Functional NHE1 expression is critical to blood brain barrier integrity and sumatriptan blood to brain uptake

Erika Liktor-Busa; Kiera T. Blawn; Kathryn L. Kellohen; Beth M. Wiese; Vani Verkhovsky; Jared Wahl; Anjali Vivek; Seph M. Palomino; Thomas P. Davis; Todd W. Vanderah; Tally M. Largent-Milnes

doi:10.1371/journal.pone.0227463

Peer Review History

Original SubmissionDecember 16, 2019
20 Jan 2020 Decision Letter - Eliseo A Eugenin, Editor PONE-D-19-34794 Functional NHE1 Expression is Critical to Blood Brain Barrier Integrity and Sumatriptan Blood to Brain Uptake PLOS ONE Dear Largent, 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. We would appreciate receiving your revised manuscript by Mar 05 2020 11:59PM. When you are 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. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols 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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'. 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. We look forward to receiving your revised manuscript. Kind regards, Eliseo A Eugenin, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (if provided): Dear Dr. Largent Thank you for submit your manuscript to PLOSone. The reviewers had positive comments about your work and suggest some changes that need to be included. Please send your manuscript back with the changes requested Best Regards Eliseo 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 http://www.plosone.org/attachments/PLOSOne_formatting_sample_main_body.pdf and http://www.plosone.org/attachments/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions. [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: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes 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: 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 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 is an interesting work evaluating the mechanisms of cortical spreading depolarization, a phenomenon linked to migraines. The work is solid and provides a number of significant insights that will be of interest to a broad neuroscience community. I have a few comments that require attention prior to further consideration of the work for publication in PLOS One. a) The following statement outlining the overall goal of the study “Given that pH is a known regulator of 1) BBB integrity [30-32] and 2) transport of xenobiotics across the BBB [33,34] and that CSD induces regional perturbations in pH [35], the overall goals of the studies herein focused on NHE1 as a molecular target implicated in cortical KCl-induced deficiencies in BBB integrity resulting from pH dysfunction and regulation of sumatriptan blood to brain uptake” is challenging to parse out. It would be helpful to clarify in series the specific hypothesis of the paper. b) As I understood it, the work is primarily focused on the role of NHE1 on the BBB integrity. However, the bulk of the mechanistic observations are made using cell culture assays, not in freshly dissected intact tissue or in vivo. There is a certain value of these model systems. In particular, induction of depolarization with KCl pulse along with a sucrose assay in endothelial cell monolayers is robust and reproducible. Having said that, this simplistic model system fails to replicate the precise architecture of the endothelial/pericyte system of the intact BBB and how it may be affected in the pathology. This limitation is my major concern and at least should be acknowledged and addressed in the Discussion. At best, the authors may want to address this by adding new experiment within a section starting on line 420 by testing the BBB integrity in vivo with either live Evan Blue assay or postmortem evaluation of plasma albumin, following the induction of CSD with KCl. I understand that these are extensive and technically challenging experiments that may be outside the scope of this work. Nevertheless, these would establish a direct link between CSD –BBB failure – a therapeutic action of NHE1 blockade. c) Not clear the meaning of the lines 87-88: “…highlights that comprise of NHE1 may have differential effects on BBB 88 integrity versus xenobiotic uptake.” d) Lines 357. Please provide a premise for using a sucrose uptake assay to test BBB integrity in endothelial cell monolayer. e) The use of both pharmacological and genetic targeting of NHE1 and evaluation of pain are strength. Reviewer #2: The manuscript, “Functional NHE1 Expression is Critical to Blood-Brain Barrier Integrity and Sumatriptan Blood to Brain Uptake”, Liktor-Busa et al. describe the dynamic role of sodium-hydrogen exchanger type 1 (NHE1) in blood-brain barrier (BBB) 31 integrity during cortical spreading depression events and the contributions of this antiporter on xenobiotic uptake. This is very interesting work and clinically relevant. The main strength of this study is that it is technically strong. However, I have a minor concern on the expression of NHE1 in Figure 4C and 4D. It would be in a better readable form if the authors replace these western blotting figures. Another concern is that the authors have given 4 supplementary figures. If the journal has an option to give these figures as main figures, it would be better to include as main figures. ****** 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: Yes: Botir T Sagdullaev Reviewer #2: 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 to be viewed.] 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 us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0227463.r001
