Reciprocal Regulation of Chaperone-Mediated Autophagy/Microautophagy and Exosome Release

Abstract

Autophagy-lysosome proteolysis is involved in protein quality control and classified into macroautophagy (MA), microautophagy (mA) and chaperone-mediated autophagy (CMA), by the routes of substrate delivery to lysosomes. Both autophagy-lysosome proteolysis and exosome release are strongly associated with membrane trafficking. In the present study, we investigated how chemical and small interfering RNA (siRNA)-mediated activation and inhibition of these autophagic pathways affect exosome release in AD293 cells. Activation of MA and mA by rapamycin and activation of CMA by mycophenolic acid significantly decreased exosome release. Although lysosomal inhibitors, NH₄Cl and bafilomycin A1, significantly increased exosome release, a MA inhibitor, 3-methyladenine, did not affect. Exosome release was significantly increased by the siRNA-mediated knockdown of LAMP2A, which is crucial for CMA. Inversely, activity of CMA/mA was significantly increased by the prevention of exosome release, which was induced by siRNA-mediated knockdown of Rab27a. These findings indicate that CMA/mA and exosome release are reciprocally regulated. This regulation would be the molecular basis of extracellular release and propagation of misfolded proteins in various neurodegenerative diseases.

INTRODUCTION

Intracellular protein degradation is involved in protein quality control by degrading unfolded or misfolded proteins. Especially, the removal of unnecessary proteins in neurons is vital for the neural functions and survival.¹⁾ There are two major protein degradation systems, ubiquitin-proteasome system (UPS) and autophagy-lysosome system.¹⁾ The latter is further classified into three pathways, macroautophagy (MA), microautophagy (mA) and chaperone-mediated autophagy (CMA), by the routes of substrate delivery to lysosomes²⁾ (Fig. 1). Age-related decline of these protein degradation systems is considered to be related to the pathogenesis of neurodegenerative diseases whose frequencies increase with age.³⁾ Namely, decline in protein degradation is considered to cause the accumulation of misfolded proteins to inclusion bodies, which is commonly observed in various neurodegenerative diseases. Thereafter, accumulation of misfolded proteins triggers further impairment of protein degradation.^4,5) There have been many reports about the role of UPS and MA in the pathogenesis of neurodegenerative diseases.^6,7) CMA is known to be involved in Parkinson’s and Huntington’s diseases.^8–10) We have also revealed that CMA and/or mA are related to the pathogenesis of several types of spinocerebellar ataxia.^11,12)

Fig. 1. Schematic Illustration of Three Pathways in Autophagic Lysosomal Protein Degradation

Chemicals and siRNA-mediated knockdown that affect these pathways are also indicated.

Recently, prion-like propagation of misfolded proteins between neurons has been focused as a mechanism in the pathological spread of inclusion bodies, which is related to the progression of neurodegenerative diseases.^13,14) It is controversial how misfolded proteins propagate between neurons. Exosome is one of the candidates that mediate this propagation.¹⁵⁾ Exosomes are extracellular vesicles with diameters of 40–200 nm, which contain various proteins and micro RNAs (miRNAs) and participate in intercellular communication.¹⁶⁾ Neurodegenerative disease-related proteins, including α-synuclein, tau, amyloid precursor protein and superoxide dismutase 1, are frequently found in isolated exosomes.^17–19) However, it remains unknown how misfolded proteins are incorporated into exosomes.

Since both autophagy-lysosome system and exosome release are strongly associated with membrane trafficking (Fig. 1), it is possible that the activity of autophagic protein degradation affects the exosome release. Indeed, several reports indicate the involvement of MA in the regulation of exosome release and release of mutant proteins via exosomes.^20,21) However, it remains unknown how CMA and mA affect exosome release. Multivesicular bodies (MVBs) are generated by invagination of late endosomal membrane, and intraluminal vesicles are released by the fusion of MVBs to plasma membrane as exosomes.¹⁶⁾ In mammalian mA, heat shock cognate 70 (Hsc70) delivers substrate proteins to MVBs²²⁾ (Fig. 1). In addition, Hsc70 is also crucial for the substrate degradation via CMA²³⁾ (Fig. 1). Taken together, there could be a reciprocal and close regulation between CMA/mA and exosomes. In the present study, we investigated how exosome release is regulated by the chemical activation and inhibition, or small interfering RNA (siRNA)-mediated inhibition of activities of CMA and mA. In addition, we revealed how siRNA-mediated inhibition of exosome release affects the activities of CMA and mA using our novel method to monitor them.²⁴⁾

