Physiological and pathological roles of exosomes in the nervous system

Kohei Yuyama; Yasuyuki Igarashi

doi:10.1515/bmc-2015-0033

Open Access Published by De Gruyter January 19, 2016

Physiological and pathological roles of exosomes in the nervous system

Kohei Yuyama and Yasuyuki Igarashi

From the journal Biomolecular Concepts

https://doi.org/10.1515/bmc-2015-0033

Abstract

Exosomes represent a subtype of extracellular nanovesicles that are generated from the luminal budding of limiting endosomal membranes and subsequent exocytosis. They encapsulate or associate with obsolete molecules to eliminate or to transfer their cargos in intercellular communication. The exosomes are also released and transported between neurons and glia in the nervous system, having a broad impact on nerve development, activation and regeneration. Accumulating evidence suggests that the exosomes are attributed to the pathogenesis of several neurodegenerative diseases such as prion disease, Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, as well as aging, in which the exosomes lack the capacity for cellular self-repair and spread their enclosed pathological agents among neurons. In this article, we review the current proposed functions of exosomes in physiological and pathological processes in the nervous system.

Keywords: exosome; glial cell; neurodegenerative disease; neuron

Introduction: the nature of exosomes

In the extracellular space, several types of membrane vesicle are present. These vesicles of cell origin are mainly divided into two groups: shedding vesicles and exosomes. The former represent diverse types of particle, which are generated by directly budding and pinching off from plasma membranes. These include microvesicles (also termed microparticles, ectosomes, or shedding vesicles), irregularly shaped particles of heterogeneous sizes with diameters ranging from 100 nm to 1 μm diameter, and apoptotic blebs (generally referred to as apoptotic bodies), which are 50 nm–4 μm nonuniformly shaped vesicles generated during the late stage of cell death (1). Exosomes are generated via endocytic pathways, not in the plasma membrane. They are formed by inward invagination and fission of a distinct domain of endosomal membranes. The endosomal compartments filled with intraluminal vesicles (ILVs) are termed multivesicular bodies (MVBs), which correspond to intracellular precursor organelles for exosomes. A set of MVBs fuse with the plasma membrane and ILVs are released into the extracellular space. The secreted ILVs are called exosomes. Exosomes are uniformly spherical and the smallest extracellular vesicles (40–100 nm) (2, 3). They were first reported by two groups almost at the same time as transferrin receptor-associated vesicles in reticulocytes (4–6). Since their discovery, exosomes have been reported to be released from a number of cell types in vitro, including the cells in the nervous system (3). Moreover, they have been isolated from diverse body fluids, including blood, saliva, breast milk, and cerebrospinal fluids (CSF) (7–10). Early studies on reticulocytes showed that exosomes can transport obsolete contents out of these cells during their maturation to erythrocytes, assuming that the primary function of exosomes might be the disposal of unnecessary cellular components as an alternative degradation pathway. In neuroblastoma cells, a low activity of lysosomes induced by alkalizing agents leads to the enlargement of endosomal compartments, which promotes the release of accumulated proteins and lipids with exosomes (11, 12). Niemann-Pick type C1 disease (NPC1) is an autosomal recessive lysosomal disorder. Loss of function of npc1 reduces lysosomal inactivity, resulting in the accumulation of cholesterol and sphingolipids within lysosomes and endosomes (13). In fibroblasts derived from NPC1 patients, the elevated levels of exosome secretion were observed to attenuate the toxic lipid storage (14). The exosome generation has also been associated with the autophagy system. Activation of autophagy by the LC3 overexpression promotes the fusion of MVBs with autophagic vacuoles and subsequently inhibits exosome released from erythroleukemic cells (15–17). These lines of evidence point to a close relationship between exosome secretion and other degradation pathways. On the other hand, exosomes also participate in intercellular transmission and the spread of their cargo. The information exchanges mediated by exosomes have been described as a novel form of intercellular communication in various biological phenomena (18, 19). In this article, we summarize the topics on currently proposed biological events associated with exosomes in the nervous system.

Formation of MVBs is the first step in exosome generation, which proceeds via an endosomal sorting complex required for the transporter (ESCRT) machinery (20, 21). The ESCRT protein complex is composed of four subunits, ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III, which associate with accessory proteins such as Alix. These subunits are sequentially recruited to the cytosolic surface of the endosomal membrane and drive the inward budding and fission of the membrane to form ILVs. Recent studies have also demonstrated exosome generation in an ESCRT-independent manner. The ESCRT-independent exosome biogenesis includes lipid- and tetraspanin-required pathways. Lipid-dependent formation of ILVs is required for the conversion of sphingomyelin (SM) to ceramide (Cer) (22). The polarhead group of SM, the phosphocholine structure, is degraded by neutral sphingomyelinase (nSMase)-2 at the luminal surface of endosomes, leading to Cer generation. The cone-shaped structure of Cer may physically spontaneously induce a negative curvature in the membrane and promote ILV formation. Alternatively, sphingosine-1 phosphate, a metabolite of Cer, has been reported to induce inward budding through the activation of the G protein-coupled S1P receptor (23). Hydrolyzation of phosphatidylcholine (PC) into phosphatidic acid (PA) by phospholipase D has also been proposed to promote exosome generation (24). Translocation of phospholipase D2 from the cytosol to the endosomal compartment triggers exosome release. The precise mechanism of PA-dependent exosome generation is unclarified; however, similar to the conversion of SM to Cer, the loss of the headgroup of PC might drive ILVs formation through the alternations of the physical properties of endosome membranes (25). Several studies have shown that tetraspanins participate in the generation of MVBs (26–28). In Hela cells, tetraspanin CD63 has been reported to compete with Hrs, a component of ESCRT-III, to generate distinct ILV subpopulations (27). Rab GTPases are essential regulators for vesicle transport including endosome trafficking. Several Rab proteins, Rab11, Rab27, and Rab35, have been identified to be involved in exosome secretion in different culture cell types (29–32).

