCs) in the experimental autoimmune encephalo- etiology of the disease is still unknown, but several myelitis (EAE) rat model of MS (151). Recent studies proteins have been identified to be involved in its have shown that BMSC exosomes play a crucial role pathogenesis. Some of these proteins, such as SOD1, in many autoimmune diseases and aid in tissue p-TDP-43, or FUS, have been shown to be transported repair, but the specific mechanisms are still unknown from cell to cell by the EVs pathway, contributing to (152,153). These authors discovered that exosome the propagation of prion-like misfolded isoforms and treatment significantly reduced inflammatory cell increasing the neuropathology of ALS (160). Likewise, infiltration into the CNS, neural behavioral scores, the levels of IL-6 have been shown to be increased in TGF-α and IL-12 levels, and the demyelination astrocyte-derived EVs and are positively correlated process. In contrast, mRNA expression levels of M2 with disease progression (160). Similarly, EVs-driven phenotype markers IL-10 and TGF-β were miRNAs have been shown to be altered in ALS significantly increased. Similarly, Fathollahi et al. patients, with fewer reads than healthy subjects and evaluated the therapeutic potential of MSC-derived different expression patterns (161). exosomes in an EAE mouse model of MS. In this case, In this sense, many research groups are currently the aim of the study was to evaluate, using clinical evaluating the potential of EVs as diagnostic and symptoms and histological analysis of CNS lesions, therapeutic tools in ALS (Table 4). Basso et al. the effect of intranasal administration of SEV on explored the influence of mutant SOD on the protein disease activity and antigen-specific responses (154). secretion pathway and exosome release in astrocytes The results showed that treatment with MSC- and its implications for disease spread and motor exosomes significantly decreased clinical scores and neuron pathology (162). SOD1 mutations are the was more effective in alleviating clinical scores than major genetic contributors to ALS, with more than 170 MSC alone. In addition, this decrease was associated SOD1 mutations identified (163). The obtained results with an increase in immunomodulatory responses, showed that mutant SOD1 astrocytes released a larger such as Foxp3, CD25, and regulatory T cell frequency. proportion of proteins through exosome spreading. In this regard, Hosseini Shamili et al. also evaluated Moreover, the authors found that astrocyte-derived the immunomodulatory properties of MSC-derived exosomes transferred SOD1 to spinal neurons and exosomes as a platform for reducing MS clinical induced selective motor neuronal death (162). Thus, scores (155). In this case, the LJM-3064 aptamer, which these findings suggest that astrocyte-derived has been shown to possess a specific affinity toward exosomes may have a role in disease spreading and myelin and has demonstrated re-myelination motor neuron pathology. New therapeutic properties (156), was covalently conjugated to the approaches could target exosomes to contain disease surface of the exosomes and employed as both a progression. Silverman et al. also explored the origin targeting ligand and a therapeutic agent. Their results of CNS-derived extracellular vesicles carrying showed that exosomes promote the proliferation of misfolded SOD1 in a mouse model of ALS (164). In oligodendroglia in the in vitro assays and reduce the this case, quantitative proteomics comparing inflammatory response and demyelination in the in brain-derived EVs from non-transgenic and vivo studies (155). Finally, Pusic et al. conducted an transgenic ALS mice (SOD1G93A mice) revealed that interesting study in which dendritic cell cultures were these EVs contain canonical exosomal markers stimulated with low-level IFNγ exosomes (157). These enriched in synaptic and RNA-binding proteins. The exosomes contained miRNAs that could increase complete brain EV proteome contained various baseline myelination, reduce oxidative stress, and proteins implicated in ALS, especially in EVs from improve re-myelination processes. The results also SOD1G93A mice, which were significantly depleted showed that these exosomes were preferentially taken in myelin oligodendrocyte