Oxidative Stress Impairs Autophagy via Inhibition of Lysosomal Transport of VAMP8

Abstract

Autophagy is a highly conserved intracellular degrading system and its dysfunction is considered related to the cause of neurodegenerative disorders. A previous study showed that the inhibition of endocytosis transport attenuates soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein transport to lysosomes and block autophagy. The other studies demonstrated oxidative stress, one of the inducers of neurodegenerative diseases inhibits endocytosis transport. Thus, we hypothesized that oxidative stress-induced endocytosis inhibition causes alteration of SNARE protein transport to lysosomes and impairs autophagy. Here, we demonstrated that oxidative stress inhibits endocytosis and decreased the lysosomal localization of VAMP8, one of the autophagy-related SNARE proteins in a human neuroblastoma cell line. Moreover, this oxidative stress induction blocked the autophagosome-lysosome fusion step. Since we also observed decreased lysosomal localization of VAMP8 and inhibition of autophagosome-lysosome fusion in endocytosis inhibitor-treated cells, oxidative stress may inhibit VAMP8 trafficking by suppressing endocytosis and impair autophagy. Our findings suggest that oxidative stress-induced inhibition of VAMP8 trafficking to lysosomes is associated with the development of neurodegenerative diseases due to the blocked autophagosome-lysosome fusion, and may provide a new therapeutic target for restoring the autophagic activity.

INTRODUCTION

Aging is a major risk factor for neurodegenerative disorders such as Alzheimer’s disease and Parkinson’s disease.¹⁾ These diseases are caused by the accumulation of aggregated proteins such as β-amyloid, tau, and α-synuclein.²⁾ This pathogenic protein storage is associated with impairment of the autophagy pathway in the neuron.^3,4)

Autophagy is a highly conserved intracellular degrading system from yeast to humans and plays an important role in maintaining cellular homeostasis. Autophagic substrates such as damaged organelles or abnormally aggregated proteins are internalized by autophagosomes and then degraded by the fusion of autophagosomes with lysosomes. The autophagosome-lysosome fusion step is mediated by soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) proteins, which are essential for intracellular membrane traffic including autophagy and localized in both transport vesicles and target organelles.^5–7) The membrane fusion step is induced by the complex formation between SNARE proteins of transport vesicles and that of the target organelle via their intramolecular SNARE motifs.

Accumulated evidence showed that the suppression of autophagy leads to the development of neurodegenerative disorders by failing to degrade pathogenic proteins.^3,4) Moreover, it has also been reported that autophagic degrading activity is decreased with aging. However, this underlying mechanism has been still unclear.^8–10)

Recently Genome-wide association study has identified various endocytosis-related genes as risk factors for neurodegenerative disorders.^11–13) Thus, endocytosis failure may contribute to the development of neurodegenerative diseases. Indeed, knocking down of phosphatidylinositol binding clathrin assembly protein (PICALM), one of the endocytosis-related genes, has been reported to impair autophagy by blocking autophagosome-lysosome fusion step via inhibition of endocytic transport of SNARE proteins to the lysosome and cause accumulation of intracellular tau proteins.¹⁴⁾

Another inducer of neurodegenerative disorders is age-dependent oxidative stress.¹⁵⁾ Oxidative stress is caused by an increase in intracellular reactive oxygen species (ROS). Although ROS has been implicated in the development of neurodegenerative diseases by causing neuronal cell death via DNA, proteins, lipids, or organelle damage,^16,17) it is unclear whether oxidative stress is involved in the accumulation of proteins responsible for neurodegenerative diseases. Furthermore, it has been reported that oxidative stress also attenuates endocytosis.^18–20) Therefore, we hypothesized that the oxidative stress-induced reduction of endocytosis also causes suppression of SNARE proteins transport and contributes to autophagy impairment.

In this study, we tested whether the inhibition of endocytosis or oxidative stress contributes to the impairment of autophagy via inhibition of SNARE proteins transport to lysosomes by using a human neuronal cell model.

