The Role of Exosomes/Extracellular Vesicles in Neural Signal Transduction

Abstract

Exosomes, in a broad sense extracellular vesicles (EVs), are secreted from several cells and also exist in cerebrospinal fluid (CSF); they contribute to signal transduction not only between neural cells but also among hematopoietic cells. In addition to the peripheral nervous system, the association of regeneration and EVs has also been reported in the central nervous system, for example, following a spinal cord injury. Furthermore, it has become clear that major causative factors of neurodegenerative diseases are transmitted by EVs; thus, EVs are involved in the pathogenesis of neurodegenerative diseases. In particular, we would like to outline the relationship between neurophysiology and neurological disorders centered on EV-mediated communication between neural and glial cells.

INTRODUCTION

An exosome is a membrane vesicle about 30 to 120 nm in size, secreted from various cells. It exists in the cerebrospinal fluid (CSF) and other bodily fluids (saliva, urine). Proteins, lipids, nucleic acids, and so on are contained within exosomes or on their membranes. It has become clear that exosomes are taken up by cells, and are involved in intercellular communication. An exosome is produced from an endosome, which is an intracellular organelle responsible for the screening, decomposition and reuse of various substances taken into cells. Through the multivesicular body (MVB), in which a membrane is formed inside this endosome, the MVB fuses with the cell membrane, allowing the membrane vesicle to be released from the cell (Fig. 1). Currently, exosomes are referred to as extracellular vesicles (EVs) in a broad sense, as either an apoptotic body (size of 0.5 to 5 µm, formed by apoptosis), or as microvesicles (size of 0.05 to 1 µm, created by part budding). The size of each type of EV varies, according to the literature. Because it is difficult to strictly isolate only exosomes from EVs, according to size and/or specific makers, in this review we will describe the exosome as unified to EVs (Fig. 2). Our laboratory revealed that EVs are involved in the “synapse pruning” that regulates the number of synapses between neurons and microglia.¹⁾ In particular, the major causative protein of various neurodegenerative diseases is encapsulated in EVs derived from CSF, and the relation between disease onset and exosomes has been studied intensively in recent years.

Fig. 1. Exosome–Endosomal Pathway

Exosomes are released through the endosome system to the outside of the cell by multivesicular endosomes (MVBs, multivesicular bodies) fusing with the cell membrane. Microvesicles are produced by budding of the cell membrane via necking and released to the outside of the cell. (Color figure can be accessed in the online version.)

Fig. 2. Extracellular Vesicles

EVs contain three vesicle types: apoptotic bodies; microvesicles; and exosomes. Strict isolation of exosomes is difficult as size overlaps and marker molecules are common. However, it is possible to isolate high-purity exosomes using phosphatidyl serine (PS) when almost no dead cells are present (Magcapture, Wako; 293-77601). (Color figure can be accessed in the online version.)

1. EV-MEDIATED NEURONAL REGENERATION

1.1. Axon–Schwann Cell Communication

Regeneration of the central nervous system (CNS), i.e., after spinal cord injury, is difficult, but can be repaired in the peripheral nervous system. Contributing to this peripheral axon regeneration are Schwann cells,²⁾ which form myelin sheaths in the axon. Schwann cells support dendritic regeneration by promoting dedifferentiation and proliferation, the removal of myelin and axonal debris, etc. Therefore, it was investigated whether EVs are involved in the transmission of information between Schwann cells and axons. Axonal formation and axonal regeneration of injured Dorsal Root Ganglion (DRG), a model of peripheral nerves, were promoted by Schwann cell derived EVs.³⁾ In fact these EVs were incorporated in the axons and growth cones. Injury to a rat in vivo sciatic nerve model, and injection of EVs derived from Schwann cells, confirmed the regeneration of the rat peripheral axon. In other words, axonal regeneration was thought to occur as a result of EV-mediated signal transduction between Schwann cells and axons.