glycoprotein compared up by oligodendrocytes, thus suggesting that they with those from non-transgenic mice (164). They could positively affect myelination (157). These results observed that brain- and spinal cord-derived EVs exhibit great potential for the use of these EVs as a from both NTg mouse models were positive for the re-myelination therapeutic tool not only in multiple astrocyte marker GLAST and the synaptic marker sclerosis but also in other dys-myelinating SNAP25, whereas the microglial marker CD11b was syndromes. largely absent. https://www.ijbs.com Int. J. Biol. Sci. 2023, Vol. 19 735 Table 4. Selected relevant studies of recent findings of EVs for the diagnosis and/or treatment of Amyotrophic Lateral Sclerosis. Fluid of Origin Isolation method Purpose of Target of study Type of study Main Results Reference EVs cell-type of the study EVs Culture Astrocytes Ultracentrifugation Diagnose SOD1 Pre-clinical (In Mutant SOD1 astrocytes release mutant Basso et al. medium and Vitro) SOD1-containing exosomes that are toxic for motor 2013 treatment neurons. Results suggest that astrocyte-derived exosomes may have a role in disease spreading and motor neuron pathology. Brain and Astrocytes, Ultracentrifugation Diagnose SOD1 Pre-clinical (In Brain astrocytes and neurons, but not microglia, are Silverman spinal cord neurons and and vivo) and the main EV source and are involved in the disease et al. 2019 tissue microglia treatment Clinical development thought their pathogenic disease-causing proteins. Culture ASCs PureExo® Exosome Treatment Oxidative stress Pre-clinical (In Exosomes were able to protect NSC-34 cells from Bonafede et medium isolation kit vitro) oxidative damage and increase the cell viability. al. 2016 Culture ASCs PureExo® Exosome Treatment Motor Pre-clinical (In ASC-derived exosomes targeted lesioned ALS Bonafede et medium isolation kit performance vivo) regions, protected muscle, lumbar motoneurons and el. 2020 the neuromuscular junction, improved the motor performance, and decreased the glial cells activation. Plasma Total EXOBead/EXOBuffer® Diagnosis NfL, pNfH and Clinical Exosomal TDP-43 ratio was increasing along with Chen et al. TDP-43 follow-up. 2020 CSF Total Size Exclusion Diagnosis CSF proteins Clinical Three proteins were increased and 11 proteins were Hayashi et Chromatography on Drip decreased in ALS patients in CSF exosomes. The al. 2020 Column protein with the greatest increase was INHAT repressor, which is closely associated with nucleolar function. Plasma Total Differential centrifugation Diagnosis Small EVs, large Clinical Raman spectra showed that sporadic ALS patients Morasso et and filtration protocol EVs and plasma have a different EVs lipid/protein content and less al. 2020 intense bands than healthy controls. Plasma Total Vn96 peptide isolation and Diagnosis miRNA Clinical Elevated levels of 5 miRNAs and reduced levels of 22 Saucier et centrifugation signature in miRNAs in EVs. miRNAs with relevance to ALS were al. 2019 extracellular found to be deregulated: vesicles - Diagnosis: miR-9-5p, miR-183-5p, miR-338-3p, - Disability progresion:.miR-1246. MiR-15a-5p and miR-193a-5p ALS, amyotrophic lateral sclerosis; ASCs, adipose-derived stromal cells; CSF, Cerebrospinal fluid; EVs, extracellular vesicles; lEVs, large extracellular vesicles; NIR, INHAT repressor; NFL, Neurofilament light chain; PALS, persons diagnosed and living with ALS; pNfH, phosphorylated neurofilament heavy chain; SOD1, Copper-Zinc Superoxide Dismutase; TDP-43, exosomal TAR DNA-binding protein-43. Moreover, EVs from the brains and spinal cords cells, lumbar motoneurons, neuromuscular junction, of the SOD1G93A ALS mice and human SOD1 and muscle. Their results showed that ASC-exosomes familial ALS patients’ spinal cords contained protected muscle, lumbar motoneurons, and the abundant misfolded SOD1 (164). These results neuromuscular junction, which resulted in improved suggest that brain astrocytes and neurons, but not motor performance (166). Likewise, a decrease in glial microglia, are the main EV sources and are involved cell activation was observed in treated SOD1G93A in disease development through their pathogenic mice. Curiously, exosomes were found to target disease-causing proteins. lesioned ALS regions of