The compo differs depends on their origin, types and conditions of parental cells. Although exosomes are devoid of proteins associated with the mitochondria, nuclei, endoplasmic reticulum, or Golgi apparatus, they contain membrane and cytoplasmic proteins (2). Exosome biogenesis accounts for the loading of ESCRT proteins such as Alix and Tsg101 into exosomes. The ESCRT complex is also responsible for the binding and sequestration of ubiquitinated proteins into ILVs, resulting in the abundance of ubiquitinated cargos in exosomes (21, 33). Tetraspanins contribute to the sorting of proteins toward exosomes (34). In lymphoblast cultures, proteomic analysis of exosomes and their parental cells revealed selective exosome loading of CD81 tetraspanin-associated molecules. Protein lipidation, including the GPI (glycosylphosphatidylinositol) anchor and saturated fatty acid modification, serve as tags for targeting proteins to exosomes (35, 36). GPI-anchored proteins, such as CD55, CD58, and CD59, and palmitoylated proteins, such as Lyn, are selectively incorporated into exosomes during reticulocyte maturation (32, 37). Proteins with these lipid modifications are preferentially recruited into lipid rafts, which are microdomains of cell membranes (35, 38). The lipid composition of exosome membranes is distinct from that of cellular membranes, and the major components of lipid rafts, namely SM and cholesterol, concentrate in exosome membranes (25, 39). Just how these lipids are more preferentially packed into the exosomes than in parental cells remains unclear. However, ILV formation induced by SM conversion might result in the high loading of raft lipids into the generated vesicles. Indeed, other components of lipid rafts, glycosphingolipids (GSLs), are abundant in exosomes (40, 41). Various raft-resident proteins have also been reported to be abundant in exosomes (42). As another mode of targeting into exosomes, oligomerized membrane-anchored proteins, such as HIV Gag proteins, have been reported to be sorted in exosomes (43–45). In addition to proteins and lipids, exosome cargos include genetic materials such as mRNA and miRNA (46). They can be translated or regulate gene expression in recipient cells (47–49). To catalog accumulated data of exosome cargos, a manually curated web-based database, ExoCarta (http://www.exocarta.org) has records of proteins, lipids, and RNAs that have been identified in different studies (50).

Physiological function of exosomes in nervous systems

Exosomes are released constitutively or stimulus-dependently from almost all cell types in the nervous system, including neurons (51), astrocytes (52), oligodendrocytes (53), and microglia (54). The resultant exosomes can be collected from CSF in several animal species and humans (10, 55). Exosomes are capable of transferring their cargos between cells either homologous or heterologous (Figure 1). Here, we summarize the recent studies on the physiological roles of exosomes in neuronal development, transmission, and regeneration.

Figure 1:

Roles of exosomes via neuron-glia communication.

(A) Oligodendroglial exosomes autonomously inhibit myelin sheath formation through RhoA-actomyosin signaling. As yet unknown secreted molecules from activated neurons inhibit oligodendroglial exosome release. (B) Electrically active neurons release glutamate (Glu). Glu stimulates oligodendrocytes to release exosomes through the Glu receptor (GluR)-mediated Ca²⁺ influx. The exosomes are taken up into neurons in where the effects of their cargos such as Hcp/HSP70 are exerted. Neuronal exosomes accelerate phagocytic activity in microglia. (C) Exosomes released from Schwann cells are selectively incorporated into axons. The exosomes activate growth cones, which leads to the elongation of injured axons.

Development

During development in the central nervous system (CNS), oligodendrocytes change their morphology as they enwrap neuronal axons as the myelin sheath for rapid impulse conduction. Extension of the myelin membrane sheath is regulated by signals from both the oligodendrocytes themselves and targeted neurons (56). The exosomes released from primary cultured oligodendrocytes have the ability to prevent membrane expansion (57). The feedback inhibitory effect of the exosomes is dependent on src-family kinase Fyn and RhoA-ROCK-myosin signaling. The molecules in the exosomes that are involved in actomyosin contractility have not been identified yet. It has been reported that the conditioned medium from primary cultured neurons inhibits the exosome release from oligodendrocytes. The signals from neurons that regulate glial exosome secretion are also as yet unknown. However, these signals could provide a novel mode of neuron-glia communication, in which neurons control myelin sheath formation by modulating the inhibitory activity of exosomes against oligodendrocytes.

