Alpha-Synuclein Inhibits the Secretion of Extracellular Vesicles through Disruptions in YKT6 Lipidation

Abstract

Parkinson's disease is characterized by the presence of alpha-synuclein (α-syn) primarily containing Lewy bodies in neurons. Despite decades of extensive research on α-syn accumulation, its molecular mechanisms have remained largely unexplored. Recent studies by us and others have suggested that extracellular vesicles (EVs), especially exosomes, can mediate the release of α-syn from cells and inhibiting this pathway could result in increased intracellular α-syn levels. In this study, we have discovered that elevated levels of α-syn themselves lead to reduced α-syn -containing EVs in α-syn–inducible H4 cells and induced pluripotent stem cell-derived dopaminergic (DA) neurons from both sexes. Our investigations have revealed that the impairment in EV secretion is not due to their generation but rather a consequence of changes in a soluble N-ethylmaleimide–sensitive factor attachment protein receptor protein, YKT6. Specifically, as α-syn levels increase, membrane-associated YKT6 is reduced. Pharmacological inhibition of farnesylation using FTI has led to decreased EV secretion and subsequent elevated levels of α-syn. In summary, our findings suggest that increased levels of α-syn impair YKT6-mediated EV secretion, establishing a detrimental cycle of intracellular α-syn accumulation in human DA neurons.

Significance Statement

Neurodegenerative disorders, including Parkinson's disease (PD), are characterized by the pathological accumulation of insoluble proteins, primarily in neurons. Regulating intracellular levels of these proteins is critical. Despite extensive research for decades, the precise mechanism of these protein deposits remains unexplained. In this study, we discovered that extracellular vesicles (EVs) play a pivotal role in regulating alpha-synuclein (α-syn) levels through release from neurons. Furthermore, increased levels of α-syn impede its EV secretion, creating a pathological loop. Elevated levels of α-syn interfere with the localization of YKT6, a sensitive factor attachment protein receptor protein, to the vesicle membrane by inhibiting YKT6 palmitoylation. These findings illustrate a novel mechanism of α-syn accumulation in neurons and provide a target to potentially mitigate PD pathology.

Introduction

Parkinson's disease (PD) is characterized by selective dopaminergic (DA) neuronal cell death and deposition of alpha-synuclein (α-syn) containing Lewy bodies and neurites within remaining neurons. Increased expression of α-syn, a presynaptic protein with an unknown function, has been implicated in both sporadic and familial PD pathogenesis (Burbulla et al., 2017). Notably, hereditary PD can result from the duplication or triplication of alpha-synuclein (SNCA) gene encoding α-syn, highlighting a dose- and time-dependent relationship (Singleton et al., 2003; Chartier-Harlin et al., 2004; Ibanez et al., 2004). Additionally, impaired degradation of α-syn is implicated in various forms of hereditary PD, where Lewy bodies and neurites are observed in the patient's brains, suggesting a role of reduced degradation in the α-syn accumulation (Gan-Or et al., 2015).

Recent studies have emphasized the significance of exosomes and other exocytotic pathways in α-syn secretion, potentially influencing intracellular α-syn levels within neurons (Tsunemi et al., 2014, 2019). We have discovered that dysfunctional secretory pathways resulted in increased levels of α-syn, whereas enhanced secretion leads to reduced α-syn levels. This indicates that α-syn secretion significantly impacts intracellular α-syn levels. Furthermore, we also observed that the reduction of exosomal secretion is dependent on α-syn levels in induced pluripotent stem cell (iPSC)-derived DA neurons. However, the relationship between the exosomal secretory pathway and intracellular α-syn levels has not been explored comprehensively. Importantly, identifying the cellular pathways and targets that regulate exosome-mediated α-syn secretion could offer relevant therapeutic strategies for addressing α-syn–mediated neurotoxicity in various synucleinopathies.

In our study, we utilized H4 cells and iPSC-derived DA neurons obtained from PD patients expressing SNCA A53T mutations. Our research revealed that intracellular α-syn levels play a pivotal role in regulating exosome secretion. While neurons from PD patients could generate normal amounts of intraluminal vesicles (ILVs), which eventually become exosomes upon secretion, they exhibited impaired exosome secretion, resulting in ILV accumulation.

We identified YKT6, a soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) protein, as a target of α-syn. Elevated levels of α-syn impaired its association with the membrane of multivesicular bodies (MVBs) and impeded the fusion of MVB and plasma membrane. This disruption ultimately resulted in decreased exosomal release of α-syn and the subsequent intracellular α-syn accumulation.

Materials and Methods

Plasmids

pHLuroin-CD63 was generated by inserting CD63 into the LAMP-1 construct of the pHluorin-LAMP1, enabling luminal expression of pHluorin when expressed within the cells (Cheng et al., 2018). The expression plasmids for RAB11 (RC200352), RAB27B (RC206519), RAB5A (RC203873), and YKT6 (RC200260) were purchased from OriGene. YKT6 cDNA was inserted in the pEGFP-N1 or pET21 vector.

Extracellular vesicle isolation and quantification

Extracellular vesicles (EVs) were purified as described previously (Tsunemi et al., 2014). Briefly, after media were replaced with cell-conditioned media which had been made free of EVs by ultracentrifugation overnight, the cells were culture for specific periods. EVs were collected from the cultured media using a basic differential centrifugation method (200 × g for 5 min, 1,200 × g for 10 min, and 16,500 × g for 30 min), followed by ultracentrifugation at 110,000 × g for 60 min. After washing in PBS, EVs were collected by centrifugation at 110,000 × g for 60 min.

Analysis of EVs was conducted by two procedures. First, we utilized NanoSight LM10 system (NanoSight), configured with a 405 nm laser and a high-sensitivity digital camera system (OrcaFlash2.8, Hamamatsu C11440, NanoSight). Samples were administered and recorded for 1 min under sustained flow controlled by a script control system equipped with the NanoSight syringe pump. Videos were analyzed by the NTA software (v2.3). Second, EVs were quantified using the ExoCounter (JVCKENWOOD) following the manufacturer's protocol. The ExoCounter captures and quantifies EVs through a sandwich immunoassay, utilizing a disc with a primary antibody attached and secondary antibodies conjugated to nanobeads (Jiang et al., 2023). Briefly, the disc's surface was coated with the capturing antibody (CD36), allowing small EVs <160 nm to be captured at the bottom of the trench. After blocking, 50 μl of culture media were applied. Captured EVs were subsequently detected by the secondary antibody (CD9) conjugated to magnetic nanobeads. The number of beads was counted by an optical pickup equipped with a laser diode and a photodetector. The light from the beads was transformed into pulses and counted by a pulse counter circuit.

α-Syn detection

α-Syn ELISA was conducted as described previously (Tsunemi et al., 2014). α-Syn oligomers/fibrils were formed as described previously (Mazzulli et al., 2011). Briefly, after α-syn monomers were incubated at 37°C for 10 d under continuous agitation of 1,000 rpm, α-syn n oligomers/fibrils were centrifuged at 10,000 × g for 30 min. The pellets were resuspended in PBS, and fibril formation was assessed by Thioflavin T spectroscopic assay and electron microscopic analysis.

