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. 2023 Feb 16;18(2):e0281957. doi: 10.1371/journal.pone.0281957

Ethyl pyruvate protects SHSY5Y cells against 6-hydroxydopamine-induced neurotoxicity by upregulating autophagy

Yuening Luo 1, Kazuichi Sakamoto 1,*
Editor: Khuen Yen Ng2
PMCID: PMC9934379  PMID: 36795720

Abstract

Parkinson disease is a chronic progressive neurodegenerative disorder with a prevalence that increases with age. The glycolytic end-product pyruvate, has antioxidant and neuroprotective feature. Here, we investigated the effects of ethyl pyruvate (EP), a pyruvic acid derivative, on 6-hydroxydopamine-induced SH-SY5Y cell apoptosis. Ethyl pyruvate decreased protein levels of cleaved caspase-3, phosphorylated endoplasmic reticulum kinase (pERK), and extracellular signal-regulated kinase (ERK), suggesting that EP reduces apoptosis via the ERK signaling pathway. Ethyl pyruvate also decreased oxygen species (ROS) and neuromelanin contents, suggesting that it suppresses ROS-mediated neuromelanin synthesis. Furthermore, increased protein levels of Beclin-1 and LC-II, and LC-II:LC-I ratios indicated that EP upregulates autophagy.

Introduction

Parkinson disease (PD) is the second most common progressive neurodegenerative disease after Alzheimer disease [1]. Parkinson disease is closely associated with age; it affects ~ 0.1% of the global population and increases to 1% and 2%‒4% of the those aged over 60 and 80 years, respectively [2, 3]. Parkinson disease is characterized by a loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc), striatal dopamine depletion, and abnormal α-synuclein aggregation [4, 5]. Other manifestations such as mitochondrial dysfunction, neuroinflammation, and oxidative stress have been identified in models of PD [6–8].

The role of α-synuclein aggregation and oxidative stress in PD pathogenesis has been established [5, 9]. Alpha synuclein is easily misfolded and polymerized, and unlike unfolded proteins, the primary degradation of aggregated α-synuclein is autophagy [10–12]. Rapamycin increases autophagy and protects against PD in models [11]. These findings indicate that autophagy might function as a neuroprotective strategy for PD by eliminating aggregated α-synuclein.

Oxidative stress is a risk factor for PD. Oxidative stress damages proteins, nucleic acids, and lipids that leads to cell dysfunction and death [7]. Numerous compounds with antioxidant activities have potential for treating PD according to findings in vitro and in vivo [13, 14].

The dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA) is selectively uptaken by dopaminergic neurons via the dopamine transporter (DAT) and stored in mitochondria, where it promotes the generation of free radicals and decreases ATP synthesis [15]. It is widely used to develop PD models in vivo and in vitro [16, 17]. It is easily oxidized to generate the superoxide anion, para-quinone, as well as hydrogen peroxide [18]. This is followed by intracellular reactive oxygen species (ROS) that eventually cause neuronal cell death [19]. The major advantage of the PD model induced by 6-OHDA is that the range of dopaminergic lesions can be controled and motor deficits can be quantified [20].

The final product of glycolysis is pyruvate, an anti-oxidant and ROS scavenger in vitro and in vivo [21, 22], but it is unstable in aqueous solution, which limits its application as a therapeutic agent. In contrast, the ethyl ester form of pyruvate, ethyl pyruvate (EP), is relatively stable and also has anti-oxidative, anti-inflammatory, and anti-apoptotic properties [22–24]. Ethyl pyruvate prevents the degeneration of nigrostriatal dopamine (DA) neurons, increases striatal dopamine levels, and improves motor function in PD models in vivo [25]. It also protects rat pheochromocytoma PC12 cells from dopamine-induced cytotoxicity in vitro [24].

However, the protective effect of EP on 6-OHDA induced SH-SY5Y cell death remains unclear. The present study aimed to determine whether EP is neuroprotective against 6OHDA-induced cell death in vitro and evaluated its potential for treating PD.

Materials and methods

Cell culture

The human neuroblastoma cell line SH-SY5Y (Riken Cell Bank, Tsukuba, Japan) was cultured and maintained in 5% CO2 at 37°C in DMEM/F12 medium (Sigma-Aldrich Corp., St. Louis, MO, USA) supplemented with 10% FBS (Hyclone Laboratories, Logan, UT, USA), penicillin (Wako, Tokyo, Japan), and streptomycin (Wako, Tokyo, Japan). Cells (6.25 ×104 cells/cm2) were seeded in 96-well plates and 6-cm dishes for 24 h, then incubated with 1, 2.5, and 5 mM EP (Wako, Tokyo, Japan) for 24 h followed by 75 μM 6-OHDA (50 mM; Sigma-Aldrich Corp.) dissolved in (dDW) for 6 h to assay apoptosis or 24 h for MTT assays, western blotting and melanin quantitation.