The Wnt signaling pathway provides crucial positional information that regulates a number of developmental processes such as pattern formation, cell proliferation, and migration (58). Wnt proteins play important roles in axon guidance and synaptogenesis in the nervous system. Exosomes are found to be involved in intracellular transmission of Wnt proteins in the neuromuscular junction (59). Moreover, Wnt signaling plays a pivotal role in neuromuscular development in Drosophila (60). Fly Wingless (Wg), a Wnt homolog, is secreted from neurons with exosomes in an activity-dependent manner (61). The secreted Wg protein recognizes the Wg receptor Frizzled-2 (Fz2) at the postsynaptic surface and transduces signals in muscle cells. The neuronal protein Evi is of the protein required for the intracellular transport of the Wg protein and the subsequent exosome loading. Knockdown of YKT6, a SNARE protein, prevents the exosome-associated Wnt/Evi secretion; thus, it effectively blocks Wnt secretion with the exosomes and simultaneously induces adult wing notching in Drosophila (62). To date, the exosome-associated Wnt secretion has been confirmed in many other cell cultures including rat microglia and human HEK cells (62, 63). These findings have important implications regarding a universal role of exosomes in Wnt morphogenic signaling during nervous system development.

Neuronal activity

Neuronal exosome release was firstly observed in primary cultured cortical neurons (51). Proteomic analysis revealed that the neuronal exosomes contain the glutamate receptor subunit GluR2/3 and the adhesion molecule L1CAM, which is mainly expressed in neurons (64), together with exosomal marker proteins such as Alix, Tsg101, and flotillin-1. The exosomes are released from both soma and dendrites of neurons. Exosome secretion from neurons is promoted by Ca²⁺ influx or treatment with GABA receptor antagonists. In contrast, it is prevented by treatment with GluR antagonists (65), implicating that neuronal exosomes are released in a synaptic-activity-dependent manner. Neuroblastoma-derived exosomes are preferentially incorporated into glial cells rather than to neurons, whereas the exosomes released from stimulated cortical neurons are selectively bound and endocytosed into neurons (66). The machinery for exosome targeting selectivity remains unclear; however, these findings suggest a novel role of exosomes in interneuronal transport such as recycling of synaptic components.

Oligodendrocytes play critical roles not only in axon myelination but also in supporting the maintenance of long-term axonal integrity and control of nervous activity. Recent studies have shown that oligodendroglial exosomes have broad effects on neuronal physiology (53, 67, 68). The exosomes released from oligodendrocytes are triggered by Ca²⁺ entry or glutamate receptor stimulation similarly to those released from neurons (67). Hence, the glutamate that is released from the stimulated neurons might affect oligodendrocytes to promote exosome generation. The secreted oligodendroglial exosomes are in turn incorporated into neurons either from axons or somata. The protein and RNA cargos that are enclosed in exosomes are functionally retrieved and increase cell viability under the conditions of oxidative stress, nutrient deprivation, and ischemia (67, 69). The nature of the neuroprotective effect of oligodendrocytic exosomes remains obscure; however, multiple sets of signaling alterations in oligodendrocytes have been identified after exosome treatment using phosphorylation arrays (68). These include Akt, Erk1/2, CREB, GSK-3a/β, and JNK, which may contribute to the neuroprotective function. Another cargo protein, Hsc/Hsp70, might also be involved in cell robustness. The protein is derived from glial cells and shows neuroprotection (70). Hsc/Hsp70 is reported to be loaded onto the oligodendroglial exosomes and then taken up by neurons (67). In addition, the transferred oligodendroglial exosomes also enhance spontaneous electrical neuronal activity, as revealed by electrophysiological analysis (68). On the other hand, the exosomes derived from neurons support the microglia-dependent removal of degenerating neurites (71). In this case, serum depletion induces neurite degeneration in differentiated neuroblastoma, and cocultured microglial cells in turn remove the degenerative neurites by phagocytosis. It was found that neurite removal is accelerated when microglial cells are preincubated with the exosomes derived from stimulated neuroblastoma, indicating a role of neuronal exosomes as a regulator of neuritic elimination through microglial activation. The removal of degenerative neurites and inactivated synapses is required for the reorganization of complex neuronal circuits in the brain. A future work will be necessary to elucidate the exosome functions in vivo; nevertheless, it is obvious that exosomes act as a crucial player in the physiological state of the nervous system.

Neuronal regeneration

In the peripheral nervous system, Schwann cells (SCs) are the principal glial cells that wrap motor and sensory neurons to form the myelin sheath and provide these neurons trophic support. SCs are also involved in neuronal regeneration after nerve injury. Nerve repair proceeds through the interaction between neurons and glia, which is not under the control of neuronal soma (72). SC-derived exosomes were found to promote axonal regeneration (73, 74). The exosomes derived from rat primary SC cultures are internalized into the axons of dorsal root ganglion neurons, not fibroblasts, and are especially concentrated in the axon terminal growth cones. The SC-exosomes facilitate axonal elongation after mechanical transection of dorsal root ganglion explant cultures. Following the exosome treatment, growth cones morphologically represent the active state, and the activity of RhoA, a GTPase whose activation inhibits axonal elongation, is significantly reduced. The effect of SCs-exosomes on axonal elongation is further confirmed in an in vivo study (73). Administration of SC-exosomes to rats results in the acceleration of nerve fiber regeneration after sciatic nerve crush.

