PONE-D-19-29517

Starvation-induced autophagy via calcium-dependent TFEB dephosphorylation is  suppressed by Shigyakusan.

PLOS ONE

Dear Dr. Noda,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Academic Editor’s comments:

1) Why there is no LC3-I band in the LC3 blot? Considering the proximity of LC3-I and LC3-II bands, how can the authors be sure that the visible band is LC3-II?

2) Total 4EBP1 should be analyzed together with phosphor-4EBP1.

Reviewer 1:

This manuscript by Ikari et al. describes the effects of TJ-35, a Kampo medicine herbal extract, on autophagy.  Upon starvation, TFEB and ULK dephosphorylation, as well as calcium concentration influx, were suppressed in the presence of TJ-35.  While these observations were interesting and potentially provide mechanistic insight on the action of TJ-35, there are some concerns that need to be addressed.

1. All image quantification should be described in more detail: e.g. how many experiments were performed, how many cells were counted and quantified…

2. ANOVA statistical analysis should be used for experimental groups >2 in figs 2, 3, 4, 5, 7, 8.

3. Many control experiments were missing.  e.g. Fig 2A, 2B should include DMEM+TJ-35, Fig 2C should include no treatment, as well as Bafilomycin A treatment alone; Figs 3A, 3B, 3C, 4A need DMEM+TJ-35.  These controls (or experimental) are important and can provide some useful information, especially considering TJ-35 was identified to have effects on tf-LC3 GFP/RFP ratio under medium rich (no starvation) condition in Fig 1B.

4. Fig 2A, could the authors provide the RFP image (of tf-LC3) on each panel and analyze the ratio of GFP/RFP as well?  It can provide more information on which step autophagy is affected by TJ-35.

5. Fig 4C, it’s clear that TJ-35 had effect on TFEB dephosphorylation (based on the mobility shift). However, the effect on p-ULK1 was less obvious.  It should be normalized with total ULK1 (the amount of ULK1 was not equal among the samples).

6. The title on Fig 8 legend is not appropriate.  “TJ-35 prevents starvation induced calcium influx from the ER mediated by the IP3 receptor”, while this set of experiments did not involve TJ-35 at all.  

7. In Fig 8, 0.5 micro M of EDTA was used to chelate calcium to conclude that the medium (or EBSS) was not the source of starvation-induced calcium influx.  However, the concentration of calcium in EBSS and DMEM is at the range of milli M, the EDTA added was not sufficient to chelate all the calcium.  Higher concentration of EDTA would need to be used for this experiment.  

8. In Fig 6, have the authors tested if addition of TJ-35 has similar effect as cyclosporin A on inhibiting TFEB dephosphorylation through calcineurin? Similarly in Fig 7A, have the authors tested DMEM + ionomycin + TJ-35 condition?

Reviewer 2:

The report presents an interesting observation that “Shigyakusan” inhibits starvation-induced autophagy by promoting calcium dependent TFEB phosphorylation. Particularly, the elevation of cytosolic calcium upon starvation and involvement of ER calcium export to cytosol is exciting. The findings could be of interest to basic research in autophagy. However, more works needs to be carried out, this study could promise a discovery of novel autophagy modulator. For now, there are number of aspects that needs to be clarified before presentation in the PLOS One. Once these concerns are addressed, I recommend publication of this paper in PLOS One.

Major/Minor concerns are listed as follows.

1) Authors should include the exact ratio in which the different ingredients of “Kampo” (at least of the Shigyakusan/TJ-35) has been prepared.

2) Authors should explain on why they came up with certain concentration of positive control (Torin-1), negative control (Bafilomycin A) of autophagy for screening purpose. Importantly, author should explain how they choose specified concentration of each “Kampo” and how they were able to titrate its effect in relation to positive and negative control.

3) Representative images of at least for Control, Torin-1, BafilomycinA and TJ-35 should be included from the screening results.

4) After screening, authors already have an idea that autophagosome numbers might be decreased by TJ-35, therefore, authors should compare its effect with known inhibitors of autophagosome formation (possibly quantifying LC3 puncta). This is important to classify the effect of TJ-35 on autophagosome biogenesis. Comparing TJ-35 with the agents blocking autophagosome-lysosome fusion or agents preventing lysosomal acidification would only provide limited information that TJ-35 is an autophgay inhibitor as GFP/RFP intensity ratio is not affected by autophagosome population. Alternatively, authors should better show the GFP and RFP puncta per cell at least for Control, TJ-35, Torin-1, and Bafilomycin A.

5) Combined effect of TJ-35 with Chloroquine or Bafilomycin A is needed to suggest that TJ-35 prevent autophagosome formation. (Possibly TJ-35 pre-treatment followed by addition of chloroquine or Bafilomycin A).

6)  Is this effect of TJ-35 limited to starvation-induced autophagy?

