Abstract
Graphical abstract
Schematic representation of lysosomal damage-induced PARK7 secretion. Upon L-leucyl-L-leucine methyl ester (LLOMe)-induced acute lysosomal damage, PARK7 and galectin-3 (LGALS3) are recruited to damaged lysosomes. Following LLOMe washout, lysophagy is activated, during which autophagosomes selectively engulf the damaged lysosomes containing translocated PARK7 and LGALS3. However, instead of fusing with functional lysosomes for degradation, autophagosomes containing SEC22B bypass the degradative route. This process is facilitated by a specific SNARE complex comprising SEC22B, STX3/4 and SNAP23/29, enabling the autophagosomes to fuse with the plasma membrane, producing PARK7 into the extracellular space.
Abstract
Objective
PARK7/DJ-1 is a multifunctional protein and redox sensor essential for cellular survival and oxidative stress defense. PARK7 secretion is linked to pathophysiologies, including neurodegenerative diseases, cancer and inflammation. This study investigates the mechanisms of PARK7 secretion in response to lysosomal membrane permeabilization induced by L-leucyl-L-leucine methyl ester (LLOMe).
Methods
HeLa and mouse embryonic fibroblasts cells were treated with LLOMe followed by washout with serum-free Dulbecco’s modified Eagle’s medium. PARK7 secretion was analyzed by assessing the intracellular and extracellular protein levels.
Results
Upon LLOMe treatment, PARK7 translocates to damaged lysosomes, colocalizing with galectin-3 (LGALS3), a marker of lysosomal damage. Following LLOMe washout, an increase in lysophagy flux was observed, enhancing the secretion of both PARK7 and LGALS3. The knockdown of TANK-binding kinase 1, a critical lysophagy regulator, suppresses LLOMe-induced PARK7 secretion, confirming a lysophagy-dependent mechanism. Notably, while the inhibition of autophagy initiation blocks PARK7 secretion, the disruption of autophagosome–lysosome fusion does not affect its release. In addition, the SEC22B-mediated SNARE complex, comprising STX3/4 and SNAP23/29, is essential for the fusion of secretory autophagosomes with the plasma membrane during PARK7 secretion.
Conclusion
These findings reveal a novel lysophagy-dependent mechanism of PARK7 secretion, where acute lysosomal damage facilitates the recruitment of PARK7 to damaged lysosomes, subsequent autophagosome encapsulation and nondegradative release into the extracellular space through a dedicated SNARE complex.
Significance statement
The findings of this study broaden the understanding of unconventional protein secretion under lysosomal stress and highlight the potential therapeutic targets for diseases linked to lysosomal dysfunction, including neurodegeneration and inflammation.
Introduction
Parkinson’s disease (PD) protein PARK7, also known as DJ-1, is a multifunctional protein that plays critical roles in various molecular processes, including cell survival and gene regulation (Yokota et al. 2003, Taira et al. 2004, Ariga & Iguchi-Ariga 2017, Dash et al. 2022). PARK7 is remarkably recognized for its antioxidant properties, where it scavenges reactive oxygen species (ROS), protects mitochondrial integrity and promotes cellular resilience (Taira et al. 2004, Takahashi-Niki et al. 2004). Notably, PARK7 lacks a conventional signal peptide, enabling its secretion through an unconventional pathway (Urano et al. 2018). PARK7 has been detected in multiple biological fluids, including cerebrospinal fluid, blood and breast milk, indicating its systemic relevance (Yanagida et al. 2009, Kaneko et al. 2014, Ariga 2015, Zhao and Zhang 2019, Kim et al. 2021, Ahat et al. 2022, Yamada et al. 2022). In pathological contexts such as cancer, early-stage PD and stroke, PARK7 secretion is upregulated in response to cellular stressors, including oxidative damage and inflammation. Once released, PARK7 modulates various biological processes: it acts as an anti-inflammatory agent; promotes metastasis, angiogenesis and osteogenesis; and facilitates the degradation of toxic protein aggregates, such as transthyretin (Koide-Yoshida et al. 2007, Yanagida et al. 2009, Kim et al. 2012, Kaneko et al. 2014, Gu et al. 2022). These diverse functions underscore the crucial roles of PARK7 in cellular defense mechanisms and highlight the need for a deeper understanding of its secretion mechanisms.