Exosomes in neurodegenerative diseases and aging

It has been apparent that several pathogenic proteins that are involved in neurodegenerative diseases, including prion disease, Alzheimer’s disease (AD), Parkinson’s disease (PD), and amyotrophic lateral sclerosis (ALS), are loaded into ILVs in MVBs then subsequently secreted extracellulary via exosomes. Endocytic disturbances such as abnormalities of MVB formation commonly found in these diseases suggest that impairment of exosome generation is associated with the development of such diseases (Table 1). In the following, we summarize the accumulated lines of evidence on the bidirectional roles of exosomes, that is, they function in the clearance of intracellular pathogens as well as the intercellular spread of their toxicity in disease pathogenesis.

Table 1

Neurological disease proteins found in exosomes and MVBs/endocytic disturbance.

Human disease	Exosome-asscosiated protein	References	MVB/endocytic impairment	References
Creutzfeldt-Jakob disease	PrPc, PrPsc	(10, 75–79)	MVB and endosome enlargement	((80), (81) [BSE in cattle])
Alzheimer’s disese	Aβ	(82–86)	Endosome enlargement	(81, 87–89)
	APP	(12, 82–84)	Overexpression of RabGTPases
	BACE	(83, 84)	CHMP2B high immunoreactivity
	Presenilin	(83)	PICALM, BIN1 mutation
Alzheimer’s disese, frontal temporal dementia	Tau	(55, 86, 90, 91)	Same as above	Same as above
Parkinson’s disease	α-synuclein	(92–98)	CHMP2B mutation	(100, 101)
	LRRK2	(99)	CHMP2B-positive inclusions
Amyotrophic lateral sclerosis	SOD1	(102–105)	CHMP2 mutation	(108–110)
	TDP-43	(106, 107)	[2)Axonal exdosome transport deficits
Polyglutamine diseae	Heat shock protein	(111)
(Huntington disease)	(HSP40, HSP70, HSP90)
Schizophrenia	Dysbindin-1B	(112)	Mutations in BLOC-1 subunits, dysbindin and muted	(113, 114)

Prion disease

Exosomes in the nervous system were first studied in prion diseases, in which exosomes contained prion proteins (75). Prion diseases include scrapie in sheep, bovine spongiform encephalopathy in cattle, and Creutzfeldt-Jakob disease in humans. They are transmissible neurodegenerative disorders in which the cellular prion protein PrPc is abnormally folded into the neurotoxic pathogenic form PrPsc. In an infectious prion disease, when PrPsc proteins enter the host through the gastrointestinal tract, they replicate themselves in peripheral organs and then invade into CNS. Approximately 80% of all cases of prion diseases are sporadic and are characterized by the propagation of endogenously generated PrPsc into the brain. Aging is reported as one of the risk factors for a sporadic prion disease, since the onset of the disease is usually at the ages of 45–75 years, with the average of approximately 65 years of age. From recent in vitro studies, exosomes have been suggested to contribute to the release of membrane-bound PrP from host cells and to the incorporation of the pathogen into other cells. Both PrPc and PrPsc have been observed in MVBs and secreted into the culture medium with exosomes (75–78). PrP was also detected in the exosomes isolated from ovine CSF and PrP-inoculated mouse plasma (10, 79). The mechanism by which PrP is loaded into exosomes is still obscure. Recent findings suggest that the GPI membrane anchor of PrP might facilitate PrP sorting. PrP is predominantly localized at lipid rafts in the plasma membrane of culture cells (115). Inhibition of nSMase activity induces in turn inhibits both exosome release and PrP packaging (77, 116). These suggest that PrP might attach to lipid rafts through its GPI membrane anchor and then subsequently directly enter into the exosomes. Furthermore, it has been demonstrated that the exosome-bound PrP is infectious in vitro and in vivo. Exosomes released from infected cells can be transferred to other cells including neurons (76). Infectious PrPsc, which is contained in the exosomes, induces the conversion of PrPc into PrPsc in recipient cells. Intracerebral inoculation of infected cell-derived exosomes into normal mice for 3 months produced several symptoms of prion disease such as weight loss and locomotor disturbance in the inoculated mice (76).

If PrP in the exosomes spreads via body fluid, there might be no distance restriction in its propagation. Thus, exosomes could play an important role in transmitting the pathogenic protein from the peripheral to CNS. Several types of cell in the peripheral tissues have been reported to secrete PrP associated with exosomes as well as neurons (78). However, normal membrane vesicles including exosomes are generally considered to be not capable of passing through the blood-brain barrier (BBB). Recent studies shown that malignant alternation can change the miRNA profiles in the exosomes derived from the brain metastatic cancer cells, and the exosomes that hold specific miRNAs can promote the destruction of BBB (117). PrP infection has also been shown to cause changes in miRNA ultrastructural features and signature in exosomes (118, 119). The study of the efficiency of exosomes to target the CNS might be one of the challenging issues in the future.

