Extracellular Vesicles in the Treatment of Neurological Disorders

Abstract

Extracellular vesicles (EVs) are small, cell-derived membranous particles containing various nucleic acids, proteins, and lipids that play essential roles in intercellular communication through the transfer of their cargos. Current evidence suggests that part of the regenerative benefit from stem cell therapy arises through EVs released from transplanted cells, which generated interest in using EVs for clinical applications. EVs from various cellular sources, including mesenchymal stem cells, neural stem cells, and glia, are efficacious in models of neurological disease. In these models, EVs attenuate reactive gliosis, neuronal death, pro-inflammatory signaling, as well as reduce cognitive, behavioral, and motor deficits. An advantage of using EVs is that they are naturally permeable to the blood-brain barrier and can be modified to contain molecules of interest, thereby serving as a vehicle to transport therapeutics into the brain. This review summarizes the current state of research using EVs as a therapeutic system in models of neurological disorders and highlights considerations for future research.

1. Introduction

Disorders of the central nervous system (CNS) represent a leading cause of death worldwide (Feigin et al., 2019). Unique challenges associated with treating neurological disorders include the limited permeability of the blood-brain barrier (BBB) and the wide range of clinical features associated with CNS disorders. Over the last several years, extracellular vesicles (EVs) have emerged as a potential treatment for CNS disorders. EVs are small, membranous particles released by most cells and can facilitate intercellular communication. The existence of EVs has been documented for decades (Anderson, 1969; Benz and Moses, 1974; Dalton, 1975), but more recent research demonstrated that EVs isolated from specific sources could have anti-inflammatory and neuroprotective properties (Doeppner et al., 2015; Jarmalaviciute et al., 2015; Xin et al., 2013a; Zhang et al., 2014; Zhang et al., 2015). Thus, research leveraging EVs as a treatment for CNS disorders is progressing rapidly. This review focuses on current research using EVs and highlights important considerations for future research.

2. Extracellular Vesicles

2.1. Biogenesis, Classification, and Function of Extracellular Vesicles

EVs are cell-derived, membranous vesicles released into the extracellular space (Hessvik and Llorente, 2018; Raposo and Stoorvogel, 2013; Yanez-Mo et al., 2015). EVs are highly heterogeneous but can be broadly divided into three categories: exosomes, microvesicles, and apoptotic bodies (Table 1). Exosomes are the smallest type of EV and are released when multivesicular bodies (MVBs), a type of late endosome with internal vesicles formed by the inward budding of the membrane, fuse with the cellular membrane (Hessvik and Llorente, 2018) (Figure 1a). In contrast, microvesicles are shed directly from the outer plasma membrane by its outward budding and, on average, tend to be larger than exosomes (Raposo and Stoorvogel, 2013) (Figure 1b). Apoptotic bodies are the largest EV type and contain degraded fragments of cells undergoing programmed cell death (Yanez-Mo et al., 2015). Unlike exosomes and microvesicles released from healthy cells, apoptotic bodies are released during apoptosis. Apoptotic bodies will not be discussed in this review.

Table 1:

Characteristics of EV Subtypes

EV Subtype	Average Size (diameter)	Biogenesis	Functions	Contents
Exosomes	50–150 nm	Released when MVBs fuse with the outer plasma membrane	Intercellular communication	Proteins, nucleic acids (commonly small RNAs), and lipids
Microvesicles	100–1000 nm	Shed directly from the outer plasma membrane	Intercellular communication	Proteins, nucleic acids (commonly small RNAs), and lipids
Apoptotic Bodies	Up to 5 μm	Shed from cells undergoing programmed cell death	Facilitation of phagocytosis	Cellular debris, organelle fractions

Abbreviations: Extracellular Vesicle (EV), Multivesicular Bodies (MVBs)

Figure 1: Biogenesis of EVs.

A: Exosomes arise from the endosomal system when multivesicular bodies (MVBs) fuse with the plasma membrane. The intralumenal vesicles of MVBs, upon their release into the extracellular space, become exosomes. B: Microvesicles arise by budding directly off the plasma membrane into the extracellular space. Created with BioRender.com

Early research on EVs documented their role in shuttling unneeded or excess proteins out of cells as a mechanism to eliminate cellular waste (Johnstone et al., 1987; Pan et al., 1985). More recently, EVs are being recognized for their role in intercellular communication (Delpech et al., 2019; Kourembanas, 2015; Raposo and Stoorvogel, 2013; Yanez-Mo et al., 2015; Zappulli et al., 2016). The contents of EVs, which include lipids, proteins, and nucleic acids (Table 1), are highly heterogeneous and dependent on the type and condition of the parental cell (van Niel et al., 2018). However, EVs are typically enriched for particular classes of molecules, such as microRNAs (miRNAs) (Raposo and Stoorvogel, 2013; Yanez-Mo et al., 2015). Cargo can be passively incorporated or actively targeted to EVs, and the mechanism of loading varies based on the type of cargo and EV subtype (Raposo and Stoorvogel, 2013; van Niel et al., 2018).

After EVs are released, they move into the extracellular space and can exert their effect on target cells via multiple mechanisms. Surface molecules on EVs can interact with extracellular receptors on targets cells (van Niel et al., 2018; Yanez-Mo et al., 2015). EVs can also fuse with the cellular membrane, releasing their contents, or be taken up via processes such as endocytosis (van Niel et al., 2018; Yanez-Mo et al., 2015). Components of the EV not integrated into receiving cells can enter the endosomal pathway or be degraded via endogenous cellular mechanisms (van Niel et al., 2018).

