Promise of extracellular vesicles for diagnosis and treatment of epilepsy

Highlights

• Evaluation of brain-derived EVs in the blood may help in identifying biomarkers for distinct types of epilepsies.

• Investigation of brain-derived EVs in the blood could help in identifying miRNAs involved in epileptogenesis.

• EVs released from mesenchymal stem cells have therapeutic properties.

• Stem cell-derived EV therapy appears attractive for counteracting epileptogenic changes after SE.

• EVs can be engineered to deliver specific miRNAs, proteins, or antiepileptic drugs to the brain.

Abstract

Extracellular vesicles (EVs) released from cells play vital roles in intercellular communication. Moreover, EVs released from stem cells have therapeutic properties. This review confers the potential of brain-derived EVs in the cerebrospinal fluid (CSF) and the serum as sources of epilepsy-related biomarkers, and the promise of mesenchymal stem cell (MSC)-derived EVs for easing status epilepticus (SE)-induced adverse changes in the brain. Extracellular vesicles shed from neurons and glia in the brain can also be found in the circulating blood as EVs cross the blood–brain barrier (BBB). Evaluation of neuron and/or glia-derived EVs in the blood of patients who have epilepsy could help in identifying specific biomarkers for distinct types of epilepsies. Such a liquid biopsy approach is also amenable for repeated analysis in clinical trials for comprehending treatment efficacy, disease progression, and mechanisms of therapeutic interventions. Extracellular vesicle biomarker studies in animal prototypes of epilepsy, in addition, could help in identifying specific micro ribonucleic acid (miRNAs) contributing to epileptogenesis, seizures, or cognitive dysfunction in different types of epilepsy. Furthermore, intranasal (IN) administration of MSC-derived EVs after SE has shown efficacy for restraining SE-induced neuroinflammation, aberrant neurogenesis, and cognitive dysfunction in an animal prototype. Clinical translation of EV therapy as an adjunct to antiepileptic drugs appears attractive to counteract the progression of SE-induced epileptogenic changes, as the risk for thrombosis or tumor is minimal with nanosized EVs. Also, EVs can be engineered to deliver specific miRNAs, proteins, or antiepileptic drugs to the brain since they incorporate into neurons and glia throughout the brain after IN administration.

This article is part of the Special Issue “NEWroscience 2018"

Introduction

A variety of vesicles released by cells into the extracellular space are generically referred to as extracellular vesicles (EVs) [1]. Extracellular vesicles, found in all body fluids, are delimited by a phospholipid bilayer and contain nucleic acids and proteins from donor cells [2]. Extracellular vesicles partake in intercellular communication and likely also influence many cellular processes. Studies characterizing the composition of EVs in various body fluids have suggested that an analysis of EVs from the serum or the cerebrospinal fluid (CSF) could provide information on specific biomarkers in various neurodegenerative diseases. Investigation of EVs isolated from the serum or CSF has advantages because such characterization allows the analysis of the composition of EVs secreted from specific brain cells (e.g., EVs derived from neurons vis-à-vis astrocytes). Also, the configuration of EVs is likely more stable than soluble biomarkers found in the CSF or the serum.

Extracellular vesicles are broadly classified as microvesicles (MVs) or exosomes (EXs) based on their intracellular origin. Microvesicles bud off from the plasma membrane and are rich in negatively charged phospholipids such as phosphatidylserine at their surface [2] (Fig. 1). The size of MVs can vary from 100 to 1000 nm [3]. The composition of MVs depends on the cell type from which they are generated and the type of stimulation causing their creation. However, proteins specific to MVs are yet to be identified [2]. In contrast, EXs originate in endosomes as intraluminal vesicles through inward invagination of the endosomal membrane, which eventually results in the formation of multivesicular bodies (MVBs) containing a constellation of EXs (Fig. 1). While some MVBs fuse with lysosomes to degrade their cargo, others fuse with the plasma membrane to secrete EXs into the extracellular space [4] (Fig. 1). The creation of EXs, particularly the formation of intraluminal vesicles, requires the involvement of intricate protein machinery termed the endosomal sorting complex required for transport (ESCRT). Nonetheless, the formation of EXs can also occur in an ESCRT-independent manner, which involves the participation of syndecan/syntenin/ALG-2-interacting protein X (ALIX) pathway [5]. Besides, CD9- and CD63-dependent pathways have also been implicated in EX formation [2], [6], [7]. The EXs exhibit well-demarcated round morphology, and their size can vary from 30 to 100 nm, depending on the type of cells from which they are released [8], [9], [10], [11]. While the kinetics, the composition, and the biological properties of EXs vary significantly in health and disease conditions [12], EXs from various sources have been isolated, characterized, and customized for specific applications. Analyses of the composition of EXs derived from multiple sources have implied that EXs are rich in proteins, lipids, and nucleic acids derived from their parental cells.