Alzheimer’s disease

In addition to prion disease, the exosomes in relation to the pathogenesis of AD have been intensively investigated. Although the exact pathological significance of exosomes remains to be conclusively resolved, several fascinating schemes have recently been proposed. AD is a neurological disorder with progressive loss of memory and cognitive function, and is characterized by extracellular deposits of amyloid-β peptide (Aβ), called senile plaques, mainly in the hippocampus and cortex. Aβ is a short peptide composed of about 40 amino acids and is generated mainly in neurons and released extracellulary. The levels of Aβ are maintained at a steady state in normal brains. However, impairment of the balance of Aβ metabolism is attributed to the formation of its toxic assemblies, which are connected to the AD pathology. In neurons, Aβ is derived from the sequential processing of amyloid precursor protein (APP) with β- and γ-secretases. Both of these enzymes are localized in endocytic compartments; hence, Aβ generation mainly occurs in the endocytic pathway (120). Neuronal endocytic dysfunction such as MVB enlargement occurs in the early stage of AD, suggesting that it might contribute to pathological Aβ accumulation (121–123). Aβ is found to accumulate in MVBs in AD brains (124). This is similarly found in APP-transfected neuroblastoma in which Aβ is localized in MVBs and secreted in association with the exosomes (82). APP and its metabolite CTFs are loaded into the exosomes(83). Exosomal proteins such as Alix and flotillin-1 are abundant in senile plaques in AD patients, suggesting a potential role of the exosomes in the formation of Aβ deposits (82).

Accumulated lines of evidence indicate that glycan-linked sphingolipids, especially sialic acid-containing GSLs, serve as sites for Aβ attachment (125, 126). Aß-bound GM1, a GSL with sialic acid, has been found in brains exhibiting early pathological changes of AD (127). In neuroblastoma cell cultures, we found that synthetic Aβ binds to exosomes and the degradation of GSL-glycans prevents the binding between Aβ and exosomes (128). We next examined the GSLs-derived glycans in exosomes by quantitative GSLs-glycomics and found that GSLs are more highly abundant in the exosomes than in the parental cells (41). In particular, the levels of sialylated GSLs are much higher in the exosomes. These indicate that Aβ can associate with accumulated and clustered GSL-glycans, which are localized at the exosomal surface and exposed to the outer space. Later, the exosomes that are released from primary cultured neurons are also found to have much high levels of GSLs than those from primary microglia and astrocytes (85). Accordingly, Aβ can binds to only neuronal exosomes, not glial exosomes. It remains to be clarified how high levels of GSLs are packed into exosomes; however, it is reasonable to consider that GSLs might be integrated into the exosome membrane as components of lipid rafts through ceramide-dependent ILV formation. Indeed, the exosome release from neurons is almost prevented by SMase inhibition (128). Intriguingly, Aβ cannot only attach to exosome, but can also form nontoxic fibril assemblies at the exosomal surface in a GSLs-dependent manner. Microglia are brain-resident phagocytes, which contribute to the removal of dead cells and debris. Several studies including ours showed that neuronal and glial exosomes are preferentially internalized by microglia (128, 129). Aβ-bound exosomes are also actively taken up into microglia and then Aβ is degraded within microglia by the lysosomal system (128). These suggest that exosomes support Aβ clearance by trapping Aβ with their surface GSL and transporting it into microglia (Figure 2). An in vivo study showed that the neuron-derived exosomes injected into APP transgenic mouse brains also associate with endogenous Aβ in the brains and are incorporated into microglia (41). Furthermore, continuous infusion of the exosomes for two weeks decreases Aβ, inhibits amyloid depositions, and attenuates synaptic toxicity (41, 85). These studies clearly demonstrated that intracerebral exosome administration ameliorates Aβ-related AD pathogenesis. Improvement of Aβ clearance by exosome supplementation or enhancement of exosome generation might provide a novel therapeutic approach for AD. However, these indicate that normal phagocytic function is important for exosome-dependent Aβ clearance. If the clearance function of microglia is impaired or absent, the exosome-hoboing Aβ assemblies would trigger pathological events (i.e. senile plaque formation and interneuronal transfer of Aβ). Further careful in vivo studies are required to clarify the roles of exosomes in AD pathogenesis.

Figure 2:

Roles of neuronal and microglial exosomes related to Aβ in Alzheimer’s disease.

Neuronal exosomes are associated with intracellular or extracellular Aβ through GSLs abundant on the exosomal surface. Exosomes promote the assembly of nontoxic Aβ. Aβ-exosome complex is subsequently taken up into microglia to degrade Aβ. Neuronal exosomes likely promote Aβ clearance. In the absence of microglial phagocytic activity, exosome-associated Aβ might act as a seed for Aβ amyloid plaque. In turn, exosomes that are released from Aβ-stimulated microglia are autonomously incorporated into microglia and induces caspase-3-dependent apoptosis. Enriched Cer and PAR-4 in the microglial exosomes are responsible for the toxic effect.