Western blotting

Immunoblotting was conducted as described previously (Tsunemi et al., 2014; 2019; Tsunemi and Krainc, 2014). The antibodies used were anti-human α-syn 211 (ab80627, Abcam), anti-human GAPDH (ABS16, Sigma-Aldrich), human vimentin (550513, BD Biosciences), anti-human LAMP 1 (sc-20011, Santa Cruz Biotechnology), anti-human EEA1 (C45B10, Cell Signaling Technology), anti-human CD63 (H5C6, Developmental Studies Hybridoma Bank), anti-human Flotillin-1 (610820, BD Biosciences), anti-human β-III tubulin (MMS-435P, Covance), anti-human TH (AB152, Merck Millipore), anti-human YKT6 (#97076, Cell Signaling Technology), anti-human LDH (sc-133123, Santa Cruz Biotechnology), anti-human ALIX (sc-53540, Santa Cruz Biotechnology), anti-human TSG101 (14497-1-AP, Proteintech), anti-LC3 (14600-1-AP, Proteintech), anti-P62 (18420-1-AP, Proteintech), anti-vimentin (ab137321, Abcam), and anti-β-actin (sc-47778, Santa Cruz Biotechnology).

Cell culture

Inducible human neuroglioma H4 cells expressing wild-type α-syn under the control of a tetracycline-inducible promoter (tet-off) have been described previously (Tsunemi et al., 2014).

We maintained iPSCs and differentiated them into DA neurons as described previously (Tsunemi et al., 2019). Briefly, human iPSCs were taken from male and female control, and a male PD patient carrying SNCA A53T mutations (kindly gifted from Dr. Rudolf Jaenisch; Soldner et al., 2011) and isogenic controls were cultured and reprogrammed, and differentiation toward DA neurons was conducted (Mazzulli et al., 2016; Tsunemi et al., 2019). Neuralization efficiency in DA neurons was analyzed immunocytochemically using neuron-specific, β-III tubulin, and midbrain-specific markers (TH, FOXA2, and LMX1a; data not shown; Tsunemi et al., 2019). At 40 d after the initiation of differentiation, we conducted experiments. After 24 h of 10 nM of FTI-277 treatment for H4 cells that were cultured in 96-well plates, the cytotoxicity of FTI was assessed by caspase activation (CellEvent Caspase3/7 Green Detection Reagent, Thermo Fisher Scientific), cell counting (Cell Counting Kit-8, Dojindo Laboratories), and LDH activities in the medium (Cytotoxicity LDH Assay Kit-WST, Dojindo Laboratories) following the manufacture's protocols. For analyzing autophagic activity, the treatment of 1 μM ionomycin (Iono), 200 nM Bafilomycin A1 (BafA1), and 100 nM rapamycin (Rapa) for 4 h in H4 cells before harvesting the cells. For YKT6 knock down, YKT6 Human siRNA Oligo Duplex (Locus ID 10652, SR307262, OriGene) was used following the manufacture's protocol. All chemicals were purchased from Sigma-Aldrich unless otherwise specified.

pHLuorin assay

pHLyorin-CD63 was transfected in H4 cells or DA neurons carrying SNCA A53T mutations that were cultured on glass-bottom dishes. At 24 h post-transfection, the cells were treated with 1 μM LysoSensor Yellow/Blue DND-160 and 50 nM LysoTracker Red DND-99 for 30 min. After being washed with culture media, the cells were observed under the Zeiss LSM 880 confocal system with the Zeiss AX10 inverted microscope equipped with α Plan-Apochromat 63× (1.4 numerical aperture) oil-immersion objective. The cells were maintained at 37°C and 5% CO₂ on the temperature-controlled heating stage in a CO2-controlled incubator. MVB and plasma membrane fusion were induced by the treatment with Iono (1 μM), BafA1 (200 nM), and NH4Cl (2.5 mM). BafA1 and were used for inhibiting acidification of intracellular vesicles. To make the extracellular space Ca²⁺-free, PBS was replaced with culture media. Absorbances at 440/525 nm (for LysoSensor Yellow/Blue), 488 nm (for pHLuorin), and 592 nm (for LysoTracker Red) were recorded in an interval of 1 s for 5–20 min depending on the experiments.

Quantitative RT-PCR

Total RNA was extracted from cultured cells using an RNeasy Plus Mini kit (74134, QIAGEN) and was reverse-transcribed to cDNA using a PrimeScript RT (2670A, Takara Bio), according to the manufacturer's protocols. The cDNA was stored at −80°C until used. Quantitative PCR was performed using an SYBR Green Master Mix for qPCR (A46012, Thermo Fisher Scientific) and a 7,500 Fast Real-Time PCR System (Applied Biosystems). Relative mRNA expression was calculated using the comparative 2^−ΔΔCT method and normalized to ACTB.

Measuring pH of acidic vesicles

The pH of acidic vesicles was measured as described previously (Tsunemi and Krainc, 2014). Briefly, cells were plated in 96-well plates at a density of 30,000/well. After staining with LysoSensor Yellow/Blue DND-160 (1 mM; Invitrogen), fluorescence was measured using SpectraMax i3 multimode microplate reader (Molecular Devices). To obtain a calibration curve, cells were equilibrated with MES buffers adjusted with pH 3.5–8.0, containing 10 μm nigericin and 10 μm monensin, for 5 min before staining with LysoSensor. A standard curve was created by plotting the fluorescence ratio (Yellow⁵³⁵/Blue⁴⁶⁰) against the pH of MES buffers. We determined the pH of acidic vesicles exposed to various concentration of Iono using this standard curve.

Lysosomal GCase activity assays

Lysosomal glucocerebrosidase (GCase) activity within acidic subcellular compartments was assessed in living DA neurons as described previously (Mazzulli et al., 2016). Briefly, neurons were loaded with cascade blue dextran at 1 mg/ml (#D-1976, Life Technologies) for 24 h. On the day of the assay, neurons were treated with dimethyl sulfoxide (DMSO) or 200 nM BafA1 (EMD Millipore) for 1 h. After the removal of dextran blue, cells were exposed to 5-(pentafluoro-benzoylamino)fluorescein di-ß-D-glucopyranoside (PFB-FDGluc; Life Technologies) at a concentration of 100 µg/ml. Subsequently, the cells underwent three washes with warm media and were replaced with phenol red-free neurobasal media. Fluorescence intensity was monitored every 30 min for 3 h in a SpectraMax i3 plate reader (Molecular Devices) with excitation/emission wavelengths set at 485/530 nm for PFB-FDGluc and 400/430 nm for cascade dextran blue.

Fluorescence intensities of PFB-FDGluc were normalized to dextran blue, presented as normalized fluorescence intensity over time, and analyzed by calculating the area under the curve (Chartier-Harlin et al.) for both DMSO and BafA1 treatments. The lysosomal activity was determined by the subtraction of the AUC of BafA1 from the DMSO curves. The AUC values were graphically represented in a column format, and error bars depict variations among different wells within the same culture set.