Our previous work demonstrated the upregulation of PARK7 secretion in 6-hydroxydopamine (6-OHDA)-induced cell culture models, leading to the hypothesis that autophagic machinery may regulate its secretion (Urano et al. 2018). Autophagy, a fundamental cellular degradation and recycling pathway, is essential for maintaining cellular homeostasis by targeting damaged organelles, misfolded proteins and pathogens for lysosomal degradation (Mizushima et al. 2008). Beyond its degradative role, autophagy has been increasingly recognized for facilitating unconventional protein secretion, which allows cytosolic proteins lacking traditional secretory signals to be exported from the cell. This secretory autophagy pathway involves repurposing core autophagy machinery, including autophagosomes and lysosomes, which enclose and transport specific cargoes for extracellular release rather than degradation (Dupont et al. 2011, Ponpuak et al. 2015, Cavalli & Cenci 2020).
Lysosomal damage, characterized by lysosomal membrane permeabilization (LMP), results in the release of lysosomal contents into the cytoplasm, including proteolytic enzymes, which trigger oxidative stress, autophagic dysfunction and cell death (Xu & Ren 2015). In this context, PARK7 is crucial for responding to oxidative stress by scavenging ROS and maintaining mitochondrial functions, thereby preserving cellular homeostasis. Importantly, lysosomal damage is a hallmark of various disorders, including neurodegenerative diseases such as PD and Alzheimer’s disease, lysosomal storage diseases such as Gaucher’s and Niemann–Pick disease and certain cancers, where it disrupts autophagy, cellular metabolism and immune responses (Wang et al. 2018, Zoncu & Perera 2022, Yang & Tan 2023). Understanding the role of PARK7 in lysosomal damage-related pathologies may provide novel therapeutic insights, particularly under conditions characterized by lysosomal dysfunction and oxidative stress.
In the present study, we explore the role of lysophagy, the selective autophagic removal of damaged lysosomes, in the unconventional secretion of PARK7 under conditions of severe lysosomal damage. Using L-leucyl-L-leucine methyl ester (LLOMe), a potent LMP inducer, we demonstrate that PARK7 translocates to damaged lysosomes, which are subsequently engulfed by autophagosomes, marking the initiation of lysophagy. Intriguingly, LLOMe-induced PARK7 secretion depends on autophagosome formation but does not require the completion of autophagic turnover or lysosomal degradation. Instead, autophagosomes containing damaged lysosomes evade fusion with functional lysosomes and, through the mediation of the R-SNARE protein SEC22B, fuse with the plasma membrane to facilitate the extracellular release of PARK7. These findings reveal a novel role of lysophagy in regulating PARK7 secretion and offer insights into the broader mechanisms of autophagy-based unconventional protein secretion under lysosomal stress.
Materials and methods
Antibodies and reagents
The antibodies used were as follows: anti-PARK7 (5933 for western blot, Cell Signaling Technology, USA, sc-27006 and sc-55572 for immunofluorescence, Santa Cruz Biotechnology, USA), anti-ACTB (A5441, Sigma, USA), anti-FN1 (610077, BD Bioscience, USA), anti-MAP1LC3B (L7543, Sigma), anti-SQSTM1 (PM045, MBL Life Science, Japan), anti-SNAP29 (111303, Synaptic Systems, Germany), anti-SEC22B (186003, Synaptic Systems), anti-STX3 (ab133750, Abcam, UK), anti-STX4 (110042, Synaptic Systems), anti-SNAP23 (111202, Synaptic Systems), anti-LAMP1 (ab24170, Abcam), anti-TFEB (4240, Cell Signaling Technology), anti-LGALS3 (sc-32790, Santa Cruz Biotechnology), anti-TBK1 (ab40676, Abcam) and anti-pTBK1 (5483, Cell Signaling Technology). Horseradish peroxidase-conjugated secondary antibodies for western blot were obtained from Jackson ImmunoResearch. The secondary antibodies for immunofluorescence staining purchased from Invitrogen included Alexa Fluor 488-conjugated goat anti-mouse IgG (H+L, A11029), Alexa Fluor 546 goat anti-rabbit IgG (H+L, A11035), Alexa Fluor 546 donkey anti-goat IgG (H+L, A-11056) and Alexa Fluor 488-conjugated goat anti-rabbit IgG (H+L, A11008). The reagents used were as follows: LLOMe (16008, Cayman Chemical, USA), bafilomycin A1 (BafA1, 19-148, Sigma), chloroquine (CQ, 083-10581, FUJIFILM, Japan) and brefeldin A (BFA, 203729, Selleck Chemicals, USA).