On the other hand, recent findings in primary astrocyte cultures indicated that synthetic Aβ accelerates exosome release. The exosomes derived from Aβ-treated-astrocytes, which contain prostate apoptosis response-4 (PAR-4) and abundant Cer, especially C18:0 Cer, cause apoptosis of normal astrocytes (130). Cotreatment with a specific antibody against PAR-4 is found to prevent the exosome-induced apoptosis. In addition, exosomes collected from nSMase-2-deficient (fro/fro) astrocytes do not show astroglial toxicity. These indicate that enriched Cer and PAR-4 in the exosomes are involved in the apoptosis of astrocytes. In addition to neuronal impairment, astrocytic dysfunction has been reported to be associated with AD pathogenesis, e.g. the elevated number of senile plaques (131). Thus, the astrocyte-derived exosomes might contribute to AD development. As challenging issues in the future, we need to confirm whether the toxic exosomes are generated within brains and examine how the exosomes affect other types of cell including neurons.

AD is a tauopathy which is characterized by the presence of neurofibrillar tangles composed of phosphorylated tau. Exosome-associated tau has been detected in culture media and CSF (55, 86, 91). Tau has been found in the exosomes derived from human tau-expressed neuroblastoma cells and to possess phosphoepitopes such as AT180, AT8, and PHF1, associated with AD (55). The levels of exosomal phosphorylated tau (AT270) increase in the CSF at an early stage of AD. The mechanism by which tau is load into exosomes remains unresolved. Electron microscopy showed that tau appears to attach to the surface of CSF exosomes. However, from the exosome topology, cytosolic protein tau would be expected to reside in the exosomal lumen. Further additional examination is required under conditions that tau leaked from degenerated cells is excluded. Recently, tau transmissibility between neurons and microglia has been proposed to be based on the propagation of tau during AD development (91). In a tau propagation mouse model, microglia, which phagocyte tau-containing neuronal debris, release tau with exosomes, which efficiently transmit tau to neurons. Depletion of microglia in the mice suppressed tau propagation.

As AD-related agents have been found to be included in blood exosomes, the neuron-derived exosomes are isolated by L1CAM immunoabsorption from human serum from the subjects of healthy control and mild cognitive impairment (MCI), and patients with sporadic AD (69, 86). As a result, higher levels of Aβ_1-42 and phosphorylated tau (S396, T181) are demonstrated in the neuronal exosomes from MCI subjects and AD patients than in healthy control. In addition, several lysosome-associated proteins, such as cathepsin D and lysosome-associated membrane protein-1 (LAMP-1), also show significantly increased levels in the exosomes in AD compared with the control, indicating that impairment of the endosome-lysosome pathway occurs in neurons in early AD brains. The findings implicate that the above proteins may be potential biomarkers for predicting AD development before clinical diagnosis.

Parkinson’s disease

PD is a progressive neurological disorder pathologically characterized by dopaminergic neuronal loss and presence of intraneuronal inclusions called Lewy bodies (LB) in the midbrain. LB is formed from abnormally folded aggregates of α-synuclein (α-syn), which also forms toxic oligomeric intermediates that are directly linked to neuronal damage (132). α-Syn is generally considered to exert its toxic effect only inside cells; however, recent findings showed that α-syn is secreted extracellulary and transmits its toxicity to neighboring cells both in vitro and in vivo (133, 134). In α-syn-transfected neuroblastoma cells, either monomeric or oligomeric α-syn is released from the cells in association with exosomes (92). The exosome-bound α-syn induces the cell death upon their exposure of differentiated neuroblastoma cells to this α-syn. Moreover, the exosome-associated α-syn oligomers are more prone to be uptaken into neuroblastoma cells, exerting greater toxic effects to the recipient cells than free α-syn (94). The report that the microglial capacity for uptaking exosome-associated α-syn oligomers decreases age-dependently suggests the enhancement of α-syn toxicity against neurons at an old age (135). The exosomes might function in α-syn oligomerization, not only in transporting preformed α-syn aggregates (136). The aggregation of α-syn was accelerated by either the exosomes derived from neuroblastoma cells or the synthetic liposomes formed from extracted exosomal lipids, both of which contain exosomal GSLs, such as GM1 and GM3. These raise the possibility that glycolipids on the exosome surface have the potential to transform monomeric α-syn into toxic assemblies, opposing the effect of Aβ that forms nontoxic fibrils (128). α-Syn has been detected in human plasma and CSF (137). Part of plasma α-syn is reported to associate with exosomes (96, 137). Radiolabeled α-syn included in exosomes is transported to blood as soon as it is intracerebrally injected into mouse brains. α-Syn is also detected in the exosomes of human plasma and there is a significantly higher levels of exosomal α-syn in the blood of PD patients than in that of age-matched control. Elevated levels of exosomal α-syn in blood might reflect the enhancement of α-syn-bound exosome generation in PD brains. Dysfunction of several proteins that are involved in exosome generation has been reported to be linked to PD pathogenesis both in cellular models and PD patients. Rab11 GTPase and CHMP2B, two components of ESCRTIII, colocalize with α-syn-positive intracellular inclusions, and Rab11 activity modulates α-syn secretion and aggregation (98, 100). Loss of the expression of the ATPase ion pump ATP13A2 (PARK9) decreases the number of ILVs in MVB. Inversely, elevated ATP13A2 expression levels, enhance exosome-related α-syn secretion and decrease α-syn levels inside cells, which consequently attenuate intracellular α-syn toxicity in neuroblastoma cells (95, 97). The above findings suggest the dual roles of exosomes, that is, protective and pathological effects on PD development. Thus, the promotion of exosome generation combined with the prevention of exosome incorporation into neighboring neurons might be necessary for exosome-mediated therapeutic strategies for PD.