MATERIALS AND METHODS

Cell Culture

Human neuroblastoma cell line (SH-SY5Y) was grown in Dulbecco’s modified Eagle’s medium (DMEM)/Nutrient mixture F-12 Ham (Sigma-Aldrich, U.S.A) containing 10% fetal bovine serum (FBS) (Biosera, France), 100 µg/mL Streptomycin (Sigma-Aldrich), and 70 µg/mL Penicillin G (Sigma-Aldrich) at 37 °C humidified incubator in 5% CO₂. Before each experiment, SH-SY5Y cells were differentiated into neuronal cells by 10 µM retinoic acid (Sigma-Aldrich) in DMEM/Nutrient mixture F-12 Ham containing 1% FBS for 8–10 d with changing the medium every 3 d.

Reagent Treatment

Chlorpromazine (Santa Cruz Biotechnology, U.S.A) was treated as an endocytosis inhibitor to SH-SY5Y cells at a final concentration of 12 µM in DMEM/Nutrient mixture F-12 Ham containing 1% FBS for 24 h.

For oxidative stress induction, SH-SY5Y cells were exposed to 0.5 mM H₂O₂ (Wako, Japan) in DMEM/Nutrient mixture F-12 Ham containing 1% FBS for 30 min at 37 °C. Then, the medium was replaced with DMEM/Nutrient mixture F-12 Ham containing 1% FBS, and the cells were incubated for 12, 24, or 48 h.

For autophagy induction, SH-SY5Y cells were treated with 1 µM Torin-1 (Santa Cruz Biotechnology) in DMEM/Nutrient mixture F-12 Ham containing 1% FBS for 2 h.

Preparation of Cell Lysates

SH-SY5Y cells were washed with ice-cold phosphate-buffered saline (PBS), then removed with a scraper and collected in PBS. The collected cell suspension was centrifuged at 2000 × g for 5 min at 4 °C, and the pellet was resuspended with a RIPA buffer (50 mM Tris–HCl (pH 7.6), 150 mM NaCl, 1% Nonidet P40, 0.5% Sodium Deoxycholate, 0.1% sodium dodecyl sulfate (SDS)) containing protease inhibitor cocktail (1 µM Pepstatin A, 20 µM Leupeptin, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM phenylmethylsulfonyl fluoride (PMSF)) and phosphatase inhibitor cocktail (Nacalai Tesque, Japan, 1/100 dilution). The cell suspensions were sonicated and centrifuged at 12000 × g for 15 min at 4 °C. The supernatants were collected and used in experiments. The protein concentration of cell lysates was determined by DC™ protein assay kit (Bio-Rad, U.S.A) using bovine serum albumin (BSA) as a standard.

Antibodies

Primary Antibodies

A mouse anti-LAMP2 antibody (ab25631,1/200 dilution for immunocytochemistry) was purchased from Abcam (U.K). A rabbit anti-VAMP8 antibody (HPA006882, 1/200 dilution for immunocytochemistry) was purchased from Sigma-Aldrich. A rabbit anti-LC3B antibody (NBP100-2220, 1/200 dilution for immunocytochemistry) and a rabbit anti-Syntaxin7 antibody (NBP1-87497, 1/200 dilution for immunocytochemistry) were purchased from NOVUS (U.S.A). A rabbit anti-LC3B antibody (#3868, 1/1000 dilution for Western blotting) was purchased from CST (U.S.A). A mouse anti-p62 antibody (610833, 1/1000 dilution for Western blotting) and a mouse anti-GM130 antibody (610822, 1/200 dilution for immunocytochemistry) were purchased from BD Biosciences (U.S.A). A mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (sc-32233, 1/1000 dilution for Western blotting) was purchased from Santa Cruz Biotechnology.

Secondary Antibodies

A goat anti-mouse immunoglobulin G (IgG) H&L (Alexa fluor^® 555) antibody (ab150114, 1/1000 dilution for immunocytochemistry) and a goat anti-rabbit IgG H&L (Alexa fluor^® 488) antibody (ab150077, 1/1000 dilution for immunocytochemistry) were purchased from Abcam. A goat anti-mouse IgG, HRP linked antibody (#7076, 1/1000 dilution for Western blotting) and a goat anti-rabbit IgG, HRP linked antibody (#7074, 1/1000 dilution for Western blotting) were purchased from CST.

