Dear Dr Lim,

Thank you for submitting the revision of your manuscript entitled "LSM12 -EPAC1 Defines a Neuroprotective Pathway that Sustains the Nucleocytoplasmic RAN Gradient" for consideration as a Research Article by PLOS Biology.

I have checked the revision and consulted with the Academic Editor, and I am writing to let you know that we would like to send your submission out for external peer review. As this is a new submission, we need you to complete your submission by providing again the metadata that is required for full assessment. To this end, please login to Editorial Manager where you will find the paper in the 'Submissions Needing Revisions' folder on your homepage. Please click 'Revise Submission' from the Action Links and complete all additional questions in the submission questionnaire.

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Kind regards,

Ines

--

Ines Alvarez-Garcia, PhD

Senior Editor

PLOS Biology

ialvarez-garcia@plos.org

Carlyle House, Carlyle Road

Cambridge, CB4 3DN

Dear Dr Lim,

Thank you very much for submitting a revised version of your manuscript "LSM12-EPAC1 Defines a Neuroprotective Pathway that Sustains the Nucleocytoplasmic RAN Gradient" for consideration as a Research Article at PLOS Biology. This revised version of your manuscript has been evaluated by the PLOS Biology editors, the Academic Editor and the three original reviewers.

You will see that the reviewers think the manuscript is very much improved and both Reviewers 1 and 3 are mostly satisfied. Reviewer 2, however, still raises two concerns that should be addressed before we can consider the manuscript for publication. One is the fact that a single C9 iPS cell line is used in the experiments and that this is below the standards in the field, thus we deem essential that more than one iPSC line is used for the experiments. In addition, you will need to show that LSM12-EPAC1 rescue neuronal death or neurodegenerative phenotypes, as this reviewer requests.

In light of the reviews (attached below), we will not be able to accept the current version of the manuscript, but we would welcome re-submission of a much-revised version that takes into account the reviewers' comments. We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent for further evaluation by the reviewers.

We expect to receive your revised manuscript within 3 months.

Please email us (plosbiology@plos.org) if you have any questions or concerns, or would like to request an extension. At this stage, your manuscript remains formally under active consideration at our journal; please notify us by email if you do not intend to submit a revision so that we may end consideration of the manuscript at PLOS Biology.

**IMPORTANT - SUBMITTING YOUR REVISION**

Your revisions should address the specific points made by each reviewer. Please submit the following files along with your revised manuscript:

1. A 'Response to Reviewers' file - this should detail your responses to the editorial requests, present a point-by-point response to all of the reviewers' comments, and indicate the changes made to the manuscript.

*NOTE: In your point by point response to the reviewers, please provide the full context of each review. Do not selectively quote paragraphs or sentences to reply to. The entire set of reviewer comments should be present in full and each specific point should be responded to individually, point by point.

You should also cite any additional relevant literature that has been published since the original submission and mention any additional citations in your response.

2. In addition to a clean copy of the manuscript, please also upload a 'track-changes' version of your manuscript that specifies the edits made. This should be uploaded as a "Related" file type.

*Re-submission Checklist*

When you are ready to resubmit your revised manuscript, please refer to this re-submission checklist: https://plos.io/Biology_Checklist

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Please make sure to read the following important policies and guidelines while preparing your revision:

*Published Peer Review*

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. Please see here for more details:

https://blogs.plos.org/plos/2019/05/plos-journals-now-open-for-published-peer-review/

*PLOS Data Policy*

Please note that as a condition of publication PLOS' data policy (http://journals.plos.org/plosbiology/s/data-availability) requires that you make available all data used to draw the conclusions arrived at in your manuscript. If you have not already done so, you must include any data used in your manuscript either in appropriate repositories, within the body of the manuscript, or as supporting information (N.B. this includes any numerical values that were used to generate graphs, histograms etc.). For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5

*Blot and Gel Data Policy*

We require the original, uncropped and minimally adjusted images supporting all blot and gel results reported in an article's figures or Supporting Information files. We will require these files before a manuscript can be accepted so please prepare them now, if you have not already uploaded them. Please carefully read our guidelines for how to prepare and upload this data: https://journals.plos.org/plosbiology/s/figures#loc-blot-and-gel-reporting-requirements

*Protocols deposition*

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosbiology/s/submission-guidelines#loc-materials-and-methods

Thank you again for your submission to our journal. We hope that our editorial process has been constructive thus far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Ines

--

Ines Alvarez-Garcia, PhD,

Senior Editor,

ialvarez-garcia@plos.org,

PLOS Biology

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Reviewers’ comments

Rev. 1:

The authors have addressed all my concerns. I am happy with the manuscript in its form.