Amyotrophic lateral sclerosis

ALS is an adult-onset neurodegenerative disease characterized by the progressive loss of upper and lower motor neurons mainly in the spinal cord and brainstem. Mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1) are predominantly associated with familial ALS (138). Misfolded SOD1 has been observed in neurons in sporadic and familial ALS (105). Recent findings suggest that misfolded SOD1 converts other SOD1 proteins including the WT to misfolded forms that are released into the extracellular space similar to pathogenic prion proteins (103). Exosome- associated SOD1 secretion has been reported in either WT or mutant SOD1 (G93A)-overexpressed motor neurons (102). Mutant SOD1 (G93A)-overexpressing primary astrocytes also release SOD1 extracellulary with exosomes (104). The mutant SOD1-associated astrocytic exosomes potentiate neuronal death in a coculture system of primary spinal neurons and astrocytes, suggesting that the exosomes transfer toxic molecules including G93A SOD1 to neighboring neurons, which might contribute to the spatiotemporal propagation of the ALS pathology. The mechanism by which SOD1 is loaded into exosomes remains unclear. Mutant SOD1 (G127X, G85R) is detected on the exosome surface (105), which is different from WT SOD1, which is usually localized inside exosomes (139). Thus, treatment of the exosomes with specific antibodies against mutant SOD1, which could recognize their epitopes on the exosomes and then block their toxicity, inhibits the conversion of SOD1 into its misfolded form in the recipient cells (105). TAR DNA-binding protein of 43 kDa (TDP-43) has been reported to be included in the exosomes (106). TDP-43 is a protein composed of an intraneuronal inclusion found in ALS patients. Pathogenic TDP-43 such as hyperphosphorylated and ubiquitinated species, tends to aggregate and represent cellular toxicity. Neurons containing TDP-43-positive inclusions are deficient in normal TDP-43 functions. Loss of normal function and gain of toxic function are both proposed to contribute to the ALS pathology (140). Phosphorylated TDP-43 aggregates are released from neuroblastoma cells and incorporated into recipient cells, in which the toxic assemblies operate as templates for new aggregate formation (106). Extracellular TDP-43 is observed in exosomes, which suggests the roles of exosomes against toxic aggregates similar to those in AD and PD, in which the exosomes are exploited to decrease the intracellular toxicity of SOD1 and TDP-43 by facilitating their release from cells and concomitantly supporting their intraneuronal propagation in development of ALS pathology.

Aging

Aging has been considered as the most common risk factor among neurodegenerative disorders such as AD, PD, ALS, and sporadic prion disease, which all show rising rates of morbidity with aging. Several groups including ours analyzed the exosomes derived from CSF in animal models and human subjects in relation to aging. Such studies provide clues to clarifying the roles of exosomes in disease pathogenesis. We isolated CSF exosomes from APP transgenic mice during aging from 2 to 23 months of age and measured the exosome concentration by nanoparticle analysis (85). We found that the number of CSF exosomes markedly declined during aging. Similarly, we purified the exosomes from CSF of cymologus monkeys and found significant reductions in the levels of the exosome marker proteins Alix and Tsg101 in the aged monkeys compared with the young (our unpublished observation). Furthermore, extracellular vesicles including the exosomes have been reported to undergo temporal reduction in levels with age in human CSF (141). In accordance with reductions in the levels of mouse and monkey CSF exosomes, Aβ in the CSF exosomes keep decreasing with age (85). However, the Aβ levels continue to increase from 6 to 23 months of age in the brain tissues of both APP transgenic mice and cymologus monkeys. The data raise the possibility that the generation of endogenous exosomes from brain cells might modulate Aβ metabolism in the brain.