the brain. These results Bonafede et al. investigated the neuroprotective together highlight the potential of exosomes as a properties of exosomes in several in vitro models of novel treatment strategy for this incurable disease. ALS. In their first work, the authors hypothesized that Regarding diagnostic purposes, Chen et al. the administration of exosomes from adipose-derived conducted a longitudinal clinical trial in which 18 ALS stromal cells (ASCs) could ameliorate ALS symptoms patients were recruited, plasma samples were since ASCs have been demonstrated to promote extracted at different time points, and exosomes were neuroprotection and neurodegeneration. For this isolated by immunoprecipitation (167). Neurofilament purpose, they used NSC-34 cells transfected with light chain (NfL), phosphorylated neurofilament different human mutant SOD1 transgenes, which are heavy chain (pNfH) by ELISA, and exosomal TAR commonly used to mimic a motoneuron-like behavior DNA-binding protein-43 (TDP-43) ratio were selected in ALS (165). Obtained results showed that, in both as the study biomarkers. TDP-43 is the major naïve and over expressing mutant SOD1 NSC-34 cells, component of the ubiquitinated inclusions that the addition of ACS-derived exosomes rescued cells appear in the brains of 95%–97% of ALS patients from H2O2 oxidative stress-induced death (165). In a (168,169). Their results showed that the exosomal later study, the same authors evaluated the influence TDP-43 ratio significantly changed in 3- and 6-month of ASC-derived exosomes on disease progression in a follow-up and that NfL (but not pNfH) was SOD1G93A ALS mouse model (166). In this case, significantly higher in the rapid progression group at exosomes were administered intranasally and baseline and 3-month follow-up (167). This suggests intravenously, and their effects were tested on glial that plasma NfL and exosomal TDP-43 ratio could be https://www.ijbs.com Int. J. Biol. Sci. 2023, Vol. 19 736 potential biomarkers for ALS long-term follow-up forms and are responsible for substantial morbidity studies. Similarly, Hayashi et al. developed a clinical and mortality worldwide. Their incidence has trial in which a proteomic analysis of significantly increased in the last three decades (173). exosome-enriched fractions derived from the CSF of The last Global Burden of Disease report indicated amyotrophic lateral sclerosis patients was performed 330,000 incident cases of CNS cancer and 227,000 (170). They used liquid chromatography-tandem mass deaths globally (174). Moreover, it was estimated that spectrometry (LC-MS/MS) for the proteomic analysis, the incidence rate of all brain tumors was 10.82 per and a wide variety of proteins were compared to 100,000 person-years (175). Glioblastoma multiforme idiopathic normal-pressure hydrocephalus (iNPH) (GBM) is the most common malignant primary brain patients as disease controls. Their results revealed that tumor. This form comprises 27% of all tumors, and a novel INHAT repressor (NIR) was increased in 80% of malignant tumors (173). It is estimated that exosome-enriched fractions of the CSF. Likewise, more than two-thirds of adults diagnosed with these findings confirmed that NIR expression was glioblastoma will die within 2 years of diagnosis (176). reduced in motor neurons of sporadic ALS patients, Gliomas are primary brain cancers originating from thus suggesting NIR as a candidate biomarker protein progenitor cells, neural stem cells, or differentiated for ALS (170). Other techniques have been explored to mature neural cells that are transformed into tumor analyze the protein content of EVs in ALS patients. stem cells (177). In turn, they can be classified Thus, in a recent study by Morasso et al., Raman according to their aggressiveness, ranging from 2 to 4, spectroscopy was used to evaluate the molecular or according to their origin, which could be differences in plasma-derived EVs between sporadic oligodendroglioma, astrocytoma, or oligoastrocy- ALS patients and sex- and age-matched healthy toma. controls (171). In this case, Raman spectroscopy A growing body of evidence suggests that provided an overview of the biochemical composition gliomas can influence cells both nearby and beyond of EVs. The EVs of ALS patients