Western Blotting

The cell lysates were mixed with 6 × SDS sample buffer (125 mM Tris–HCl (pH 6.8), 4% SDS, 20% Glycerol, 0.01% BPB, 10% 2-Mercaptoethanol) and heated at 100 °C for 3 min. SDS-polyacrylamide gel electrophoresis (PAGE) has performed on 7.5 or 15% acrylamide gels in SDS buffer (25 mM Tris, 192 mM Glycine, 0.1% SDS). Electrophoresed acrylamide gels were transferred to polyvinylidene difluoride (PVDF) membranes using TRANS-Blot SD SEMI-DRY TRANSFER CELL (Bio-Rad) in the blotting buffer (25 mM Tris, 192 mM Glycine, 20% Methanol). After blocking in 5% skim milk/0.1% Tween20/ TBS-T for 1 h at room temperature, a primary antibody was treated overnight at 4 °C. The PVDF membrane was washed with TBS-T and treated with a secondary antibody for 1 h at room temperature. The PVDF membrane was washed again with TBS-T and treated with Western Lightning Plus-ECL (PerkinElmer, Inc., U.S.A) or Western Lightning Ultra (PerkinElmer, Inc.) for HRP signal detection using ChemiDoc XRS+ (Bio-Rad).

Immunocytochemistry

SH-SY5Y cells were seeded in 8-well Lab-Tek chamber slides (Thermo Fisher SCIENTIFIC, U.S.A) coated with 3 mg/mL atelocollagen (Koken, Japan) at a density of 6 × 10⁴ cells. After reagent treatments, the cells were fixed with 4% paraformaldehyde (PFA)/PBS at 4 °C overnight. Fixed cells were washed with PBS and blocked with 5% Goat serum,1% BSA/PBS at room temperature for 1 h. A primary antibody was treated at 4 °C overnight. The cells were washed with PBS-T and treated with secondary antibody and Hoechst33258 (Wako, 1/200 dilution) to stain nuclei for 1 h at room temperature with protection from light. The cells were washed again with PBS-T and mounted with 50% glycerol/PBS. The cells were imaged using LSM700 (Zeiss, Germany). For colocalization analysis, Pearson’s correlation was calculated by using Image J.

Endocytosis Assay of Cholera Toxin Subunit B (CtxB)

SH-SY5Y cells were placed at 4 °C to stop endocytosis for 5 min. Then the cells were treated with 1 µg/mL CtxB Alexa fluor™488 Conjugate (Thermo Fisher SCIENTIFIC) at 4 °C for 30 min. After PBS wash, the cells were incubated in pre-warmed DMEM/Nutrient mixture F-12 Ham containing 1% FBS at 37 °C for 10, 20, or 30 min. Then the cells were fixed with 4% PFA/PBS at 4 °C overnight and fixed cells were performed immunocytochemistry to stain the Golgi marker, GM130. After the immunocytochemistry step, the cells were imaged to detect CtxB transport to the Golgi region using LSM700.

Statistical Analysis

Data values were described as mean ± standard error of the mean (S.E.M.). Statistical significance was analyzed using Student’s t-test for comparisons between 2 groups, and a one-way-ANOVA followed by Dunnett test for 3 or more groups. A p value less than 0.05 was considered significant.

RESULTS

Endocytosis Inhibition Decreases Lysosomal Localization of VAMP8

We investigated whether the inhibition of endocytosis causes impaired autophagy-related SNARE proteins, VAMP8, or STX7 transport to lysosomes. We treated endocytosis inhibitor, chlorpromazine (CP) to SH-SY5Y cells and analyzed co-localization of VAMP8 or STX7 with the lysosome marker, lysosomal membrane-associated protein 2 (LAMP2). CP treatment significantly reduced the lysosomal localization of VAMP8 in SH-SY5Y cells (Fig. 1A). On the other hand, no changes were observed in the lysosomal localization of STX7 (Fig. 1B). These results suggest that the inhibition of endocytosis decreased VAMP8 but not STX7 transport to lysosomes. In autophagy, lysosomal VAMP8 is an essential SNARE protein to fuse autophagosomes. Therefore, the inhibition of endocytosis leads to reduced lysosomal localization of VAMP8 and affects the autophagosome-lysosome fusion step.

