Crosstalk between exosomes and autophagy: A review of molecular mechanisms and therapies

Abstract Exosomes are extracellular vesicles that primarily exist in bodily fluids such as blood. Autophagy is an intracellular degradation process, which, along with exosomes, can significantly influence human health and has therefore attracted considerable attention in recent years. Exosomes have been shown to regulate the intracellular autophagic process, which, in turn, affects the circulating exosomes. However, crosstalk between exosomal and autophagic pathways is highly complex, depends primarily on the environment, and varies greatly in different diseases. In addition, studies have demonstrated that exosomes, from specific cell, can mitigate several diseases by regulating autophagy, which can also affect the excessive release of some harmful exosomes. This phenomenon lays a theoretical foundation for the improvement of many diseases. Herein, we review the mechanisms and clinical significance of the association and regulation of exosomes and autophagy, in order to provide a new perspective for the prevention and treatment of associated diseases.


| INTRODUC TI ON
An exosome is an extracellular vesicle with a diameter of 30-100 nm that naturally exists in bodily fluids, including blood, saliva, urine and breast milk. In 1983, Pan et al 1  proteins, mRNA and miRNA that participate in the body's immune responses, antigen presentation, cell migration and differentiation, tumour invasion and autophagy. 5 Coordination between exosomes and autophagy plays an important role in maintaining intracellular homeostasis. 6,7 Autophagy is a common metabolic process in most eukaryotic cells, functioning to promote cell survival. 8 Under various stress signals, such as starvation, hypoxia or endoplasmic reticulum stress, autophagy can degrade soluble proteins and other organelles into amino acids in the cytoplasm for energy production and biosynthesis as a self-protective mechanism. In addition, autophagy clears denatured or misfolded proteins, and ageing or damaged organelles to maintain intracellular homeostasis. 9 Under severe or chronic stress, excessive or insufficient autophagy can lead to the accumulation of a large amount of self-degradation or toxic substances, ultimately leading to cell death, which is closely associated with the pathogenesis of various cancers, as well as neurodegenerative, metabolic-related and immune diseases. 10,11 Recent studies have shown that stress and pathological conditions regulate autophagy through exosomes and their cargos. For example, under hypoxic conditions, exosomes released by cardiomyocytes transferred miR-30a to adjacent cells, thereby inhibiting autophagy by interrupting the Beclin-1 pathway, which reduces myocardial injury. 12 Exosomes secreted by breast cancer cells transferred miR-126 to adipocytes, which induced autophagy through the AMPK/mTOR pathway, thereby altering adipocyte metabolism and promoting cancer progression. 13 Conversely, insufficient or excessive autophagy affects the release of exosomes.
For example, insufficient autophagy increases the amount of exosomes, which promotes the diffusion of α-synuclein and exacerbates the progression of Parkinson's disease (PD). 14 Therefore, the relationship between exosomes and autophagy warrants further investigation.
An increasing number of studies have demonstrated that exosomal and autophagic pathways are cross-regulated and affect the development of various diseases. This article reviews the following four findings to provide a new perspective for the prevention and treatment of related diseases: (a) exosomes, and their cargos, can regulate autophagic pathways via different mechanisms, which is highly dependent on the environment and cell source; (b) exosomes derived from certain cells, especially mesenchymal stem cells (MSCs), can mitigate several F I G U R E 1 Components of exosomes and trafficking. Exosomes are secreted into the extracellular space by donor cells and have commonly conserved components, including CD9, CD63, CD81, Alix, flotillin, TSG101, MHC, HSP70, HSP90 and CD47. Exosomes can carry various cargos and interact with recipient cells primarily via three pathways: (1) phagocytosis, (2) ligand-receptor binding and (3) membrane fusion. Following uptake by recipient cells, exosomes release their cargo, which can modulate autophagy diseases by regulating autophagy; (c) autophagy can affect the formation and release of exosomes; (d) intervening in the key signalling pathways and molecules of autophagy can reduce the release of harmful exosomes, thereby relieving the symptoms of several diseases.

