L444P Gba1 mutation increases formation and spread of α-synuclein deposits in mice injected with mouse α-synuclein pre-formed fibrils

Anna Migdalska‐Richards; Michal Wegrzynowicz; Ian F. Harrison; Guglielmo Verona; Vittorio Bellotti; Maria Grazia Spillantini; Anthony H. V. Schapira

doi:10.1371/journal.pone.0238075

Peer Review History

Original SubmissionMay 13, 2020
4 Jun 2020 Decision Letter - David R Borchelt, Editor PONE-D-20-14240 L444P Gba1 mutation increases formation and spread of α-synuclein deposits in mice injected with mouse α-synuclein pre-formed fibrils PLOS ONE Dear Dr. Schapira, 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. Below you will find comments from the Academic editor and an expert reviewer. The editor and reviewer are both concerned about the relatively small number of L444P/+ animals that were analyzed, and there are some questions about statistical methods or interpretation. The reviewer suggests validation of the histology data with a second antibody, which would be optional for the authors in accord with journal policy. Please submit your revised manuscript by Jul 19 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols We look forward to receiving your revised manuscript. Kind regards, David R Borchelt Academic Editor PLOS ONE Additional Editor Comments: The manuscript is approaching the bare minimum of a publishable unit of information. Can the authors provide a justification for the low "n" number for L444P/+ mice that were injected an analyzed? Four is a relatively small number. I am not entirely convinced that the differences in pathology between genotypes is so robust than an n of 4 is really sufficient to draw hard conclusions. The data in Figure 2D and E should be presented as scatter plots so that the reader can visualize the variability in the data. On page 8, line 182 the authors state that "the number of p-a-Syn deposits was elevated in the striatum of L444P/+ mice compared to WT littermates, but this was not statistically significant". These two phrases are mutually exclusive. If the statistics do not confirm that the level of deposits was elevated, then one can not make the statement as written. Because you have unequal sample sizes, it is necessary to do a post hoc test of validity. If the variances in the data sets are similar, then you can use Tukey-Kramer or Fisher's test among other possibilities. If the variances in the data are not equal, then there are other post hoc tests that must be used. With the journals making it relatively painless to provide supplementary data, it would be useful to show some images of the absence of pathology in the controls as supplemental data. Similarly, with an N of only 4, more images of pathology could be provided as supplemental figures. The resolution of the supplemental images is relatively low (even after downloading the better quality image). Journal requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ 3. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data. [Note: HTML markup is below. Please do not edit.] 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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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 ****** 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 ****** 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 is a manuscript by Migdalska-Richards and colleagues reporting that a single injection of pre-formed mouse α-synuclein fibrils into the striatum of GBA1 mice that carry a L444P knock-in mutation compared to wild-type littermates result in a greater formation and spread of α-synuclein inclusions. The following issues need to be addressed: 1) Why were only 4 L444P/+ mice used compared to 10 wild-type littermates? 2) Only one antibody to pSer129 was used for immunostaining. For proper scientific validation and rigor, the findings should be confirmed with at least one addition antibody, preferably not to pSer129. 3) In Figure 2E, the quantification of the motor and cingulate cortices were combined. These should be separate. ****** 6. 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 [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0238075.r001
Revision 1
27 Jul 2020 Author Response We thank both the Editor and Reviewer for their comments. Answers to both the Editor and Reviewer: 1. Why the relatively small number of L444P/+ animals that were analysed? We used a relatively small number of L444P/+ mice based on previous publications concerning pre-formed fibril injections and our own published data. Luk, KC, Kehm V, Carroll J, Zhang B, O’Brien P, Trojanowski JQ, et al. Pathological α-synuclein transmission initiates Parkinson’s-like neurodegeneration in nontransgenic mice. Science. 2012a 338(6109):949-53. In this paper, 3–7 animals were examined per group. Luk, KC, Kehm VM, Zhang B, O'Brien P, Trojanowski JQ, Lee VM. Intracerebral inoculation of pathological α-synuclein initiates a rapidly progressive neurodegenerative α-synucleinopathy in mice. J Exp Med. 2012b 209(5):975-86. In this paper, 3–12 animals were examined per group. Migdalska-Richards AM, Wegrzynowicz M, Rusconi R, Deangeli G, Di Monte DA, Spillantini MG, et al. The L444P Gba1 mutation enhances α-synuclein induced loss of nigral dopaminergic neurons in mice. Brain. 2017 140:2706-2721. In this paper, 3–6 animals were examined per group. Based on the number of mice used in each of these 3 publications (please see above) and that with careful measurements, the number was (and is) expected to produce a statistically significant result. Also, as our funders (PUK) and institution (UCL) are committed to the 3Rs, the minimum number of animals was entered into the design. Answers to the Editor: 1. The data in Figure 2D and E should be presented as scatter plots so that the reader can visualize the variability in the data. The figures were changed accordingly. 