Cell culture
HeLa (wild-type (WT), syntaxin17 (STX17) knockout (KO), FIP200 KO (Nakamura et al. 2020) and PARK7 KO (Kojima et al. 2016)) and mouse embryonic fibroblast (MEF) (Kuma et al. 2004) cell lines used in this study were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM, 044-29765, FUJIFILM Wako, Japan), supplemented with 10% heat-inactivated fetal bovine serum (175012, Nichirei Bioscience, Japan) and 1% penicillin–streptomycin (168-23191, FUJIFILM Wako) in a humidified incubator with 5% CO2 at 37 °C.
RNA interference and plasmid transfection
ON-TARGETplus SMARTPool and nontargeting control pool siRNAs were purchased from Horizon Discovery. Transfection was performed using Lipofectamine RNAiMAX (Thermo Fisher, USA) for 48 h before conducting further experiments according to the manufacturer’s protocol. HeLa cells at approximately 60% confluency were transiently transfected with plasmids using Lipofectamine 2000 (Thermo Fisher) for 24 h according to the manufacturer’s protocol before subsequent treatments.
Sample preparation, lactate dehydrogenase (LDH) assay and western blotting
Cytotoxicity was assessed by measuring LDH release in the conditioned media as described previously (Urano et al. 2018). Collected conditioned media were treated with 10% trichloroacetic acid (200-08085, FUJIFILM Wako) to precipitate the secreted proteins as described previously (Urano et al. 2018). For whole-cell lysate preparation, cells were lysed on ice in radioimmunoprecipitation assay buffer (50 mM NaCl, 5 mM EDTA-2Na, 50 mM Tris–HCl pH 7.4, 0.5% Na-deoxycholate, 0.1% SDS and 1% NP-40) containing 1% EDTA-free protease inhibitor cocktail (169-26063, FUJIFILM Wako) and PhosSTOP (Roche, USA). Protein concentrations were determined using a BCA protein assay kit (297-73101, FUJIFILM Wako), with BSA as the standard. Samples were analyzed by SDS–PAGE as described previously (Urano et al. 2018). Immunoreactive band intensities were quantified using ImageJ (https://imagej.net/ij) (v1.54i, National Institutes of Health, USA).
Immunofluorescence staining
Cells grown on coverslips were fixed with 4% paraformaldehyde, permeabilized with 0.4% Triton X-100 (Sigma) and incubated with primary antibodies for 3 h at room temperature. After washing, species-specific secondary antibodies conjugated to Alexa Fluor dyes were applied. Nuclei were stained with Hoechst 33342 (346-07951, Dojindo, Japan), and coverslips were mounted with VECTASHIELD antifade mounting medium (Vector Lab.). Fluorescence images were acquired using a Zeiss Axiovert A1 fluorescence microscope or a Zeiss LSM 800 confocal microscope (Carl Zeiss, operated on ZEN Blue 3.4 (https://www.zeiss.com/)).
Statistical analysis
Data from at least three independent experiments were analyzed using GraphPad Prism (https://www.graphpad.com) version 10.3.1 and presented as mean ± SD. For statistical comparisons, an unpaired Student’s t-test was used for two groups, and one-way ANOVA followed by Tukey’s multiple comparisons test was used for multiple groups. Experiments presented with representative micrographs were independently repeated at least three times with consistent results. Statistical significance was defined as P < 0.05 (*P < 0.05, **P < 0.01 and ***P < 0.001).