MTT assays

Cytotoxicity was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylterazolium bromide (MTT) assays, (Sigma-Aldrich Corp.) as described [26]. The medium was replaced with 90% DMEM/F12 containing 10% MTT and incubated at 37°C for 4 h. Thereafter, 10% SDS was added and the cells were incubated overnight at room temperature (RT). Absorbance at 570 nm was determined using a microplate reader (BioTek, Tokyo, Japan).

Apoptosis assays

SH-SY5Y cells cultured in DMEM/F12 medium were seeded for 24 h, then incubated with various concentrations of EP for 24 h followed by 6-OHDA for 6 h. Apoptotic, necrotic, and live cells were quantified using apoptosis/necrosis detection kits (blue, green, red, respectively; Abcam, Cambridge, UK) as described by the manufacturer. Fluorescence microscopy (Keyence, Tokyo, Japan) images were acquired and fluorescence intensity was quantified using ImageJ to determine apoptosis.

Assays of ROS

SH-SY5Y cells were cultured in DMEM/F12 medium, then incubated with 1, 2.5, or 5 mM EP for 24 h followed by 75 μM 6-OHDA for 6 h. Intracellular ROS were determined using DCFDA/H2DCFDA cellular ROS assay kits (Abcam, Cambridge, UK) as described by the manufacturer. Fluorescence microscopy (Keyence) images were acquired and fluorescence intensity was quantified using ImageJ to determine ROS.

Melanin quantitition

The cells were sedimented by centrifugation, trypsinized, suspended in 1 N NaOH, then heated at 80°C for 1 h. Absorbance at 405 nm was detemined using a microplate reader (BioTek). The final relative melanin content was normalized to the total protein content measured using BCA assay kits (Thermo Fisher Scientific Inc., Waltham, MA, USA).

Western blotting

Cells sedimented by centrifugation were washed twice with cold PBS, then lysed using RIPA buffer (150 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl, 10 mM NaF, 1 mM Na3VO4, 1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS), 0.5% Na-deoxycholate, and protein inhibitor). Proteins were then resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using 4%‒20% polyacrylamide gels. The resolved proteins were blotted onto PVDF membranes and incubated with the following primary antibodies diluted 1:1,000 (Beclin (#3495), caspase-3 (#9662), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), (pERK) (#9101), p44/42 MAPK (Erk1/2; ERK (#9102), and Microtubule-associated proteins 1A/1B light chain 3B (LC3 A/B) XP (#12741) (all from Cell Signaling Technology, Danvers, MA, USA). The membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody diluted 1:1,000 (#7074) (Cell Signaling Technology) at RT for another 1 h. The secondary antibodies were labeled using the chemiluminscent substrate LumiGLO® (Cell Signaling Technology) and proteins were detected using the AE-9300 Ez-capture MG system (Atto Corporation, Tokyo, Japan) and quantified using ImageJ software (National Insititues of Health [NIH], Bethesda, MD, USA).

Statistical analysis

The results are expressed as the means ± SD of at least three experiments. Data was compared among groups by ANOVA post-hoc tests using SPSS Statistics (SPSS Inc., Chicago, IL, USA). Values with p < 0.05 were considered statistically significant.

Results and discussion

Ethyl pyruvate protected SH-SY5Y cells against 6-OHDA-induced cytotoxicity

MTT assay was using to measure the cell viability. Our results showed that 5mM EP have no cytotoxic; whereas 10 mM EP was clearly cytotoxic compared with control cells (Fig 1A). Therefore, we applied < 5 mM EP in further experiments. 75 μM 6-OHDA showed the strong cytotoxicity compared to that of the control group, However, Ethyl pyruvate pretreat group showed a protective effect against 6-OHDA-induced cytotoxicity (Fig 1B).

Fig 1. Effects of EP on cytotoxicity induced by 6-OHDA.

Fig 1

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(a) Cytotoxicity of EP. (b) Effects of EP on 6-OHDA-induced cell death determined as cell viability using MTT assays. Results are expressed as means ± standard deviation (N ≥ 3), *P < 0.05, †P < 0.01 vs. controls; ‡P < 0.01 vs. 6-OHDA.