Fig. 1. Inhibition of Endocytosis Decreases Lysosomal Localization of VAMP8

(A) Immunocytochemistry and colocalization analysis of 24 h CP treated SH-SY5Y cells. Red: LAMP2, Green: VAMP8. Scale bars indicate 10 µm. Error bars indicate mean ± S.E.M. (n = 5). Student’s t-test, *** p < 0.005 compared to Control. (B) Immunocytochemistry and colocalization analysis of 24 h CP treated SH-SY5Y cells. Red: LAMP2, Green: STX7. Scale bars indicate 10 µm. Error bars indicate mean ± S.E.M. (n = 5). Student’s t-test, *** p < 0.005 compared to Control.

Endocytosis Inhibition Impairs Autophagy by Blocking the Autophagosome-Lysosome Fusion Step

To elucidate whether CP-induced inhibition of endocytosis affects autophagy by decreasing the lysosomal localization of VAMP8, we analyzed autophagy-related molecules. CP treatment significantly increased LC3II and p62 levels known as autophagic activity indicators in SH-SY5Y cells (Fig. 2A). An increase in LC3II, an autophagosomal membrane protein means activation of autophagosome synthesis or inhibition of autophagosome degradation by fusion of lysosomes. Moreover, an increase in p62, which is selectively degraded by autophagy, generally indicates a reduction of autophagic degradation activity. We also confirmed that p62 levels in the CP-treated cells as control did not increase by the autophagy inhibitor Bafilomycin A1 (BafA1) (Supplementary Fig. S1). Next, we analyzed the autophagosome-lysosome fusion by immunocytochemistry to determine whether endocytosis inhibition by CP treatment impairs autophagy. Since autophagy occurs only at low levels under standard culture conditions and it is difficult to assess the autophagosome-lysosome fusion stage, autophagy was induced by Torin1 treatment, an autophagy inducer, during this experiment. Immunocytochemical analysis showed that CP treatment significantly reduced the co-localization of LAMP2 and LC3 (Fig. 2B). These results reveal that CP treatment defects autophagy by blocking the autophagosome-lysosome fusion step. CP treatment reduces lysosomal localization of VAMP8 by inhibiting endocytic membrane transport to lysosomes (Fig. 1). Therefore, it suggests that endocytosis inhibition affects autophagy by inhibition of VAMP8 transport to lysosomes and blocking the autophagosome-lysosome fusion step.

Fig. 2. Inhibition of Endocytosis Impairs Autophagy by Blocking Autophagosome-Lysosome Fusion Step

(A) Western blotting analysis of 24 h CP treated SH-SY5Y cells and relative band intensity of LC3II or p62/GAPDH. Error bars indicate mean ± S.E.M. (n = 3). Student’s t-test, * p < 0.05, ** p < 0.01 compared to Control. (B) Immunocytochemistry and colocalization analysis of 24 h CP treated SH-SY5Y cells. The cells were also treated Torin1 last 2 h. Red: LAMP, Green: LC3. Scale bars indicate 10 µm. Error bars indicate mean ± S.E.M. (n = 5). Student’s t-test, * p < 0.05 compared to Control.

Oxidative Stress-Induced Inhibition of Endocytosis Prevents Lysosomal Trafficking of VAMP8