Rev. 2:

This manuscript from Lee and colleagues is much improved from the initial submission. Unfortunately, though, the new and potentially exciting data in iPS neurons does not meet minimum standards of rigor for publication. Only a single C9 iPS cell line is used, far less than the typical 5 line minimum considered standard in the field. Furthermore, there is no data in this cell line characterizing the number of G4C2 repeats in the C9-HRE line or in the corrected c9orf72 gene (how many G4C2 repeats in the isogenic control?). To my knowledge, this line has not been published, even though it is commercially available from Cedars-Sinai. For example, see Coyne et al, Neuron, 2020 which used 20 iPSC cell lines, including one isogenic pair, but did not use this isogenic pair. Additional cell lines are critical given the new data suggesting that LSM12 and EPAC1 expression are reduced, suggesting for the first time in this paper, that these genes may play a role in C9 pathogenesis. If these findings were confirmed in multiple cell lines, this would greatly increase this reviewer's enthusiasm for publication.

A second major concern I still have is the lack of evidence to support the title of the paper "LSM12-EPAC1 Defines a Neuroprotective Pathway that Sustains the Nucleocytoplasmic RAN Gradient". All three reviewers of the initial submission asked the authors for a relatively simple experiment to test their hypothesis: overexpress LSM12 and/or EPAC1 in their Drosophila model of C9-ALS, but the results of this experiment has not been described by the authors - presumably they were unable to perform the experiment or it gave the opposite results that they would have predicted. Instead, they overexpressed these genes in cell lines and show that it restores the Ran gradient and TDP-43 mislocalization. However, this is NOT sufficient evidence to claim that LSM12-EPAC1 are neuroprotective - this would require showing that LSM12-EPAC1 rescue neuronal death or neurodegenerative phenotypes, neither of which are shown in this paper.

Rev. 3:

The authors have submitted a substantially revised manuscript and most of my comments have been answered. The addition of human patient-derived neurons has much improved the manuscript and in its current form the manuscript is suitable for publication in PLos Biology.

I have few minor comments, that would need to be addressed in the manuscript:

1) How long were the patient-derived neurons in culture and when were the experiments performed, DIV? would be good to include in the figure.

2) What was the infection efficiency and were any survival measurements done?

3) Please provide some low magnification images showing large field of view for infection of neurons in Supplementary figure

4) Was any Filter trap assays performed on patient-derived neurons to measure soluble versus insoluble Poly-GR aggregates

The manuscript has a found a novel and interesting pathway that serves as a disease pathway and these findings are of interest to the field.

Dear Dr Lim,

Thank you for submitting the new revised version of your Research Article entitled "LSM12-EPAC1 Defines a Neuroprotective Pathway that Sustains the Nucleocytoplasmic RAN Gradient" for publication in PLOS Biology. I have now obtained advice from the Academic Editor and discussed the revision with the team of editors.

We're delighted to let you know that we're now editorially satisfied with your manuscript. However before we can formally accept your paper and consider it "in press", we also need to ensure that your article conforms to our guidelines. A member of our team will be in touch shortly with a set of requests. As we can't proceed until these requirements are met, your swift response will help prevent delays to publication. Please also make sure to address the data and other policy-related requests noted at the end of this email.

To submit your revision, please go to https://www.editorialmanager.com/pbiology/ and log in as an Author. Click the link labelled 'Submissions Needing Revision' to find your submission record. Your revised submission must include the following:

- a cover letter that should detail your responses to any editorial requests, if applicable

- a Response to Reviewers file that provides a detailed response to the reviewers' comments (if applicable)

- a track-changes file indicating any changes that you have made to the manuscript.

*Copyediting*

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NOTE: If Supporting Information files are included with your article, note that these are not copyedited and will be published as they are submitted. Please ensure that these files are legible and of high quality (at least 300 dpi) in an easily accessible file format. For this reason, please be aware that any references listed in an SI file will not be indexed. For more information, see our Supporting Information guidelines:

https://journals.plos.org/plosbiology/s/supporting-information

*Published Peer Review History*

Please note that you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. Please see here for more details:

https://blogs.plos.org/plos/2019/05/plos-journals-now-open-for-published-peer-review/

*Early Version*

Please note that an uncorrected proof of your manuscript will be published online ahead of the final version, unless you opted out when submitting your manuscript. If, for any reason, you do not want an earlier version of your manuscript published online, uncheck the box. Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us as soon as possible if you or your institution is planning to press release the article.

*Protocols deposition*

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosbiology/s/submission-guidelines#loc-materials-and-methods

Please do not hesitate to contact me should you have any questions.