Ethyl pyruvate decreased SH-SY5Y cell apoptosis induced by 6-OHDA

Apoptosis assay was used to measure the effect of Ethyl pyruvate on apoptosis. 6-OHDA treat group showed a significant increase in cell death compared to that in the control group (Fig 2A). Then, the protein expression levels of caspase-3 were also measured. Similar results as apoptosis, cleaved caspase-3 protein levels were increased in 6-OHDA group and EP-treated group shown a decreased compared to the 6-OHDA group (Fig 2B and 2C).

Fig 2. Effects of EP on 6-OHDA induced cell death.

Fig 2

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(a) Apoptotic or necrotic (green), and live (blue) cells visualized using fluorescence microscopy. (b) Representative western blots of caspase-3 and cleaved caspase-3 (c) quantified using ImageJ. Results are expressed as means ± standard deviation (N ≥ 3. *P < 0.01 vs. control; †P < 0.05 vs. 6-OHDA. Scale bars, 200 μm.

Ethyl pyruvate reduced 6-OHDA-induced ROS levels in SH-SY5Y cells

ROS assay was performed to evaluate the effect of EP on ROS levels. 6-OHDA treat group showed a significant increase in ROS levels compared to the control group; EP treat group showed a decreased in ROS level compared to 6-OHDA group (Fig 3A).

Fig 3. Effects of ethyl pyruvate (EP) on 6-hydroxydopamine (6-OHDA)-induced intracellular reactive oxygen species (ROS) and neuromelanin contents.

Fig 3

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(a) Reactive oxygen species (ROS) determined from fluorescence microscopy images. Scale bars: 100 μm. Representative western blots (b) of pERK and extracellular signal-regulated kinase (ERK) quantified (c) using ImageJ. (d) Intracellular neuromelanin deposition. (e) Neuromelanin content and normalized the controls. Results are expressed as means ± standard deviation (N ≥ 3) *P < 0.01 vs. control; †P < 0.05 and ‡P < 0.01 vs. 6-OHDA.

Then, protein levels of pERK and ERK, were also measured. The pERK/ERK ratio increased in the 6-OHDA treat group compared to the control group and EP treat group showed a decreased in pERK/ERK ratio (Fig 3B and 3C).

Ethyl pyruvate decreased neuromelanin in SH-SY5Y cells induced by 6-OHDA

To evaluate the effect of EP on neuromelanin production, melanin content was measured. 6-OHDA treat group showed a significant increase in neuromelanin compared to the control group. EP treat group showed a decreased in neuromelanin content (Fig 3D and 3E).

Effects of EP on autophagy-related gene expression in SH-SY5Y cells

We measured protein levels of the autophagy-related genes Beclin-1 and LC3. Protein levels of Beclin-1 decreased, whereas LC3-II did not significantly change in the 6-OHDA, compared with controls. Ethyl pyruvate increased protein levels of Beclin-1 and LC3-II compared with the 6-OHDA group (Fig 4A–4C). The LC3-II/LC3-I ratio (marker of autophagy) did not significantly differ between the 6-OHDA and control groups. However, EP increased the LC3-II/LC3-I ratio compared with the 6-OHDA group (Fig 4D). These results suggested that EP upregulates autophagy.

Fig 4. Effects of EP on autophagy-associated Beclin-1 and LC3 gene expression.

Fig 4

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(a) Representative western blots of LC3 and Beclin-1. Protein bands of (b) Beclin-1 and (c) LC3-II quantified by ImageJ. (d) Relative ratios of LC3-II/LC3-I band density. Results are expressed as means ± standard deviation (N ≥ 3) *P < 0.01 vs. control; †P < 0.05 and ‡P < 0.01 vs. 6-OHDA).

Neuronal cells are susceptible to oxidative stress-induced cell damage that leads to neuronal cell death and is a risk factor for age-related neurodegenerative diseases [7]. Neurotoxic 6-OHDA can be easily oxidized to produce intracellular ROS and is used to destroy dopaminergic neurons in the brain [19]. The pyruvic acid derivative EP is an endogenous antioxidant metabolite [22]. The present study aimed to determine the effects of EP on 6-OHDA-induced neuronal death.

Differentiated SH-SY5Y cells might not be suitable for 6-OHDA-induced PD models due to their high tolerance of 6-OHDA toxicity [27]. Therefore, we investigated the effects of EP in undifferentiated SH-SY5Y cells.

We found that EP (< 5 mM) significantly increased the viability of cells incubated with 6-OHDA but did not affect that of control cells, suggesting a protective effect against 6-OHDA-induced cytotoxicity.

The results of apoptosis assays confirmed the effect of EP on 6-OHDA-induced cell death. Apoptosis rates increased in cells incubated with 6-OHDA, which was consistent with previous findings [28, 29], and EP decreased them. We then measured levels of caspase 3 and cleaved caspase 3 proteins. The increased abundance of cleaved caspase-3 in the 6-OHDA group, which was consistent with our previous findings [29], was reduced by EP, confirming the protective effect of EP against 6-OHDA-induced cell death.