7) Why there is a difference in the band size of TFEB between 4th lane (DMEM+TJ-35) and 5th lane (EBSS+TJ-35) in the Figure 4C? In another word, is phosphorylation state of TFEB is different in DMEM+TJ-35 and EBSS+TJ-35? Overall, the blot shown in Figure 4C is confusing as authors claim that the difference in the size of TFEB represent its phosphorylation state? Is it because of shorter exposure time that both Phospho-TFEB and TFEB is not visible in this blot as shown in Figure 6 (Clearly, TFEB antibody could detect both phospho TFEB and TFEB)? Authors should either show the clear TFEB bands in the Figure 4C or perform cell fraction to show endogenous TFEB in cytosol or nucleus? Authors should explain, why there is strong dephosphorylation state of TFEB (if it is), in the 6th lane as compared with 3rd and 5th lane.

8) Why 4EBP1 band size shown in the Figure 4C is different between samples (especially lane 4th, 5th and 6th? Does this antibody detect both phospho-EBP1 and total EBP1?

9) Why there is discrepancy in the Ulk1 and S6k phosphorylation by TJ-35 in DMEM and EBSS as shown in Figure 4C? Could this data suggest that activity of TJ-35 on mTOR is dependent upon nutrient availability?

10) Authors should explain the content of the reference# 35 mentioned in the line#349, page 14.

11) How Inonomycin and cyclosporin A affect autophagy flux and mTOR activity? It has been reported that Ionomycin results in massive accumulation of autophagosome (Hansen M et al., Mol Cell 2007), could be a characteristic of impaired autophagy. It is important to show that how ionomycin and cyclosporinA affects autophagy flux by using tf-LC3 system. Could they do an assay to compare the effect of Ionomycin and Cyclosporin A with TJ-35, this could be important for the claim that TJ-35 inhibits calcium dependent autophagosome formation. 

12) To support the conclusion drawn from Figure 8A, authors should include a calcium free medium in their experiment.

13) I believe that it should be “calcium efflux from the ER” not “calcium influx from the ER” as mentioned in the line #412, page 16. Author should correct this wherever possible in the manuscript text and figure legend.

14) Authors should design an assay to conclude that the effect of the TJ-35 on autophagy activity requires all the five ingredients and try to explain why exact combination of all ingredient is essential. Alternatively, authors should perform assay to find out which ingredient of TJ-35 is most essential in modulating autophagy activity by applying the principle of exclusion and discuss the direction for identification/purification of novel molecules present in the ingredients of TJ-35 that could modulate calcium dependent autophagy.

==============================

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Vladimir Trajkovic

Academic Editor

PLOS ONE

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'TN.

This work was supported by a grant from Uehara Memorial Foundation (TN) and commissioned work from the Tsumura Corporation (Tokyo, Japan). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.'

We note that you received funding from a commercial source: Tsumura Corporation

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This manuscript by Ikari et al. describes the effects of TJ-35, a Kampo medicine herbal extract, on autophagy. Upon starvation, TFEB and ULK dephosphorylation, as well as calcium concentration influx, were suppressed in the presence of TJ-35. While these observations were interesting and potentially provide mechanistic insight on the action of TJ-35, there are some concerns that need to be addressed.

1. All image quantification should be described in more detail: e.g. how many experiments were performed, how many cells were counted and quantified,…

2. ANOVA statistical analysis should be used for experimental groups 2 in figs 2, 3, 4, 5, 7, 8.

3. Many control experiments were missing. e.g. Fig 2A, 2B should include DMEM+TJ-35, Fig 2C should include no treatment, as well as Bafilomycin A treatment alone; Figs 3A, 3B, 3C, 4A need DMEM+TJ-35. These controls (or experimental) are important and can provide some useful information, especially considering TJ-35 was identified to have effects on tf-LC3 GFP/RFP ratio under medium rich (no starvation) condition in Fig 1B.

4. Fig 2A, could the authors provide the RFP image (of tf-LC3) on each panel and analyze the ratio of GFP/RFP as well? It can provide more information on which step autophagy is affected by TJ-35.

5. Fig 4C, it’s clear that TJ-35 had effect on TFEB dephosphorylation (based on the mobility shift). However, the effect on p-ULK1 was less obvious. It should be normalized with total ULK1 (the amount of ULK1 was not equal among the samples).

6. The title on Fig 8 legend is not appropriate. “TJ-35 prevents starvation induced calcium influx from the ER mediated by the IP3 receptor”, while this set of experiments did not involve TJ-35 at all.

7. In Fig 8, 0.5 micro M of EDTA was used to chelate calcium to conclude that the medium (or EBSS) was not the source of starvation-induced calcium influx. However, the concentration of calcium in EBSS and DMEM is at the range of milli M, the EDTA added was not sufficient to chelate all the calcium. Higher concentration of EDTA would need to be used for this experiment.

8. In Fig 6, have the authors tested if addition of TJ-35 has similar effect as cyclosporin A on inhibiting TFEB dephosphorylation through calcineurin? Similarly in Fig 7A, have the authors tested DMEM + ionomycin + TJ-35 condition?