The details on the composition of proteins, lipids, and RNA in EVs or EXs can be seen in three different web-based resources, either at http://www.exocarta.org/ or Vesiclepedia or EVpedia. These databases suggest frequently observed protein components in EVs and EXs, which include annexins, cytoskeletal proteins, heat shock proteins, integrins, metabolic enzymes, ribosomal proteins, tetraspanins, and vesicle trafficking-related proteins [13]. The major lipid components of EXs derived from various cells are recently reviewed [14], which includes cholesterol, sphingomyelin, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylethanolamine ethers, diacylglycerol, phosphatidylcholine ethers, hexosyl ceramide, ceramides, phosphatidylglycerol, phosphatidic acid, phosphatidylinositol, lactosylceramide, lysophosphatidylinositol, lysophosphatidylethanolamine, cholesteryl ester, globotriaosylceramide, etc. [14]. The nucleic acids in EXs comprise mRNAs, microRNAs (miRNAs), and other noncoding RNAs. While the composition of EXs varies depending on the type of cell from which EXs are released, the exact mechanism by which a variety of molecules are loaded into EXs is still unknown. Interestingly, the protein composition of EXs is more likely to be different from their mother cells than the protein composition in MVs [15]. Moreover, EXs tend to be enriched with proteins of extracellular matrix, heparin-binding receptors, and immune response and cell adhesion functions. Conversely, MVs are enriched with proteins from the endoplasmic reticulum, proteasome, and mitochondria [2]. Following their release by donor cells, EXs can enter recipient cells, influence their function by activating intracellular signaling pathways through the transfer of their cargo. The uptake of EXs or its cargo by recipient cells can occur through several mechanisms. For instance, endocytosis of EXs depends on caveolae in epithelial cells [16], clathrin in neurons [17], and cholesterol and lipid raft in endothelial cells [18]. The incorporation of EXs by microglia and macrophages involves macropinocytosis [19] or phagocytosis [20].

Since the size range of MVs and EXs overlaps, a rigorous characterization of EV properties is necessary for classifying them as either MVs or EXs. Initially, an evaluation of at least three specific markers was considered essential to classify EVs as EXs [21]. The markers include tetraspanins CD9, CD63, CD81, the endosomal proteins tumor susceptibility gene 101 (TSG101), and ALIX. Because some of these markers can also be found in MVs [22], recent guidelines from the International Society for Extracellular Vesicles (ISEV) suggest the use of operational terms unless the release of EXs by cells is caught in the act through live imaging techniques. The suggested operational terms are based on physical characteristics of EVs such as the size (small EVs, medium/large EVs), biochemical composition (e.g., CD63 +/CD81 +-/CD9 +- EVs), or cells of origin (e.g., neuron-derived EVs, astrocyte-derived EVs, or microglia-derived EVs) [1]. Therefore, we employed the phrase “EVs” for all EV or EX studies discussed in this review in the subsequent sections.

The goal of this article was to confer two specific issues about EVs. The first segment of the review deliberates the potential of EVs in body fluids, particularly in the serum and the CSF, as sources of specific epilepsy biomarkers. The second part of the review confers the promise of mesenchymal stem cell (MSC)-derived EVs for treating status epilepticus (SE)-induced brain dysfunction and chronic epilepsy.