A significant role of oxidative stress in PD has been identified [7, 30]. Antioxidant compounds such as asiaticoside and Ginsenoside-Rg1 are promising candidates for treating PD [13, 14]. The present and our previous [29] findings revealed EP decreased the elevated ROS levels in cells incubated with 6-OHDA.

Extracellular signal-regulated kinases (ERK1/2) are crucial regulators of neuronal responses associated with cell death [31, 32], and their activation also plays important roles in several models of 6-OHDA-induced cell death [33, 34]. Cell death induced by 6-OHDA involves ERK activation [33], but not SAPK/JNK or p38 kinase [35]. Ethyl pyruvate attenuates p-ERK expression in formalin-induced neuronal models [36] and inhibits ERK phosphorylation in those of LPS-induced inflammation [37].

Therefore, we measured levels of pERK and ERK proteins and found significantly increased ERK phosphorylation in cells incubated with 6-OHDA, which was consistent with previous findings [29, 31]. Furthermore, EP decreased ERK phosphorylation, indicating that it reduces apoptosis via the ERK signaling pathway.

Neuromelanin is produced in human SNpc dopaminergic neurons over a lifetime and accumulates with age until it occupies most of the neuronal cytoplasm [38]. Exceeding the threshold amount of accumulated NM is associated with an age-dependent PD phenotype, and enhanced lysosomal proteostasis can reduce intracellular neuromelanin and prevent neurodegeneration [39]. Oxidative stress is also associated with neuromelanin synthesis [32]. We previously showed that EP decreased intracellular neuromelanin levels increased by 6-OHDA [40]. This suggested that EP suppresses ROS-mediated neuromelanin synthesis.

Abnormal α-synuclein aggregation is also a feature of PD [5]. Autophagic proteolysis is an important degradation pathway for α-synuclein, and that increased autophagy is also relative to cell survival [10, 11]. Autophagy blocked with chloroquine induces increased α-synuclein accumulation, whereas autophagy activation by rapamycin results in α-synuclein clearance [41]. These findings showed that autophagy might play an important role in PD therapy. One study found that 6-OHDA can cause cell apoptosis, decrease autophagy markers (LC3-II/LC3-I, Beclin-1) and increase phosphate mTOR/mTOR [42]. The mTOR inhibitor rapamycin can restore increased mTOR activity caused by overexpressed α-synuclein [43], and the autophagy inhibitor chloroquine can block this protect effect [42]. Both MPTP and 6-OHDA increase α-synuclein [35, 44], and consequently inhibit autophagy [45], whereas Ethyl pyruvate decreases α-synuclein abundance [46].

We measured levels of the autophagy-related proteins, Beclin-1, and LC3. We found that 6-OHDA significantly decreased Beclin-1 expression but did not significantly alter LC3-II levels, which was consistent with our previous findings [29]. Ethyl pyruvate significantly increased Beclin-1 and LC3-II protein levels. These results suggested that EP protects SH-SY5Y cells against 6-OHDA-induced cell apoptosis by upregulating autophagy. However, 5 mM EP did not induce any significant differences in levels of Beclin-1 and LC3-II proteins compared with 2.5 mM EP, indicating that the antioxidant capacity of EP in cells is limited.

Conclusions

We showed that EP reduced apoptosis, ROS, and neuromelanin levels, and improved autophagy. We believe that further therapeutic interventions targeting EP might prove beneficial and improve the etiology of neurodegeneration.

Supporting information

S1 Raw data

(ZIP)

Click here for additional data file. (310.9KB, zip)

Acknowledgments

We afre gratefull to the partical support from the University of Tsukuba, Japan.

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

The author(s) received no specific funding for this work.

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Decision Letter 0

Khuen Yen Ng

28 Sep 2022

PONE-D-22-21944Ethyl pyruvate protects SHSY5Y cell line against 6-hydroxydopamine-induced neurotoxicity by upregulation Autophagy.PLOS ONE

Dear Dr. Sakamoto,

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Reviewer #1: Partly

Reviewer #2: Yes

********** 

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Reviewer #1: Yes

Reviewer #2: N/A

********** 

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Reviewer #1: Yes

Reviewer #2: No

********** 

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Reviewer #2: No

********** 

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Reviewer #1: The present manuscript by Luo and Sakamoto describes the neuroprotective effects of ethyl pyruvate (EP) against the 6-OHDA induced toxicity in SH-SY5Y cells, potentially mediated by the upregulation of autophagy. The current study appears similar to a recently published work by the same authors to some extent, including the in vitro model used, biochemical assays, presentation, etc. However, there are concerns in the manuscript that needs to be addressed and clarified.