MATERIALS AND METHODS

Materials

Mycophenolic acid (MPA), anti-β-tubulin mouse monoclonal and anti-tumor susceptibility gene 101 (TSG101) rabbit polyclonal antibodies were obtained from Sigma-Aldrich (St. Louis, MO, U.S.A.) LAMP2A (sense: 5′-GGC AGG AGUACUUAUUCUAGU-3′, antisense: 5′-UAGA AUAAGUACUCCUGCC AA-3′), TSG101 (sense: 5′-CUAGUUCAAUGACUAUUAAT T-3′, antisense: 5′-UUAAUAGUCAUUGAA CUAGT T-3′) and Rab27a (sense: 5′-GAUGCAUGCAUAUUGUGAA TT-3′, antisense: 5′-UUCACAAUAUGCAUGCAUCTT-3′) siRNAs and MISSION siRNA Universal Negative Control were also obtained from Sigma-Aldrich. Penicillin/streptomycin solution and ammonium chloride (NH₄Cl) were obtained from Nakalai Tesque (Kyoto, Japan). The HaloTag ligand fused with tetramethylrhodamine (TMR-HT ligand) was obtained from Promega (Madison, WI, U.S.A.). ScreenFect siRNA was obtained from FUJIFILM Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Advanced Dulbecco’s modified Eagle’s medium (DMEM) and Total Exosome Isolation kit were obtained from ThermoFisher Scientific (Waltham, MA, U.S.A.). Glass-bottomed culture dishes (35-mm diameter) were from MatTek (Ashland, MA, U.S.A.). Anti-CD63 goat polyclonal antibody was obtained from LifeSpan BioSciences (Seattle, WA, U.S.A.). Anti-Alix mouse monoclonal antibody was obtained from Cell Signaling Technologies (Danvers, MA, U.S.A.). Anti-LAMP2A rabbit polyclonal antibody was obtained from AbCam (Cambridge, U.K.). Anti-Rab27a rabbit polyclonal antibody was obtained from Santa Cruz Biotechnology (Dallas, TX, U.S.A.). Bafilomycin A1 was obtained from Toronto Research Chemicals (Toronto, Canada). Rapamycin and 3-methyladenine (3MA) were obtained from AdipoGen Life Sciences (San Diego, CA, U.S.A.).

Cell Culture and siRNA Transfection

We used AD293 cells stably expressing glyceraldehyde-3-phosphate dehydrogenase fused with HaloTag (GAPDH-HT), a marker of CMA and mA activities.²⁴⁾ AD293/GAPDH-HT cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS), 100 units/mL of penicillin and 100 mg/mL of streptomycin in a humidified atmosphere containing 5% CO₂ at 37°C.

To knockdown LAMP2A, TSG101 or Rab27a, AD293/GAPDH-HT cells (2 × 10⁵ cells/24 well plate) were transfected with nontargeting control, LAMP2A, TSG101 or Rab27 siRNAs (20 pmol each) 1 d after cell spread using ScreenFect siRNA (1 µL). Next day, cells were detached and spread onto glass-bottomed culture dishes for fluorescence observation and 12 well plates for immunoblotting.

Isolation of Exosomes

For the isolation of exosomes from cells treated with chemicals, AD293/GAPDH-HT cells were spread onto 12 well plates (1 × 10⁵ cells/well). Next day, culture media were exchanged with Advanced-DMEM containing vehicle (0.1% dimethyl sulfoxide (DMSO)) or various chemicals (15 mM NH₄Cl, 10 nM Bafilomycin A1, 10 mM 3-methyladenine (3MA), 500 nM MPA or 500 nM rapamycin), followed by the cultivation for 24 h. Cells were collected in 0.2 mL of sample buffer containing 0.5 M Tris–HCl (pH 6.8), 10% sodium dodecyl sulfate (SDS), 5 mM dithiothreitol, 10% glycerol and 1% bromophenol blue, followed by heating at 100°C for 15 min. Exosomes in culture media were isolated using Total Exosome Isolation kit according to the manufacturer’s protocol. Exosome pellets were suspended in 30 µL of sample buffer, followed by heating at 100°C for 15 min.