Results
LLOMe-induced LMP promotes unconventional PARK7 secretion
LLOMe has been reported to induce LMP within 30 min, as evidenced by the cytoplasmic accumulation of punctate LGALS3 and its colocalization with lysosomes (Eriksson et al. 2020). HeLa cells were treated with 1 mM LLOMe for varying durations, and the formation of LGALS3 puncta, a hallmark of lysosomal damage, was analyzed as indicated in Fig. 1A. Immunofluorescence data demonstrated that LLOMe exposure resulted in a time-dependent LGALS3 puncta formation, peaking around 1.5 h and persisting throughout the treatment period, indicating sustained LMP (Fig. 1B). These LGALS3 puncta predominantly colocalized with the lysosomal membrane protein LAMP1 (Fig. 1C). To investigate the effect of LLOMe-induced LMP on PARK7 secretion, HeLa cells were treated with increasing concentrations of LLOMe for 3 h. Following treatment, the cells were washed and incubated in serum-free DMEM for an additional 2 h without LLOMe (washout phase). Conditioned media and whole-cell lysates were subsequently collected for analysis. Western blotting revealed that treatment with 1 mM LLOMe significantly increased PARK7 secretion (Fig. 1D). Given that high concentrations of LLOMe can induce severe lysosomal damage and cell death, the LDH assay was performed to evaluate cytotoxicity. The LDH assay confirmed that 1 mM LLOMe enhanced PARK7 secretion without causing significant plasma membrane disruption. A similar response was observed in MEF, confirming that 1 mM LLOMe promotes PARK7 secretion without cytotoxic effects (Fig. 1E).
LLOMe induces unconventional PARK7 secretion. (A) Schematic representation of experimental plans. (B) HeLa cells were treated with 1 mM LLOMe at the indicated time and subjected to immunofluorescence analysis of LGALS3 puncta (green). (C) HeLa cells were treated with 1 mM LLOMe for 3 h and subjected to immunofluorescence analysis of punctate structure for LAMP1 (red) and LGALS3 (green). (D and E) HeLa and MEF cells were treated with LLOMe at the indicated concentrations and incubated in serum-free DMEM. Whole-cell lysates (lysate) and the supernatant (sup) were analyzed by western blot for PARK7 and β-actin (ACTB). The bar graph represents the relative secreted PARK7 and LDH released in the conditioned media. ns, nonsignificant. (F) WT HeLa and PARK7 KO HeLa cells transfected with PARK7-3XFLAG were treated with 1 mM LLOMe followed by culture in serum-free DMEM. Lysate and sup were analyzed by western blot for PARK7 and ACTB. KO, knock out. (G) HeLa cells were pretreated with 2 μg/mL BFA for 3 h and cotreated with 1 mM LLOMe followed by culture in serum-free DMEM. Lysate and sup were analyzed by western blot for PARK7, FN1 and ACTB.
Citation: Redox Experimental Medicine 2024, 1; 10.1530/REM-24-0014
To further validate LLOMe-induced PARK7 secretion, PARK7 KO cells were reconstituted with PARK7-3XFLAG plasmids. The results indicated that both endogenous and exogenous PARK7 secretion increased following LLOMe treatment in WT and PARK7 KO cells (Fig. 1F). To determine whether PARK7 secretion occurs via a Golgi-independent pathway, cells were treated with BFA, an inhibitor of ER-to-Golgi trafficking. As shown in Fig. 1G, BFA treatment suppressed the secretion of fibronectin-1 (FN1), a conventional secretory cargo, confirming the inhibition of the conventional secretory pathway. However, PARK7 secretion was unaffected by LLOMe treatment in the presence of BFA, indicating that PARK7 follows an unconventional, Golgi-independent secretory route in response to LLOMe-induced lysosomal damage.
LLOMe-induced LMP is essential but not sufficient for PARK7 secretion
Given the cytoprotective roles of PARK7 under distinct stress conditions, PARK7 localization in LLOMe-treated cells was assessed. PARK7 puncta formation was observed upon LLOMe treatment, with significant colocalization with LGALS3 punctate structures, indicating translocation of PARK7 to damaged lysosomal sites (Fig. 2A). In addition, PARK7 puncta colocalized with the autophagosomal marker MAP1LC3B at 3 h of LLOMe treatment (Fig. 2B). These findings indicate that severe lysosomal damage facilitates the recruitment of PARK7 to damaged lysosomal sites, where autophagosomes selectively engulf the compromised lysosomes. Assessment of PARK7 secretion at different time points revealed a slight increase at 1.5 h, with significant secretion observed at 3 h (Fig. 2C). LGALS3 secretion exhibited a similar pattern, significantly increasing at 3 h of LLOMe treatment. These results indicate that while LMP induction is necessary, severe lysosomal damage is crucial for promoting the secretion of both PARK7 and LGALS3.