In the CNS, EVs participate in a range of neurobiological processes (Budnik et al., 2016; Zappulli et al., 2016). For example, EVs shuttle between pre-synaptic and post-synaptic neurons in a synaptic activity- dependent manner and contribute to signaling and protein expression modifications that underlie synaptic plasticity (Korkut et al., 2013; Lachenal et al., 2011). Conversely, EVs also contribute to pathological processes. For example, EVs from glioblastoma tumor cells facilitate the spread of pathology by promoting tumor proliferation and remodeling the surrounding microvasculature (Skog et al., 2008).

2.2. Isolation and Nomenclature of Extracellular Vesicles

Most EV preparations destined for use as an experimental therapy are isolated from cell culture media, but EVs can also be isolated from biofluids and tissue (Konoshenko et al., 2018). Many different methods can be used to isolate and purify EVs, and considerations in selecting an appropriate method include cost, the downstream application, and the desire to enrich a particular subtype of EV (Buschmann et al., 2018; Konoshenko et al., 2018). Several different methods, including ultracentrifugation polymer-based precipitation, and filtration, can be used to isolate EVs based on their size or density. In contrast, affinity-based methods, like immunoprecipitation, can be used to purify EVs based on their expression of specific molecules (Konoshenko et al., 2018; Li et al., 2017). As EV isolation protocols can vary widely, professional societies like the International Society of Extracellular Vesicles (ISEV) have created guidelines that encourage transparency in publication and standardization in validation procedures (Thery et al., 2018). Typically, a combination of techniques is used in parallel to verify EV properties. Techniques commonly used include immunoblotting for positive and negative EV markers, nanoparticle tacking analysis to determine the concentration or size distribution of the preparation, and electron microscopy to verify EV morphology.

As our knowledge of EV biology rapidly progresses, the nomenclature and classification of EVs also change. Additionally, exosomes and microvesicles possess several overlapping features, including protein expression and size (See Table 1). Thus, while it is possible to isolate a population of EVs enriched for certain properties, it is difficult to achieve complete separation between exosomes and microvesicles (van Niel et al., 2018). Therefore, for clarity and consistency in nomenclature, ISEV currently recommends that the terms “extracellular vesicles” or “EVs” be used to describe most EV preparations unless the biogenesis pathway is confirmed (Thery et al., 2018). In line with the current ISEV recommendations, this review will use the term “EV.”

3. Extracellular Vesicles as a Therapy

EVs garnered attention as a potential therapeutic system when evidence emerged suggesting that they were responsible for some of the beneficial effects of stem cell therapies (Cantaluppi et al., 2012; Lai et al., 2010; Tomasoni et al., 2013; Xin et al., 2013b; Zhu et al., 2014). Stem cell therapy still has significant safety concerns, including the possibility of tumorigenesis and the immunological rejection of transplanted cells (Herberts et al., 2011; Volarevic et al., 2018). EVs represent a potential cell-free alternative that can recapitulate benefits of cell therapies (Rani et al., 2015). EVs from stem cells, as well as from some terminally differentiated cell types, show anti-inflammatory and regenerative properties in many different tissues, such as the lung, heart, and brain (Lai et al., 2010; Xin et al., 2013a; Zhu et al., 2014). The efficacy of EVs has been evaluated in several models of neurological disease, including animal models of neurological injury and neurodegenerative disease (An et al., 2013; Xin et al., 2013a; Zhang et al., 2015). In these models, EVs reduce neuropathology and ameliorate behavioral and cognitive deficits (Doeppner et al., 2015; Xin et al., 2013a; Zhang et al., 2015). Furthermore, EVs are an attractive potential treatment for neurological disorders as they are endocytosed by the endothelial cells of the brain microvasculature, allowing them to cross the BBB (Chen et al., 2016a; Garcia- Romero et al., 2017). This property also allows EVs to be used as a tool to transport exogenous molecules across the BBB (Rufino-Ramos et al., 2017).

Mesenchymal stem cells (MSCs) are often used for cell-based therapies due to their regenerative properties, ability to expand ex vivo, and ethical suitability (Liu et al., 2016; Trounson et al., 2011). Thus, as EVs garnered attention as a therapeutic alternative to cell-based therapies, MSC-derived EVs became the most common type of EVs used as a treatment in neurological disease models (Doeppner et al., 2015; Xin et al., 2013a; Zhang et al., 2015). More recently, EVs isolated from various sources, particularly cells from the CNS, are of growing interest in treating neurological diseases. For example, EVs isolated from neural stem cells (NSCs) (Webb et al., 2018a; Webb et al., 2018b), astrocytes (Pei et al., 2019), and endothelial cells (Pan et al., 2016) have been shown to reduce pathological features common in neurological disorders.

4. Neurological Insults and Injuries

In neurological disorders such as stroke, traumatic brain injury (TBI), and some types of epilepsy, an initial brain injury causes neurological damage that precipitates disease symptoms. While each of these disorders has unique features, they share many characteristics. For example, widespread neuroinflammatory signaling is associated with stroke (Iadecola and Anrather, 2011; Jin et al., 2013), TBI (Corps et al., 2015; Dashnaw et al., 2012), and epilepsy (Crespel et al., 2002; Ravizza et al., 2008; Vezzani et al., 2011). While this immune response can initiate reparative processes, aspects of these inflammatory cascades can lead to further neuronal damage and cell death, contributing to the deficits associated with the disorder (Jayaraj et al., 2019; Waisman et al., 2015). Thus, the ability of EVs to suppress these inflammatory cascades and reduce cell death makes them a promising treatment.