Immunocytochemistry

Immunocytochemical analysis was conducted as described previously (Tsunemi et al., 2014; Tsunemi and Krainc, 2014). Briefly, after fixation in 4% paraformaldehyde, the cells were permeabilized/blocked in PBS containing 0.1% saponin, 1% BSA, and 5% normal goat serum for 20 min. Specimens were then incubated with primary antibodies overnight, washed in PBS, and then incubated with Alexa Fluor-conjugated anti-rabbit or anti-mouse antibodies at 1:400 dilution for 1 h. Confocal imaging was conducted on the Zeiss LSM 880 confocal system. For quantification analysis, 10,000 cells/well were plated in triplication, and fluorescence intensity was measured using SpectraMax i3 multimode microplate reader (Molecular Devices). Epifluorescence imaging was performed on a Leica DMI3000 B inverted microscope. Live cell imaging was conducted on the Zeiss LSM 780 confocal microscope system with the Zeiss AxioObserver. Z1 inverted microscope equipped with an alpha Plan-Apochromat 100×/1.46 Oil DIC M27 objective. Cells were maintained at 37°C and 5% CO₂ on the temperature-controlled heating stage in a CO₂-controlled incubator.

Electron microscopical studies

Cryo-immunogold electron microscopy was conducted as previously described by Tsunemi et al. (2014). Briefly, after being fixed with 4.0% paraformaldehyde plus 0.2% glutaraldehyde in 0.1 m sodium cacodylate buffer, pH 7.4 for 1 h, the cells were pelleted. The pellet was mixed with warm 2.0% agarose and cut into small pieces, which were then cryoprotected in 2.3 m sucrose in PBS. Ultrathin sections were cut on a Leica EM FCS at −80°C. Double-immunogold staining was performed by incubating with mouse-anti-CD63 for 1 h at RT, followed by 1 h on drops of goat-anti-mouse 10 nm IgG gold (Ted Pella). The grids were incubated on drops of the second primary, rabbit-anti-FLAG, rinsed, and then incubated on drops of goat-anti-rabbit 15 nm IgG gold. Transmission electron microscopic analysis was conducted with the Hitachi HT7700 transmission electron microscope.

Correlative light electron microscopy (CLEM) analysis of green fluorescent protein (GFP)-YKT6–expressing H4 cells was conducted following the procedures described in the previous study (Sasazawa et al., 2022). Briefly, H4 cells were grown on gridded glass-bottom 35 mM dishes under doxycycline (Dox) treatment and transfected with the GFP-YKT6 plasmid. The cells were fixed with 0.1 M phosphate buffer, pH 7.4, containing 100 mM sucrose, 2% paraformaldehyde, and 0.2% glutaraldehyde 24 h after transfection. We observed cells under the Zeiss LSM 880 confocal system and then were refixed with 0.1 M phosphate buffer containing 100 mM sucrose and 2% glutaraldehyde with 1% osmium tetroxide, followed by embedding in epoxy resin. The grid pattern was utilized to locate the areas observed by fluorescence microscopy.

In vitro invagination assay

In vitro invagination assay was performed as described previously (Falguieres et al., 2008). Briefly, cells were grown in 10 cm dishes, washed with cold PBS, and scraped with a rubber policeman in PBS, followed by centrifugation at 150 × g for 5 min at 4°C. Pellets were washed with 8.5% sucrose, centrifuged at 750 × g for 10 min at 4°C, resuspended in 8.5% sucrose with protease inhibitors, and homogenized by passing four times through a G22 needle, followed by centrifugation at 750 × g for 10 min at 4°C. Postnuclear supernatants (PNS) were collected, and protein concentration was determined using Bradford protein assay. PNS were incubated with 1 mM HPTS (the nonpermanent fluorophore 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt); salts (in mM:12.5 HEPES, 1.5 MgOAc₂, and 1 DTT), pH 7.0; 100 mM KCl; and ATP regeneration cassette (2 mM ATP, 0.08 mg/ml creatine kinase, and 16 mM creatine phosphate) at 37°C for 20 min. Finally, uninternalized HPTS was quenched by 50 mM p-xylene-bis-pyridinium bromide in HEPES/NaCl, pH 7.4. Light membranes were obtained by flotation in sucrose step gradient. Fluorescence was measured with Sprectromax i3.

Fractionation assay

Cytosol and membrane fractionation was conducted as described previously (Mazzulli et al., 2011). H4 cells were harvested in 0.25 M sucrose buffer containing 10 mM HEPES, pH 7.4, and 0.1 M EDTA (SHB), homogenized, and centrifuged at 6,800 × g at 4°C, for 5 min. After the pellet was saved (P1), the supernatant was centrifuged at 17,000 × g at 4°C, for 10 min. The supernatant was removed (S), and the remaining pellet (P2) was saved. Fraction S was centrifuged at 100,000 × g for 1 h to obtain P3. Pellets were extracted in 1% Triton X-100 lysis buffer and then 2% SDS buffer. P3 (microsomes) and S (cytosol) fractions were analyzed by immunoblotting or by conducting Triton X-114 phase separation.

Data sharing

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Experimental design and statistical analysis

All data were prepared for analysis with standard spreadsheet software (Microsoft Excel). Statistical analysis was performed by Student's t test, two-way ANOVA Sidak's multiple-comparison test, one-way ANOVA Dunnett's multiple-comparison test, or Tukey's multiple-comparison test. All error bars represent SEM in the figures.

Results

Increased levels of α-syn resulted in the reduction of EV release in H4 and DA neurons carrying SNCA A53T mutations

To investigate the relationship between α-syn levels and EV release, we initially employed inducible H4 human neuroglioma cell lines expressing α-syn under the control of the tet-off system (Tsunemi et al., 2014). The addition of 1 µg/ml of Dox to the media effectively suppressed α-syn expression, and withdrawal of Dox induced α-syn expression (Fig. 1A–D). Subsequently, we quantified the number of EVs released into the media using nanoparticle tracking analysis (Tsunemi et al., 2014). The results consistently demonstrated a decrease in the number of EVs as α-syn levels increased, indicating an inversed correlation between intracellular α-syn levels and extracellular EVs, which aligns with our previous findings (Fig. 1E,F; Tsunemi et al., 2014). Because the traditional exosome isolation through ultracentrifugation cannot eliminate the contamination of cell debris and other vesicles, we used ExoCounter which can quantify only EVs that are <160 nm in diameter and positive for CD36 and CD9 through a sandwich immunoassay (Jiang et al., 2023; Fig. 1G). The results revealed that the number of EVs is significantly less when measured using ExoCounter, the inversed correlation between α-syn levels and EVs was similarly observed in NTA (Fig. 1E) and in ExoCounter (Fig. 1G).

Figure 1.