1.2. Axon–Axon Communication

Axonal outgrowth is regulated by guidance factors, extracellular matrix (ECM), cell adhesion molecule (CAM), cadherin, netrin / slit family, semaphorins, ephrin, etc.⁴⁾ For instance, ephrin binds with a transmembrane Eph receptor and acts as a bilateral “ephrin–Eph signal,” primarily as a repulsive axon guidance factor on the growth cone of the axon. Ephrin is also involved in the proliferation of neural precursor cells, as well as the formation of a neural tissue boundary in early developmental stages. The endosomal sorting complexes required for transport (ESCRT) protein group was identified by MS as a molecule interacting with EphB.⁵⁾ Since this ESCRT protein group plays an important role in MVB formation,⁶⁾ the contribution of EVs in axon outgrowth via the ephrin–Eph signal was analyzed. EphB was present on the membrane surface in motor neuron-derived EVs, and these EphB-expressed EVs promoted the collapse of the growth cone. In addition, this EphB was efficiently encapsulated into EVs by depolarization. Since Eph and ephrin are localized in dendrites and are involved in synapse formation and plasticity, it was suggested that stimulus-dependent EVs contribute to the remodeling of the synapse, which is the junction of axon ends and dendrites.

1.3. Neuron–Astrocyte Communication: EVs and Spinal Cord Injury

Three conditions, a) inflammatory response, b) axonal guidance factors, and c) the control of glial scarring, are important for axonal regeneration in the CNS, such as cure of spinal cord injury. In the inflammatory response (a), immune cells invaded following Blood–Brain Barrier (BBB) collapse due to injury strongly inhibits regeneration. Repulsive axon guidance factors (b), such as the previously described ephrin–Eph signal, are involved. In terms of scarring (c) it is also known that reactive astrocytes migrate and and congregate densely around the injured area, thereby inhibiting neurite extension in axonal regeneration and releasing an elongation inhibitory factor such as chondroitin sulfate proteoglycan.⁷⁾ Among these three, the relationship between glial scarring and EVs was analyzed.⁸⁾ Injection of an agonist of retinoic acid receptor β into rat spinal cord injury model (cervical avulsion) resulted in locomotion and sensory recovery. This agonist slightly promotes Phosphatase and tensin homolog (PTEN) encapsulation in neuronal EVs; the EVs are taken up into astrocytes, and the effect of PTEN, which negatively regulates cell division, decreases astrocyte proliferation. Namely, it contributes to the mitigation of spinal cord injury by lowering glial scar formation via EV-mediated neuron-to-astrocyte signaling.

1.4. Neuron–Oligodendrocyte Communication

When a lacZ reporter was expressed with an oligodendrocyte tissue specific promoter, the lacZ was expressed in some neurons.⁹⁾ It thus suggests the propagation of proteins between oligodendrocytes and neurons. In fact, the MVB of oligodendrocytes is present in the vicinity of the axon, and the amount of EV derived from oligodendrocytes increased due to the stimulation of glutamate, which is an excitatory transmitter. Interestingly, except microglial cells these EVs were more easily taken up by neurons (not astrocytes or oligodendrocytes), namely, they showed cell selective uptake ability. Furthermore, the quantity of EVs did not change in the knockout mice of glutamate receptors (N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA)). In fact, even in the co-culture of oligodendrocytes treated with an inhibitor (bicuculline) of γ-aminobutyric acid (GABA), which is an inhibitory transmitter of inhibitory neurons, the amount of EVs increased. Additionally, such EV-mediated signaling between neurons and oligodendrocytes showed a protective function of neurons during oxidative stress. Glutamate is released from axons due to electrical stimulation, is transferred to NMDA or AMPA receptors in oligodendrocytes, calcium flows into oligodendrocytes, and EVs are then released from MVB near the axon. With this incorporation of EVs into axons, it is possible EVs may be involved in neuroprotection (Fig. 3).

Fig. 3. Information Transmission from an Oligodendrocyte-Derived EVs to an Axon

Glutamate is transmitted via NMDA and the AMPA receptor in oligodendrocytes by stimulation such as depolarization, and EVs production and secretion are promoted by MVBs located in the vicinity of nerve axons due to the influx of calcium. Finally, such EVs derived from oligodendrocytes exhibit neuronal protection against stimuli such as stress. (Color figure can be accessed in the online version.)