exhibited a pattern of the BBB through the regulated excretion of EVs (178). Raman spectra that was distinct from that of the In GBMs, the EV content reflects the tumor's unique controls. Moreover, amino acids like tryptophan or signature of EGFRvIII mutation, EGFR amplification, carotenoids showed different intensities of selected TGF-β, IDH1 mutation R132H and podoplanin status peaks in the spectra of both groups of patients in the (7). In addition, EVs are also implicated in the EV compartment (171). transfection of receptors such as PDGFR and HER 2 to The study of miRNA content in EVs is also of cells that do not have them, thus contributing to interest in ALS research for diagnosis purposes. In tumor proliferation. Interestingly, glioma stem cells this sense, Saucier et al. conducted a study to identify are able to resist chemotherapy and transfer some an ALS-associated miRNA signature in EVs, which resistance characteristics through EVs, for instance, can cross the BBB and enter the circulatory system, the surface markers CD133 and CD44 or the obtained from plasma samples of people diagnosed adenosine-producing enzyme, which is a cell receptor with ALS (172). Next-generation sequencing was used responsible for the cells’ drug tolerance (179). to identify differentially expressed miRNAs Furthermore, GBM-derived EVs have also been recovered from EVs of ALS patients and healthy described to induce angiogenesis during hypoxia by controls. Their results revealed elevated levels of 5 delivery of proteins (such as IL-8, MMP 8, or PDGF) miRNAs and reduced levels of 22 miRNAs in the EVs and mRNA transcripts to pericytes and vascular of ALS patients compared with healthy controls. endothelial cells (180). Moreover, not only in GBM but Moreover, miRNAs with relevance to ALS were also in other tumors, EVs have also been shown to found to be deregulated. A few miRNAs were also contribute to immunosuppression. Notably, serum identified as potential diagnostic tools for their EVs from patients with advanced melanoma or colon association with disability progression in ALS. In carcinoma inhibited monocyte precursor differen- addition, functional assessment of transcripts targeted tiation into dendritic cells and programmed the by select ALS-associated miRNAs revealed processes precursors to become myeloid suppressive cells, such as transcriptional regulation and protein which suppress T lymphocytes through TGF-β ubiquitination in these patients (172). All these secretion, down-regulation of surface MHC class II findings highlight the miRNA signature in EVs as a expression, and persistent CD14 pattern recognition potential diagnostic strategy for this condition. receptor positivity. In contrast, EVs derived from healthy controls encouraged T lymphocyte stimula- 6.5 EVs in brain cancer tion and myeloid maturation (181). Brain tumors are one of the most feared cancer https://www.ijbs.com Int. J. Biol. Sci. 2023, Vol. 19 737 Table 5. Selected relevant studies of recent findings of EVs for the diagnosis and/or treatment of brain tumors. Fluid of Origin Isolation method Purpose of Target of study Type of study Main Results Reference EVs cell-type of the study EVs Culture GBM Ultracentrifugation Diagnosis Glioma-derived Pre-clinical (In Hypoxic glioma-derived exosomes (H-GDEs) markedly Xu et al. medium and exosomes Vitro and In facilitated autophagy and M2-like macrophage polarization 2021 treatment Vivo) compared with normoxic glioma-derived exosomes (N-GDEs), which subsequently promoted glioma proliferation and migration in vitro and in vivo. In addition, interleukin 6 (IL-6) and miR-155-3p were highly expressed in H-GDEs. Culture GBM Ultracentrifugation Diagnosis GDEs ad Pre-clinical (In The results suggest that malignant glioma-derived Iorgulescu medium and filtration and peripheral immune Vitro) exosomes are restricted in their capacity to directly prime et al. 2016 treatment effectors peripheral immunosuppression. Culture GBM ExoQuick®TC TM Treatment VEGF Pre-clinical (In VEGF-C isoform in GDEs had a role in GBM angiogenesis, Wang et al. medium Vitro and In stimulation of endothelial cell viability, migration, and 2021 Vivo) tubulation. Plasma GBM ExoQuick® one-step Diagnosis/ miRNA-148a Clinical / miR-148a delivered by exosomes may