Extracellular vesicles in CNS developmental disorders

International Journal of Molecular Sciences Review Extracellular Vesicles in CNS Developmental Disorders Ana Rita Gomes 1,2,3,4 , Nasim Bahram Sangani 3,4 , Tiago G. Fernandes 1, M. Margarida Diogo 1, Leopold M. G. Curfs 4and Chris P. Reutelingsperger 3,4,* 1Department of Bioengineering and IBB—Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, 1049-001 Lisboa, Portugal; [email protected] (A.R.G.); [email protected] (T.G.F.); [email protected] (M.M.D.) 2Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina da Universidade de Lisboa, 1649-028 Lisboa, Portugal 3Department of Biochemistry, Maastricht University, Cardiovascular Research Institute Maastricht, 6200 MD Maastricht, The Netherlands; [email protected] 4GKC-Rett Expertise Centre, Maastricht University Medical Centre, 6229 ER Maastricht, The Netherlands; [email protected] *Correspondence: [email protected] Received: 18 November 2020; Accepted: 9 December 2020; Published: 11 December 2020   Abstract: The central nervous system (CNS) is the most complex structure in the body, consisting of multiple cell types with distinct morphology and function. Development of the neuronal circuit and its function rely on a continuous crosstalk between neurons and non-neural cells. It has been widely accepted that extracellular vesicles (EVs), mainly exosomes, are effective entities responsible for intercellular CNS communication. They contain membrane and cytoplasmic proteins, lipids, non-coding RNAs, microRNAs and mRNAs. Their cargo modulates gene and protein expression in recipient cells. Several lines of evidence indicate that EVs play a role in modifying signal transduction with subsequent physiological changes in neurogenesis, gliogenesis, synaptogenesis and network circuit formation and activity, as well as synaptic pruning and myelination. Several studies demonstrate that neural and non-neural EVs play an important role in physiological and pathological neurodevelopment. The present review discusses the role of EVs in various neurodevelopmental disorders and the prospects of using EVs as disease biomarkers and therapeutics. Keywords: neurodevelopmental disorders; extracellular vesicles; exosomes; microvesicles; CNS; neurons; astrocytes; glia 1. Introduction 1.1. Extracellular Vesicles Cell-to-cell communication is a fundamental process in coordinating the functions and interactions between the diverse neural cell populations in the central nervous system (CNS) and is mainly organized through secretion of molecules in the intercellular space [ 1 ]. Extracellular vesicles (EVs) have been recognized as communication vehicles playing an important role in neural cell proliferation and differentiation, and also in immune modulation and senescence [2]. EVs can be classified and distinguished according to their biogenesis, sub-cellular origin, cargo, size and method of isolation. A subset of EVs, the exosomes, originate from the inward budding of endosomal membranes, giving rise to the formation of multivesicular bodies (MVBs). MVBs typically depict a diameter between 250–1000 nm and contain intraluminal vesicles (ILVs), which are released into the extracellular Int. J. Mol. Sci. 2020,21, 9428; doi:10.3390/ijms21249428 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020,21, 9428 2 of 20 space as exosomes after the fusion of MVBs with the plasma membrane [ 3 ]. Exosomes are the smallest EVs and range from 30 to 100 nm in diameter [ 4 ]. The microvesicles (MVs) form another subset of EVs. They are larger than exosomes, with a diameter between 0.1 and 1 µ m. MVs are released from cells by plasma membrane budding [ 5 ]. The largest subset of EVs are the apoptotic bodies, which are shed from a dying cell executing apoptosis [ 6 ]. The apoptotic bodies can vary in size between 1 and 5 µ m in diameter. EVs have been isolated from a great variety of fluids, including supernatants of cultured cells, blood, urine, cerebrospinal fluid (CSF) and serum [7]. Isolation of the different EV subtypes has been accomplished using several methods, such as isolation by size, immunoaffinity capture or precipitation. Isolation by differential ultracentrifugation is widely considered the gold standard method [ 8 – 10 ]. It should be noted, however, that physical and molecular overlap between the EV subsets has precluded the definition of specific EV subtype marker proteins to date [11]. 