2. EV-MEDIATED NEURODEGENERATIVE DISOR-DERS

2.1. Neuron–Astrocyte Communication: Apoxosomes

The characteristic pathology of neurodegenerative disorders is due to aggregates in the brain. Intercellular spreading and aggregation of proteins responsible for the major constituent components of this aggregation is drawing attention.¹⁰⁾ It is known that Aβ (amyloid β protein) forms senile plaques, that Tau protein forms neurofibrillary tangles in Alzheimer’s disease (AD), that PrP (prion) forms amyloid plaques in Prion disease, that TDP-43 forms an inclusion body in amyotrophic lateral sclerosis (ALS), that α-synuclein forms Lewy body in Parkinson’s disease (PD), and that glial cytoplasmic inclusion is involved in multiple system atrophy.

Aβ is produced when the membrane protein Amyloid-β precursor protein (APP) is cleaved by extracellular protease (β-, γ-secretase). This Aβ peptide acts on astrocytes to promote ceramide production via nSMase.¹¹⁾ Since ceramide is generally considered to enhance EV production,¹²⁾ the Aβ peptide enhanced the production and secretion of EVs in which Prostate apoptosis response-4 (PAR-4) interacted. This EV, named “apoxosome,” induced apoptosis in astrocyte-incorporated EVs in vitro and in the mouse brain. In Alzheimer’s disease model mouse, the reduction of EV production through a nSMase-ceramide pathway led to the alleviation of Alzheimer’s disease. Unfortunately, the specific astrocyte selective uptake mechanism of apoxosome was not shown. One of the binding factors of PAR-4 is binding immunoglobulin protein (BiP), which is involved in endoplasmic reticulum (ER) stress molecular chaperoning, and exists in the cell membrane.¹³⁾ BiP also has a strong correlation with Alzheimer’s disease from the viewpoint of protein misfolding and aggregation,¹⁴⁾ and BiP on the cell membrane may be involved in the uptake mechanism of apoxosomes, since it is also involved in Purkinje cell death in the cerebellum.

2.2. Neuron–Astrocyte Communication: Prion and EVs

The prion is an anchor type membrane protein of glycosylphosphatidylinositol (GPI). There are both normal type (PrP^c) and a protease resistant cell infective type (PrP^sc) prions caused by a structural change. Neuronal cell death is induced when neurons are subjected to oxidative stress, but neuronal cell death decreases by co-culturing astrocytes and neurons. However, in the prion-deficient astrocyte, the co-culture neuronal death mitigation effect was not maintained.¹⁵⁾ Neuronal cell death is also suppressed by adding EVs prepared from oxidative stress-loaded astrocytes. Analysis of the components of EVs derived from astrocytes has revealed that PrP^c and laminin receptors were recruited on EV membranes, depending on oxidative stress. In other papers, prions have interacted with the membrane surface of EVs.¹⁶⁾ Since PrP^c functions to alleviate the oxidative stress of astrocytes, it is thought that by altering the constituent components of protein molecules accompanying EVs, dependent on stress stimuli, may suppress neuronal cell death. When PrP^c interacts with PrP^sc, it changes into a cytotoxic and infectious type (Fig. 4). Aβ interaction with PrP^c caused both the inhibition of long-term potentiation of hippocampal neurons and cytotoxicity.¹⁷⁾ Recently, it has been shown that PrP^c interacts with Aβ on the surface of exosome membrane.¹⁸⁾ This interaction between PrP^c and Aβ reduced the uptake of Aβ into neurons and decreased neurotoxicity, compared to the addition of Aβ alone. Thus, EVs could alleviate the toxicity of soluble Aβ by fibrillating Aβ on the EV membrane surface via PrP^c.