4.1. Stroke

A stroke occurs when the blood supply to part of the brain is disrupted, preventing cells from receiving adequate amounts of oxygen and nutrients (Aho K, 1980; Sacco et al., 2013). Stroke can be broadly classified as ischemic or hemorrhagic due to the blockage of a cerebral artery or a ruptured blood vessel in the brain, respectively. (Amarenco et al., 2009). The resulting neuronal damage and cell death can lead to gliosis and the activation of neuroinflammatory cascades, furthering neuronal injury (Iadecola and Anrather, 2011; Jin et al., 2013).

A considerable amount of literature exists on the effects of EV administration on neuroinflammatory cascades, cell death, and behavioral deficits in animal models of stroke (Table 2). In an early report, Xin et al. administered a single intravenous dose of MSC-EVs following middle cerebral artery occlusion (MCAO) in rats. Four weeks later, they observed decreased motor coordination deficits, increased neurogenesis, increased angiogenesis, and increased neurite remodeling (Xin et al., 2013a). MSC-EV treatment in stroke models has also been shown to reduce the expression of pro-inflammatory cytokines, such as IL-1β and TNF-α (Chen et al., 2016b; Dabrowska et al., 2019; Zhao et al., 2020), as well as decrease oxidative stress and cell death markers, such as NOX-2 and cleaved caspase proteins (Chen et al., 2016b). Furthermore, Deng et al. showed that intracerebroventricular (ICV) MSC-EV treatment at the time of transient global cerebral ischemia in mice partially rescued deficits in basal synaptic transmission and long-term potentiation and was associated with improved learning and memory performance (Deng et al., 2017). Intravenous (IV) treatment with MSC-EVs in rodent stroke models has been shown to promote neuron myelination (Otero-Ortega et al., 2017) as well as reduce peripheral immune system dysregulation (Doeppner et al., 2015). Intravenous MSC-EV administration was also associated with improved motor skills recovery and reduced microgliosis in a non-human primate model of cortical injury (Go et al., 2020; Medalla et al., 2020; Moore et al., 2019). These studies suggest that MSC-EVs can attenuate various pathological features of stroke, including cellular, electrophysiological, and behavioral deficits.

Table 2:

EVs as a treatment in models of neurological injury

Source	Disorder	Effects of EV Treatment	Citations
MSC	Stroke	↑ motor performance ↑ neurogenesis ↑ angiogenesis ↑ normal LTP ↑ neuronal density ↑ learning and memory ↑ axonal sprouting ↑ white matter and myelin regeneration ↓ neuroinflammatory markers ↓ peripheral immune dysregulation ↓ gliosis	Xin et al., 2013a; Doeppner et al., 2015; Otero-Ortega et al., 2017; Moore et al., 2019 Xin et al., 2013a; Doeppner et al., 2015 Xin et al., 2013a; Doeppner et al., 2015; Chen et al., 2016 Deng et al., 2017 Doeppner et al., 2015 Deng et al., 2017 Otero-Ortega et al., 2017 Otero-Ortega et al., 2017 Chen et al., 2016; Deng et al., 2017; Dabrowska et al., 2019; Zhao et al., 2020 Doeppner et al., 2015 Chen et al., 2016; Dabrowska et al., 2019; Go et al., 2020
	TBI	↑ motor performance ↑ angiogenesis ↑ learning and memory ↑ speed of recovery ↓ neuroinflammatory markers ↓ gliosis ↓ lesion size	Patel et al., 2018; Williams et al., 2019 Zhang et al., 2015 Zhang et al., 2015; Kim et al., 2016 Williams et al., 2019 Kim et al., 2016 Zhang et al., 2015 Ni et al., 2019; Williams et al., 2020
	MTLE	↑ neurogenesis ↑ neuronal density ↑ learning and memory ↓ pro-inflammatory markers ↓ gliosis	Long et al., 2017; Xian et al., 2019
NSC	Stroke	↑ motor performance ↓ lesion size	Mahdavipour et al. 2020; Webb et al., 2018a; Webb et al., 2018b Webb et al., 2018a; Webb et al., 2018b; Mahdavipour et al. 2020
	TBI	↑ motor performance ↓ lesion size	Sun et al., 2020
	MTLE	↓ neuroinflammatory markers	Upadhya et al., 2020
Astrocytes	Stroke	↓ apoptosis ↓ lesion size ↓ neuroinflammatory markers	Pei et al., 2019; Pei et al., 2020 Pei et al., 2019
	TBI	↑ mitochondrial function ↑ neuronal recovery ↓ apoptosis	Chen et al., 2020b
Endothelial Cells/ECFC)	Stroke	↑ motor performance ↑ white matter and myelin regeneration ↑ BBB integrity ↓ apoptosis	Pan et al., 2016; Venkat et al. 2019 Venkat et al. 2019 Pan et al., 2016
	TBI	↑ motor performance ↑ BBB integrity	Gao et al., 2018

Abbreviations: Extracellular Vesicle (EV), Mesenchymal stem cell (MSC), Neural Stem Cell (NSC), Traumatic brain injury (TBI), Mesial temporal lobe epilepsy (MTLE), Blood-brain barrier (BBB), Long-term potentiation (LTP), Endothelial colony-forming cells (ECFC)

EVs isolated from other sources were also evaluated for their ability to reduce disease pathology in stroke models. Intravenously delivered endothelial cell-derived EVs attenuate neurological and cognitive deficits in rodent models of stroke (Pan et al., 2016; Venkat et al., 2019). Furthermore, EVs derived from glial cells (Pei et al., 2019) and neural stem cells (NSCs) (Mahdavipour et al., 2020; Spellicy et al., 2020; Webb et al., 2018a; Webb et al., 2018b) also show neuroprotective effects in animal stroke models. Despite accumulating evidence that EVs from different sources may be therapeutic, the relative efficacy of EVs based on the cell type of origin remains poorly characterized. For example, Webb et al. directly compared the effects of MSC-EV and NSC-derived EVs (NSC-EVs) treatment in a mouse stroke model. Their results showed that NSC-EVs administered intravenously had a greater ability to improve functional recovery than MSC-EVs (Webb et al., 2018b), suggesting that treatment efficacy may depend on the origin of the EVs.