1.2. Molecular Composition of EVs EVs carry a diverse set of molecules that can be transported over short and long distances to recipient cells. There, they execute defined biological functions, which contribute to health and disease. The composition of EVs is determined by their biogenetic pathway and the microenvironment of the parental cell [ 12 ]. The composition may also contribute as a fingerprint for establishing the origin and type of EVs, which is relevant if EVs are to be considered as biomarkers. However, this is not as unambiguous as suggested by many papers on EV research. The endosomal sorting complex required for transport (ESCRT) and accessory proteins are necessary for MVB biogenesis; hence, ESCRT proteins and Alix and TSG101 are considered standard markers of exosomes, regardless of the parental cell type [ 13 ]. It has been shown that cells depleted of the ESCRT machinery are still able to produce CD63-positive exosomes by utilizing the sphingomyelinase—ceramide machinery [ 14 ]. A recent study with exosomes extracted from neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (hiPSCs) expressed lower levels of Alix, TSG101, Hsp70 and also CD63, in comparison with hiPSC-derived cardiac cells [ 15 ]. This might suggest a different protein machinery for neural derived-exosome biogenesis and tracking (i.e., ESCRT-independent pathways) [13]. Other membrane proteins commonly found in exosomes and enriched when compared with cell lysate content are integrins and tetraspanins (such as CD9, CD81, CD82, CD63 and CD37). Exosomes also contain cytosolic proteins, such as heat-shock proteins (Hsp70, Hsp90), in addition to cytoskeletal proteins, like tubulin and actin. Moreover, exosomes contain small GTPases, such as RAB27A, RAB11 and RAB35, which play an important role in intracellular trafficking in secretory pathways during vesicle formation and also in exosome release [16]. Besides the abovementioned typical protein cargo, primary cortical neuron-derived exosomes have been characterized and identified with synaptic proteins, such as L1 cell adhesion molecule (L1CAM), glycosylphosphatidylinositol (GPI)-anchored prion protein and glutamate receptor subunit GluR2/3 [ 17 ]. EV types carrying specific neuronal protein cargo will be discussed further in the following sections. Comprehensive analyses of the composition of EV subtypes derived from different cell types revealed a substantial difference in lipidomics and proteomics between exosomes and MVs [ 17 ]. MVs encompass more proteasomes, and endoplasmic reticulum and mitochondrial proteins, whereas exosomes express relatively more proteins that function at the interface with the environment [ 18 ]. The lipid content also differs between MVs and exosomes. MVs are enriched in ceramides and sphingomyelins, and exosomes carry more glycolipids and free fatty acids [ 19 ]. Interestingly, apoptotic bodies have a content that resembles those of cell lysates, yet they also express unique features within their cargo, such as enrichment of thioredoxin peroxidase II, Alix, 14-3-3 and galectin-3 [ 20 ]. Apoptotic bodies express more specific surface markers such as phoshatidylserine (annexin A5-binding), thrombospondin and C3b [7,21]. Int. J. Mol. Sci. 2020,21, 9428 3 of 20 EV types also carry a wide range of genetic material including DNA, mitochondrial DNA (mtDNA), and coding and non-coding RNAs (long non-coding RNAs, micro (mi)RNAs and circular RNAs) [ 22 ]. Experimental evidence has demonstrated that the genetic information transferred by EVs can be used by the transcriptional and translational machineries of the recipient cell [ 22 ]. Morel et al. identified miR-124a to be abundantly expressed by neuronal exosomes and demonstrated, both in vivo and in vitro , that the neuronal exosomes transfer miR-124a to astrocytes, which consequently upregulated the expression of GLT1 [23]. In a more recent breakthrough, Men et al. have demonstrated that the miRNA profile of secreted exosomes is different from the one observed in live neuronal cells [ 24 ]. By generating a cell-type-specific ILVs/exosome reporter (CD63-GFPf/f) in mice, the authors observed that an undescribed neuron-specific miRNA, miR-124-3p, was internalized into astrocytes, also upregulating the glutamate transporter GLT1 [24]. Interestingly, the aforementioned study using vesicles isolated from hiPSC-derived cells from distinct lineage, revealed differential expression of the analyzed miRNA, suggesting the lineage specificity of miRNA cargo. For example, miR-34a-3p was observed to be highly expressed in the hiPSC-derived NPCs, in addition to miR-133a and miR-133b, which may be involved in neurite growth [ 15 ]. Nonetheless, miRNAs observed in exosomes from non-neuronal cells, such as mesenchymal stem cells (MSCs), could function as promotors of neurogenesis and neurite remodeling, similar to miR-133b [ 25 ]. A significant number of studies have