promote cancer cell Cai et al. precipitation solution Treatment Pre-clincal (In proliferation and metastasis via targeting CADM1 to 2020 Vitro) activate STAT3 pathway. This highlight the predictor and therapeutic target role of exosomal miR- 148a for GBM patients. Serum GBM ExoQuick® Diagnosis & miRNA signature Clinical Exosomes were isolated before and after radiotherapy and Li et al. precipitation monitoring exosomes-derived miRNAs sequencing was performed. A 2020 total of 18 up-regulated differentially expressed miRNAs and 16 down-regulated differentially expressed miRNAs were identified. These miRNAs were shown to play a crucial role in glioma by regulating targets and affect the occurrence and development of the disease. Serum / GBM ExoQuick exosome Prognosis ® Exosomal Clinical / Pre-clinical : Ding et al. Culture precipitation kit CircNFIX on TMZ Pre-clinical (In In Vitro: Exosomal CircNFIX confers TMZ resistance in 2020 medium chemoresistance Vivo and In glioma cells Vitro) In Vivo: Exosomal CircNFIX enhances tumor growth and TMZ resistance in vivo Clinical: - Serum exosomal CircNFIX expression is up-regulated in TMZ-resistant patients Bone Neutrophils Ultracentrifugation Treatment Exosomes as drug Pre-clinical (In Exosomes were isolated from neutrophils extracts of mice Wang et al. marrow from bone carriers vivo) bone marrow. NEs-Exos penetrated the BBB and migrated 2021 marrow into the brain. NEs-Exos responded chemotactically to inflammatory stimuli and targeted infiltrating tumor cells. DOX-NEs-Exos suppressed tumor growth and prolong survival time in the glioma in vivo models. BBB, blood brain barrier; CADM1, Cell adhesion molecule 1; CDK6, cyclin dependent kinase 6; CircNFIX, circRNA nuclear factor I X; DE, differentially expressed; DOX, doxorubicin; GBM, glioblastoma; GDEs, glioma-derived exosomes: INF-PA, invasive NF-PA; NEs-Exos, neutrophil-exosomes; NF-PAs, invasiveness of non-functioning pituitary adenomas; NNF-PA, noninvasive NF-PA; RHOU, ras homolog family member U; SPIRE2, spire type actin nucleation factor 2; STAT3,Signal transducer and activator of transcription 3;TMZ, temozolomide;VEGF, vascular endothelial growth factor Many authors have studied the potential of EVs Iorgulescu et al. also explored the immunosuppressive as diagnostic tools in GBM and their role in disease properties of exosomes in the established glioma cell spreading (Table 5). Xu et al. investigated the effects lines U87 and U251. Primary GBM lines, obtained of glioma-derived exosomes on autophagy, tumor- from fresh tumor tissue of two patients, were also associated macrophages (TAMs) polarization, and included in the study (183). Results showed that glioma progression in the human GBM cell lines GDEs displayed exosome-specific markers, but failed U87MG and U251 and the human monocyte cell lines to recapitulate the antigen-presentation machinery, U937 and THP-1. Subsequently, they evaluated the surface co-modulatory signals, or immunosup- roles of hypoxic glioma-derived exosomes (H-GDEs) pressive mediator status of their parent tumor cells. in vivo through tumor xenografts in a BALB/c nude This suggests that GDEs are restricted in their mouse model (182). Their results indicated that capacity to directly prime peripheral immunosup- H-GDEs markedly facilitated autophagy and M2-like pression (183). macrophage polarization compared to normoxic The role of EVs miRNAs on tumor growth and glioma-derived exosomes (N-GDEs). Furthermore, metastasis has gained interest in recent years. Cai et al. IL-6 and miR-155-3p were highly expressed in tried to elucidate the mechanisms through which H-GDEs and induced M2-like macrophage polariza- exosomal miRNAs contribute to GBM cell metastasis tion through the IL-6-pSTAT3-miR-155-3p- by analyzing the effects of exosomes from 30 GBM autophagy-pSTAT3 positive feedback loop. These patients and 30 healthy volunteers in the human alterations promoted glioma proliferation and glioblastoma cell line T98G and the human kidney cell migration in vitro and in vivo. These findings suggest line HEK (184). They identified miR-148a as an IL-6 and miR-155-3p as novel biomarkers for diag- exosomal risk factor for metastasis. Results showed nosis and antitumor immunotherapy of GBM (182). that circulating exosomal