1. Materials and Methods – Perhaps provide a detail description on how 6-OHDA and EP is dissolved/prepared prior to the treatment. Was there any solvent/vehicle control included in the cell viability assay?

2. Method and Materials – For the cell apoptosis (Section 2.3) and ROS (Section 2.4) assays, the authors mentioned a post-treatment of 6-OHDA for only 6 hrs, which is different from the MTT assay and melanin content measurement. Why is there a difference in the 6-OHDA treatment among the different assays? Is this a typo or did the authors perform prior screening assays (different concentration and time-point) to justify the extent of toxicity induced by 6-OHDA in SH-SY5Y cells?

3. Results – Section 3.2 – The authors mentioned a significant increase in cell death induced by 6-OHDA treatment, which is only presented in the photomicrographs. Perhaps the authors should present the quantification data, since this is also mentioned in the Methods and Materials (Pg. 4) to quantify the apoptotic, necrotic and live cells. Furthermore, the fluorescence images presented in Figure 2a appears to be of poor quality and dim throughout, which should not be the case.

4. Results – Section 3.3 – Similarly, measurement of the intracellular ROS levels should be presented instead of showing only the photomicrographs in the Results, as mentioned in the Methods section (Pg. 5).

5. There have been several prior studies on neuroprotective effects and mechanisms of EP in both in vitro and in vivo PD models. Perhaps the authors could improve the Introduction and/or Discussion of this manuscript to also discuss how this study fit into the current literature and strengthen the Discussion section.

6. Minor grammatical errors present throughout the manuscript and please check thoroughly (E.g., Pg.2, L. 1.; Pg.6, L.18; etc).

Reviewer #2: This is an interesting manuscript examining the effect of ethyl pyruvate (EP) on 6-hydroxydopamine-induced SH-SY5Y cell apoptosis. Authors stated that EP could decrease cleaved caspase-3, pERK, and ERK protein levels, suggesting that EP reduces apoptosis via the ERK signaling pathway. Moreover, this study demonstrated that EP increased the protein levels of Beclin-1, LC-II and the ratio of LC-II/LC-I, suggesting that EP can upregulating autophagy. This is totlly a "descriptive" observation. However, still I have some concerns about the methods and data.The authors would have to provide additional experimental evidences about MAPK signaling pathway and autophagy as well as have multiple rising questions. Authors should provide the answers of those questions.

Comments:

1. Details method should need to be clarified by the authors. Example in Fig 1B, authors used 6-OHDA and EP but in method section autors did not explain anything about their drugs and also concentrations. The drug and concentrations should also be stated in the method part.Moreover, in MTT method authors added 10 % SDS solution, author needs to explain the reason. Author should provide the ref. of this method.

2. In Fig. 3B, it seemed that pERK and ERK did not match with the quantitative values. There were some details that need to be clarified. The authors should provide the all raw protein bands of three independent experiments.

3. mTOR promotes anabolic metabolism and inhibits autophagy induction. Therefore, the regulation of autophagy with mTOR inhibitors provides a new therapeutic strategy for a variety of diseases. Thus, in this study author should observe the expression of mTOR complaxes such as mTORC1 and mTORC2 and also evaluate the effect of EP on mTORC1 and mTORC2.

4. Because authors focused on autophagy, thus to make a more influential study, authors should use Chloroquine and rapamycin in this study. Because Rapamycin promoted autophagy by blocking the mTOR pathway, and chloroquine enhanced apoptosis by blocking autophagy. Authors need to compare the effect of EP and Chloroquine and rapamycin on autophagy.

5. Authors should provide more information about 6-hydroxydopamine (6-OHDA) and Ethyl pyruvate (EP) in the introduction part. Example how 6-OHDA developes PD model? Are there any mechanism link with this disease?

6. Authors need to explain why they selected only ERK among the three proteins of MAPK pathway. How ERK plays an important role in this study? For more clarification authors should check p38 and JNK.

7. Authors stated that α-synuclein aggregation and oxidative stress in PD pathogenesis. Authors also stated the importance of α-synuclein in autophagy. Are there any reports about the α-synuclein and EP? If not, how authors explain this link between EP and α-synuclein?

10. The authors should indicate the specific cat. number and dilution factor of the antibodies used in the Methods section to improve the reproducibility of their findings.

11. Most of the images quality are very poor such as Fig 2A, 3A etc. Authors should provide high-quality images and blots.