LLOMe-induced severe lysosomal damage upregulates PARK7 and LGALS3 secretion. HeLa cells were treated with 1 mM LLOMe for 3 h and subjected to immunofluorescence analysis of punctate structure for (A) PARK7 (red) and LGALS3 (green) and (B) PARK7 (red) and MAP1LC3B (green). (C) HeLa cells were treated with 1 mM LLOMe at the indicated time and incubated in serum-free DMEM. Lysate and sup were analyzed by western blot for PARK7, LGALS3, and ACTB. The bar graph represents the relative secreted PARK7 and LGALS3. (C) HeLa cells were treated with 1 mM LLOMe at the indicated time and incubated in serum-free DMEM. Lysate and sup were analyzed by western blot for PARK7, LGALS3 and ACTB. The bar graph represents the relative secreted PARK7 and LGALS3.
Citation: Redox Experimental Medicine 2024, 1; 10.1530/REM-24-0014
LLOMe-induced lysophagy induction, not turnover, regulates PARK7 secretion
Our previous findings demonstrated that oxidative stress, such as that induced by 6-OHDA, enhances the autophagy-based unconventional secretion of PARK7 (Urano et al. 2018). Since lysophagy is a selective autophagy pathway for clearing damaged lysosomes (Maejima et al. 2013, Skowyra et al. 2018), we investigated whether LLOMe-induced lysophagy mediates PARK7 secretion. After 3 h of LLOMe treatment, MAP1LC3B-II accumulation was observed without significant changes in SQSTM1 levels compared to untreated cells (Fig. 3A). During the subsequent washout phase, MAP1LC3B-II accumulation was accompanied by a reduction in SQSTM1, indicating effective canonical autophagy flux. The transcription factor EB (TFEB), activated by calcium efflux during lysosomal stress promoting lysosomal biogenesis and homeostasis (Chauhan et al. 2016, Nakamura et al. 2020), showed increased dephosphorylation during the washout phase. This activation was further confirmed by the clearance of LGALS3 puncta after LLOMe washout (Fig. 3B). These findings suggest an upregulation of TFEB-mediated lysosomal biogenesis and autophagy/lysophagy in response to lysosomal damage under experimental conditions (Fig. 3C).
LLOMe-induced lysophagy induction regulates PARK7 secretion. (A) HeLa cells were treated with 1 mM LLOMe for 3 h followed by washout for 2 h. Lysate was analyzed by western blot for MAP1LC3B, SQSTM1, TFEB and ACTB. (B) HeLa cells were treated with 1 mM LLOMe for 3 h, washed out for 2 h and subjected to immunofluorescence analysis of LGALS3 puncta (green). (C) Schematic diagram of clearance of LLOMe-induced damaged lysosomes by lysophagy. (D) WT or FIP200 KO HeLa cells were treated with 1 mM LLOMe for 3 h followed by washout for 2 h. Lysate and sup were analyzed by western blot for PARK7 and ACTB. The bar graph represents the relative secreted PARK7. (E) WT or STX17 KO HeLa cells were treated with 1 mM LLOMe for 3 h followed by washout for 2 h. Lysate and sup were analyzed by western blot for PARK7 and ACTB. The bar graph represents the relative secreted PARK7. (F) HeLa cells were pretreated with 100 nM BafA1 or 5 μM CQ for 2 h and cotreated with 1 mM LLOMe for 3 h followed by washout for 2 h. Lysate and sup were analyzed by western blot for PARK7 and ACTB. The bar graph represents the relative secreted PARK7. (G) HeLa cells were treated with 1 mM LLOMe for 3 h followed by washout for 2 h. Lysate was analyzed by western blot for phosphorylated TBK1 (pTBK1) and ACTB. (H) HeLa cells transfected with siRNAs targeting NC or TBK1 were treated with 1 mM LLOMe and washed out for 2 h. Lysate and sup were analyzed by western blot for PARK7, TBK1 and ACTB. The bar graph represents the relative secreted PARK7.