Inducible H4 cells and PD patient DA neurons develop increased levels of α-syn and decreased EV secretion. A, Representative images of α-syn immunostaining in H4 cells when cultured with 1 µg/ml of Dox (left) and when cultured 24 and 48 h after withdrawal of Dox (middle and right). B, The α-syn expression levels in α-syn–inducible human neuroglioma H4 cells. Immunoblotting analysis of α-syn protein levels in H4 cells when cultured with 1 µg/ml of Dox (left) and when cultured 24 and 48 h after withdrawal of Dox (middle and right). Vimentin was used as a loading control. C, Densitometric analysis of the α-syn protein levels at 17 kDa (n = 5; p = 0.13; p < 0.0001; left). D, Densitometric analysis of the α-syn protein levels at 34 kDa (n = 5; p = 0.58; p < 0.0001; right). E, Quantification of EVs released from H4 cells for 24 h. H4 cells were cultured on six-well plates with or without Dox. The EVs were collected using a conventional ultracentrifuge followed by nanoparticle tracking analysis (n = 5; p < 0.0001). F, Representative results of nanoparticle tracking analysis (Valotassiou et al.) show decreased amount of EVs in response to Dox withdrawal (n = 5). G. Quantification of EVs from the medium (2 ml) that was used for culturing H4 cells for 24 h using ExoCounter. The total protein levels were used for normalization (n = 5; p < 0.0001). H, Representative images of α-syn and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) immunostaining of DA neurons differentiated from iPSCs taken from two healthy controls (Cont1 and Cont 2) and two lines from a patient carrying SNCA A53T mutation (A53T-1 and A53T-2). I, Top, Immunoblotting analysis of α-syn in DA neurons carrying SNCA A53T mutations. At 40 d after differentiating into DA neurons from iPSCs taken from two healthy controls and two lines from a patient carrying SNCA A53T mutation, neurons were subject to immunoblotting. The syn211 was used for detecting α-syn and GAPDH was used as a loading control. J, Left, Densitometric analysis of the α-syn protein levels at 17 kDa (n = 3; p = 0.04). K, Left, Densitometric analysis of the α-syn protein levels at 34 kDa (n = 3; p = 0.01). L, Quantification of EVs released from DA neurons carrying SNCA A53T mutations. The EVs were collected using a conventional ultracentrifuge followed by ExoCounter from the medium (2 ml) that was used for culturing DA neurons for 72 h (n = 4; p = 0.006). Scale bar, 50 μm (A, H). Values are the mean ± SEM. All experiments were performed with at least three separate culture sets. Statistical analysis was performed by one-way ANOVA Dunnett's multiple-comparison test (C, D, E, and G) or one-way ANOVA Sidak's multiple-comparison test (J, K, and L).

To confirm this inversed correlation, we extended our investigation to iPSC-derived DA neurons obtained from PD patients carrying the SNCA 53A mutation along with isogenic controls (Soldner et al., 2011). The differentiation into DA neurons followed the established protocols (Tsunemi et al., 2019). The percentage of tyrosine hydroxylase-positive DA neurons among the lines showed no significant difference (60–70%; data not shown). After 60 d of differentiation, we observed elevated levels of α-syn in patients’ neurons (Fig. 1H–K) and impaired EV secretion (Fig. 1L).

Increased α-syn levels do not affect the generation of ILVs but reduce the EV secretion

MVBs are specialized late endosomes that contain numerous ILVs generated by the inward budding of the limiting membrane (Kalluri and LeBleu, 2020). Once MVBs are fused with the plasma membrane, ILVs are released into the extracellular space, becoming known as exosomes. To investigate the mechanism of α-syn–mediated EV reduction, we conducted an in vitro invagination assay that can analyze the generation of ILVs within MVBs (Falguieres et al., 2008). The results revealed that ILV generation remains the same regardless of the expression levels of α-syn in H4 cells (Fig. 2A). Subsequently, we analyzed the quantity of ILVs and observed that as α-syn levels increased, the immunointensity of CD63, a marker for MVBs containing ILVs, also increased (Fig. 2B,C). This indicates an impairment in the secretion of MVBs. To further validate this finding, we analyzed DA neurons carrying the SNCA A53T mutation alongside isogenic controls and discovered that ILV generation did not vary among the lines (Fig. 2D). However, MVBs increased (Fig. 2E,F). In line with this, the number of α-syn puncta increased in DA neurons carrying the SNCA A53T mutation (Fig. 2G,H).

Figure 2.

Increased α-syn levels do not affect the generation of ILVs but reduce EV secretion. A, The results of in vitro invagination assay using H4 cells. PNS obtained from H4 cells were incubated with HPTS either in the presence of an ATP-regenerating system or an ATP-depleting system. The excessive dye present in the solution was quenched with p-xylene-bis-pyridinium bromide (DPX). Quantification of fluorescence intensities of HPTS is shown (n = 3; p = 0.81). B, Representative images of CD63 immunostaining of H4 cells that were cultured with 1 µg/ml of Dox (left) and when cultured 24 and 48 h after withdrawal of Dox (middle and right). C, Quantification of CD63 immunointensities normalized by DAPI immunointensities are shown (n = 5; p = 0.48; p < 0.0001). D, The results of in vitro invagination assay using Cont 1 and Cont 2 DA neurons and two DA neurons carrying SNCA A53T mutations (Mut1 and Mut 2) at 40 d after differentiation (n = 4). E, Representative images of CD63 and tyrosine hydroxylase immunostaining of Cont 1 (Cuddy et al.) and Mut 1 (bottom) DA neurons carrying SNCA A53T mutations. F, Quantification of CD63 immunostaining intensities normalized by DAPI immunointensities is shown (n = 20; p < 0.0001). G, Representative images of α-syn (red) and tyrosine hydroxylase (green) immunostaining of Cont 1 (Cuddy et al.) and Mut 1 (bottom) DA neurons carrying SNCA A53T mutations. H, Quantification of the number of α-syn puncta in each neuron is shown (n = 20; p < 0.0001). Scale bar, 50 μm (B, E, G). Values are the mean ± SEM. All experiments were performed with at least three separate culture sets. Statistical analysis was performed by Student's t test (A), one-way ANOVA Dunnett's multiple-comparison test (C), or Tukey's multiple-comparison test (C, D, F).

These results indicate that elevated levels of α-syn reduce the number of EVs not by inhibiting the generation of ILVs but by impairing the process of EV secretion.

Increased α-syn levels inhibit lysosomal calcium-dependent fusion of MVBs with the plasma membrane

We proceeded to examine the release of MVBs by generating a plasmid capable of expressing a pH-sensitive variant of GFP fused with CD63. At 24 h after transfection of this plasmid into H4 cells, we stained the cells with LysotrackerRed, a marker for late endosomes/MVBs. When Iono, a membrane-permeable calcium ionophore capable of triggering the fusion of MVB and the plasma membrane, was introduced to the media, GFP signals were detected in H4 cells where α-syn expression was not induced (Fig. 3A). However, in α-syn overexpressing H4 cells, GFP signals were barely detected upon Iono treatment, indicating an inhibition of the fusion between MVB and the plasma membrane (Fig. 2B,C). We confirmed that this fusion occurs in the Ca²⁺-free media, suggesting the importance of intracellular Ca²⁺ gradient, probably between the lumen of MVBs and cytoplasm, in this process (Fig. 3D, Extended Data Movie 1, and Fig. 1-1A) Then, we conducted basic analysis of autophagic activities by LC3 immunoblotting (Fig. 3E,F). The results demonstrated that Iono does not induced autophagy in H4 cells. Importantly, 1 μM Iono along with 200 nM BafA1 and 100 nM Rapa treatments for 4 h increased EV secretion (Fig. 3G), consistent with the previous results (Zou et al., 2019; Sagini et al., 2021). To determine whether Iono treatment increased the pH in MVBs or not, we used LysoSensor Yellow/Blue DND-160, a ratiometric probe that has been employed to measure the pH of acidic vesicles, including MVBs (Tsunemi and Krainc, 2014). In this experiment, NH4Cl was used as a positive control due to its rapid ability to disrupt vesicular acidity. The results showed that treatment with 1 μM Iono elevated the pH within acidic vesicles in H4 cells with normal a-syn expression (Extended Data Movies 2, 3, 4) although the extent of the increase was less pronounced than that observed with 2.5 mM NH4Cl treatment, as demonstrated by live-cell analysis (Fig. 3H,I, Extended Data Movies 5, 6, 7, and Fig. 2-1A,B) and a cell-based assay (Fig. 3J). These findings indicate that Iono-mediated EV release partially depends on an increase in pH within acidic vesicles. Importantly, a-syn overexpression elevated the pH in these vesicles (Fig. 3H,I).