2. On page 8, line 182 the authors state that "the number of p-a-Syn deposits was elevated in the striatum of L444P/+ mice compared to WT littermates, but this was not statistically significant". These two phrases are mutually exclusive. If the statistics do not confirm that the level of deposits was elevated, then one cannot make the statement as written. The statement was changed to: “There was a trend for an increase in p-αSYN deposits in the striatum of L444P/+ mice compared to WT littermates, but this was not statistically significant.” 3. Because you have unequal sample sizes, it is necessary to do a post hoc test of validity. If the variances in the data sets are similar, then you can use Tukey-Kramer or Fisher's test among other possibilities. If the variances in the data are not equal, then there are other post hoc tests that must be used. Typically, a post hoc test of validity is applied when ANOVA test is used to calculate a statistical significance. However, post hoc tests are not commonly used when t-Test is used to calculate a statistical significance. t-Test is commonly used to compare statistical changes between two groups, while ANOVA is typically to compare statistical changes among more than two groups. As we only had two groups, we chose t-Test for our analysis. We checked the equality of variances using Levene's test and we did not find any statistically significant changes between any analysed groups (p=0.304 for cortex and p=0.884 for striatum). We checked the data normality using Shapiro-Wilk test and found the normal distribution in all analysed groups (p=0.579 for WT/cortex, p=0.716 for L444P/cortex, p=0.052 for WT/striatum and p=0.959 for L444P/striatum). Thus, after confirming the normal distribution and the equality of variances, we could compare all the groups. 4. With the journals making it relatively painless to provide supplementary data, it would be useful to show some images of the absence of pathology in the controls as supplemental data. Similarly, with an N of only 4, more images of pathology could be provided as supplemental figures. The Supplementary Figure 3 was generated to show the absence of p-αSYN pathology in PBS-injected control mice. The Supplementary Figure 4 and 5 were generated to provide additional images showing the presence of p-αSYN pathology in αSYN-PFF-injected L444P/+ mice. 5. The resolution of the supplemental images is relatively low (even after downloading the better quality image). The better-quality images were generated. Answers to the Reviewer: 1. Only one antibody to pSer129 was used for immunostaining. For proper scientific validation and rigor, the findings should be confirmed with at least one addition antibody, preferably not to pSer129. We used a well validated antibody for pSer129 (see publications above), and we are unclear as to why another antibody not to pSer129 would have been of value. 2. In Figure 2E, the quantification of the motor and cingulate cortices were combined. These should be separate. In the mouse, the cingulate and motor cortices are adjacent and the border between them often indistinct, making separate counting inappropriate. Therefore, for the purpose of illustrating pathology (see Figure 2) we selected representative sections taken from the periphery of the respective areas away from their border. However, for the purpose of quantitation we combined the two adjacent areas of cingulate and motor cortices to avoid mis-appropriation of data between the two. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0238075.r002
10 Aug 2020 Decision Letter - David R Borchelt, Editor L444P Gba1 mutation increases formation and spread of α-synuclein deposits in mice injected with mouse α-synuclein pre-formed fibrils PONE-D-20-14240R1 Dear Dr. Schapira, 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, David R Borchelt Academic Editor PLOS ONE Additional Editor Comments (optional): Although Reviewer 1 remains dissatisfied, the authors have addressed all of the technical comments and provide an adequate justification for the animal numbers. Personally, I would still like to have seen a larger N for the transgenics, but I can't argue with the justification. 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: (No Response) ******** 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: Partly ****** 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: This is a resubmitted manuscript by Migdalska-Richards and colleagues. Some simple but scientifically important changes were requested that have not been addressed. 1) It was requested that for proper scientific validation and rigor, the findings be confirmed with at least one additional antibody that would preferably be against a difference epitope thus not another pSer129 specific antibody. Using more than one antibody for immunocytochemical validation is a standard pathological practice. The authors simply refused this request. 2) It was requested that the quantification of the motor and cingulate cortices be separated as these are distinct neuroanatomical structures with distinct functions. The authors refused to do this simple request and their explanation is that it is not appropriate as they claim that there can distinguish between these regions when taking images but that they could not do the same for quantification. 3) It is still not explained why N=10 wild-type littermates and only N =4 L444P/+ mice was used. Why the imbalance in the number of mice used? ****** 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 https://doi.org/10.1371/journal.pone.0238075.r003
Formally Accepted
13 Aug 2020 Acceptance Letter - David R Borchelt, Editor PONE-D-20-14240R1 L444P Gba1 mutation increases formation and spread of α-synuclein deposits in mice injected with mouse α-synuclein pre-formed fibrils Dear Dr. Schapira: 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. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Prof. David R Borchelt Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0238075.r004

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