Reviewer #2: The report presents an interesting observation that “Shigyakusan” inhibits starvation-induced autophagy by promoting calcium dependent TFEB phosphorylation. Particularly, the elevation of cytosolic calcium upon starvation and involvement of ER calcium export to cytosol is exciting. The findings could be of interest to basic research in autophagy. However, more works needs to be carried out, this study could promise a discovery of novel autophagy modulator. For now, there are number of aspects that needs to be clarified before presentation in the PLOS One. Once these concerns are addressed, I recommend publication of this paper in PLOS One.

Major/Minor concerns are listed as follows.

1) Authors should include the exact ratio in which the different ingredients of “Kampo” (at least of the Shigyakusan/TJ-

35) has been prepared.

2) Authors should explain on why they came up with certain concentration of positive control (Torin-1), negative control

(Bafilomycin A) of autophagy for screening purpose. Importantly, author should explain how they choose specified

concentration of each “Kampo” and how they were able to titrate its effect in relation to positive and negative control.

3) Representative images of at least for Control, Torin-1, BafilomycinA and TJ-35 should be included from the screening

results.

4) After screening, authors already have an idea that autophagosome numbers might be decreased by TJ-35, therefore,

authors should compare its effect with known inhibitors of autophagosome formation (possibly quantifying LC3 puncta).

This is important to classify the effect of TJ-35 on autophagosome biogenesis. Comparing TJ-35 with the agents blocking

autophagosome-lysosome fusion or agents preventing lysosomal acidification would only provide limited information

that TJ-35 is an autophgay inhibitor as GFP/RFP intensity ratio is not affected by autophagosome population.

Alternatively, authors should better show the GFP and RFP puncta per cell at least for Control, TJ-35, Torin-1, and

Bafilomycin A.

5) Combined effect of TJ-35 with Chloroquine or Bafilomycin A is needed to suggest that TJ-35 prevent autophagosome

formation. (Possibly TJ-35 pre-treatment followed by addition of chloroquine or Bafilomycin A).

6) Is this effect of TJ-35 limited to starvation-induced autophagy?

7) Why there is a difference in the band size of TFEB between 4th lane (DMEM+TJ-35) and 5th lane (EBSS+TJ-35) in the

Figure 4C? In another word, is phosphorylation state of TFEB is different in DMEM+TJ-35 and EBSS+TJ-35? Overall, the

blot shown in Figure 4C is confusing as authors claim that the difference in the size of TFEB represent its

phosphorylation state? Is it because of shorter exposure time that both Phospho-TFEB and TFEB is not visible in this blot

as shown in Figure 6 (Clearly, TFEB antibody could detect both phospho TFEB and TFEB)? Authors should either show

the clear TFEB bands in the Figure 4C or perform cell fraction to show endogenous TFEB in cytosol or nucleus? Authors

should explain, why there is strong dephosphorylation state of TFEB (if it is), in the 6th lane as compared with 3rd and

5th lane.

8) Why 4EBP1 band size shown in the Figure 4C is different between samples (especially lane 4th, 5th and 6th? Does this

antibody detect both phospho-EBP1 and total EBP1?

9) Why there is discrepancy in the Ulk1 and S6k phosphorylation by TJ-35 in DMEM and EBSS as shown in Figure 4C?

Could this data suggest that activity of TJ-35 on mTOR is dependent upon nutrient availability?

10) Authors should explain the content of the reference# 35 mentioned in the line#349, page 14.

11) How Inonomycin and cyclosporin A affect autophagy flux and mTOR activity? It has been reported that Ionomycin

results in massive accumulation of autophagosome (Hansen M et al., Mol Cell 2007), could be a characteristic of

impaired autophagy. It is important to show that how ionomycin and cyclosporinA affects autophagy flux by using tf-

LC3 system. Could they do an assay to compare the effect of Ionomycin and Cyclosporin A with TJ-35, this could be

important for the claim that TJ-35 inhibits calcium dependent autophagosome formation.

12) To support the conclusion drawn from Figure 8A, authors should include a calcium free medium

in their experiment.

13) I believe that it should be “calcium efflux from the ER” not “calcium influx from the ER” as mentioned in the line #412,

page 16. Author should correct this wherever possible in the manuscript text and figure legend.

14) Authors should design an assay to conclude that the effect of the TJ-35 on autophagy activity requires all the five

ingredients and try to explain why exact combination of all ingredient is essential. Alternatively, authors should

perform assay to find out which ingredient of TJ-35 is most essential in modulating autophagy activity by applying the

principle of exclusion and discuss the direction for identification/purification of novel molecules present in the

ingredients of TJ-35 that could modulate calcium dependent autophagy.

**********

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Reviewer #1: No

Reviewer #2: No

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Starvation-induced autophagy via calcium-dependent TFEB dephosphorylation is  suppressed by Shigyakusan.

PONE-D-19-29517R1

Dear Dr. Noda,

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

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

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With kind regards,

Vladimir Trajkovic

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No