shown the potential of exosomal miRNA as biomarkers, both for diagnostic purposes and for studying several neurodevelopmental and neurodegenerative disorders [ 26 ]. A study using genome-wide next-generation sequencing revealed substantial differences in exosomal miRNA profiles between CSF and serum when compared with miRNA found in the brain [ 27 ]. Half of the miRNAs already reported in the brain were only found in CSF exosomal fractions. Particularly, miR-1911-5p was detected in both brain tissue and CSF. Therefore, brain pathophysiology could be inferred by the analysis of exosomal pathogenic proteins and miRNA extracted from CSF and other biological fluids. As we will discuss next, EVs, predominantly exosomes, could also provide novel mechanisms of intercellular communication during nervous system development, offering new clues on the progression of neurodevelopmental pathologies. 2. EVs Mediate Communication in CNS—During and Post-Development Several studies have demonstrated that the various EV types play a role in mediating critical interactions during CNS development, mainly in cellular connection and circuit maintenance. A study by Marzesco et al. was one of the first reports describing the existence of EVs during neurodevelopment. The vesicles positive for the stem cell marker prominin-1 (CD133) were found in the luminal fluid of the neural tube in embryonic mouse brains [ 28 ]. Moreover, it was also observed that primary cultures of cortical neurons were able to secrete exosomes containing specific neural proteins [ 17 ]. Similarly, mature cortical and hippocampal neurons also secrete exosomes [ 29 ]. These studies highlight the role of EVs in regulating synaptic activity during development, particularly their role in neuronal communication mediated by glutamatergic synaptic activity, 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl) propanoic acid (AMPA) and N-methyl-d-aspartate (NMDA) receptors [29]. In the specific case of neurons, exosomes are released from post-synaptic soma or dendrites [ 24 ], and they mediate several processes, such as the maintenance of homeostasis by triggering synapse pruning by microglial cells [ 30 ], or the outflow of molecular information to neighboring cells, mediated by miRNAs. These miRNAs may induce gene expression in recipient cells in an activity-dependent manner [ 31 ]; as previously described, miR-124 internalized by astrocytes is capable of regulating the glutamate transporter (GLT1) levels, as well as glutamate uptake in the brain [23]. Several reports also describe the release of EVs, mainly exosomes types, by astrocytes during brain development under normal neuronal activity or during oxidative stress or other stressful insult conditions. Neuroactive substances, like Hsp70 [ 32 ] or synapsin I, are released from Int. J. Mol. Sci. 2020,21, 9428 4 of 20 non-neural cells via exosomes and ultimately promote neurite outgrowth and neuronal survival [ 33 ]. Oligodendrocytes, responsible for myelin sheath production, have been demonstrated to release exosomes with myelin proteins, which could be triggered by glutamate release from active neurons. Moreover, these oligodendrocyte secreted-exosomes are responsible for increasing the firing rate activity of neurons [34]. Finally, microglia, the immune cells present in the brain, play a relevant role in immune regulation, mainly by propagating vesicles with cytokines, chemokines and reactive oxygen species, which will mediate the inflammatory response. For example, stimulation of cortical microglia with lipopolysaccharides (LPS) resulted in the release of exosomes with an enriched cargo of pro-inflammatory cytokines, such as IL-1 β , which are responsible for the propagation of inflammation [ 35 ]. Microglia can also release immunomodulatory exosomes containing major histocompatibility complex (MHC) Class II receptors. Furthermore, another study proved that microglia-derived MVs are responsible for stimulating synaptic vesicle release in presynaptic terminals, by involving neuronal sphingosine and ceramide production [ 36 ]. Another study showed that stimulators of serotonin (5-HT) receptors increased microglial exosome release and that exosomes enclose an insulin-degrading enzyme that is known to ultimately degrade the neurotoxic peptide amyloidβ [ 37 ]. Actually, this study aimed to demonstrate the functional signaling between neurons (serotonergic neurons) and microglia. Taken together, these reports show the important role of EVs, mainly exosomes, in CNS development, in further modulation of neuronal activity, and cell-to-cell communication (Figure 1), neuroprotection and repair. Figure 1. Schematic illustration