Figure 3.

Increased α-syn levels inhibit lysosomal calcium-dependent fusion of MVBs with the plasma membrane. A, Representative images of red (acidic vesicles) and green fluorescence (pHLuorin-CD63) before and after 1 μM Iono treatment in H4 cells in which α-syn levels were normal (top; Dox addition), and expression of α-syn was induced (bottom; Dox withdrawal). B, Representative results of the change of green fluorescence (pHLuorin) by 1 μM Iono treatment in H4 cells. C, Quantification of the change of green fluorescence intensities is shown (n = 6; p < 0.0001). D, Quantification of the change of green fluorescence intensities which was conducted under Ca²⁺-free media (PBS; n = 6; p < 0.0001). E, The effect of 1 μM Iono, 200 nM BafA1, and 100 nM Rapa for 4 h on autophagy status. Representative images of immunoblotting analysis of p-62, LC3-I, and LC3-II in H4 cells where α-syn expression was not induced (far left) and was induced (right four). GAPDH was used as a loading control. F, Densitometric analysis of the LC3-II protein levels (n = 3; p = 0.95 and p > 0.99). G, Quantification of EVs isolated from the medium (2 ml) that was used for culturing H4 cells for 4 h of 1 μM Iono, 200 μM BafA1, and 100 nM Rapa. The total protein levels were used for normalization (n = 5; p < 0.0001). H, Representative change of LysoSensor Yellow/Blue fluorescence ratios of 440/525 nm in acidic vesicles were observed following treatments in H4 cells with suppressed (Dox +1–3) or induced (Dox −1 to 3) α-syn expression: 1 μM Iono treatment, followed by a wash; 2.5 mM NH4Cl treatment, followed by a wash; and 1 μM Iono treatment, followed by a wash. I, Fluorescence intensity ratios (440/525 nm) in acidic vesicles were measured in H4 cells, where a-syn expression was not induced (Dox +1–3) and induced (Dox −1 to 3), under the following conditions: in media (leftmost two columns), during treatment with 1 μM Iono (middle-left two columns), during treatment with 2.5 mM NH4Cl (middle-right two columns), and during treatment with 1 μM Iono (rightmost two columns). The values represent the average measured 1–2 min after each treatment (n = 5). J, Changes of lysosomal pH induced by treatments with Iono (0–5 μM), BafA1 (200 nM), and NH4Cl (2.5 mM) in H4 cells where α-syn levels were normal (n = 5). K, Representative green fluorescence (pHLuorin) change induced by 1 μM Iono treatment in DA neurons carrying SNCA A53T mutations and isogenic controls. L, Quantitative evaluation of MVEs—the plasma membrane fusion in DA neurons from isogenic control (Cont 1) and DA neurons carrying SNCA A53T mutations (A53T-1; n = 6; p = 0.0002). M, Quantitative evaluation of MVEs—the plasma membrane fusion in DA neurons which was conducted under Ca²⁺-free media (PBS; n = 6; p < 0.0001). N, Representative change of LysoSensor Yellow/Blue fluorescence ratios of 440/525 nm in acidic vesicles were observed following sequential treatments: 1 μM Iono, followed by a wash; 2.5 mM NH4Cl, followed by a wash; and 1 μM Iono, followed by a wash. The analysis was performed on DA neurons carrying the SNCA A53T mutation (A53T-1–3) and their isogenic controls (Iso cont 1–3). O, Fluorescence intensity ratios (440/525 nm) in acidic vesicles were measured in DA neurons carrying the SNCA A53T mutation (A53T) and isogenic controls (Iso cont; n = 5), under the following conditions: in media (leftmost two columns), during treatment with 1 μM Iono (middle-left two columns), during treatment with 2.5 mM NH4Cl (middle-right two columns), and during treatment with 1 μM Iono (rightmost two columns). The values represent the average measured 1–2 min after each treatment. P, Quantification of EVs released from Dox-treated or Dox-untreated (α-syn induced) H4 cells. The cells were treated with 1 μM Iono for 1 h (n = 5). Q, Quantification of EVs released from isogenic controls and DA neurons carrying SNCA A53T mutations that were treated with 1 μM of Iono for 1 h (n = 5). R, Quantification of EVs released from Dox-treated H4 cells. The cells were treated with 1 μM Iono, 200 nM BafA1, or 2.5 mM NH4Cl for 4 h (n = 5). Scale bar, 50 μm (A). Values are the mean ± SEM. All experiments were performed with at least three separate culture sets. Statistical analysis was performed by Student's t test (C, D, L, K), one-way ANOVA Dunnett's multiple-comparison test (F, G, J, R), or Tukey's multiple-comparison test (I, O, P, Q).

Movie 1

Representative fluorescence movies of absorbance at 488  nm (for pHLuorin). The cells were treated with 1 μM ionomycin for 5  min, followed by a wash for 5  min, treated with 2.5  mM NH4Cl for 5  min, followed by a wash for 5  min, treated with 1 μM ionomycin for 5  min, and followed by a wash. Download Movie 1, MOV file ^{(22.4MB, mov)} .

Movie 2

Representative fluorescence movies of absorbance at 525  nm during the same procedures as described for Movie 1. Download Movie 2, MOV file ^{(50.6MB, mov)} .

Movie 3

Representative fluorescence movies of absorbance at 440  nm during the same procedures as described for Movie 1. Download Movie 3, MOV file ^{(28.2MB, mov)} .

Movie 4

Representative changes in fluorescence movies of absorbance at 488  nm (for pHLuorin). The cells were treated with 1 μM ionomycin for 5  min, followed by a wash for 5  min, treated with 2.5  mM NH4Cl for 5  min, followed by a wash for 5  min, treated with 1 μM ionomycin for 5  min, and followed by a wash. Download Movie 4, MOV file ^{(28.6MB, mov)} .

Movie 5

Representative fluorescence movie of absorbances at 525  nm during the same procedures as described for Movie 4. Download Movie 5, MOV file ^{(47.1MB, mov)} .

Movie 6

Representative fluorescence movie of absorbances at 440  nm during the same procedures as described for Movie 4. Download Movie 6, MOV file ^{(43.8MB, mov)} .

Movie 7

Representative fluorescence movies of absorbance at 488  nm (for pHLuorin). The cells were treated with 2.5  mM NH4Cl for 5  min, followed by a wash for 5  min, treated with 1 μM ionomycin for 5  min, followed by a wash for 5  min, treated with 2.5  mM NH4Cl for 5  min, and followed by a wash. Download Movie 7, MOV file ^{(27.6MB, mov)} .

Extended figures

Download Extended figures, PDF file ^{(521.4KB, pdf)} .