12. Please check English grammar and sentence structure.

13. Authors need to use molecular weight in the western blot images.

14. In Fig 2 B cleaved caspase-3 showed 2 bands, Are there any reason, authors need to explain or provide another image?

15. In western blot images Beta actin should be equal but in Fig 2B,3B and 4A why in all groups beta-actins are not equal?

16. In western blot method, authors needs to explain about the dilution of antibodies. And also provide the how many percentage of gel authors used during WB?

17. In discussion part authors demonstrated that Extracellular signal-regulated kinases (ERK1/2) are crucial regulators of neuronal responses associated with cell death [26] [27]. In several different 6-OHDA-induced cell death model, ERK1/2 activation also plays an important role [28][29]. Therefore, we measured the protein levels of pERK and ERK. Cells exposed to 6-OHDA showed a significant increase in ERK phosphorylation consistent with previous studies[24][26], and pretreatment with EP decreased the ERK phosphorylation. This suggests that EP reduces apoptosis via the ERK signaling pathway. But authors did not explain how 6-OHDA -induced cell death model ? and how to make link with ERK. Additionally, authors should provide some ref. about the EP and ERK in other diseases. In overall author should re-arrage the some paragraphs of discussion part and make it more clear.

18. Apoptosis assay, ROS assay, Melanin Content first two lines are very similar, why? Authors needs to write in different ways. And in method section authors needs to stated their EP concentrations.

19. Authors should state their seeded plate (example 6 well plate or 60 mM dish or 24 well plate etc) and also cells density.

********** 

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Reviewer #1: Yes: Wei Ling Lim

Reviewer #2: No

**********

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PLoS One. 2023 Feb 16;18(2):e0281957. doi: 10.1371/journal.pone.0281957.r002

Author response to Decision Letter 0

6 Dec 2022

Dear Editors and Reviewers

Thank you very much for reviewing our manuscript and offering valuable advice.

We have addressed your comments with point-by-point responses and revised the manuscript accordingly.

Replies to Reviewers

Reviewer #1:

The present manuscript by Luo and Sakamoto describes the neuroprotective effects of ethyl pyruvate (EP) against the 6-OHDA induced toxicity in SH-SY5Y cells, potentially mediated by the upregulation of autophagy. The current study appears similar to a recently published work by the same authors to some extent, including the in vitro model used, biochemical assays, presentation, etc. However, there are concerns in the manuscript that needs to be addressed and clarified.

Reply:

We appreciate for your constructive comments.

1. Materials and Methods – Perhaps provide a detail description on how 6-OHDA and EP is dissolved/prepared prior to the treatment. Was there any solvent/vehicle control included in the cell viability assay?

Reply:

Thank you for your comment. 6-OHDA was dissolved in dDW, and EP is a solution state. In this study, we used max 5mM EP, so we didn’t diluted it. We added this to the Materials and Methods section, please refer to Page 4 Line 17.

2. Method and Materials – For the cell apoptosis (Section 2.3) and ROS (Section 2.4) assays, the authors mentioned a post-treatment of 6-OHDA for only 6 hrs, which is different from the MTT assay and melanin content measurement. Why is there a difference in the 6-OHDA treatment among the different assays? Is this a typo or did the authors perform prior screening assays (different concentration and time-point) to justify the extent of toxicity induced by 6-OHDA in SH-SY5Y cells?

Reply:

Thank you for your comment. In our pervious study, we measured the different time-point, and we found that 6h treatment for ROS and apoptosis showed the best results. In this study we also used 6h treatment for ROS and apoptosis.

3. Results – Section 3.2 – The authors mentioned a significant increase in cell death induced by 6-OHDA treatment, which is only presented in the photomicrographs. Perhaps the authors should present the quantification data, since this is also mentioned in the Methods and Materials (Pg. 4) to quantify the apoptotic, necrotic and live cells. Furthermore, the fluorescence images presented in Figure 2a appears to be of poor quality and dim throughout, which should not be the case.

Reply:

Thank you for your comment. We have added the quantification data, please refer to Fig.2. The resolution of all Figures was increased to 550 dpi. Please refer to Fig.1-4.

4. Results – Section 3.3 – Similarly, measurement of the intracellular ROS levels should be presented instead of showing only the photomicrographs in the Results, as mentioned in the Methods section (Pg. 5).

Reply:

Thank you for your comment. We added the quantification data, please refer to Fig. 3.

5. There have been several prior studies on neuroprotective effects and mechanisms of EP in both in vitro and in vivo PD models. Perhaps the authors could improve the Introduction and/or Discussion of this manuscript to also discuss how this study fit into the current literature and strengthen the Discussion section.