For the isolation of exosomes from cells treated with siRNAs, culture media were exchanged to Advanced DMEM 1 d after cell spread onto 12 well plates. After the cultivation for 24 h, cells and exosomes in culture media were collected as described above.

Cell and exosome lysates were analyzed by immunoblotting as described previously¹²⁾ using anti-Alix (1 : 1000), anti-CD63 (1 : 3000, goat polyclonal), anti-LAMP2 (1 : 2000), anti-TSG101 (1 : 500), anti-Rab27a (1 : 500) and anti-β-tubulin (1 : 5000) antibodies as primary antibodies.

Observation of Chaperone-Mediated Autophagy and Microautophagy Activities

Activities of CMA and mA were assessed by the punctate accumulation of GAPDH-HT as described previously.²⁴⁾ After 24 h cultivation from the spread on glass-bottomed culture dishes, AD293/GAPDH-HT cells on glass-bottomed culture dishes were labeled with 100 nM TMR-HT ligand for 10 min, followed by the further cultivation for 18 h. Fluorescence of TMR was observed using a confocal laser microscope (TCS SP5, Leica Biosystems, Nussloch, Germany).

RESULTS

We first investigated whether chemical activation of autophagy affects exosome release. AD293/GAPDH-HT cells were treated with mycophenolic acid (MPA, 500 nM), which is an selective inhibitor of inosine monophosphate dehydrogenase,²⁵⁾ inhibits de novo synthesis of guanine ribonucleotide and activates CMA²⁶⁾ (Fig. 1), and rapamycin (500 nM), which is a selective inhibitor of mammalian target of rapamycin complex 1 (mTORC1)²⁷⁾ and activates MA²⁸⁾ and mA²⁹⁾ (Fig. 1), for 24 h. Exosome release was assessed by the amounts of exosome markers, Alix and CD63 in exosome lysates. MPA significantly decreased CD63 and tended to decrease Alix in exosome lysates, while it significantly increased CD63 in cell lysates (Fig. 2). Rapamycin significantly decreased Alix and CD63 in exosome lysates, while it did not significantly affect the amounts of 2 exosome proteins in cell lysates (Fig. 2). These findings suggest that autophagic activation prevents extracellular release of exosomes.

Fig. 2. Effects of Autophagic Activators on Exosome Markers in Exosome and Cell Lysates in AD293/GAPDH-HT Cells

A. Representative immunoblots of exosome markers (Alix and CD63) in exosome and cell lysates from cells treated with vehicle (Veh, 0.1% DMSO), mycophenolic acid (MPA, 500 nM) and rapamycin (Rap, 500 nM) for 24 h. β-Tubulin was detected as an internal control in cell lysates. B. Quantitative analyses of exosome markers in exosome lysates. The amount of each band was normalized by the amount of β-tubulin in cell lysates. Data was represented as mean ± standard error of the mean (S.E.M.) C. Quantitative analyses of exosome markers in cell lysates. The amount of each band was normalized by the amount of β-tubulin in cell lysates. Data were represented as mean ± S.E.M. * p < 0.05 vs. vehicle (Tukey multiple comparisons test, n = 5).

Next, we investigated whether chemical inhibition of autophagy affects exosome release. AD293/GAPDH-HT cells were treated with ammonium chloride (NH₄Cl, 15 mM) or bafilomycin A1 (10 nM), inhibitors of lysosomal proteolysis (Fig. 1), or 3-methyladenine (3MA, 10 mM), which is a nonselective inhibitor of phosphoinositide 3-kinase, slightly preferable to Vps34, and is frequently used as a selective inhibitor of MA³⁰⁾ (Fig. 1), for 24 h. NH₄Cl significantly increased Alix in exosome lysates, while it significantly increased CD63 in cell lysates (Fig. 3). Bafilomycin A1 prominently increased Alix and CD63 in exosome lysates, while it significantly increased CD63 in cell lysates (Fig. 3). Compared with these 2 lysosomal inhibitors, 3MA did not significantly affect the amounts of 2 exosome markers in exosome and cell lysates (Fig. 3). These findings suggest that impairment of lysosomal proteolysis, especially CMA and mA, enhances extracellular release of exosomes.