Conflicting reports exist on the ability of EV treatment to reduce lesion size as some studies reported reduced lesion size with treatment (Chen et al., 2016b; Nalamolu et al., 2019), while others found no effect (Moore et al., 2019; Otero-Ortega et al., 2017). These variable results might be explained by differences in the animal models and the intervals between injury, treatment, and lesion measurement. Similarly, while several studies showed that intravenously delivered MSC-EVs can improve motor and sensory function after stroke (Chen et al., 2016b; Doeppner et al., 2015; Otero-Ortega et al., 2017; Xin et al., 2013a), Nalamolu et al. did not observe similar benefits from IV MSC-EV treatment when measured seven days after injury (Nalamolu et al., 2019). Differences between reports on the effect of EVs on recovery may be explained by the timing of symptom evaluation as several studies detected an effect of treatment 14 days or later after injury but did not observe any treatment effect after seven days (Otero-Ortega et al., 2017; Xin et al., 2013a). Overall, current evidence supports the ability of EVs to reduce pathological features of stroke in animal models, but the efficacy of such treatments depends on the relative timing between the injury, symptom assessment, and EV administration.

4.2. Traumatic Brain Injury

A traumatic brain injury (TBI) occurs when a sudden trauma to the brain leads to disrupted brain function (Menon et al., 2010; Wang et al., 2018). TBI symptoms can range from mild, temporary alternations of consciousness to severe injury and possible death (Dixon, 2017; Saatman et al., 2008). After TBI, widespread inflammatory signaling, such as gliosis and cytotoxic pro-inflammatory cytokine release, can further increase neuronal death and degradation of the BBB (Corps et al., 2015; Dashnaw et al., 2012). Thus, neurological injury after a TBI arises from both the trauma of the initial event, as well as secondary damage caused by the activation of cytotoxic inflammatory responses (Corps et al., 2015; Dashnaw et al., 2012).

MSC-EVs were found to reduce associated pathology in TBI models (Table 2). For example, Zhang et al. intravenously administered MSC-EVs one day after injury in a controlled cortical impact (CCI) rat TBI model and observed increased vascular density, increased hippocampal neurogenesis, and reduced reactive gliosis. The treated rats also showed functional and behavioral recovery, as reflected in lower neurological deficit scores, and improved learning and memory (Zhang et al., 2015). Other studies using rodent TBI models that received IV MSC-EVs also observed improved cognitive outcomes, reduced expression of inflammatory cytokines and apoptotic markers, and a shift in microglial polarization towards a protective anti-inflammatory phenotype (Kim et al., 2016; Ni et al., 2019).

EVs also show efficacy in large animal models of TBI. MSC-EV-treated swine showed less impairment on a neurological severity rating scale after combined TBI and hemorrhagic shock (Williams et al., 2019). A subsequent study in this swine model demonstrated that IV MSC-EV treatment also attenuated brain swelling, lesion size, and BBB breakdown (Williams et al., 2020).

EVs isolated from CNS-derived cells may also reduce pathology and attenuate behavioral deficits in TBI animal models. For example, IV NSC-EV administration after TBI reduced lesion size and improved motor recovery in male rats after CCI, although less recovery was observed in female rats (Sun et al., 2020). In another study, Chen et al. reported that astrocyte-derived EVs reduced apoptosis and mitochondrial dysfunction after neuronal injury in vitro and ICV injection of these EVs improved neuronal recovery after a fluid percussion injury in rats (Chen et al., 2020b). Intravenous treatment with endothelial colony-forming cell- derived EVs improved motor recovery and BBB integrity after TBI in mice (Gao et al., 2018). These studies show that EVs from multiple cellular sources can reduce pathological and behavioral features of TBI in animal models.

4.3. Mesial Temporal Lobe Epilepsy

Temporal lobe epilepsy (TLE) is the most common form of treatment-resistant epilepsy in adults, and mesial TLE (MTLE) is the most common form of TLE (Engel, 1996; Engel, 2001). MTLE is associated with recurrent spontaneous seizures, neuropsychological deficits, and hippocampal sclerosis (Blumcke et al., 2013; Engel, 2001; Falconer et al., 1964; Thom, 2014). Patients often experience an initial neurological insult, such as a TBI or complex early-life febrile seizures, that contribute to the eventual development of TLE (Engel, 1996; Falconer et al., 1964). Rodent models of MTLE can be generated by administering chemiconvulsants, such as pilocarpine or kainic acid, to cause an initial period of status epilepticus (SE) that is followed by neuropathological changes, including a robust immune response and neuronal cell loss. These changes contribute to the development of behavioral abnormalities and spontaneous seizures in these models (Jiang et al., 2015; Mazzuferi et al., 2012; Wang et al., 2015).