Accumulating evidence demonstrated that the interaction of the two axonal transport proteins, dynactin and dynein, is impaired in the axons in the brains of aged monkeys (142). Dysfunction of the motor protein complex leads to axonal transport impairment and endocytic pathologies including Rab GTPases overexpressions and endosome enlargements (143). Endocytic impairment has been found in dynein-siRNA-treated neuroblastoma cells, which are used as a model of aged neurons, concomitant with a significant inhibition of exosome release from the cells. Intracellular accumulations of Aβ and tau have been identified in neurons in the brains of aged monkeys, suggesting that endocytic disturbance might prevent the exosome-mediated secretion of pathogenic proteins in aged brain cells. However, it has been reported that aging accelerates exosome release in other tissues except those in the nervous system. In the retina pigment epithelium (RPE), decrease in lysosome activity during aging results in the impairment of intracellular organelles such as mitochondria, with damaged DNA accumulating inside cells. Thus, exocytic activity is elevated in the cells to discard obsolete components, which promotes exosome secretion (144). The exosomes form extracellular, amorphous deposits called drusen on Bruch’s membrane in the macula of the retina, which leads to the development of age-related macular degeneration (AMD) (145). Age-related changes in the endosome-lysosome pathway might modulate exosome secretion differently in individual cells. Further studies would contribute to better understanding of the endocytic machinery for modulating exosome generation according to age-dependent endocytic alternations.

Conclusions and perspectives

In this review we have illustrated that new roles of exosomes are emerging, which cover a broad range of neuronal physiological functions and diseases. It appears that exosomes in the nervous systems act more than just in the disposal of cellular contents, but also in intercellular exchanges of their functional cargos. These would enable development to proceed normally and maintain normal function of the nervous systems; on the other hand, these exacerbate the pathologies through the spread of pathogenic agents over time throughout the nervous systems. Exosome studies in this area have been carried out mostly with cell cultures; thus, their degree of contribution to elucidating the in vivo functions has not been fully clarified. Further studies are required including the development of techniques to analyze exosomes in small volumes of fluids and the generation of animal models with the altered levels of exosome secretion. Deepening our understanding of the fundamental process such as biogenesis, cargo sorting and recipient targeting and the identification of specific markers of each origin would open the way to solving the problems.

Potential values of exosomal miRNA have been validated as the biomarkers of many types of cancer (146). Alterations of exosomal miRNA profiling have been reported in neurodegenerative diseases such as AD and PD, and also in some mental disorders such as schizophrenia and bipolar disorder (147, 148). Although the involvement of these identified exosomal miRNAs in the pathogenesis of these diseases is unclear, the miRNA profiles of CSF- or blood-derived exosomes might be available as novel diagnostic or prognostic biomarkers for the diseases. Understanding of the significant pathological roles of exosomes would lead to the development of novel therapeutic approaches for the diseases. For example, with neuronal exosomes found to clear Aβ, exosome administration might provide a new approach to attenuating the AD pathology. Exosomes have been used as a delivery platform for targeting encapsulated reagents or siRNAs into the brain (149, 150). Peripheral injection of exosomes carrying siRNA succeeded in brain targeting and specific gene knockdown in AD and PD mouse models (149, 151). Development of engineered nanovesicles in combination with the understanding of the pathological roles of exosomes could lead to the exploitation of exosomes as a clinical tool.

Corresponding author: Kohei Yuyama, Laboratory of Biomembrane and Biofunctional Chemistry, Graduate School of Advanced Life Science, Hokkaido University, Kita21 Nishi11, Sapporo 001-0021, Japan, e-mail: kyuyama@pharm.hokudai.ac.jp

List of abbreviations
Aβ: amyloid-β peptide
AD: Alzheimer’s disease
ALS: amyotrophic lateral sclerosis
APP: amyloid precursor protein
α-syn: α-synuclein
BBB: blood-brain barrier
Cer: ceramide
CSF: cerebrospinal fluids
CNS: the central nervous system
ESCRT: endosomal sorting complex required for transporter
Fz2: Frizzled-2
GPI: glycosylphosphatidylinositol
GSLs: glycosphingolipids
ILVs: intraluminal vesicles
LAMP-1: lysosome-associated membrane protein-1
LB: Lewy bodies
MCI: mild cognitive impairment
MVBs: multivesicular bodies
NPC1: Niemann-Pick type C1 disease
nSMase: neutral sphingomyelinase
PA: phosphatidic acid
PAR-4: prostate apoptosis response-4
PD: Parkinson’s disease
PC: phosphatidylcholine
SCs: Schwann cells
SM: sphingomyelin
SOD: Cu/Zn superoxide dismutase
TDP-43: TAR DNA-binding protein of 43 kDa
Wg: Wingless

Acknowledgments

We are grateful for the support by Creation of Innovation Centers for Advanced Interdisciplinary Research Areas Program, Ministry of Education, Culture, Sports, Science and Technology, Japan.

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Received: 2015-11-25

Accepted: 2015-12-22

Published Online: 2016-1-19

Published in Print: 2016-2-1

This article is distributed under the terms of the Creative Commons Attribution Non-Commercial License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Physiological and pathological roles of exosomes in the nervous system

Abstract

Introduction: the nature of exosomes

Physiological function of exosomes in nervous systems

Development

Neuronal activity

Neuronal regeneration

Exosomes in neurodegenerative diseases and aging

Prion disease

Alzheimer’s disease

Parkinson’s disease

Amyotrophic lateral sclerosis

Aging

Conclusions and perspectives

Acknowledgments

References

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