Fig. 3. Effects of Autophagic Inhibitors on Exosome Markers in Exosome and Cell Lysates in AD293/GAPDH-HT Cells

A. Representative immunoblots of exosome markers (Alix and CD63) in exosome and cell lysates from cells treated with vehicle (Veh, 0.1% DMSO), NH₄Cl (15 mM) and bafilomycin A1 (Baf, 10 nM) for 24 h. β-Tubulin was detected as an internal control in cell lysates. B. Quantitative analyses of exosome markers in exosome lysates. The amount of each band was normalized by the amount of β-tubulin in cell lysates. Data was represented as mean ± S.E.M. C. Quantitative analyses of exosome markers in cell lysates. The amount of each band was normalized by the amount of β-tubulin in cell lysates. Data were represented as mean ± S.E.M. * p < 0.05, *** p < 0.001 vs. vehicle (Tukey multiple comparisons test, n = 4).

To reveal which impairment of CMA or mA is mainly involved in the enhancement of exosome release, we investigated the effect of siRNA-mediated knockdown of CMA- and mA-related proteins on the exosome release in AD293/GAPDH-HT cells.²⁴⁾ We have previously revealed that the activities of CMA and mA are selectively decreased by siRNA-mediated knockdown of LAMP2A,²³⁾ a CMA-related protein, and TSG101, a mA-related protein,²²⁾ respectively²⁴⁾ (Fig. 1). As we previously reported,²⁴⁾ LAMP2A and TSG101 siRNAs significantly decreased LAMP2A and TGS101, respectively, in cell lysates (Figs. 4A, C). siRNA-mediated knockdown of LAMP2A significantly increased the amounts of exosome markers in exosome lysates, while it also significantly increased their amounts in cell lysates (Fig. 4). In contrast, siRNA-mediated knockdown of TSG101 prominently decreased the amounts of exosome markers in exosome lysates, while it significantly increased CD63 in cell lysates (Fig. 4). These findings indicate that LAMP2A knockdown enhances extracellular release of exosomes, whereas TSG101 knockdown prevents exosome release.

Fig. 4. Effects of siRNA-Mediated Knockdown of CMA and mA-Related Proteins on Exosome Markers in Exosome and Cell Lysates in AD293/GAPDH-HT Cells

A. Representative immunoblots of exosome markers (Alix and CD63) in exosome and cell lysates from cells transfected with control (Cont), LAMP2A (L2A) and TSG101 siRNAs. LAMP2A and TSG101 were detected in cell lysates to confirm siRNA-mediated knockdown. β-Tubulin was detected as an internal control in cell lysates. B. Quantitative analyses of exosome markers in exosome lysates. The amount of each band was normalized by the amount of β-tubulin in cell lysates. Data were represented as mean ± S.E.M. C. Quantitative analyses of exosome markers, LAMP2A and TSG101 in cell lysates. The amount of each band was normalized by the amount of β-tubulin in cell lysates. Data was represented as mean ± S.E.M. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control siRNA (Tukey multiple comparisons test, n = 5).

We finally investigated the effect of the inhibition of exosome release on activities of CMA and mA. Rab27a mediates the fusion of MVBs and plasma membrane and is involved in the exosome release.³¹⁾ Rab27a siRNA successfully decreased Rab27a in cell lysates (Figs. 5A, C). siRNA-mediated knockdown of Rab27a significantly decreased the exosome release, which was indicated by the decreased Alix and CD63 in exosome lysates (Figs. 5A, B). We evaluated CMA/mA activities using AD293/GAPDH-HT cells. After the labeling of cytosolic GAPDH-HT with HT ligands fused with fluorescent dye, labeled GAPDH-HT is accumulated to late endosomes and lysosomes via the CMA and mA pathways.²⁴⁾ Therefore, the punctate accumulation of GAPDH-HT reflects the activities of CMA and mA. siRNA-mediated knockdown of Rab27a significantly increased the punctate accumulation of GAPDH-HT (Figs. 5D, E). These findings suggest that impairment of exosome release triggers the activation of CMA and/or mA.