of extracellular vesicles (EVs) in cell–cell communication in the CNS. EVs mediate intercellular communication between neurons, oligodendrocytes, astrocytes and glial cells. The EV is composed of lipids, proteins and polynucleotides. The cellular origin of EVs will define the cargo content and signaling capacity. For example, neural-derived exosomes carry synaptic cell adhesion molecules: neuronal-specific cell membrane marker L1CAM, GPI-anchored prion protein and GluR2/3. Proteins involved in vesicular trafficking, such as Rab proteins, annexins and cytoskeletal proteins, are present in EVs derived from neuronal and non-neuronal cells. Int. J. Mol. Sci. 2020,21, 9428 5 of 20 3. EVs in Developmental Pathology of the Nervous System Alterations in brain-derived EV composition (Table 1) have been associated with changes in the crosstalk between neural cells, some of which have been linked to neurodegenerative disorders. Given their role in mediating intercellular communication at different stages of normal and pathological CNS development [ 2 ], including neural cell proliferation and differentiation, synaptic formation, and learning and memory processes [ 38 ], neurodevelopmental pathologies will benefit from in-depth knowledge about EVs in the CNS. Table 1. EV cargo alterations associated with various models of human neurodevelopmental disorders. Disease EVs—Type and Source EVs—Cargo Alterations in Disease Context Reference Rett syndrome (RTT) Exosomes from hiPSC-derived neurons (from both isogenic control and MeCP2LOF-disesase cell lines) Proteomic analysis revealed a downregulation in neurodevelopmental signaling proteins associated with neuronal maturation, axonal guidance and synaptogenesis. [39] Autism spectrum disorder (ASD) EVs isolated from ASD children’s serum Significant increase in total protein concentration and in the amount of mtDNA in EVs. [40] Down syndrome (DS) Exosomes from a Ts2 mice model with DS-like phenotype Increased levels of exosomes in DS models influenced by larger and more abundant number of MVBs and more ILVs per neuron; Neuronal exosomes with a homeostatic role for neurotoxic material release in response to chronic endosomal dysfunction. [41] Exosomes isolated from DS patients, Ts2 mouse brains and human DS fibroblasts Enriched in APP carboxyl-terminal fragments (APP-CTFs) and in full-length APP (flAPP). [42] Exosomes from blood samples from DS patients Increased CD81 levels (more abundant neuronal exosomes secreted). Neuronal exosomes contained Aβpeptide products and hyper-phosphorylated species of Tau (P-Tau). [43,44] Exosomes from blood samples of DS patients DS neuronal exosomes showed higher levels of A β 1-42, phosphorylated P-T181-Tau and P-S396-Tau. [45] Fetal alcohol syndrome (FAS) EVs from cultured neurons and astrocytes (ethanol-treated) EVs from the treated condition are increased in number, with higher content of inflammatory-related proteins, such as TLR4, NFκB-p65, IL-1R, caspase-1 and NLRP3, as well as miRNAs (miR-146a, miR-182 and miR-200b). [46] Exosomes from microglia BV-2 cell lines (exposed to ethanol during biogenesis) Decreased levels of CD18 (a microglial and immune cell marker). Both Hsp70 and Hsp90 were increased (preventing damaging pro-inflammatory responses). Decreased expression of Rab 7 protein, (important role in vesicle trafficking and exosome biogenesis). [47] EVs from an in vitro model of NSCs (exposed to ethanol) miR-140-3p was identified to be increased during ethanol treatment, which could influence neurogenesis inhibition and neuronal alterations. [48] Acute bilirubin encephalopathy (ABE) EVs isolated from the CSF of ABE patients Differentially expressed proteins associated with immune-inflammatory response, such as SAA1, APP, LBP, CRP, immunoglobulin and complement components (C4B and C5). Altered levels of specific EVs cargo, mainly S100A9, S100A7, DEFA1 and LTF. [49] EVs are projected to offer novel therapeutic avenues (Table 2) to treat CNS diseases and play a role as biomarkers of disease status and progression (Table 3). The analysis of EVs’ molecular signals such as mRNA, miRNA, lipid or protein content, and their correlation with human brain developmental pathologies will be discussed next and summarized in Figure 2. In the following sections, the terminology of EV subtypes is in accordance with the original work. Int. J. Mol. Sci. 2020,21, 9428 6 of 20 Table 2. EVs cultured or administrated, and their therapeutic effect. Disease EVs—Type and Source EVs—Culture/Administration EVs—Therapeutic Effect Reference Rett syndrome (RTT) Exosomes extracted from IC hiPSC-derived neurons Exosomes cultured with MeCP2LOF hiPSC-derived neurons