To date, only a few studies have investigated the efficacy of EV treatment in models of MTLE. Long et al. intranasally administered MSC-EVs after pilocarpine-induced SE and observed decreased expression of pro-inflammatory cytokines and increased expression of anti-inflammatory cytokines 24 hours post-SE (Long et al., 2017). MSC-EV treatment was also associated with decreased density of reactive microglia and reduced hippocampal neuron loss four days post-SE. When tested five to six weeks after SE, MSC-EV-treated mice showed increased hippocampal neurogenesis and decreased cognitive and memory impairments. In another study using the pilocarpine model, Xian et al. intracerebroventricularly (ICV) administered MSC-EVs after SE and similarly observed decreased pro-inflammatory cytokine expression four days post-SE, and attenuation of learning and memory deficits eight weeks post-SE (Xian et al., 2019). They also noted decreased expression of astrogliosis markers, including GFAP and C3 (Xian et al., 2019). Recently, Upadhya et al. demonstrated that intranasal treatment with NSC-EVs after pilocarpine-induced SE reduced expression of inflammatory proteins, including TNF-α, IL-1β, and IFN-γ, when measured twenty-four hours later (Upadhya et al., 2020). These results suggest EV treatment might be efficacious in mouse models of MTLE; however, the ability of EVs to reduce the development of spontaneous seizures in MTLE models has yet to be reported.

5. Neurodegenerative Diseases

Neurodegenerative diseases (NDs) are a diverse group of disorders in which there is progressive degeneration or death of neurons. Clinical features depend on the function of the primarily affected cells but typically involve a decline in cognitive ability or motor control that worsens over time. Although each disorder has distinct characteristics and neurobiology, NDs share several features. For example, many NDs display pathological accumulation of proteins, such as tau and amyloid-β in Alzheimer’s disease, α-synuclein in Parkinson’s disease, and SOD1 or TMP-34 in amyotrophic lateral sclerosis (Peng et al., 2020; Ross and Poirier, 2004). Also, aberrant immune activation or dysregulation in many NDs is associated with increased neuronal damage (Heneka et al., 2014; Stephenson et al., 2018).

Interestingly, endogenous EVs have been implicated in the pathological accumulation of proteins in several NDs (Gomes et al., 2007; Iguchi et al., 2016; Shi et al., 2014). Toxic, aggregation-prone proteins can be packaged into EVs that are then released, resulting in the transfer of toxic cargo to other cells (Coleman and Hill, 2015; You and Ikezu, 2019). Thus, targeting or selectively inhibiting the biogenesis or release of EVs carrying potentially toxic proteins is an area of active research (You and Ikezu, 2019). For example, inhibition of nSMase 2, an enzyme that synthesizes ceramide and promotes exosome release, has been studied in the context of neurodegenerative disorders. In preclinical models, nSMase 2 inhibitors were capable of reducing amyloid-β and tau pathology in Alzheimer’s disease (Bilousova et al., 2018; Dinkins et al., 2014; Dinkins et al., 2016) and α-synuclein pathology in Parkinson’s disease (Sackmann et al., 2019; Zhu et al., 2021). However, it is possible that broadly interfering with EV release could adversely alter intercellular communication, and the consequences of this are not yet understood. While endogenous EVs may contribute to disease progression in NDs, exogenous EVs are a potential treatment as they can aid in the clearing of aggregated proteins (An et al., 2013; Ding et al., 2018; Lee et al., 2016) and the reduction of immune dysregulation (Ding et al., 2018; Laso-Garcia et al., 2018; Rajan et al., 2016).

5.1. Alzheimer’s Disease

Alzheimer’s disease (AD) is the leading cause of dementia worldwide and is characterized by progressive cognitive decline and memory loss (Reitz et al., 2011; Scheltens et al., 2016). AD is associated with neurofibrillary tangles of phosphorylated tau protein and plaques composed of amyloid-β protein (Aβ), which can spread throughout the brain and contribute to neuronal degeneration (Jack et al., 2018; Reitz et al., 2011).

Katsuda et al. showed that MSC-EVs carry neprilysin, an enzyme that can degrade Aβ (Katsuda et al., 2013). When tested in the APP/PS1 mouse model of AD, intracerebral or repeated IV MSC-EV treatment, beginning before or during the early stages of the disease, resulted in fewer Aβ plaques in the brain (Ding et al., 2018; Elia et al., 2019; Yang et al., 2020) and improved performance in learning and memory tasks (Ding et al., 2018; Yang et al., 2020). In line with research from other neurological disorders, MSC-EV treatment in rodent models of AD reduces the expression of markers of inflammation and oxidative stress, including IL-1β, TNF-α, Iba1, and HO-1 (Ding et al., 2018; Yang et al., 2020), and promotes neurogenesis (Reza-Zaldivar et al., 2019).

Also, EVs isolated from CNS-derived cells were shown to reduce the synaptotoxicity of Aβ. For example, EVs isolated from either N2a neuroblastoma cell cultures or human cerebral spinal fluid were demonstrated to reduce deficits in synaptic plasticity and restore normal long-term potentiation (LTP) when intracerebrally administered to a rat AD model (An et al., 2013). Repeated IV administration of EVs from endothelial cells have been shown to reduce Aβ deposits in the brain and improve cognitive functioning (Pan et al., 2020). Also, ICV administration of NSC-EVs enhanced cognitive function and protected synapses from degeneration in AD rodent models (Li et al., 2020; Micci et al., 2019). The ability of EVs to reduce both protein aggregation and convey neuroprotection suggests the possibility of comprehensive therapeutic benefits in AD (Table 3).