Reply:

Thank you for your comment. We added more references into the Introduction and discussion section, please refer to Page 4 Line 3-9, Page 12 Line 10-13, Page 13 Line 11-16.

6. Minor grammatical errors present throughout the manuscript and please check thoroughly (E.g., Pg.2, L. 1.; Pg.6, L.18; etc).

Reply:

Thank you for your comment. We checked the manuscript again.　

Reviewer #2:

This is an interesting manuscript examining the effect of ethyl pyruvate (EP) on 6-hydroxydopamine-induced SH-SY5Y cell apoptosis. Authors stated that EP could decrease cleaved caspase-3, pERK, and ERK protein levels, suggesting that EP reduces apoptosis via the ERK signaling pathway. Moreover, this study demonstrated that EP increased the protein levels of Beclin-1, LC-II and the ratio of LC-II/LC-I, suggesting that EP can upregulating autophagy. This is totlly a "descriptive" observation. However, still I have some concerns about the methods and data.The authors would have to provide additional experimental evidences about MAPK signaling pathway and autophagy as well as have multiple rising questions. Authors should provide the answers of those questions.

Reply:

Thank you for your constructive comments.

Comments:

1. Details method should need to be clarified by the authors. Example in Fig 1B, authors used 6-OHDA and EP but in method section autors did not explain anything about their drugs and also concentrations. The drug and concentrations should also be stated in the method part.Moreover, in MTT method authors added 10 % SDS solution, author needs to explain the reason. Author should provide the ref. of this method.

Reply:

Thank you for your comment. We added the relevant concentrations in the Method and Materials section, please refer to Page 4 Line 16-17. We used 10% SDS as a formazan solubilization solution and we added the relevant references, please refer to Page 5 Line 4.

2. In Fig. 3B, it seemed that pERK and ERK did not match with the quantitative values. There were some details that need to be clarified. The authors should provide the all raw protein bands of three independent experiments.

Reply:

Thank you for your comment. The quantitative values in Figure 3B are the ratio of pERK to ERK. we observed a decrease in pERK, but at the same time, the level of ERK also decreased significantly, so the ratio of pERK to ERK finally resulted in a large increase compared to the control group. raw data is attached to the additional images, please refer to Supplementary Figure.

3. mTOR promotes anabolic metabolism and inhibits autophagy induction. Therefore, the regulation of autophagy with mTOR inhibitors provides a new therapeutic strategy for a variety of diseases. Thus, in this study author should observe the expression of mTOR complaxes such as mTORC1 and mTORC2 and also evaluate the effect of EP on mTORC1 and mTORC2.

Reply:

Thank you for your comment. We gratefully appreciate for your valuable suggestion. Currently, we are unable to conduct experiments because our lab is being moved. Therefore, we added some ref about EP and mTOR.

The previous studies showed the relationship between EP and mTOR, and EP decreases mTOR phosphorylation and thus promotes autophagy. We added this section to the discussion. Please refer to Page 13 Line 11-16.

4. Because authors focused on autophagy, thus to make a more influential study, authors should use Chloroquine and rapamycin in this study. Because Rapamycin promoted autophagy by blocking the mTOR pathway, and chloroquine enhanced apoptosis by blocking autophagy. Authors need to compare the effect of EP and Chloroquine and rapamycin on autophagy.

Reply:

Thank you for your comment. We gratefully appreciate for your valuable suggestion. Currently, we are unable to conduct experiments because our lab is being moved. Therefore, we added some ref about EP and Chloroquine and rapamycin.

The previous studies showed that rapamycin can restore increased mTOR activity (Gao et al., 2015). And chloroquine can block these protect effect of some medicine(He et al., 2022). We added this section to the discussion, please refer to Page 13 Line 11-15.

5. Authors should provide more information about 6-hydroxydopamine (6-OHDA) and Ethyl pyruvate (EP) in the introduction part. Example how 6-OHDA developes PD model? Are there any mechanism link with this disease?

Reply

Thank you for your comment. We added more references into the Introduction section, please refer to Page 3 Line 11-14,16-17, Page 4 Line 3-6.

6. Authors need to explain why they selected only ERK among the three proteins of MAPK pathway. How ERK plays an important role in this study? For more clarification authors should check p38 and JNK.

Reply:

Thank you for your comment. ERK and autophagy have a strong relative and have a strong connection with ROS, so we have mainly studied ERK in this experiment. About JNK and p38, previous study showed that JNK and p38 are not affected (Gómez-Santos et al., 2002), so in this experiment we didn’t measure JNK and p38 protein level. This was added in discussion section, please refer to Page 12 Line 10-13.