Increased/improved: Puncta densities; Synaptogenesis; Neuronal activity (higher network synchronization); Proliferation; Neuronal fate in developing neural cultures. [39] Autism spectrum disorder (ASD) EVs isolated from ASD children’s serum EVs cultured with human microglia cells Increased Secretion of IL-1β, a pro-inflammatory cytokine. [40] Exosomes secreted by mesenchymal stem cells (MSCs) Exosomes were intranasally administrated in a BTBR mouse model (presents autistic-like behaviors and ASD symptoms) Increased: Male to male social interaction; Reduced: Repetitive behaviors; miRNA-143 cargo (an immunomodulatory effector in the host cells). [50] Exosomes from adipose-derived MSCs Exosomes were intranasally and intravenously administrated into BTBR and Shank3 mice models (ASD cognitive and motor impairments) Improved: ASD behavioral phenotype (mainly by non-invasive intranasal administration). [51] Exosomes from adipose-derived MSCs Exosomes cultured with primary neuronal cell cultures, prepared from a newborn SHANK3 homozygote mouse model of autism. Upregulated: Proteins related to anti-inflammatory processes; Proteins related to immunomodulation; BDNF (neuroprotection and neurogenesis mediator). [52] Down syndrome (DS) Neuronal-derived exosomes purified from the blood of individuals with DS-AD and controls Exosomes were injected into a control mouse model Increased: P-Tau amount in pyramidal neurons and in the dentate gyrus of the hippocampus; Spread of toxic P-Tau species via exosome mediation (unpublished work) [53] Fetal alcohol syndrome (FAS) EVs from cultured neurons and astrocytes (ethanol-treated) Ethanol-treated EVs from astrocytes were incubated with cortical neural cultures Increased: Levels of the inflammatory protein COX-2; miRNAs: miR-146a (regulation of genes related to inflammatory pathways) [46] Table 3. EVs as potential biomarkers for neurodevelopment-related disorders. Disease EVs—Type and Source EVs—Biomarker Potential Reference Down syndrome (DS) Exosomes from DS patients’ blood samples Increased levels of: CD81; Aβpeptide products; Hyper-phosphorylated species of Tau (P-Tau) [43,44] Neural origin exosomes extracted from DS patients’ blood samples Elevated levels of: Aβ1-42; Phosphorylated P-T181-Tau; P-S396-Tau [45] Fetal alcohol syndrome (FAS) Fetal neural exosomes isolated from maternal plasma Significantly lower levels of: HSF1; Bcl–XL; REST; Synaptophysin; Synaptotagmin; Synaptopodin; GAP–43 [54] Acute bilirubin encephalopathy (ABE) EVs isolated from the CSF of ABE patients Upregulated: LTF and DEFA1 C4B and C5 (complement-associated proteins) Downregulated: S100A7 and S100A9 [49] Int. J. Mol. Sci. 2020,21, 9428 7 of 20 Figure 2. Schematic illustration summarizing the main changes observed in the cargo of EVs in the neurodevelopmental disorders that are discussed in this review. Therapeutic possibilities using or targeting exosomes are also indicated. 3.1. Rett Syndrome Rett syndrome (RTT) is a severe neurological disorder affecting brain development and function and is caused by mutations in the gene encoding the methyl-CpG-binding protein 2 (MeCP2), localized in the X chromosome [ 55 ]. MeCP2 mutations lead to developmental regression, with a range of neurodevelopmental defects, including loss of speech, acquired movement skills and severe cognitive impairment after an apparently normal development [ 56 ]. In vitro studies using RTT hiPSC-derived neurons showed impaired neuronal maturation, supported by the presence of fewer synapses, smaller soma size, altered calcium signaling, functional defects in firing activity and excitatory/inhibitory (E/I) imbalance [ 57 , 58 ]. These phenotypic alterations observed in RTT could also be closely related to changes in the biological content of exosomes, as discussed next. Recently, Sharma et al. used neurons derived from hiPSCs lacking MeCP2 to analyze the effect of MeCP2 deficiency on the protein content of exosomes [ 39 ]. This study showed that the loss of MeCP2 function (MeCP2LOF) was associated with a dramatic change in the protein content of exosomes and the signaling bioactivity of the exosomes. Proteomic analysis revealed that some neurodevelopmental signaling proteins, mainly associated with neuronal maturation, axonal guidance and synaptogenesis, were upregulated in isogenic control (IC) exosomes when compared with MeCP2LOF. Neuronal RTT cultures were then treated with healthy exosomes, which increased puncta densities (Synapsin1 staining), resulting in an increase in synaptogenesis. Furthermore, spike recordings revealed an improvement of neuronal activity with higher network synchronization. In this context, exosomes displayed