To corroborate these findings, we examined DA neurons and observed that the fusion between MVB and the plasma membrane was inhibited in DA neurons carrying SNCA A53T mutations (Fig. 3K,L). This fusion also occurred in the Ca²⁺-free media in DA neurons (Fig. 3M, Extended Data Movie 8, and Fig. 3-1A). Treatment with 1 μM Iono elevated the pH within acidic vesicles in DA neurons of the isogenic control, although the extent of the increase was less pronounced than that observed with 2.5 mM NH4Cl treatment. (Fig. 3N,O, Extended Data Movies 9, 10, 11). Iono-induced EV secretion was significantly reduced in α-syn overexpressing H4 cells (Fig. 3P) and DA neurons carrying SNCA A53T mutations (Fig. 3Q). Importantly, treatment with 1 μM Iono induces EV secretion more effectively than treatment with either 2.5 mM NH4Cl or 200 nM BafA1 (Fig. 3R). Given that pH elevation is more pronounced with NH4Cl treatment (Fig. 3J), Ca²⁺ release from MVBs, rather than an increase in pH within MVBs, likely plays a key role in the fusion of MVBs with the plasma membrane. Taken together, these data suggest that elevated α-syn levels impair fusion between MVB and the plasma membrane, likely due to the inhibition of Ca²⁺ release and dysregulated acidification of MVBs, ultimately leading to a reduction in EV secretion.

Movie 8

Representative fluorescence movies of absorbance at 525  nm during the same procedures as described for Movie 7. Download Movie 8, MOV file ^{(20.8MB, mov)} .

Movie 9

Representative fluorescence movies of absorbance at 440  nm during the same procedures as described for Movie 7. Download Movie 9, MOV file ^{(38.3MB, mov)} .

Movie 10

Representative fluorescence movie of an H4 cell treated with 1 uM Ionomycin in PBS. Download Movie 10, MOV file ^{(25.1MB, mov)} .

Movie 11

Representative fluorescence movie of a DA neuron (isogenic control1) treated with 1 uM Ionomycin in PBS. Download Movie 11, MOV file ^{(25.3MB, mov)} .

YKT6 is a key molecule for α-syn–mediated EV reduction

To further analyze the molecular mechanism underlying α-syn–mediated impaired MVBs fusion with the plasma membrane, we conducted a screening of several proteins that might be targeted by α-syn. To this end, we overexpressed RAB11 (Savina et al., 2005), RAB27B (Ostrowski et al., 2010), RAB5A (Ostrowski et al., 2010), and YKT6 (Gross et al., 2012; Sun et al., 2020), all of which have been implicated in EV secretion. Interestingly, we discovered that YKT6, a soluble N-ethylmaleimide-SNARE protein that is involved in vesicle fusion with their targeted membrane, had the capacity to reverse the α-syn–mediated EV reduction in H4 cells (Fig. 4A). Significantly, the overexpression of YKT6 also mitigated α-syn levels in H4 cells (Fig. 4B,C). Importantly, YKT6 did not reduce α-syn at the mRNA level (Fig. 4D). We also tried to reduce YKT6 levels in H4 cells using human YKT6-specific siRNA (Fig. 4E–H). YKT6 levels successfully decreased both when a-syn was induced and when it was suppressed (Fig. 4E,F). Consequently, the reduction of YKT6 levels led to increased α-syn levels (Fig. 4G) and decreased number of EVs (Fig. 4H), corroborating the important role of YKT6 in EV release and α-syn levels. YKT6 is primarily cytosolic under the physiological conditions but translocates to intracellular vesicles following lipid modifications (Fukasawa et al., 2004). To assess the intracellular localization of YKT6, we expressed GFP-YKT6 in H4 cells. After fixation, the cells were stained against CD63, a marker for MVBs. Our results revealed that numerous CD63-positive vesicles, particularly those in close proximity to the plasma membrane, exhibited positive for GFP-YKT6 (Fig. 4I). We further corroborated these findings through cryo-electron microscopy (Fig. 4J) and CLEM (Fig. 4K), verifying the localization of YKT6 on MVBs. Notably, we detected YKT6 proteins in EVs isolated from both H4 cells and DA neurons (Fig. 4L), indicating that YKT6 is indeed localized on MVBs and plays a pivotal role in EV secretion. Immunocytochemistry focused on YKT6 revealed that YKT6, which is typically found on acidic vesicles, exhibited a reduction in abundance upon α-syn overexpression in H4 cells (Fig. 4M,N) and also in iPSC-derived DA neurons carrying SNCA A53T mutations (Fig. 4O,P). To confirm this observation, we conducted immunoblot analysis using fractionated samples and observed that the total YKT6 increased with α-syn induction (Fig. 4Q,R), while membrane-associated YKT6 decreased (Fig. 4Q,S), aligning with the results obtained from immunohistochemistry. In summary, our findings indicate a correlation between YKT6 expression levels and α-syn levels. However, the increased levels of α-syn may inhibit the membrane association of YKT6.

Figure 4.

YKT6 is a key molecule for α-syn–mediated EV reduction. A, The effect of overexpression of proteins that are associated with EV production and release (RAB11, RAB 27, RAB5, or YKT6) on EV release from H4 cells. Several plasmids that express RAB11, RAB 27, RAB5, or YKT6 were overexpressed in H4 cells cultured without Dox (α-syn induced). Medium changed to EV-free medium 24 h after transfection; EVs were collected from the medium that culture for the next 24 h and quantified by the ExoCounter (n = 5, p = 0.01). B, The effect of overexpression of RAB11, RAB 27, RAB5, or YKT6 in H4 cells where α-syn expression is induced. Representative images of immunoblotting analysis of α-syn in H4 cells where α-syn expression is not induced (far left) and is induced (right five). Vimentin was used as a loading control. C, Densitometric analysis of the α-syn protein levels (n = 3; p = 0.01). D, The mRNA expression levels of α-syn in H4 cells where YKT6 is overexpressed. Twenty-four hours after transfection of YKT6 in H4 cells, cells were harvested, and RNA was extracted. The mRNA expression levels of α-syn were assessed by real-time PCR. E–H, The effect of siRNA-mediated YKT6 knockdown for 24 h on EV secretion in H4 cells. E, Representative images of immunoblotting analysis of YKT6 and α-syn in YKT6-knockdowned H4 cells where α-syn expression was induced (left) and was not induced (right). GAPDH was used as a loading control. F, Densitometric analysis of the YKT6 protein levels (n = 3; p < 0.0001). G, Densitometric analysis of the α-syn protein levels (n = 3; p = 0.01; p = 0.003). H, Quantification of EVs secreted from H4 cells where α-syn expression was induced (left) and was not induced (right; n = 5; p < 0.0001). Medium changed to the conditioned (EV-free) medium 4 h after siRNA transfection; EVs were collected from the medium that culture for the next 24 h and quantified by the ExoCounter. I, Representative images of YKT6-GFP–expressing H4 cells that were stained with anti-CD63 (red). Arrowheads highlight colocalization between YKT6 and CD63. J, Representative immune-EM image of a MVB in FLAG-YKT6–expressing H4 cells. Double-staining was conducted with anti-CD63 antibody (10 nm gold particles) and anti-FLAG antibody (15 nm gold particles, indicated by an arrow). K, Representative images of CLEM analysis. H4 cells were grown under Dox treatment and transfected with the GFP-YKT6 plasmid. After fixation of 2% paraformaldehyde, we observed cells under the Zeiss LSM 880 confocal system (a, b). The cells were refixed and were conducted EM analysis (c, d). L, Representative images of immunoblot analysis of EVs that were isolated from the media that were cultured with H4 cells for 24 h (left) or DA neurons for 72 h (Cont 1; right). Alix and TSG101 were used as exosome markers. M, Representative images of YKT6 immunostaining in H4 cells where α-syn expression was not induced (Dox +, top) and was induced (Dox −, bottom). LysoTracker red was used as the marker for late endosomes. M, Colocalization analysis was conducted using the ZEN software (n = 10; p < 0.0001). O, Representative images of YKT6 immunostaining in DA neurons carrying SNCA A53T mutation (bottom) or isogenic controls (Cuddy et al.). CD63 was used as the marker for multivesicular vesicles. P, Colocalization analysis was conducted using the ZEN software (n = 10; p = 0.0006). Q, Representative images of immunoblot analysis of YKT6 levels in the cytosol and membrane fractions from H4 cells before (left two lanes) and after withdrawal of Dox (right four lanes). R, Densitometric analysis of the total (cytosol plus membrane YKT6 protein levels before and after withdrawal of Dox (n = 3; p = 0.009; p = 0.002). S, Densitometric analysis of the YKT6 protein ratio between the membrane and cytosol fractions before and after withdrawal of Dox (n = 3; p = 0.03; p = 0.003). Scale bar, 5 μm (K), 100 μm (I, M), and 200 nm (J). Values are the mean ± SEM. All experiments were performed with at least three separate culture sets. Statistical analysis was performed by one-way ANOVA Dunnett's multiple-comparison test (C, R, S), or Student's t test (F, G, H, N, P).