Table 3:

EVs as a treatment in models of neurodegenerative diseases

Source	Disorder	Effects of EV Treatment	Citations
MSC	AD	↑ learning and memory ↑ expression of anti-inflammatory cytokines ↑ neurogenesis ↓ dysmorphic neurites ↓ expression of pro-inflammatory cytokines ↓ oxidative stress ↓ Aβ accumulation	Wang et al., 2018; Ding et al., 2018; Reza-Zaldivar et al., 2019; Yang et al., 2020 Ding et al., 2018; Yang et al., 2020 Reza-Zaldivar et al., 2019 Elia et al., 2019 Ding et al., 2018 Yang et al., 2020 Ding et al., 2018; Elia et al., 2019; Yang et al., 2020
	PD	↓ apoptosis	Chen et al., 2020a
	ALS	↑ mitochondria function ↑ motor performance ↓ motoneuron death ↓ gliosis ↓ apoptosis ↓ SOD1 accumulation	Calabria et al., 2019 Bonafede et al., 2020 Bonafede et al., 2019; Bonafede et al., 2016 Lee et al., 2016
	MS	↑ restoration or retention of myelin ↑ regulatory T cells ↓ expression of pro-inflammatory cytokines	Li et al., 2019b; Clark et al., 2019; Jafarinia et al., 2020 Fathollahi et al., 2021 Laso-Garcia et al., 2018; Li et al., 2019b
NSC	AD	↑ learning and memory ↓ expression of inflammatory cytokines ↓ synapse loss/damage ↓ electrophysiological deficits	Micci et al., 2019; Li et al., 2020 Li et al., 2020 Li et al., 2020; Micci et al., 2019 Micci et al., 2019
Dental Stem Cells	MS (PDLSC-EVs)	↑ spine density ↓ disease severity ↓ expression of inflammatory cytokines	Rajan et al., 2016
	PD (SHED-EVs)	↑ motor function ↓ expression of inflammatory cytokines	Narbute et al., 2019
Astrocytes	PD	↓ apoptosis	Shakespear et al., 2020
Microglia	AD	↓ neuronal death	Zhang et al., 2020
	MS	↑ OPC migration	Lombardi et al., 2019
Oligo	MS	↓ disease severity	Casella et al., 2020
N2a cells	AD	↓ Aβ accumulation ↓ electrophysiological deficits	An et al., 2013
CSF	AD	↓ electrophysiological deficits	An et al., 2013
Endothelial Cells	AD	↑ learning and memory ↓ Aβ accumulation	Pan et al., 2020

Abbreviations: Extracellular vesicle (EV), Mesenchymal stem cell (MSC), Cerebrospinal fluid (CSF), Neural stem cell (NSC), Oligodendrocyte (Oligo), Oligodendrocyte precursor cell (OPC), Periodontal ligament stem cells (PDLSC), Stem cells from human exfoliated deciduous teeth (SHED), Alzheimer’s Disease (AD), Parkinson’s Disease (PD), Blood-brain barrier (BBB), Long-term potentiation (LTP), Amyloid-beta protein (Aβ), Multiple sclerosis (MS), Amyotrophic lateral sclerosis (ALS)

5.2. Parkinson’s Disease

Parkinson’s disease (PD) is a progressive movement disorder characterized by bradykinesia (slowed movement), tremor, and impaired balance (Jankovic, 2008). PD is associated with the loss of dopaminergic neurons in the substantia nigra region of the basal ganglia and deposits of α-synuclein protein in the brain (Kalia and Lang, 2015; Rocha et al., 2018). In addition to genetic animal models, PD can be modeled through the administration of neurotoxins such as 6-OHDA and MPTP, which are taken up by neurons through the dopamine transporter, leading to the loss of dopaminergic neurons (Tieu, 2011).

EVs isolated from stem cells from human exfoliated deciduous teeth (SHEDs) attenuated motor deficits in rats when intranasally administered one week after 6-OHDA treatment (Narbute et al., 2019). MSC-EVs also show anti-apoptotic effects in in vitro and in vivo 6-OHDA PD models (Chen et al., 2020a) (Table 3). Additionally, Shakespear et al., showed that astrocyte-derived EVs also have anti-apoptotic effect in in vitro PD models (Shakespear et al., 2020) but whether these neuroprotective effects will translate to in vivo PD models is still unknown. Published research evaluating EVs as a treatment for PD has primarily focused on the effects of modified or engineered EVs to enhance their therapeutic efficacy. Expression and aggregation of α-synuclein was successfully reduced using selectively engineered EVs, such as those that help direct EVs or their content to α-synuclein (Ren et al., 2019) or those that alter expression of α-synuclein (Cooper et al., 2014; Izco et al., 2019).

5.3. Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is caused by the progressive degeneration of motor neurons, leading to a loss of motor control (Kiernan et al., 2011; Rowland and Shneider, 2001). While most ALS cases are sporadic, mutations in several genes, including SOD1 and C9ORF72, are found in familial ALS (Renton et al., 2014).