Several studies have shown that oxidative stress induces impairment of endocytosis.^18–20) Additionally, our results have suggested that altered endocytosis affects VAMP8 transport to lysosomes and lysosome-autophagosome fusion. We next analyzed whether the inhibition of endocytosis by oxidative stress, which has been reported in previous studies also inhibits VAMP8 trafficking to lysosomes. To determine whether oxidative stress suppresses endocytosis in a neuronal cell model, we evaluated endocytosis transport using a fluorescence-labeled cholera toxin b subunit (CtxB). CtxB is taken up by endocytosis by binding to the cell surface GM1 ganglioside and transported to the Golgi apparatus via recycling endosomes.²¹⁾ We evaluated endocytic CtxB transport to the Golgi apparatus by co-localization analysis of CtxB, and GM130 as Golgi apparatus markers. Although colocalization of CtxB and GM130 increased in control cells within 10 min, oxidative stress-induced cells required 30 min to show colocalization of CtxB and GM130 (Fig. 3A). This result shows that hydrogen peroxide-induced oxidative stress inhibits endocytosis transport. Since we confirmed that oxidative stress suppressed endocytosis, we analyzed whether the inhibition of endocytosis by oxidative stress also changes in the localization of lysosomal SNARE proteins. As a result of co-localization analysis of LAMP2 and VAMP8 or STX7, oxidative stress-loaded SH-SY5Y cells observed a decrease of lysosomal localization of VAMP8 but not STX7 same as the results of CP induced endocytosis inhibition (Figs. 3B, C). These results showed that oxidative stress-induced endocytosis inhibition also suppresses the transport of VAMP8 to lysosomes.

Fig. 3. Oxidative Stress-Induced Endocytosis Impairment Alters VAMP8 Transport

(A) Endocytosis assay of CtxB by immunocytochemistry of SH-SY5Y cells 48 h after H₂O₂ treatment. Red: GM130, Green: CtxB. Scale bars indicate 10 µm. Error bars indicate mean ± S.E.M. (n = 5). Student’s t-test, *** p < 0.005 compared to Control of the same time points. (B) Immunocytochemistry and colocalization analysis of SH-SY5Y cells 12–48 h after H₂O₂ treatment. Red: LAMP2, Green: VAMP8. Scale bars indicate 10 µm. Error bars indicate mean ± S.E.M. (n = 5). One-way-ANOVA followed by Dunnett test, * p < 0.05, ** p < 0.01 compared to Control. (C) Immunocytochemistry and colocalization analysis of SH-SY5Y cells 12∼48 h after H₂O₂ treatment. Red: LAMP2, Green: STX7. Scale bars indicate 10 µm. Error bars indicate mean ± S.E.M. (n = 5).

Oxidative Stress Inhibits Autophagy by Blocking the Autophagosome-Lysosome Fusion Step

To examine whether the oxidative stress-induced inhibition of VAMP8 localization of lysosomes also affects the autophagosome-lysosome fusion step, we performed a quantitative analysis of LC3II and p62 as indicators of autophagy. Oxidative stress-loaded SH-SY5Y cells showed significant increases in LC3II and p62 protein levels, which may be due to a decrease in autophagy-degrading activity (Fig. 4A). We also showed that the p62 level did not increase in oxidative stress-induced cells by co-treatment with BafA1 (Supplementary Fig. S2). In addition, immunocytochemistry analysis showed that oxidative stress significantly decreased the colocalization of LAMP2 and LC3 in autophagy induced by treatment of Torin-1 (Fig. 4B). Our results have shown that oxidative stress decreases VAMP8 transport to lysosomes via endocytosis inhibition. Therefore, these results suggest that oxidative stress-induced inhibition of endocytosis blocked the autophagosome-lysosome fusion step in autophagy.

Fig. 4. Oxidative Stress Impairs Autophagy by Blocking Autophagosome-Lysosome Fusion Step

(A) Western blotting analysis of SH-SY5Y cells 48 h after H₂O₂ treatment and relative band intensity of LC3II or p62/GAPDH. Error bars indicate mean ± S.E.M. (n = 3). Student’s t-test, * p < 0.05, *** p < 0.005 compared to Control. (B) Immunocytochemistry and colocalization analysis of SH-SY5Y cells 48 h after H₂O₂ treatment. The cells were also treated Torin1 last 2 h. Red: LAMP2, Green: LC3. Scale bars indicate 10 µm. Error bars indicate mean ± S.E.M. (n = 5). Student’s t-test, * p < 0.05 compared to Control.

DISCUSSION

The decrease in the autophagic activity is thought to be closely related to the onset of neurodegenerative diseases.^3,4) In this study, we have analyzed the mechanisms by which endocytosis and oxidative stress suppress autophagy, both of which are thought to be involved in the pathogenesis of neurodegenerative diseases.