Inhibition of farnesylation of YKT6 reduced EVs and increased α-syn levels

YKT6 undergoes a translocation to the vesicular membrane through a series of lipid modifications including farnesylation followed by the palmitoylation (Fukasawa et al., 2004). To further dissect the impact of these lipidations on YKT6 and the involvement in EV release and α-syn, we employed FTI-277, an inhibitor of YKT6 farnesylation. Initially, we conducted immunoblot analysis using fractionated samples, confirming that FTI treatment significantly reduced the levels of membrane-associated YKT6 in H4 cells (Fig. 5A–C). Importantly, we confirmed that FTI-277 treatment did not exhibit cytotoxicity to the cells (Fig. 5D–F). As anticipated, FTI treatment led to a significant reduction in the number of EVs released from H4 cells, regardless of α-syn induction (Fig. 5G). Consequently, α-syn levels increased within H4 cells (Fig. 5H,I) and in the EVs released from these cells (Fig. 5J). Furthermore, we extended our analysis to iPSC-derived DA neurons and observed that FTI treatment significantly decreased the number of EVs in both controls and DA neurons carrying SNCA A53T mutations (Fig. 5K). As a result, α-syn levels increased within DA neurons (Fig. 5L,M). A recent study has demonstrated that FTI treatment enhances the trafficking of the lysosomal hydrolases (Cuddy et al., 2019). To validate this finding, we measured β-GCase activities within lysosomes using 5-PFB-FDGluc (Mazzulli et al., 2016). The results consistently showed an increase in GC activities upon FTI treatment in all four lines, suggesting elevated lysosomal activities (Fig. 5N). These data collectively indicate that the inhibition of the farnesylation impairs the membrane association of YKT6, resulting in reduced EV release and increased intracellular α-syn levels, despite heightened lysosomal activities.

Figure 5.

FTI-mediated inhibition of YKT6 farnesylation results in reduced EV secretion and increased intracellular α-syn levels. A, Representative images of immunoblot analysis of YKT6 levels in the cytosol and membrane fractions from DA neurons taken from an isogenic control treated with DMSO (left two lanes) or 10 nM of N-[4-[2(R)-amino-3-mercaptopropyl]amino-2-phenylbenzoyl]methionine methyl ester trifluoroacetate salt (FTI-277) for 7 d (right two lanes). B, Densitometric analysis of YKT6 membrane/cytosol ratios in DA neurons treated with DMSO or FTI (n = 3; p = 0.0005). C, Densitometric analysis of YKT6 membrane/cytosol ratios in DA neurons treated with DMSO or FTI-277 (n = 3; p = 0.0008). D, Caspase 3/7 activation was assessed in H4 cells that were cultured in 96-well plates after 24 h of 10 nM of FTI-277 treatment by measuring absorbance in wells at 490 nm (n = 3; p = 0.85). E, Viable cell numbers of H4 cells were assessed after 24 h of 10 nM of FTI-277 treatment by measuring absorbance in wells at 450 nm (n = 3; p = 0.55). F, LDH activities in the medium that were cultured in H4 cells were assessed after 24 h of 10 nM of FTI-277 treatment by measuring absorbance in wells at 490 nm (n = 3; p = 0.49). G, The number of EVs that were released from H4 cells that were cultured with Dox (α-syn was not induced; left two lanes) and cultured without Dox (α-syn induced; right two lanes; n = 5). The cells were treated with DMSO (white boxes) or 10 nM of FTI-277 (black boxes) for 24 h. H, Immunoblotting analysis of α-syn in H4 cells that were cultured without Dox (α-syn induced; left) with DMSO and with FTI-277 (right). Vimentin was used as a loading control (n = 3; p = 0.001). I, Densitometric analysis of the α-syn protein levels relative to GAPDH (n = 3). J, Quantitative analysis of α-syn in EVs isolated from culture media of H4 cells that were cultured with Dox (α-syn was not induced; left two lanes; n = 5; p = 0.45) and cultured without Dox (α-syn induced; right two lanes; n = 3; n = 5; p = 0.03). The H4 cells were treated with DMSO (white boxes) or FTI-277 (gray boxes) for 24 h. K, The number of EVs isolated from the media in which Cont 1, Cont 2 DA neurons, and two DA neurons from the patient carrying SNCA A53T mutations (Mut 1 and Mut 2) that were cultured with DMSO or 10 nM of FTI-277 for 7 d (n = 5; p < 0.0001). The EVs were collected from the media that were cultured last 3 d. L, Immunoblot analysis of α-syn in Cont 1, Cont 2 DA neurons, and two DA neurons from the patient carrying SNCA A53T mutations (Mut 1 and Mut 2) after 7 d with DMSO or 10 nM of FTI-277. GAPDH was used as a loading control (n = 3). M, Densitometric analysis of the α-syn protein levels relative to GAPDH in Cont 1, Cont 2 DA neurons, and two mutants (Mut 1 and Mut 2) after 7 d of treatment with DMSO or 10 nM of FTI-277 (n = 3). N, β-GCase activities within lysosomes were measured by qualifying the degradation of a fluorescent-tagged substrate (PFB-FDGluc) in DA neurons taken from isogenic controls and the two patients carrying SNCA A53T mutations that were treated with DMSO or 10 nM of FTI-277 for 7 d (n = 3). Values are the mean ± SEM. All experiments were performed with at least three separate culture sets. Statistical analysis was performed by Student's t test (B–F and I) or two-way ANOVA Sidak's multiple comparison (G, J, K, M, and N).

In summary, our findings suggest that increased levels of α-syn hinder the lipidation of YKT6, leading to decreased membrane-associated YKT6 and reduced fusion between multivesicular endosome (MVE) and the plasma membrane. This reduction in EV release, in turn, contributes to further elevating intracellular α-syn levels.