Fig. 5. Effects of siRNA-Mediated Knockdown of Rab27a on Exosome Release and CMA/mA Activity in AD293/GAPDH-HT Cells

A. Representative immunoblots of exosome markers (Alix and CD63) in exosome and cell lysates from cells transfected with control (Cont), Rab27a (Rab) siRNAs. Rab27a was detected in cell lysates to confirm siRNA-mediated knockdown. β-Tubulin was detected as an internal control in cell lysates. B. Quantitative analyses of exosome markers in exosome lysates. The amount of each band was normalized by the amount of β-tubulin in cell lysates. Data was represented as mean ± S.E.M. C. Quantitative analyses of exosome markers and Rab27a in cell lysates. The amount of each band was normalized by the amount of β-tubulin in cell lysates. Data was represented as mean ± S.E.M. ** p < 0.01, *** p < 0.001 vs. control siRNA (unpaired t-test, n = 4). D. Representative fluorescent images of GAPDH-HT puncta in AD293/GAPDH-HT cells transfected with control (upper) and Rab27a (lower) siRNAs. Scale bars are 20 µm. E. Quantitative analyses of GAPDH-HT puncta in each cell. Data were represented as mean ± S.E.M. ** p < 0.01 vs. control siRNA (unpaired t-test, control siRNA; n = 65, Rab27a siRNA; n = 70).

DISCUSSION

In the present study, we revealed that changes in autophagic activity, especially CMA and mA, affect exosome release. Although MPA and rapamycin decreased exosome release (Fig. 2), these chemicals also regulate other cellular functions than autophagic activity. MPA inhibits GTP synthesis and DNA replication.²⁵⁾ Rapamycin inhibits mTORC1,²⁷⁾ which is involved in cell growth and negatively regulates macroautophagy. Therefore, it is possible that these chemicals impair exosome release through the other mechanism than autophagic activation. However, the finding that a lysosomal inhibitor, bafilomycin A1, prominently enhanced exosome release (Fig. 3) strongly suggest that there is a close relationship between autophagic activity and exosome release. On the other hand, another lysosomal inhibitor, NH₄Cl, significantly increased only Alix in exosome lysates (Fig. 3). This difference might be due to the mechanism how these chemicals inhibit lysosomal proteolysis. Bafilomycin A1 inhibits vesicular ATPase (V-ATPase) and sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA), leading to neutralization of lysosomal lumen and inhibition the fusion between lysosomes and autophagosomes/endoscopes, respectively.^32,33) On the other hand, NH₄Cl just neutralizes acidic pH of lysosomal lumen. Because V-ATPase-deficient lysosome can fuse with autophagosomes and endosomes,³²⁾ NH₄Cl-mediated neutralization of lysosomes would not hamper this fusion. Taken together, although exosome release is enhanced by the impairment of lysosomal proteolysis through the neutralization of lysosomes, it is more strongly enhanced by the accumulation of late endosomes/MVBs through the inhibition their fusion with lysosomes.

Why did NH₄Cl differentially regulate Alix and CD63 in exosome lysates? A recent report indicates that sphingosine 1-phosphate is involved in the sorting of cargo proteins to exosomes but does not affect the formation of exosomes.³⁴⁾ This finding strongly suggests that the cargo sorting to exosomes is differently regulated from the exosome formation. It is possible that NH₄Cl differentially regulates the sorting of Alix and CD63 to exosomes. Although we could not determine whether NH₄Cl increases exosomes with low Alix or increases the sorting of CD63 to exosomes, it enhances the extracellular release of exosomes and/or cargo proteins of exosomes.

MA has been especially focused as the regulator of exosome release, because several MA-related proteins are reported to modulate the process of exosome release.²⁰⁾ However, 3MA, a MA inhibitor, did not affect the exosome release in the present study (Fig. 3). Although there are several reports indicating that activation and inhibition of MA alter the exosome release,^35–37) rapamycin and bafilomycin A1 were mainly used as an activator and an inhibitor of MA, respectively. Rapamycin is reported to activate mA as well as MA in yeast.²⁹⁾ Our preliminary study revealed that rapamycin also activates the mammalian mA (under submission). Indeed, we confirmed that rapamycin significantly prevents exosome release in MA-deficient Atg5-knockout mouse embryonic fibroblast cells (Supplementary Fig. 1). Bafilomycin A1 inhibits all pathways of autophagic protein degradation.³³⁾ Therefore, our present findings indicate that MA less contributes to the regulation of exosome release than CMA and mA.