We found that inhibition of endocytosis interferes with the transport of VAMP8, one of the SNARE proteins essential for the progression of autophagy, to lysosomes and impairs the autophagosome-lysosome fusion step (Fig. 1). Interestingly, the treatment of endocytosis inhibitor increased the lysosomal localization of STX7, the same lysosomal-localized SNARE protein. Since previous studies have shown that VAMP8 is transported by endocytosis,²²⁾ this result may indicate that STX7 may be transported to lysosomes by a different pathway than VAMP8. Several endocytosis-related genes have been identified as risk factors for neurodegenerative diseases.^11–13) Mutations in these genes may suppress autophagy through decreased transport of VAMP8, as shown in this study, and lead to the development of neurodegenerative diseases. In fact, one of the endocytosis-related genes, PICALM knockdown, has been reported to inhibit autophagy by altering VAMP8 localization.¹⁴⁾

We also demonstrated that oxidative stress inhibits endocytosis, decreases lysosomal localization of VAMP8, increases that of STX7, and inhibits autophagosome-lysosome fusion (Fig. 3). Similar phenomena were observed in the treatment of endocytosis inhibitors. These results suggest that oxidative stress-induced changes in the localization of VAMP8 and STX7 are due to the inhibition of endocytosis. Oxidative stress is also considered as a cause of neurodegenerative diseases.^15,16) Our results suggest that oxidative stress leads to the development of neurodegenerative diseases by a mechanism of endocytosis inhibition that is different from the previously reported ROS-mediated damage to DNA, proteins, lipids, and organelles.^15–17)

Our findings show that decreased VAMP8 transport to lysosomes inhibits autophagy (Fig. 2). Thus, improving VAMP8 transport to lysosomes may be a novel therapeutic strategy for neurodegenerative diseases by recovering the autophagy activity. Indeed, overexpression of VAMP8 in the neurodegenerative disorder model cell has been reported to inhibit the decline of autophagic activity.²³⁾

Abnormal autophagy and increased oxidative stress are known to be involved not only in neurodegenerative diseases but also in the pathogenesis of diseases such as nonalcoholic steatohepatitis (NASH)²⁴⁾ and chronic kidney disease (CKD).²⁵⁾ Therefore, our findings that oxidative stress reduces the lysosomal transport of VAMP8 may be related to the pathogenesis of these diseases.

Since we could not explain what mechanisms that oxidative stress inhibits endocytosis trafficking of VAMP8 to lysosomes in this study, further consideration will be needed to elucidate its detailed molecular mechanisms. One study showed the oxidative stress induces inhibition of endocytosis via p38 mitogen-activated protein kinase (MAPK) activation.²⁰⁾ Because p38 MAPK regulates endocytosis by modulating Rab5 localization,²⁶⁾ dysregulated p38 MAPK activity may alter endocytosis transport. It has also been reported that oxidative stress activates CDK5 and GSK3β.^27,28) These kinases inhibitory regulate endocytosis by phosphorylating one of the endocytosis executors, dynamin1.^29,30) Therefore, changes in the activity of these kinases may be involved in the inhibition of VAMP8 transport by oxidative stress that we have shown in this study. We also could not prove whether autophagy dysfunction by oxidative stress is solely due to endocytosis suppression because the co-treatment experiment with H₂O₂ and CP caused cell death. Therefore, in future research, we would like to elucidate the mechanism of oxidative stress-induced endocytosis dysfunction and conduct experiments to rescue endocytosis suppression caused by oxidative stress to clarify this.

In conclusion, this study demonstrates that oxidative stress-induced inhibition of endocytosis suppresses the lysosomal transport of VAMP8 and impairs autophagy in SH-SY5Y cells. This phenomenon seems to be associated with the development of neurodegenerative diseases due to attenuated autophagy and lead novel therapeutic targets for restoring the autophagic activity.

Acknowledgments

This study was financially supported by AMED ACT-M (Issue No. 21im0210116h0002) and JSPS KAKENHI Grant No. 18K06658.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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