miR-148a levels were https://www.ijbs.com Int. J. Biol. Sci. 2023, Vol. 19 738 significantly higher in serum from GBM patients chemoresistance in glioma. Results showed that compared with serum from healthy volunteers. In exosomal CircNFIX conferred TMZ resistance to addition, they also found that both protein and recipient sensitive cells through the enhancement of mRNA levels of cell adhesion molecule 1 (CADM1) cell migration, invasion and repression of cell were decreased in the tissues of GBM patients. In apoptosis under TMZ exposure. Exosomal CircNFIX T98G cells, inhibition of miR-148a suppressed cell also promoted tumor growth and its depletion proliferation and metastasis. Interestingly, there was a enhanced TMZ sensitivity in glioma cells in vivo. strong negative correlation between exosomal Furthermore, the authors found that CircNFIX miR-148a and CADM1 mRNA levels in samples of directly interacted with miR-132, whereas CircNFIX patients (184). These findings suggest that exosomal knockdown enhanced TMZ sensitivity in resistant miR-148a could be a potential treatment target against glioma cells by up-regulating miR-132 (188). This proliferation and metastasis in GBM patients. suggests that exosome-mediated transfer of CircNFIX GBM is one of the most highly vascularized partially enhances TMZ resistance in glioma through human cancers. As previously stated, the role of EVs the miR-132 pathway, thus highlighting a potential in cancer angiogenesis has recently gained attention, prognostic biomarker for improving the clinical but the proangiogenic molecules carried within have benefits of TMZ treatment. Wang et al. also explored yet to be identified. In this regard, Wang et al. the influence of exosomes in chemoresistance in investigated the role of vascular endothelial growth gliomas, but in this case, in terms of improving the factor (VEGF) in GDEs in GBM cell lines and primary efficacy of drugs. Based on the intrinsic inflammatory cultures (185). Their findings revealed that VEGF-C chemotaxis and excellent BBB-crossing capability of from GDEs, by binding to VEGF receptor 2, neutrophils, the authors designed a bioinspired stimulated Tafazzin expression in endothelial cells (a neutrophil-exosomes (NEs-Exos) system with loaded mitochondrial enzyme that exchanges fatty acids doxorubicin (DOX) and evaluated it in two in vivo between phospholipids) (186). This event is derived models of zebrafish and C6-Luc glioma-bearing mice from stimulating endothelial cell viability, migration, (191). In the first step, the authors extracted NEs from and tubulation. In addition, they demonstrated that the bone marrow of mice. Subsequently, exosomes the inhibition of exosomal release, together with were isolated from these NEs. Their results showed bevacizumab treatment, had a combined inhibitory that NEs-Exos rapidly penetrated the BBB and effect on angiogenesis (185). Similarly, Li et al. studied migrated into the brain. Additionally, NEs-Exos the miRNA signatures in exosomes to evaluate the responded chemotactically to inflammatory stimuli effect of radiotherapy in glioma patients (187). Exo- and targeted infiltrating tumor cells. Moreover, somes were isolated before and after radiotherapy, DOX-NEs-Exos efficiently suppressed tumor growth and exosome-derived miRNA sequencing was per- and prolonged survival time in glioma in vivo models formed. A total of 18 upregulated differentially (191). These results represent a promising expressed miRNAs and 16 down-regulated chemotherapeutic approach for the clinical treatment differentially expressed miRNAs were identified. of glioma and other brain diseases. Following radiotherapy, qPCR analysis revealed that the hsa-miR-6731-5p and hsa-miR-208b-3p were 7. Conclusion overexpressed, while the hsa-miR-2116-3p was The biogenesis of EVs is initiated by an underexpressed. Moreover, target genes of identified invagination of the plasma membrane of the origin miRNAs were shown to be primarily involved in cell. This invagination includes a cell-surface metabolic processes, p53 signaling pathways, and protein-specific fingerprint and soluble molecules cancer pathways. This suggests that these miRNAs associated with the extracellular matrix, such as ions, could play a crucial role in glioma development (187). metabolites, peptides, or lipids. This gives EVs a Ding et al. evaluated the role of circular RNAs crucial role as promising biomarker tools for many (circRNA) in the resistance to chemotherapy diseases, in addition to their biological relevance and treatments, a major obstacle for glioma management easy accessibility from a broad range of body fluids. (188). circRNAs are a novel class of endogenous They have been identified in various human diseases, non-coding RNAs that form covalently closed loops such as AD, cancers, cardiovascular diseases, (189). These molecules have been found to be sarcoidosis, or prion diseases. In addition, they have associated with the initiation and progression of been described to be involved in many physiological different human diseases, including cancers (190). In functions, such as cellular intercommunication, this work, the authors explored the effect and maintenance and improvement of synaptic plasticity, underlying mechanism of exosomal circRNA nuclear neurotransmission in the adult brain, innate immune factor I X (CircNFIX) on temozolomide (TMZ) responses, human reproduction, pregnancy, and https://www.ijbs.com Int. J. Biol. Sci. 2023, Vol. 19 739 embryonic development or wound healing. Since EVs ISCIII Acción Estratégica en Salud, integrated in the are released into body fluids, this affords us the Spanish National R+D+I Plan and financed by ISCIII opportunity to use these vesicles as promising Subdirección General de Evaluación and the Fondo candidates for diagnostic and therapeutic purposes. Europeo de Desarrollo Regional (FEDER “Una Specifically, they have been shown to possess critical manera de hacer Europa") grant PI17/01474, information about pathological processes underlying PI19/00335, PI22/01403 and PI22/00258. The support major brain diseases. In AD, it has been described that of CIBERNED (ISCIII) under the grants EVs play an important role in spreading hallmarks CB06/05/2004 and CB18/05/00010. The support across the brain and are involved in APP cleavage, from PREADAPT project. Joint Program for delivery of pathogenic mRNA, and an increase in Neurodegenerative Diseases (JPND) grant Nº Aβ1-42 production. In PD, EVs have been shown to AC19/00097, from HARPONE Project, Agency for contain α-synuclein, which has been related to the Innovation and Entrepreneurship (VLAIO) grant Nº acceleration of the α-synuclein cytotoxic effects and PR067/21, and from DESCARTES project, German the pathogenesis severity. In MS, EVs secreted by Research Foundation (DFG). The support of oligodendrocytes negatively contribute to the Fundación bancaria “La Caixa” and Grífols SA homeostasis of myelin biogenesis and induce (GR@ACE project). disintegration of the BBB, thus facilitating lymphocyte and myeloid cell transmigration into the CNS. In ALS, Author Contributions several proteins involved in its pathogenesis have AC performed the conceptualization, biblio- been shown to be transported from cell to cell by the graphic search and writing of the original draft, EV pathway, contributing to the propagation of figures and tables. ME, MB, RP, EEA and ESL prion-like misfolded isoforms and increasing the contributed in the bibliographic search and writing of neuropathology of ALS. Finally, in GBM, a growing sections. EBS, AC, MM, MIP and MB contributed in body of evidence suggests that gliomas can influence the supervision, writing/review and critical editing. cells both nearby and beyond the BBB through the AR contributed in the supervision, writing/review regulated excretion of EVs. The EV content of GBMs and critical editing, project administration, resources reflects the tumor’s unique signature and is and funding acquisition. All authors have made a implicated in the transfection of receptors such as substantial contribution to the work. All authors have PDGFR and HER2 to non-tumoral cells. In conclusion, read and agreed to the published version of the EVs have a promising future as drug targets, manuscript. monitoring tools, and diagnostic tools for many diseases, especially those with difficult access and Competing Interests complex etiologies, such as major brain diseases. The authors have declared that no competing interest exists. 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