Evidence from several in vitro ALS models shows that EV treatment may be beneficial (Table 3). Treatment of NSC-34 motor neuron-like cells expressing SOD1 mutations with MSC-EVs increased cell viability (Bonafede et al., 2019; Bonafede et al., 2016) and decreased the expression of apoptotic proteins (Bonafede et al., 2019). Additionally, when neuronal cells from mutant mice expressing the SOD1 p.G93A mutation (p.G93A mouse model) were treated with MSC-EVs, the treated cells exhibited reduced expression of SOD1 protein, indicative of decreased protein aggregation, and restored expression of mitochondrial proteins, including PGC-1a, (Lee et al., 2016). MSC-EV treatment also rescued the dysfunction of mitochondrial membrane potential and coupling efficiency in NSC-34 SOD1 mutant cells (Calabria et al., 2019). MSC-EVs were tested in the p.G93A mouse model of ALS and were found to improve motor performance, protect motoneurons, and decrease glial cell activation with repeated intranasal or IV administration (Bonafede et al., 2020). As MSC-EVs have only been tested in SOD1 models of ALS, it is not clear if the therapeutic potential of MSC-EVs would extend to ALS cases associated with other genetic mutations (e.g., C9ORF72 mutations) or sporadic ALS cases.

5.4. Multiple Sclerosis

Multiple sclerosis (MS) is associated with axon demyelination and impaired axonal signaling in the CNS (Karussis, 2014; Oh et al., 2018). The most common type of MS is relapsing-remitting MS, characterized by periods of new or worsening symptoms along with periods of remission; however, many patients eventually develop a progressive form of MS (Lublin and Reingold, 1996). An autoimmune response is believed to underlie MS, and the development of therapeutics to modulate this immune response is an area of active research (Correale et al., 2017; Martin et al., 2016). Animal models of MS, such as the experimental autoimmune encephalomyelitis (EAE) model, are developed by the immunization of animals against CNS or myelin-related proteins (Robinson et al., 2014).

Intravenous MSC-EV administration (Table 3) in an EAE mouse model was associated with reduced expression of the pro-inflammatory cytokines IL-1β and TNF-α (Laso-Garcia et al., 2018; Li et al., 2019b) and increased expression of the anti-inflammatory cytokine IL-10 (Li et al., 2019b). Likewise, Li et al. showed that IV administration of MSC-EVs in a rat EAE model not only attenuated inflammation but also reduced demyelination in the CNS (Li et al., 2019b), possibly via the ability of MSC-EVs to shift microglial polarization towards an anti-inflammatory phenotype (Laso-Garcia et al., 2018; Li et al., 2019b). Other studies showed that intranasal MSC-EV treatment could increase the number of regulatory T cells (Fathollahi et al., 2021; Jafarinia et al., 2020), which can suppress the inflammation seen in autoimmune diseases like MS. (Fletcher et al., 2010). Intravenous MSC-EV treatment also promotes oligodendrocyte maturation and helps myelin formation and retention (Clark et al., 2019). Similarly, microglial-derived EVs promoted oligodendrocyte precursor cell migration in vitro (Lombardi et al., 2019). In addition, EVs isolated from periodontal ligament stem cells (PDLSC-EV) were demonstrated to reduce expression of inflammatory markers and decrease disease severity when administered intravenously (Rajan et al., 2016). Casella et al. showed that oligodendrocyte-derived EVs (Ol-EVs) directly injected into the cisterna magna, which express myelin-related proteins, induced immunosuppressive monocytes and apoptosis of autoreactive T cells, restoring immune tolerance for myelin-related proteins and decreasing disease severity (Casella et al., 2020).

6. Engineering and Modifications of EVs

A significant advantage of EVs is their ability to be engineered (Figure 2). For example, before isolation, EV content can be altered by changing the physiology of parental cells (Figure 2A)(Vader et al., 2016). During isolation, a desired subpopulation of EVs can be collected through the choice of isolation and purification method, such as immunoprecipitation (Figure 2B), which enables a subpopulation of EVs to be isolated based on their expression of a specific surface protein (Konoshenko et al., 2018; Li et al., 2017). Alternatively, the content or surface of EVs can be altered after isolation (Figure 2C). For example, EVs can be loaded via electroporation or lipid-based methods, and molecules can be added to the surface through covalent or non-covalent bonds (Vader et al., 2016).

Figure 2: Common EV Modifications.

A: There are several strategies to engineer EVs before their isolation. This includes treating parent cells with a genetic construct or drug and changing the cellular source of EVs. B: The choice of isolation and purification methods can alter the population of EVs that are collected as different methods separate EVs based on distinct properties. C: After their isolation, EVs can be further modified through the addition of molecules to the surface or loading with a desired cargo. Created with BioRender.com.

One application of EV modification is to enable them to function as vehicles for therapeutic molecules. EVs can be altered to hold specific nucleic acids, often small RNAs, as well as proteins or drugs (Kalani et al., 2016; Liu et al., 2019; Mattera et al., 2020). For example, EVs have been used to stabilize and deliver the microRNA, miR-124, which is known to have anti-inflammatory properties. EVs enriched in miR-124 provided greater suppression of neuroinflammation and improved neurological recovery in TBI models (Ge et al., 2020; Li et al., 2019a; Yang et al., 2019). Using similar approaches, EVs may also have applications in targeted genetic therapies. Didiot et al. used EVs to deliver a small interfering RNA (siRNA) to target mRNA of the huntingtin gene, which, when expanded, gives rise to Huntington’s disease (The Huntington’s Disease Collaborative Research Group, 1993). When injected into the mouse striatum, the siRNA-loaded EVs resulted in reduced expression of the huntingtin protein compared to injection of the siRNA alone (Didiot et al., 2016). While this study only examined wild-type huntingtin mRNA and protein expression, these results suggest that packaging siRNAs in EVs may increase their effectiveness.