In the present study, both activation (rapamycin) and inhibition (TSG101 knockdown) of mA impair exosome release (Figs. 2, 4). We knocked TSG101 down because it is involved in the substrate uptake to MVBs in mA.²²⁾ TSG101 is well-known as a component of the endosomal sorting complexes required for transport (ESCRT) complexes, which are crucial for MVB biogenesis.³⁸⁾ As mentioned above, MVBs are related to both mA and exosome biogenesis.^16,22) Therefore, siRNA-mediated knockdown of TSG101 impairs MVB biogenesis, resulting in the inhibition of both mA and exosome release. Indeed, it has been reported that exosome release is significantly decreased by the knockdown of several ESCRT components including TSG101 in HeLa cells.³⁹⁾ To validate the involvement of mA in the regulation of exosome release, it is necessary to block mA in the later steps than MVB biogenesis. However, it remains unknown how mA is regulated after MVB synthesis in mammalian cells.

In addition to the result that MPA impairs exosome release, siRNA-mediated knockdown of LAMP2A increased exosome release (Fig. 4). These findings strongly suggest the involvement of CMA in the regulation of exosome release. It is difficult to speculate how CMA regulates exosome release because vesicular transport is not necessary for the substrate delivery to lysosomes in CMA.⁴⁰⁾ Emerging evidence reveals that mutant proteins are delivered to exosomes and extracellularly released under impairment of lysosomal proteolysis.^35,41,42) In addition, CMA is involved in degradation of several proteins related to neurodegenerative diseases, like α-synuclein and huntingtin.⁴³⁾ Taken together, there is a possibility that accumulation of mutant proteins enhances exosome release. This might be a mechanism that neurons protect from the toxicity of mutant or misfolded proteins under age-related decline of CMA.

If exosome release is a mechanism that cells protect themselves from misfolded proteins, prevention of exosome release would trigger the accumulation of misfolded proteins, which could activate protein degradation systems. In the present study, we revealed that prevention of exosome release by siRNA-mediated Rab27a activates CMA and/or mA (Fig. 5). Because Rab27a is involved in the fusion of MVBs and plasma membrane,³¹⁾ Rab27a knockdown would cause the accumulation of MVBs in cytoplasm. However, CD63 was not increased in cell lysates by Rab27a knockdown (Figs. 5A, C). It is possible that Rab27a knockdown activates mA, which delivers substrate proteins by the fusion of MVBs to lysosomes²²⁾ (Fig. 1). Because a previous report indicates that CD63 is degraded by lysosomes,⁴⁴⁾ CD63 would be degraded by the activation of mA. Although further studies are necessary to elucidate which of CMA or mA is activated by Rab27a knockdown, it is reasonable that Rab27a knockdown would trigger the accumulation of MVBs, leading to the activation of mA.

In conclusion, our present findings strongly suggest that there is a machinery to reciprocally regulate autophagy-lysosome proteolysis, especially CMA and mA, and exosome release. These two pathways are considered to cooperatively participate in the cellular protection against the accumulation of mutant and misfiled proteins.^3,45) Age-related and misfolded protein-triggered decline of autophagy-lysosome proteolysis might enhance the extracellular release of misfolded proteins via exosome for the protection of neurons.⁴⁶⁾ The released exosomes containing misfolded proteins could cause the propagation of toxic proteins and disease progression of neurodegenerative disease.¹⁵⁾ Therefore, activation of CMA and mA could contribute to the prevention of propagation of toxic proteins as well as the neural survival in neurodegenerative diseases. It is expected to identify specific activators of CMA and/or mA as the candidates of therapeutics for neurodegenerative diseases.

Acknowledgments

This work is financially supported by Japan Society for the Promotion of Science KAKENHI [Grant number 16K08276] and Takeda Science Foundation. We appreciate Prof. Noboru Mizushima, The University of Tokyo, for giving us Atg5-knockout MEF cells.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

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