a prominent role in regulating important molecular pathways. The involvement of RNA, miRNA and circRNA needs further investigation. Other experimental models of RTT revealed impairments in the length and type of dendritic spines causing abnormalities in synaptic communication. A study with a Mecp2-deficient male mice showed thalamo-cortical axon arbor failure, resulting in reduced complexity and density of the dendritic branches in neurons [ 59 ]. Another study using 3D forebrain organoids derived Int. J. Mol. Sci. 2020,21, 9428 8 of 20 from RTT hiPSCs demonstrated a decrease in the number of more mature branched spines and an altered electrophysiological profile characterized by defects in spontaneous synaptic transmission and connection [ 60 ]. It has been hypothesized that synaptic physiology is, at least partially, mediated by exosome release [ 29 ], implying that RTT pathology may be associated with aberrant exosome biology. Both in vivo and in vitro models may help to provide a mechanistic understanding of the role of exosomes in RTT pathology of the different brain regions. In addition, exosomes were revealed to be potential agents for translational research, presenting themselves as treatment options for targeting pathological features of RTT, particularly synaptic activity regulation. Strong evidence suggests that brain-derived neurotrophic factor (BDNF) is significantly reduced in the brains of RTT patients [ 61 ] and RTT mouse models [ 62 ]. MeCP2 mutations affect BDNF gene transcription, mRNA translation and protein trafficking, contributing to the RTT symptomatology. BDNF binds to a specific membrane-bound receptor, tropomyosin-related kinase B (TrkB), organizing signaling cascades that modulate neuronal differentiation, survival in early development and synaptic transmission [ 63 ]. A promising diagnosis approach could rely on EV isolation from the peripheral blood of RTT patients. In a study by Suire et al., it was reported that adults with aging-associated walking speed decline showed higher levels of proBDNF and BDNF in isolated EVs, specifically an enriched subpopulation of neuronal origin, expressing the neuronal marker L1CAM [ 64 ]. In addition, mRNA levels of BDNF transcripts were observed to be lower in brain samples from RTT patients. Thus, the identification and quantification of specific miRNAs present in circulating brain-derived EVs could contribute to the diagnosis and also to reveal important cues about the affected pathways and mechanisms associated with the pathology [ 63 ]. BDNF overexpression in hippocampal neurons was shown to rescue several RTT-associated phenotypes and dendritic atrophy [ 62 ]. However, the use of the natural form of this neurotrophic factor is not a useful clinical approach due to its short half-life and inability to cross the blood–brain barrier (BBB) [ 62 ]. Nevertheless, understanding the role of exosomes in RTT can open therapeutic avenues based on exosomes as carriers of therapeutic molecules; for example, BDNF or miRNAs that regulate BDNF expression [63]. 3.2. Autism Spectrum Disorder The autism spectrum disorder (ASD) is a lifelong neurodevelopmental pathology with core abnormalities in social and communication capacity, and stereotyped behaviors and interests [ 65 ]. Brain development defects present in ASD are associated with disruption of cell proliferation and differentiation, neuronal maturation, neurite outgrowth, altered synaptogenesis and reduced neural network functionality [ 65 – 67 ]. Indeed, inflammation in the CNS and neuro-immune crosstalk dysregulation are prevalent in ASD patients. The described neural dysfunction could be directly related to neuroinflammation, which is characterized by the activity and proliferation of glial cells, astrocytes and microglia [ 68 ]. Robust studies have shown that microglia and astrocytes can release exosomes with pro-inflammatory mediators, such as cytokines, chemokines, reactive oxygen species (ROS) and secondary messengers that are important players in the inflammatory process [ 69 ]. In human brain samples from ASD patients, microglial and astroglial activation was found to be increased, particularly in the cerebellum. More importantly, the upregulation of macrophage chemoattractant protein-1, MCP-1 and pro-inflammatory interleukin IL-6 were observed in high amounts in reactive astrocytes in the cerebellum and the cortical and subcortical white matter regions. The anti-inflammatory cytokine tumor growth factorβ 1 (TGFβ 1) was also strongly increased, not only in neuroglia but also in the granular cell layer and Purkinje cell population, presenting significant degeneration [70]. A recent study