Sincerely,

Ines

--

Ines Alvarez-Garcia, PhD,

Senior Editor,

ialvarez-garcia@plos.org,

PLOS Biology

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DATA POLICY: IMPORTANT - PLEASE READ

You may be aware of the PLOS Data Policy, which requires that all data be made available without restriction: http://journals.plos.org/plosbiology/s/data-availability. For more information, please also see this editorial: http://dx.doi.org/10.1371/journal.pbio.1001797

Note that we do not require all raw data. Rather, we ask that all individual quantitative observations that underlie the data summarized in the figures and results of your paper be made available in one of the following forms:

1) Supplementary files (e.g., excel). Please ensure that all data files are uploaded as 'Supporting Information' and are invariably referred to (in the manuscript, figure legends, and the Description field when uploading your files) using the following format verbatim: S1 Data, S2 Data, etc. Multiple panels of a single or even several figures can be included as multiple sheets in one excel file that is saved using exactly the following convention: S1_Data.xlsx (using an underscore).

2) Deposition in a publicly available repository. Please also provide the accession code or a reviewer link so that we may view your data before publication.

Regardless of the method selected, please ensure that you provide the individual numerical values that underlie the summary data displayed in the following figure panels as they are essential for readers to assess your analysis and to reproduce it:

Fig. 1B-E; Fig. 2B, D; Fig. 3B, D, F, G; Fig. 4B, D, F; Fig. 5A, B, C, D, F, H; Fig. 6B, D, F, H, J; Fig. 7A, B, D, F, H; Fig. 8D, F; Fig. S1A, C; Fig. S2B, C; Fig. S3A, B; Fig. S4B, C; Fig. S5B; Fig. S6; Fig. S7A, C; Fig. S8B, D, E; Fig. S9B, C; Fig. S10A; Fig. S11C; Fig. S12 and Fig. S14B

We note that you were planing to deposit the RNA and TRAP sequencing data in GEO, so please do so and let us know the GEO number.

NOTE: the numerical data provided should include all replicates AND the way in which the plotted mean and errors were derived (it should not present only the mean/average values).

Please also ensure that figure legends in your manuscript include information on WHERE THE UNDERLYING DATA CAN BE FOUND, and ensure your supplemental data file/s has a legend.

Please ensure that your Data Statement in the submission system accurately describes where your data can be found.

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BLOT AND GEL REPORTING REQUIREMENTS:

For manuscripts submitted on or after 1st July 2019, we require the original, uncropped and minimally adjusted images supporting all blot and gel results reported in an article's figures or Supporting Information files. We will require these files before a manuscript can be accepted so please prepare and upload them now. Please carefully read our guidelines for how to prepare and upload this data: https://journals.plos.org/plosbiology/s/figures#loc-blot-and-gel-reporting-requirements

Dear Dr Lim,

On behalf of my colleagues and the Academic Editor, Gillian P Bates, I am pleased to inform you that we will be delighted to publish your Research Article in PLOS Biology.

PRODUCTION PROCESS

Before publication you will see the copyedited word document (within 5 business days) and a PDF proof shortly after that. The copyeditor will be in touch shortly before sending you the copyedited Word document. We will make some revisions at copyediting stage to conform to our general style, and for clarification. When you receive this version you should check and revise it very carefully, including figures, tables, references, and supporting information, because corrections at the next stage (proofs) will be strictly limited to (1) errors in author names or affiliations, (2) errors of scientific fact that would cause misunderstandings to readers, and (3) printer's (introduced) errors. Please return the copyedited file within 2 business days in order to ensure timely delivery of the PDF proof.

If you are likely to be away when either this document or the proof is sent, please ensure we have contact information of a second person, as we will need you to respond quickly at each point. Given the disruptions resulting from the ongoing COVID-19 pandemic, there may be delays in the production process. We apologise in advance for any inconvenience caused and will do our best to minimize impact as far as possible.

EARLY VERSION

The version of your manuscript submitted at the copyedit stage will be posted online ahead of the final proof version, unless you have already opted out of the process. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.

PRESS

We frequently collaborate with press offices. If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximise its impact. If the press office is planning to promote your findings, we would be grateful if they could coordinate with biologypress@plos.org. If you have not yet opted out of the early version process, we ask that you notify us immediately of any press plans so that we may do so on your behalf.

We also ask that you take this opportunity to read our Embargo Policy regarding the discussion, promotion and media coverage of work that is yet to be published by PLOS. As your manuscript is not yet published, it is bound by the conditions of our Embargo Policy. Please be aware that this policy is in place both to ensure that any press coverage of your article is fully substantiated and to provide a direct link between such coverage and the published work. For full details of our Embargo Policy, please visit http://www.plos.org/about/media-inquiries/embargo-policy/.

Thank you again for submitting your manuscript to PLOS Biology and for your support of Open Access publishing. Please do not hesitate to contact me if I can provide any assistance during the production process.

Kind regards,

Erin O'Loughlin

Publishing Editor,

PLOS Biology

on behalf of

Ines Alvarez-Garcia,

Senior Editor

PLOS Biology