Discussion

The deposition of Lewy bodies and Lewy neurites in neurons is a pathological hallmark of PD, highlighting the critical role of α-syn accumulation (Lang and Lozano, 1998) in Parkinson's pathogenesis (Rubinsztein, 2006). In our previous work, we demonstrated reduced secretion of exosomes and α-syn in cellular models of Kufor–Rakeb syndrome (KRS), a condition characterized by loss-of-function mutations in ATP13A2 which codes endolysosomal protein ATP13A2 (Tsunemi and Krainc 2014). In this study, we extended our investigation to more general PD models characterized by α-syn accumulation. By examining α-syn–inducible neuroglioma H4 cells and genetic forms of PD, carrying mutations in SNCA genes encoding α-syn, we have reaffirmed that EV secretion plays a pivotal role in regulating α-syn levels in human neurons. Elevated α-syn levels impaired EV secretion, leading to increased α-syn accumulation and establishing a detrimental feedback loop.

The α-syn is predominantly located at the presynaptic terminals in healthy neurons and plays a crucial role in the synaptic transmission (Burre et al., 2010). However, when its expression level surpasses a certain threshold, α-syn mislocalizes to the cytosol and exerts toxicity toward neurons through various mechanisms (Burbulla et al., 2017). For instance, elevated levels of α-syn hindered the trafficking of GCase, a lysosomal hydrolase (Mazzulli et al., 2011). Oligomeric species of α-syn impede ER to Golgi trafficking of GCase, leading to lysosomal dysfunction and subsequent accumulation of α-syn. The toxic variants of α-syn interfere with multiple steps in vesicular fusion to the Golgi apparatus. One of the targeted proteins is the N-ethylmaleimide–sensitive factor attachment protein receptors (SNARE) protein YKT6 (Thayanidhi et al., 2010), which can associate with various vesicles due to its lack of a transmembrane domain (Thayanidhi et al., 2010). YKT6 primarily localizes in the cytosol and translocates to the Golgi apparatus upon stimulation. It has also been detected in exosomes (ExoCarta) and is essential for the process of exosomal Wints secretion (Karuna et al., 2020). Furthermore, palmitoylation of YKT6 is crucial for the fusion of MVBs with the plasma membrane (Sun et al., 2020). More recently, YKT6 plays a crucial role in autophagosome–lysosome fusion which is targeted by α-syn (Pitcairn et al., 2023).

We confirmed the localization of YKT6 in MVBs, where it facilitates vesicular fusion between MVBs and plasma membrane (Fig. 4I,J). Our subsequent investigations revealed that elevated levels of α-syn impair this process (Fig. 4Q–S). YKT6's membrane association is regulated by a conformational change, forming a closed cytosolic form to an open membrane-bound form through lipid modification, including farnesylation and palmitoylation (Wang et al., 2017). Recently, the inhibition of YKT6 farnesylation was reported to induce YKT6 membrane association and subsequent vesicular fusion at the Golgi apparatus (Cuddy et al., 2019) or autophagosomes (Pitcairn et al., 2023). We validated this by demonstrating increased GCase activities upon FTI treatment, an inhibitor of YKT6 farnesylation (Fig. 5N). However, we also observed that FTI reduced YKT6 membrane association in MVBs, indicating that farnesylation of YKT6 exerts opposing effects on vesicular fusion at the Golgi apparatus and MVBs. Importantly, FTI treatment enhanced lysosomal proteolysis (Fig. 5N) but exaggerated α-syn accumulation (Fig. 5L,M), indicating that exosomal α-syn secretion surpasses lysosomal α-syn degradation in terms of reducing α-syn levels in DA neurons. A recent study demonstrated that pathogenic α-syn inhibits vesicle-binding of microtubule-associated protein 6 (MAP6) by accelerating its palmitate turnover. The α-syn enhances the activity of the de-palmitoylase, acyl-protein-thioesterase-1 (APT1), which is responsible for the MAP6 depalmitoylation (Ho et al., 2021). These findings illustrate another example of α-syn's toxicity through the targeting of protein palmitoylation. Our results are clearly opposite to the findings of Tang et al. who reported that elevated levels of α-syn result in increased EV secretion without affecting YKT6 levels (Tang et al., 2021). One possible explanation of these discrepancies may be the levels of α-syn. When it exceeds certain levels, cell membranes become damaged and loosen, allowing release of vesicles within the cells.

Exosomes are one of the EVs that carry proteins, lipids, and mRNAs, allowing them to convey information to distant organs similar to hormones (Kalluri and LeBleu, 2020). They also play a role in various disorders. For example, a relatively high number of exosomes are secreted from cancer cells. These exosomes carry miRNAs that promote vascularization at distant sites, facilitating cancer metastasis (Wortzel et al., 2019). Exosomes released from lung cancer contain specific miRNA that guides them to particular organs, implying that exosomes can target specific organs (Hoshino et al., 2015). Additionally, exosomes are implicated in the development of various neurodegenerative disorders. Microglia uptake Tau proteins and release them in exosomes. This process facilitates the neuronal transmission of Tau (Balogun et al., 2015). We and others have discovered that exosomes carry α-syn and may regulate its levels in neurons (Tsunemi et al., 2014). Dysfunctional ATP13A2 results in decreased exosomal secretion in KRS, a rare form of neurodegenerative disorder primarily characterized by parkinsonism (Ramirez et al., 2006). ATP13A2 is localized on MVBs containing numerous ILVs, which transform into exosomes upon cell release. Dysfunctional ATP13A2 impairs ILV formation. In this study, we observed reduced exosome release as intracellular α-syn levels increased in α-syn–inducible H4 neuroglioma cell lines and DA neurons carrying a SNCA mutation, demonstrating an inverse correlation between exosomal release and intracellular α-syn levels. This relationship creates a pathological loop of significant importance in the pathomechanism of PD.

Our results suggest that the EV pathway is important for neurons to reduce α-syn levels. EV α-syn is continuously secreted even under physiological conditions as evidenced by the presence in the cerebrospinal fluid of both PD patients and the healthy controls (Borghi et al., 2000). When the neuronal machinery for α-syn degradation exceeds its limit, neurons may resort to secretory pathways as a last effort to mitigate toxic protein accumulation (Rubinsztein, 2006). Indeed, EVs from PD patients contain more toxic forms of α-syn in blood compared with healthy individuals (Ishiguro et al., 2024; Yan et al., 2024). However, enhancing EVs could potentially exacerbate PD pathology by spreading the toxic form of α-syn in brains. Indeed, inoculation of EVs that carry α-syn in mouse brains induces Lewy pathology throughout the entire brain (Ngolab et al., 2017). Recently, microglial exosomes have been identified as crucial contributors to the development of PD pathology in mice (Guo et al., 2020; Xia et al., 2021). Further studies are necessary to thoroughly investigate the contribution of EVs to PD pathology.

In conclusion, our study demonstrates that intracellular α-syn levels can be regulated through EV secretion. Notably, heightened α-syn levels impair EV secretion, creating a pathological loop that exacerbates further α-syn accumulation. Importantly, impaired EV secretion surpasses increased lysosomal proteolysis in determining the intracellular α-syn levels, underscoring the significance of this pathway. Targeting EV secretion could therefore represent a vital therapeutic strategy for mitigating α-syn accumulation across various synucleinopathies, including PD.