Citation: Redox Experimental Medicine 2024, 1; 10.1530/REM-24-0014
To further investigate the role of autophagy in LLOMe-induced PARK7 secretion, RB1CC1/FIP200 KO HeLa cells, which are defective in autophagosome formation (Hara et al. 2008), were used. PARK7 secretion was significantly diminished in FIP200 KO cells compared to WT cells (Fig. 3D), confirming that autophagosome formation is essential for LLOMe-induced PARK7 secretion. However, in STX17 KO HeLa cells, which lack the autophagosomal SNARE necessary for autophagosome–lysosome fusion (Itakura et al. 2012), LLOMe-induced PARK7 secretion remained largely unaffected (Fig. 3E). In addition, treatment with autophagosome–lysosome fusion and autolysosomal degradation blockers BafA1 or CQ (Mauthe et al. 2018) did not significantly impact PARK7 secretion (Fig. 3F). These results suggest that autophagosome–lysosome fusion and degradation processes are not critical for PARK7 secretion under these conditions.
TANK-binding kinase 1 (TBK1), activated by calcium release from damaged lysosomes, is essential for lysophagy initiation (Eapen et al. 2021, Shima et al. 2023) through the recognition and sequestration of autophagosome substrates (Heo et al. 2015, Richter et al. 2016). In the present study, LLOMe treatment triggered TBK1 phosphorylation, which decreased during the washout phase (Fig. 3G), indicating diminished calcium signaling as the damaged lysosomes were repaired or removed. Further experiments with TBK1 knockdown via siRNA revealed significant suppression of PARK7 secretion under LLOMe treatment (Fig. 3H). These findings demonstrate that lysophagy initiation, specifically autophagosome formation and maturation, is critical for LLOMe-induced PARK7 secretion.
Dedicated SNAREs regulate LLOMe-induced PARK7 secretion
Recent studies suggest that autophagosomes play a role not only in degradation but also in the secretion of cytosolic proteins via autophagy-mediated secretory pathways (Kimura et al. 2017, Yamada et al. 2022). Given our findings that autophagosomes mediate LLOMe-induced PARK7 secretion, we investigated the role of SEC22B, an R-SNARE located on autophagosomal membranes that facilitates the fusion with the plasma membrane for interleukin-1β secretion (Kimura et al. 2017). SEC22B knockdown via siRNA in MEF cells significantly diminished PARK7 secretion in response to LLOMe (Fig. 4A), indicating its essential role in this unconventional secretory pathway. Building on previous findings that dedicated SNAREs regulate secretory autophagy under lysosomal stress or nutrient starvation (Kimura et al. 2017), the specific Qabc-SNARE partners of SEC22B that mediate the fusion of autophagosomes with the plasma membrane during PARK7 release were assessed. The knockdown of STX3, STX4, SNAP23 and SNAP29 in MEF cells resulted in a marked decrease in LLOMe-induced PARK7 secretion (Fig. 4B and C). These findings indicate that a SEC22B-containing SNARE complex, including STX3/4 and SNAP23/29, regulates LLOMe-induced PARK7 secretion by mediating autophagosome-plasma membrane fusion.
A dedicated SNARE complex regulates LLOMe-induced PARK7 secretion. MEF cells transfected with siRNAs targeting (A) NC or SEC22B, (B) NC or STX3 or STX4 or in combination and (C) NC or SNAP23 or SNAP29 or in combination were treated with 1 mM LLOMe followed by washout for 2 h. Lysate and sup were analyzed by western blot for the target proteins indicated in the figures. The bar graphs represent the relative secreted PARK7. NC, negative control.