reported that EVs isolated from the serum of ASD patients have a significant increase in total protein concentration when compared with healthy controls [ 40 ]. Moreover, when ASD serum-derived EVs were cultured with human microglia cells, secretion of IL-1 β , a pro-inflammatory cytokine, was observed. The same authors previously observed an increased amount of mtDNA in EVs from the serum of ASD patients. They suggested that EV mtDNA may trigger the pro-inflammatory Int. J. Mol. Sci. 2020,21, 9428 9 of 20 response of immune cells and may partially explain the immune dysregulation reported in autistic patients [71]. The current literature strongly points towards a role of EVs in modulating the pro-inflammatory responses of microglia and astrocytes, and neuroinflammation. Most studies have been performed with non-ASD animal models but the results of these studies can also have significance for understanding ASD pathology and for designing therapies to alleviate ASD disease features. A recent study explored the role of bone marrow MSC exosomes in LPS-induced neuroinflammation in the cerebellum of rats. MSC exosomes triggered and stimulated the release of pro-inflammatory cytokines, such as tumor necrosis factorα and interleukins IL-1 β and IL-6, by the activated astrocytes and microglia, causing neuronal cell degeneration and synaptic dysfunction. However, in addition to this paracrine pro-inflammatory activity, the exosomes exhibited a protective effect, predominantly on Purkinje neurons, as shown by a reduction in DNA damage and apoptosis [72]. Plasma levels of the endocannabinoid anandamide, produced in post-synaptic cell membranes, were shown to be reduced in ASD patients [ 73 ]. Thus, the lack of binding of anandamide to the receptor CB1 might be implicated in sustained presynaptic transmission of target GABAergic neurons in ASD. Interestingly, another study demonstrated that microglial EVs carrying anandamide on their surface promote a significant decrease in inhibitory postsynaptic currents of neurons [ 74 ]. Consequently, the use of healthy derived microglial EVs could potentially be a therapeutic strategy to restore the excitation/inhibition balance in ASD. The loading of the exosomes with specific cargos that will interfere with the host cells, such as miR-124-3p, could also alleviate the phenomenon of neuroinflammation. Recent work disclosed that miRNA-124-3p from microglial exosomes was responsible for suppressing mTOR signaling, thus inhibiting neuroinflammation, consequently improving the neurologic outcome by promoting neurite outgrowth [ 75 ]. This concept of customized exosome packaging was previously tested in vivo by encapsulating curcumin. The exosomes containing curcumin were delivered intranasally to an LPS mouse model and afforded protection against inflammation, with reduced levels of interleukin IL-1 β being produced by CD45.2 microglial cells [76]. Recently, a black and tan brachyury (BTBR) mouse model (a model with autistic-like behaviors and all the core symptoms of ASD) was used in an in vivo study for intranasal administration of exosomes secreted by MSCs [ 50 ]. Administration of MSC exosomes increased social interactions and reduced repetitive behaviors. RNA sequencing revealed upregulation of miRNAs such as miRNA-143, possibly related to the immunomodulatory effect of MSC exosomes. The same authors recently published a preclinical study in which exosomes extracted from adipose-derived MSCs were administered intranasally and intravenously to BTBR and Shank3 mutated mice [ 51 ]. The disruption of the gene Shank3 is associated with some ASD features, such as cognitive and motor impairments. In both animal models, the ASD behavioral phenotype was improved, mainly by using non-invasive intranasal administration. The same authors performed RNA sequencing and proteomics to determine the effects of MSC exosomes in cultured primary neurons [ 52 ]. They observed the upregulation of proteins related with anti-inflammatory processes and with immunomodulation. Interestingly, BDNF was amongst the upregulated growth factors, suggesting a role for BDNF as a mediator of neuroprotection and neurogenesis. More in-depth studies are needed to reveal the cause–consequence relationships between the molecular and biological cues extracted from EVs (cytokines, pro-inflammatory molecules, misfolded proteins, miRNAs) and ASD pathology. Such studies could be performed with hiPSCs derived from ASD patients. The hiPSCs can be differentiated into cortical [ 77 ] and cerebellum organoids [ 78 ], offering the possibility to study regional aspects of pathogenesis. 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