Fig. 4. EVs, Prions, Aβ, and Cytotoxicity

① When a prion (PrP^c) is combined with an infectious type (PrP^sc), structural change is induced to become the infective type. ② When interacting with Aβ oligomers, prion (PrP^c) decrease neuronal function (long-term potentiation) and induce cytotoxicity. ③ Infectious prion (PrP^sc) promote Aβ production from APP, while normal-type prion (PrP^c) inhibit Aβ production. ④ Aβ is first produced from APP by the two steps of γ- and β-secretase cleavage. ⑤ Laminin receptor can interact with prion (PrP^c) and Aβ. Therefore, it is possible for Aβ to be released to prions. ⑥ The fibrillation of Aβ on the exosomal membrane surface causes prions to mitigate the cytotoxicity of Aβ oligomers, as in ②. (Color figure can be accessed in the online version.)

2.3. Neuron–Neuron Communication: SOD1, TDP-43, and EVs

Amyotrophic lateral sclerosis (ALS) is thought to occur sporadically in more than 90% of cases, but in its familial disease pathogenesis form, the major causative molecules are superoxide dismutase 1 (SOD1), TAR DNA-binding protein 43 (TDP-43), and fused in sarcoma (FUS). SOD1 is an enzyme that detoxifies intracellular reactive oxygen; TDP-43 is an RNA binding protein normally localized in the nucleus; and FUS is also a molecule with a typical RNA binding domain. Examination of SOD1 propagation between neurons, using motor neuron-like cells, revealed that misfolded SOD1 with an abnormal structure exists on the EV membrane surface, and propagates.¹⁹⁾ Interestingly, the SOD1 point mutation with transient expression showed an aggregate-like structure in the cytoplasm, and had cytotoxicity, but in stable SOD1 expression, it was nearly as unchanged as the wild type. However, when a culture supernatant containing misfolded SOD1 was added to another cell, endogenous SOD1 was converted to the misfolded form. This induction was more effectively induced by the addition of EVs. Misfolded SOD1 was not detected in healthy subjects or in Alzheimer’s patients’ brains; it was detected only in both sporadic and familial ALS. It is thought that SOD1, which has an abnormal structure similar to “Prion-like” propagation,²⁰⁾ spreads and accumulates between motor neurons via EVs, thereby promoting cell death.

Aggregate propagation of “Prion-like” molecules is also shown in TDP-43. When aggregate-like TDP-43 derived from an ALS patient’s brain is added to another cell, aggregate-like TDP-43 was induced, and promoted cell death by inhibiting the proteasome system.²⁰⁾ It was suggested that this aggregate-like TDP-43 interacts with EVs and propagates. However, TDP-43 and FUS point mutations (P525L, R495X) did not participate in their own aggregation or propagation, respectively, but they were involved in the propagation of misfolded SOD1.²¹⁾ It is considered that TDP43 triggered the aggregation of SDO1, even in the normal type, when the expression was enhanced, and may thus accelerate the onset of ALS. In addition, EVs derived from adipose stromal cells had the effect of alleviating the cytotoxicity of H₂O₂.²²⁾ EVs are not only a negative aspect of “Prion-like” propagation, but may also present a positive aspect as a treatment tool. In summary, it is suggested that ALS-related proteins are transmitted and accumulated in normal neurons via EVs, thereby inducing neuronal cell death.

2.4. Neuron–Neuron Communication: α-Synuclein and EVs

The first familial Parkinson’s disease gene, α-synuclein, is abundant in neurons and erythrocytes. Analysis of α-synuclein encapsulated in neuron-derived EVs reveals that α-synuclein oligomers are present not only in extracellular fluid but also in membranes of EVs,²³⁾ and are secreted from neurons.²⁴⁾ Compared to the culture supernatant and the α-synuclein in EVs, EVs had higher efficiency uptake into the cells, as well as increased caspase activity.²³⁾ It was analyzed whether α-synuclein fibrillation occurs on the EV membrane surface as well as Aβ. As a result, the aggregation of α-synuclein was observed even in the presence of a small amount of EVs, compared with α-synuclein alone.²⁴⁾ PrP^c-Aβ proceeds toward detoxification on the surface of EV membranes.¹⁸⁾ Conversely, in the case of α-synuclein, EVs are involved in negative propagation in the promotion of fibrillization.