Citation: Redox Experimental Medicine 2024, 1; 10.1530/REM-24-0014
Discussion
This study offers important insights into the role of lysophagy in the unconventional secretion of PARK7 during severe lysosomal damage. Cellular responses to lysosomal damage, including ESCRT-mediated membrane repair, autophagy-based removal of damaged lysosomes (lysophagy) and TFEB-mediated lysosomal biogenesis, are primarily regulated by LGALS3 (Jia et al. 2020). In response to endomembrane damage, LGALS3, a β-galactoside-binding cytosolic lectin, rapidly recognizes damage-exposed glycans and recruits ESCRT components to the damaged sites for the restoration of membrane repair, including lysosomal damaged membrane (Jimenez et al. 2014, Scheffer et al. 2014, Radulovic et al. 2018). When lysosomal damage is extensive, LGALS3 shifts from promoting membrane repair to initiating autophagic removal of damaged lysosomes through lysophagy (Jia et al. 2020). LMP can be triggered by various pathological conditions, including infections, lipid oxidation or crystal formation, resulting in cell death and disease pathology (Xu & Ren 2015, Wang et al. 2018, Zoncu & Perera 2022, Yang & Tan 2023). By facilitating lysophagy, cells mitigate the release of harmful lysosomal contents, which could otherwise contribute to inflammation, neurodegeneration and additional cellular damage. Our findings significantly advance the understanding of PARK7 secretion during lysosomal stress. Unlike traditional lysosomal degradation, this process involves autophagosome formation and suggests a novel cellular mechanism that responds to lysosomal damage. PARK7, a protein crucial for antioxidant defense and cellular survival, follows a noncanonical secretion pathway, reflecting an adaptive response to lysosomal damage.
Unconventional secretion involves several mechanisms for cargo entry into vesicles. These strategies include specific transporters, direct incorporation into autophagic structures, such as phagophores, or engulfment by multivesicular bodies (MVBs) or endosomes (Zhang et al. 2015, Cohen et al. 2020). While we did not assess the role of exosomes or MVBs in LLOMe-induced PARK7 secretion, our data suggest that autophagosomes, rather than these pathways, capture translocated PARK7 and LGALS3 at lysosomal damage sites. The engulfment of PARK7, as part of damaged lysosomal material, reveals critical aspects of cargo targeting for secretion via secretory autophagosomes, at least under lysosomal damage. These findings differ from the LLOMe-induced secretion of IL-1β in immune cells, where galectin-8 (Gal8) facilitates cargo transfer to MAP1LC3B-positive membranes through a specialized secretory autophagy cargo receptor (TRIM16-Gal8) complex (Kimura et al. 2017). The known roles of LGALS3 in lysophagy and its cosecretion with PARK7 suggest an intriguing interplay between galectins and PARK7 in maintaining lysosomal homeostasis, which merits further investigation.
A fundamental distinction between secretory and degradative autophagy is their final destination, degradative autophagosomes fusing with lysosomes, while secretory autophagosomes bypass this step and instead fuse with the plasma membrane, as seen with IL-1β secretion (Dupont et al. 2011, Zhang et al. 2015, Kimura et al. 2017, Padmanabhan & Manjithaya 2020). One of the most significant findings of this study is the necessity of autophagosome formation for LLOMe-induced PARK7 secretion. This is evident from the decreased secretion observed in FIP200 KO cells, which lack autophagosome biogenesis. In contrast, PARK7 secretion remains unaffected in STX17 KO cells, which are deficient in autophagosome–lysosome fusion, indicating that completion of autophagic turnover is not required for PARK7 secretion. This mechanism is distinct from our previous findings on 6-OHDA-induced PARK7 secretion, necessitating autophagic flux completion, including autophagosome biogenesis, maturation and lysosomal fusion (Urano et al. 2018). Our findings align with the observations from other secretory autophagy cargoes, such as α-crystallin B (D’Agostino et al. 2019) and HMGB1 (Kim et al. 2021), which depend on autophagosome formation without requiring complete autophagic turnover. Our results suggest a specialized mechanism in which autophagosomes carrying damaged lysosomal material bypass fusion with functional lysosomes and instead fuse directly with the plasma membrane. This process appears to be mediated by the R-SNARE protein SEC22B and Qabc-SNARE partners, such as STX3, STX4, SNAP23 and SNAP29. A similar SNARE complex mediates the release of cytosolic proteins such as IL-1β and ferritin during LLOMe-induced lysosomal damage in immune cells (Kimura et al. 2017). These observations imply that secretory autophagosome-mediated protein secretion utilizes machinery for autophagosome biogenesis and maturation independent of lysosomal involvement, adaptable to various stress conditions, including oxidative stress, nutrient deprivation and lysosomal dysfunction.