The efficacy of EVs can also be improved by altering surface molecules to increase the targeting of EVs to the brain or a specific pathology. For example, the rabies virus glycoprotein (RVG) is often used to facilitate improved crossing of the BBB (Alvarez-Erviti et al., 2011; Wiklander et al., 2015). Another strategy is to add aptamers to EVs to target them or their content to regions of disease pathology. EVs have been engineered to directly target PD pathology by expressing an aptamer with an affinity towards α-synuclein, and systemic administration of these engineered EVs reduced α-synuclein aggregation and improved motor performance in a mouse PD model (Ren et al., 2019). EVs have also been modified to contain an aptamer targeting myelin, and this modification led to increased efficacy of EVs in an MS model (Hosseini Shamili et al., 2019).

The efficacy of EVs can also be changed by subjecting cells to treatments that can broadly alter EV content. For example, EVs derived from the BV2 microglial cell line treated with the anti-inflammatory cytokine IL-4 have a greater ability to suppress inflammatory signaling (Song et al., 2019; Tian et al., 2019). Furthermore, culturing cells under different conditions can affect the ability of EVs to mitigate disease features. For example, studies have observed that EVs from 3D cell cultures, instead of traditional 2D cultures, exhibit increased efficacy in neurological disease models (Yang et al., 2020; Zhang et al., 2017). This result suggests that controlling for the culture conditions of cells is critical to maximizing the efficacy of isolated EVs.

7. Conclusions and Future Directions

The literature summarized above demonstrates that EVs can attenuate pathological features, including neuron death and neuronal inflammation, and reduce functional and behavioral deficits associated with several neurological disorders. Their ability to readily cross the BBB also makes them well-suited for treating neurological disorders. Furthermore, they offer the advantage of being easily manipulated and engineered to increase their efficacy in clinical applications.

The exact mechanisms by which EVs exert their therapeutic effects are unclear. However, in preclinical studies, individual components of EVs that partially mediate some of the protective effects have been identified. For example, miR-124 was found to partially mediate the protective effects of EVs in a stroke model (Song et al., 2019), and the gap junction alpha 1 protein was shown to be important for the ability of EVs to promote neuronal recovery in a TBI model (Chen et al., 2020b). EVs with therapeutic effects have also been shown to contain growth factors and anti-inflammatory RNAs or proteins (Kim et al., 2020; Kim et al., 2012; Upadhya et al., 2020). Such observations suggest that the benefits of EV treatment may be due to the combined effect of many different components. Likewise, treatment with EVs leads to the activation of multiple cellular pathways that likely contribute to the observed benefits. Pathways which have been shown to be activated include those that facilitate autophagy (Chen et al., 2020a; Huang et al., 2018; Pei et al., 2019), inhibit apoptosis (Bonafede et al., 2019; Bonafede et al., 2016; Chen et al., 2020a; Chen et al., 2020b; Pan et al., 2016; Shakespear et al., 2020), and promote anti-inflammatory signaling (Ding et al., 2018; Kim et al., 2016; Laso-Garcia et al., 2018; Li et al., 2019b; Long et al., 2017; Ni et al., 2019). Overall, EVs seem to have broad, multimodal effects which may offer an advantage over treatments that act via a single mechanism. The complex and diverse contents of EVs might also provide therapeutic applications in neurodevelopmental or psychiatric disorders. For example, motivated by prior research showing reduced behavioral abnormalities in the BTBR mouse model of autism spectrum disorder following MSC transplantation (Perets et al., 2017; Segal-Gavish et al., 2016), Perets et al. intranasally administered MSC-EVs to the BTBR mouse model and similarly observed a reduction in repetitive behaviors and attenuation of social deficits (Perets et al., 2018).

Currently, few side-by-side comparisons have been performed between EVs isolated from diverse sources, making it difficult to determine whether one source of EVs may be more beneficial than another. For example, Branscome et al. evaluated the effects of MSC-EV and induced pluripotent stem cell-derived EV (iPSC-EV) treatment on the ability of cultured astrocytes to recover after irradiation. While increased astrocyte viability was observed with both EV types, cells treated with iPSC-EVs had few projections, while MSC-EV-treated astrocytes displayed more projections and appeared more naïve (Branscome et al., 2020). Similarly, while both NSC-EVs and MSC-EVs reduced disease phenotypes in a murine stroke model, the NSC-EVs were reported to show greater efficacy (Webb et al., 2018b). Such differences would not be revealed unless EVs from various sources are directly compared.

Different routes of administration (ROA) and treatment frequencies are also often used in studies evaluating the therapeutic potential of EVs; however, few studies have systematically compared the effect of these parameters on treatment efficacy. This has important implications for clinical translation since some ROA and dosing regimens, while feasible under research conditions, will be impractical in a clinical setting. In addition, while beneficial effects have been observed following the administration of EVs over multiple weeks in neurodegenerative disease models (Bonafede et al., 2020; Ding et al., 2018; Yang et al., 2020), it is possible that patients may require chronic treatment, highlighting the need to more thoroughly evaluate the long-term effects of EV administration.

Finally, EVs have yet to be tested across the full range of features for many disorders. For example, while EVs are shown to reduce neuroinflammation and behavioral deficits following status epilepticus in MTLE rodent models (Long et al., 2017; Xian et al., 2019), their effect on spontaneous seizures has not yet been established. To fully assess the clinical potential of EVs, it will be necessary to evaluate their efficacy against the broad range of clinically challenging features associated with each disorder. Future research addressing these open questions would help establish the therapeutic potential of EVs in neurological disorders.