| Components and trafficking of exosomes
Exosomes are small vesicles composed of lipid bilayer membranes that contain proteins, nucleic acids, lipids and other substances. They have conserved components, including tetraspanin proteins (CD9, CD63, CD81), Alix, flotillin, TSG101, immunomodulatory proteins (MHC), heat shock proteins (HSP70, HSP90), and CD47 15 ( Figure 1). CD9, CD81, CD63, flotillin, TSG101 and Alix, which are exosomes biomarkers, are involved in biogenesis, cargos clustering, and exosomes secretion. 5 MHC controls the exchange of antigen information between immune cells. 5 HSP70 and HSP90 help exosomes adapt to the extracellular environment 16 ; CD47 on exosomes produces a 'don't eat me' signal, preventing exosomes from being digested by monocytes and macrophages, thus improving their stability in the body. 17 Apart from conserved proteins, exosomes also express cell-specific proteins that reflect the origin of donor cells. For example, exosomes derived from platelets contain von Willebrand factor and integrin CD41a, whereas T cell-derived exosomes contain CD3. 18,19 Exosomes thus characterize the origin of parental cells and share some of their functional characteristics. In addition, exosomes carry various cargos, such as mRNAs, miRNAs and siRNAs, which can be transferred to recipient cells and affect the expression of the corresponding genes, several of which are associated with autophagic proteins 20 ( Figure 1). However, the type and amount of exosomal cargo depends on the physiological or pathophysiological conditions of the donor cells. For example, hypoxia induced the upregulation of miR-30a in myocardial cell-derived exosomes, 12 whereas cigarette smoke increased exosomal miR-210 in bronchial epithelial cells. 21 Exosomal miR-7-5p has also been observed to increase in irradiated cells. 22 Exosomes interact with recipient cells primarily via three path-

| Molecular mechanisms and biological effects of autophagy
The autophagic process includes five primary stages: initiation, nucleation, elongation and maturation, fusion and degradation. 27 The mammalian target of rapamycin (mTOR) is the key relator of the initiation stage, in which its activation (ie Akt and MAPK signals) inhibits autophagy, whereas its negative regulation (ie AMPK and P53 signals) induces autophagy. Under stress conditions, mTOR is inactivated, whereas the ULK complex (composed of ULK1, FIP200, and autophagy-related protein 13 [Atg13]) is activated. 28 Beclin-1 is an important molecule for autophagosome formation, 29 which then forms a complex with Vps34 and Atg14L, promoting the nucleation stage and recruiting proteins from the cytoplasm. 30,31 In the elongation and maturation stage, two ubiquitin-like conjugation systems are required to promote the extension of the autophagosome membrane. The first system involves the microtubule-associated protein light chain 3(LC3)phosphatidylethanolamine (PE) complex. LC3 is cleaved by Atg4 at its The Atg12-Atg5 conjugate interacts noncovalently with Atg16 to form a large complex. 33 In the fusion stage, autophagosomes and lysosomes fuse to form autolysosomes, whereas in the degradation stage, cargos inside the autolysosomes are degraded ( Figure 2).
Autophagy is strictly regulated to maintain homeostasis. Once autophagy is initiated, multiple Atg proteins cooperate to coordinate the next steps of autophagy. Whether autophagy imparts a protective function in diseases remains debatable. 34 For instance, insufficient autophagy contributes to the accumulation of tau and synuclein proteins, and promotes neurodegenerative disease. 35 In the context of cancer, autophagy has been shown to initially act as a tumour suppressant but later protecting tumour cells from the immune system's defence mechanisms. 36 Similarly, heart and liver diseases have been shown to be positively and negatively regulated by autophagy, respectively. Therefore, the regulation of autophagy by exosomes is complex and may produce different (or even opposite) effects in various diseases.  (Table 1 and Figure 3).

F I G U R E 2
Schematic diagram of the molecular mechanism of autophagy. The autophagic process includes five primary stages, namely initiation, nucleation, elongation and maturation, fusion and degradation. The molecular pathway, comprising the core autophagy proteins, is illustrated. PE, phosphatidylethanolamine Akt is a serine/threonine specific protein kinase that inhibits autophagy by activating mTOR. Akt1 is reportedly the direct target gene of miR-425-3p. 38 Ma et al 39