Meanwhile, the overexpression of ATP13A, which is one of familial Parkinson’s disease genes, promotes the incorporation of α-synuclein into EVs.²⁵⁾ Leucine-Rich Repeat Kinase 2 (LRRK 2), which is another familial Parkinson’s disease gene, has important functional domains (guanosine 5′-triphos-phatase (GTPase), Kinase) at the C terminus, and many Parkinson’s disease familial mutations have been reported in this C-terminal domain. The R1441C mutation of this GTPase caused abnormality in axonal transport of mitochondria, both in the forward and reverse directions.²⁶⁾ In addition, the R1441C mutation caused intracellular localization change, and caused filamentous abnormal MVB, including the R1441C form.²⁷⁾ In addition, LRRK2 interacts with Rab5b, which is one pf the EV makers. However, some behavioral abnormality has been shown in mice with the R1441G mutation.²⁸⁾ In Parkinson’s disease, combined factors such as the aggregation of degenerative disease proteins and the propagation of toxicity, various stress disorders, mitochondrial dysfunction, and so on, are being discussed; this exemplifies the difficulty of elucidating the mechanisms involved. However, EVs in CSF from patients with Parkinson’s disease have nevertheless been analyzed, although in some cases α-synuclein in EVs could not be detected.²⁹⁾ The relationship between EVs and α-synuclein regarding protein level needs to be carefully scrutinized.

2.5. Microglia–Neuron Communication: Tau Protein and EVs

Tau is a microtubule-associated protein that is abundantly expressed in neurons. It is known that the accumulation of phosphorylated tau in the entorhinal cortex occurs during early stages of Alzheimer’s disease, and in the declining ability to smell.³⁰⁾ Therefore, the propagation and accumulation of phosphorylated tau from the entorhinal cortex to the hippocampus (dentate gyrus) was analyzed using an adeno adeno-associated virus (AAV) vector.³¹⁾ When tau oligomer was added to a primary culture of neural cells, the tau oligomer were phagocytized efficiently in microglia, compared to neurons and astrocytes. Furthermore, in microglia, tau was encapsulated in EVs dependent on ATP stimulation, and these EVs were taken up in juvenile primary culture neurons. It is thus suggested that tau is propagated through neurons via microglia by EVs. The phosphorylated tau accumulation decreased in the dentate gyrus subjected to removal of microglia or inhibition of nSMase2 which promote EV production. In the dentate gyrus where phosphorylated tau was deposited using AAV, the excitability of synapses decreased. In the entorhinal cortex, microglia phagocytosed tau, and this microglia encapsulated and propagated tau via EVs caused the accumulation of tau in neurons of the dentate gyrus, which caused a deterioration in neuronal function. The mechanism of EV release from microglia, selective transport, and incorporation into the neurons of the dentate gyrus has not yet been determined. It is interesting that those microglia actively exhibiting phagocytosis action cannot fully decompose tau, thus contributing to the early onset of Alzheimer’s disease by involving microglia in spreading through EVs. However, there have also been reports that the accumulation of phosphorylated tau in the dentate gyrus do not affect cognitive function.³²⁾

3. NEUROPHYSIOLOGY VIA EVS

3.1. Hematopoietic Cell–Neuron Communication

Using transgenic mice expressing Cre recombinase, specifically in their hematopoietic lineage, the LacZ reporter gene was able to be expressed in some cerebellar neurons.³³⁾ This was thought to involve transmission from blood cells through EVs to cerebellar Purkinje cells. When the mouse was subjected to an acute phase inflammation model (peritonitis; mouse peritoneal macrophage induction load) and a chronic phase inflammation model (cancer cell transplantation or entorhinal cortex injury of the temporal lobe), the LacZ reporter gene expression in Purkinje cells increased, and expression was also observed in neurons in other cerebral and mesencephalic regions: in the cerebral cortex, a NeuN positive neuron with hippocampal ammonium horn, or in the mesencephalic substantia nigra, a tyrosine hydroxylase (TH) positive dopaminergic neuron. Also, bone marrow cell derived EVs were able to permeate the BBB using a BBB cell line model. Interestingly, in this transgenic mouse, the peripheral blood and bone marrow cell-derived EVs contained almost no Cre protein; it was abundantly encapsulated with Cre mRNA, and protein expression of reporter gene occurred in the transferred neurons. In other words, EVs released from the hematopoietic cells into the blood due to inflammation at the periphery, permeates the BBB, then mRNA is propagated to neurons, translated, and this induces reporter gene expression.