7. Authors stated that α-synuclein aggregation and oxidative stress in PD pathogenesis. Authors also stated the importance of α-synuclein in autophagy. Are there any reports about the α-synuclein and EP? If not, how authors explain this link between EP and α-synuclein?

Reply:

Thank you for your comment. The previous study showed that EP can decreases α-synuclein, and we added this to the discussion section, please refer to Page 13 Line 15-16

10. The authors should indicate the specific cat. number and dilution factor of the antibodies used in the Methods section to improve the reproducibility of their findings.

Reply:

Thank you for your comment. We added the cat. number and dilution factor of the antibodies in Method and Materials section, please refer to Page 6 Line 16-18, Page 7 Line 2.

11. Most of the images quality are very poor such as Fig 2A, 3A etc. Authors should provide high-quality images and blots.

Reply:

Thank you for your comment. The resolution of all Figures were increased to 550 dpi. Please refer to Fig.1-4.

12. Please check English grammar and sentence structure.

Reply:

Thank you for your comment. We checked the manuscript again.

13. Authors need to use molecular weight in the western blot images.

Reply:

Thank you for your comment. We revised it, please refer to Fig2-4.

14. In Fig 2 B cleaved caspase-3 showed 2 bands, Are there any reason, authors need to explain or provide another image?

Reply:

Thank you for your comment. The activation of Caspase-3 requires cleavage at Asp175, resulting in an activated p17/p19 protein fragment. And the purchased caspase-3 antibody is show 19kDa and 17kDa size bands at the cleaved caspase-3 position. Please refer to https://www.cellsignal.jp/products/primary-antibodies/caspase-3-antibody/9662

15. In western blot images Beta actin should be equal but in Fig 2B,3B and 4A why in all groups beta-actins are not equal?

Reply:

Thank you for your comment. We used BSA to determine the protein concentration of each sample, and when we did WB, used the same concentration. Some changes in other proteins may affect the final b-actin changes. To eliminate these different, we normalize the data by b-actin.

16. In western blot method, authors needs to explain about the dilution of antibodies. And also provide the how many percentage of gel authors used during WB?

Reply:

Thank you for your comment. We added the dilution factor of the antibodies and gel in Method and Materials section, please refer to Page 6 Line 13-18, Page 7 Line 2.

17. In discussion part authors demonstrated that Extracellular signal-regulated kinases (ERK1/2) are crucial regulators of neuronal responses associated with cell death [26] [27]. In several different 6-OHDA-induced cell death model, ERK1/2 activation also plays an important role [28][29]. Therefore, we measured the protein levels of pERK and ERK. Cells exposed to 6-OHDA showed a significant increase in ERK phosphorylation consistent with previous studies[24][26], and pretreatment with EP decreased the ERK phosphorylation. This suggests that EP reduces apoptosis via the ERK signaling pathway. But authors did not explain how 6-OHDA -induced cell death model ? and how to make link with ERK. Additionally, authors should provide some ref. about the EP and ERK in other diseases. In overall author should re-arrage the some paragraphs of discussion part and make it more clear.

Reply:

Thank you for your comment. We gratefully appreciate for your valuable suggestion. In the previous study, 6-OHDA-induced cell death involves ERK activation (Kulich and Chu, 2001), and in other model, EP treatment shown the effect on ERK phosphorylation(Lee and Kim, 2011) (Lee et al., 2012). We added more references into the discussion section, please refer to Page 12 Line 10-13.

18. Apoptosis assay, ROS assay, Melanin Content first two lines are very similar, why? Authors needs to write in different ways. And in method section authors needs to stated their EP concentrations.

Reply:

Thank you for your comment. We revised it, and add the concentration of EP and 6-OHDA，please refer to Page 4 Line 16-17.

19. Authors should state their seeded plate (example 6 well plate or 60 mM dish or 24 well plate etc) and also cells density.

Reply:

Thank you for your comment. We revised it, please refer to Page 4 Line 15-18,

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Decision Letter 1

Khuen Yen Ng

5 Feb 2023

Ethyl pyruvate protects SHSY5Y cell line against 6-hydroxydopamine-induced neurotoxicity by upregulation Autophagy.

PONE-D-22-21944R1

Dear Dr. Sakamoto,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Khuen Yen Ng, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Acceptance letter

Khuen Yen Ng

8 Feb 2023

PONE-D-22-21944R1

Ethyl pyruvate protects SHSY5Y cells against 6-hydroxydopamine-induced neurotoxicity by upregulating autophagy

Dear Dr. Sakamoto:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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on behalf of

Dr. Khuen Yen Ng

Academic Editor

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