F I G U R E 3
Schematic summary of the related signalling pathways in the context of exosomes-mediated autophagy regulation. Exosomes release their cargos into recipient cells that can regulate autophagy. The different effects of exosomes on autophagy regulation and the different signalling pathways affected, primarily due to the different cargos they carry. Exosomal miRNA recognizes its target mRNA, suppresses the translation of target mRNA, and reduces related proteins. The synthesis of mTOR, Beclin-1, Atgs, and their upstream proteins, are blocked, which in turn affects the process of autophagy also known as Vps34) combines with Beclin-1 at the ECD site, recruiting other autophagic regulatory proteins such as Atg14 in the cytoplasm to form protein complexes, which promotes the extension of the lipid membrane and the maturation of autophagosomes. 44,45 However, Bcl-2, an inhibitor of apoptosis, binds to Beclin-1 at the BH3 site and inhibits the formation of these complexes, thereby inhibiting Beclin-1-dependent autophagy. 45 Beclin-1 is encoded by BECN 1, which is the target gene of miR-30a. 46 Exosomal miR-30a inhibits autophagy by targeting the

| E XOSOME S DERIVED FROM MSC S MITI G ATE VARI OUS D IS E A S E S BY REG UL ATING AUTOPHAGY
MSCs, stem cells with the potential for self-renewal and multi-directional differentiation, 57 produce various bioactive substances that can regulate immunity, activate endogenous stem/progenitor cells, and promote tissue repair, angiogenesis and anti-apoptosis. 58 However, their therapeutic properties are primarily attributed to a therapeutic effect on SY5Y cells and SD rats exposed to 6-OHDA, and that autophagy played an important role in mediating the beneficial effect of exosomes on PD.

| Liver diseases
MSC-Exos can improve hepatic fibrosis, hepatic ischaemic injury and liver failure by regulating autophagy. Qu et al 73

| Other diseases
In addition to the mentioned diseases above, MSC-Exos can also

| REG UL ATI ON OF E XOSOME B I OG ENE S IS AND RELE A S E VIA AUTOPHAGY
As shown above, extracellular exosomes can regulate the intracellular autophagic processes through various signalling pathways, and play an important role in various diseases. In turn, evidence indicates that autophagy can regulate the biogenesis and release of exosomes in cells, which has implications in both physiological and pathological implications.

| Under physiological implications
The secretory process of exosomes is complex, yet orderly.  87 Murrow et al 89   promote RACs to damage endothelial cell function by releasing exosomes. 94 Inhibition of autophagy or exosomes was shown to reverse this injury, although the mechanism remains unclear.

| ROLE OF AUTOPHAGY-REG UL ATING E XOSOME S IN DEL AYING THE DE VELOPMENT OF DIS E A S E S
Under pathological conditions, abnormal autophagy can cause the release of harmful exosomes and accelerate disease progression.
Reducing the release of harmful exosomes could alleviate tissue damage, especially in liver diseases. For example, the increased expression of miR-155 induced by alcohol increases the release of harmful exosomes by targeting mTOR in the autophagic pathway. Silencing miR-155 restored autophagy and reduced exosome release, which may curtail the occurrence of alcoholic liver disease. 95 Autophagy impairment mediated by TRIB3 and the selective autophagy receptor SQSTM1 in human liver fibrosis promoted the secretion of harmful exosomes, which induced the migration, proliferation and activation of HSCs. Disrupting the TRIB3-SQSTM1 interaction reduced liver fibrosis by restoring autophagy and inhibiting exosomemediated HSC activation. 96

| CON CLUS I ON S AND PER S PEC TIVE S
Generally, exosomes and autophagy are closely associated. In this review, we highlighted new ideas for disease prevention by identifying key signalling pathways and targets with mutual influence.
Although exosomes exist in extracellular fluid (primarily blood), they can release their cargos into target cells via endocytosis, receptor ligand binding and membrane fusion. These cargos can regulate the autophagic level of target cells through the mTOR and Beclin-1 signalling pathways, and autophagy-related proteins, thus affecting the development of associated diseases. MSC-Exos can mitigate diseases by regulating autophagy, especially in cerebral ischaemia, MI, liver fibrosis, and DN. In the future, we will explore the therapeutic role of MSC-Exos and autophagy in other diseases. In addition, intracellular autophagy can affect the biogenesis and release of exosomes to maintain intracellular homeostasis under physiological conditions. Abnormal autophagy can lead to the increased release of harmful exosomes, whose resulting damage can be attenuated by interfering with the autophagic process.
However, promising exosomal and autophagic research is largely conducted using cell culture systems, which requires further validation using animal models and physiology-related experimental conditions.

ACK N OWLED G EM ENTS
This work was financially supported by grants from National Natural Science Foundation of China (No.81970085, 81670086) and the major special project for the prevention and control of chronic diseases in Tianjin (No.17ZXMFSY00080).

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.