While our findings provide valuable insights, several limitations exist. First, we have not fully elucidated how PARK7, located within the autophagosomal lumen, escapes fusion with lysosomes. One plausible explanation is that severe lysosomal damage compromises lysosomal homeostasis, allowing PARK7 to evade lysosomal degradation and be secreted as a part of the protective cellular processes, including redox sensing, antioxidative defense, inflammation, angiogenesis and metastasis (Koide-Yoshida et al. 2007, Yanagida et al. 2009, Kim et al. 2012, Kaneko et al. 2014, Gu et al. 2022). Alternatively, the secretion of PARK7 could reflect a novel cellular mechanism that preserves damaged lysosomal contents for extracellular delivery, potentially serving unknown functions. Second, lysosomal exocytosis – where lysosomes fuse with the plasma membrane to release contents extracellularly – is commonly activated under cellular stress (Andrei et al. 1999). In our study, the knockdown of SNAP23 and STX4, SNARE proteins often involved in lysosomal exocytosis, implicated these SNAREs in LLOMe-induced PARK7 secretion. However, as general membrane fusion regulators, these SNAREs may mediate autophagosome–plasma membrane fusion alongside autophagosomal SNAREs such as SEC22B. Our results showed that SEC22B and autophagosome biogenesis are essential for PARK7 secretion, supporting an autophagy-based secretion pathway rather than lysosomal exocytosis. Although we did not inhibit lysosome-specific SNAREs or employ lysosomal exocytosis inhibitors such as vacuolin-1, PARK7’s sequestration into autophagosomes suggests a predominant role for autophagy. Further studies explicitly targeting lysosomal exocytosis would help clarify its role, if any, in PARK7 release. Third, although LGALS3 is cosecreted with PARK7 during lysosomal damage, further research is needed to determine whether LGALS3 follows the same secretion pathway or employs a distinct mechanism. A promising direction for future research could explore the potential role of PARK7 and LGALS3 cosecretion as biomarkers or mechanistic players in the pathogenesis of neurodegenerative diseases such as PD and ischemic stroke, particularly where lysosomal damage is implicated. The observed autophagy-mediated secretion of PARK7 in response to lysosomal damage may reflect a compensatory mechanism to prevent toxic accumulation of lysosomal contents and mitigate oxidative stress – critical factors in neurodegeneration. This pathway could thus reveal a dual role for PARK7 as an antioxidant and regulatory molecule involved in cellular signaling during lysosomal damage. Fourth, our model focuses primarily on severe lysosomal damage, and it is unclear whether these pathways are similarly relevant under milder forms of lysosomal stress. Future research should investigate the dose- and time-dependent dynamics of PARK7 translocation to lysosomal damage sites, identify binding partners at these sites and explore the molecular pathways regulated by PARK7 in various disease contexts.
In summary, this study underscores the importance of lysophagy in the unconventional secretion of PARK7 during lysosomal damage. This mechanism represents a novel cellular response to stress, expanding our understanding of the intricate pathways involved in autophagy and lysosomal maintenance. Future research to elucidate the detailed mechanisms of PARK7 secretion and its broader physiological relevance will have significant implications for therapeutic strategies targeting diseases associated with lysosomal dysfunction.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the work reported.
Funding
This work was supported by a grant from the Research Institute for Production Development, Kyoto, Japan, to Yasuomi Urano.
Author contribution statement
Biplab Kumar Dash helped in conceptualization, methodology, investigation, data curation, formal analysis, validation, visualization, writing of the original draft, reviewing and editing. Yasuomi Urano contributed to conceptualization, formal analysis, validation, writing, review, editing, resource management project administration, funding acquisition and investigation. Noriko Noguchi helped in formal analysis, validation, writing, review, editing, resources, project administration and funding acquisition.
Acknowledgments
We thank Dr Tamotsu Yoshimori of Osaka University, Japan, for providing HeLa (WT, STX17 KO and FIP200 KO), Dr Noriyuki Matsuda of Tokyo Metropolitan Institute of Medical Science, Japan, for PARK7 KO HeLa and RIKEN BRC through the National BioResource Project of the MEXT/AMED, Japan, for MEF cell lines. We also thank Ryoma Hirose (Systems Life Sciences laboratory, Doshisha University) for preparing the pcDNA3.1-3XFLAG-Tagged-human PARK7 plasmids used in this study.
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