3.2. Hematopoietic Cell–Glia Communication

The position of the cerebral ventricle, located in the ventricle plexus, is a capillary-rich structure that produces CSF. Since a neurodegenerative disease causative molecule is present in EVs in CSF, the relationship between the choroid plexus and EVs was analyzed.³⁴⁾ Systemic inflammation caused by lipopolysaccharide (LPS) increases EVs in murine CSF, and the encapsulated amount of proinflammatory microRNA (miRNA) also increases. This is related to an increase in MVBs and EVs in the choroid plexus. EVs derived from LPS-stimulated CSF and encapsulated RNA were found to be taken up by astrocytes in the choroid plexus cell line model, and were readily taken up into microglia cells after injection into the mouse ventricle. Indeed, these EVs inhibited the expression of some of the miRNA target genes in the brain; they suppressed this miRNA inhibition of target genes as a result of the EV production inhibitor GW 4869.³⁵⁾ In other words, it is suggested that the choroid plexus communicates inflammatory stimulation signals from the periphery via the bloodstream to astrocytes and microglia via the CSF–EVs pathway.

3.3. Neuron–Microglia Communication: Synapse Pruning and EVs

Analysis of synapse removal, or the “synapse pruning” mechanism via EVs, was performed using a differentiation/neurite outgrowth system by the addition of NGF in PC12 cells.¹⁾ (Fig. 3). Neurite outgrowth was enhanced in the co-culture of PC12 differentiated neuronal cells and MG-6 microglial cells. MFG-E8 recognizes apoptotic cells by binding to dead cell plasma membrane PS (phosphatidylserine) that recognizes apoptotic cells.³⁶⁾ In the presence of a dominant negative MFG-E8 form, D89E, the removal effect by apoptosis did not affect neurite outgrowth by microglia. It was thus suggested that phagocytosis, rather than apoptosis by microglia, is involved in synaptic pruning under this condition. On the other hand, when neuronal EVs were added to microglia, EVs were efficiently taken up, and synapse removal by microglia was enhanced. Synaptic pruning by neuronal EVs was performed by neural specific signal transduction between neurons and microglia; it was not enhanced by non-neuronal EVs. Comprehensive analysis of changes in gene expression in microglia incorporating neuronal EVs revealed that the expression of complement C3, a phagocytosis-related molecule, increased. Since synaptic pruning via complement has been reported,³⁷⁾ the elevated complement expression in microglia-incorporated EVs is consistent with this report. Thus, it is suggested that EVs are capable of positively mobilizing synaptic pruning, even in microglia, away from neurons (Fig. 5).

Fig. 5. Synapse Pruning via Neuronal EVs

Microglia are efficiently taken in by EVs produced from neurons that were stimulated by KCl as a depolarization agent, the expression of complement C3 increases in microglia, and synapse removal occurs via the complement cascade. (Color figure can be accessed in the online version.)

CONCLUSION

What is common to many of these cases is that constituents such as proteins, whether encapsulated in EVs or attached to the surface of the membrane, maintain homeostasis in the nervous system by the activation of glial cells incorporating EVs. Thus, it is conceivable that the collapse of this homeostasis is largely related to the onset of neurological death disease. Neural EVs were deeply involved in the collapse of homeostasis. Therefore, it is possible that there are many examples of the involvement of EV-mediated signal transduction in the physiology of the nervous, which have not yet attracted attention.

Acknowledgment

This article has been supported by Grants-in-Aid for Scientific Research KAKENHI (to H. K. and R. H.).

Conflict of Interest

This study was funded by Ono Pharmaceutical Co., Ltd.

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