Revision 1
4 Mar 2020 Author Response Re: Revision of PONE-D-19-34794 Title: Functional NHE1 Expression is Critical to Blood Brain Barrier Integrity and Sumatriptan Blood to Brain Uptake Response to Referees: The authors wish to thank the reviewers for their remarks and support for the research/manuscript. Below we have addressed individual concerns/comments in bold. Italicized points are authors’ interpretation of key reviewer concerns. Within the text of the manuscript, we have highlighted revision in red font. Reviewer #1: A) The following statement outlining the overall goal of the study “Given that pH is a known regulator of 1) BBB integrity [30-32] and 2) transport of xenobiotics across the BBB [33,34] and that CSD induces regional perturbations in pH [35], the overall goals of the studies herein focused on NHE1 as a molecular target implicated in cortical KCl-induced deficiencies in BBB integrity resulting from pH dysfunction and regulation of sumatriptan blood to brain uptake” is challenging to parse out. It would be helpful to clarify in series the specific hypothesis of the paper. Answer: As the reviewer suggested, we clarified the specific hypothesis in the revised version of the paper. B) As I understood it, the work is primarily focused on the role of NHE1 on the BBB integrity. However, the bulk of the mechanistic observations are made using cell culture assays, not in freshly dissected intact tissue or in vivo. There is a certain value of these model systems. Induction of depolarization with KCl pulse along with a sucrose assay in endothelial cell monolayers is robust and reproducible. Having said that, this simplistic model system fails to replicate the precise architecture of the endothelial/pericyte system of the intact BBB and how it may be affected in the pathology. 1) This limitation is my major concern and at least should be acknowledged and addressed in the Discussion. At best, the authors may want to address this by 2) adding new experiment within a section starting on line 420 by testing the BBB integrity in vivo with either live Evan Blue assay or postmortem evaluation of plasma albumin, following the induction of CSD with KCl. I understand that these are extensive and technically challenging experiments that may be outside the scope of this work. Nevertheless, these would establish a direct link between CSD –BBB failure – a therapeutic action of NHE1 blockade. Answers: B1) We agree with the reviewer that in vitro model systems of BBB are not able to fully replicate the precise architecture of the intact BBB. Despite their limitations, several different forms of cell-based systems are widely used and accepted model of BBB. In the current study, we used endothelial cells cultured in astrocyte-conditioned media in order to promote endothelial growth, differentiation and mimic the in vivo neurovascular unit. Our former data (Cottier et al. Loss of Blood-Brain Barrier Integrity in a KCl-Induced Model of Episodic Headache Enhances CNS Drug Delivery. eNeuro 2018; 5:ENEURO.0116-18.2018.) indicated that cortical KCl injection induced paracellular BBB leakiness to radiolabeled sucrose in the cortex, as assessed by in situ brain perfusion. We also observed an increase in BBB permeability to sumatriptan in both the cortex as well as the brainstem. The elevated permeability of sucrose and sumatriptan, observed in those experiment corresponded well with our in vitro data, presented in the current manuscript. B2) We appreciate the recommendation of the reviewer for “adding new experiment within a section starting on line 420 by testing the BBB integrity in vivo with either live Evan Blue assay or postmortem evaluation of plasma albumin, following the induction of CSD with KCl”. We have previously found in Cottier et al., citation above, that paracellular permeability to sucrose was detected. The ringer in this experiment contained Evan’s Blue Albumin; parenchymal uptake of EBA was not detected after cortical KCl injection to induce CSD. To delineate the link between CSD-BBB failure and NHE1 blockade as requested, we investigated FITC-dextran (4000 Da) transport in aCSF and KCl injected rats in the presence of either vehicle or zoniporide. Tissue was harvested 90 after cortical injection for consistency with other data in the current submission as well as Cottier et al., 2018. Parenchymal detection of FITC fluorescence in tissue samples was below the limit of detection (20 ng/mL) for all groups (naïve, saline-aCSF, saline-KCl, zoniporide-aCSF, and zoniporide-KCl), which suggest that BBB breaches in vivo after cortical KCl injections do not allow passage of molecules larger than 4kDa (please see method at line 149, results at line 535). Moreover we investigated the expression level of NHE1 in cortical microvessels, isolated from naïve, aCSF, and KCl-treated animals (see in Fig 7D). We did not observe significant differences between aCSF and KCl-treated animals, but the total expression of NHE1 was significantly increased 90 min after cortical KCl injection compared to naïve. C) Not clear the meaning of the lines 87-88: “…highlights that comprise of NHE1 may have differential effects on BBB 88 integrity versus xenobiotic uptake.” Answer: We refined this sentence in the revised version. D) Lines 357. Please provide a premise for using a sucrose uptake assay to test BBB integrity in endothelial cell monolayer. Answer: As mentioned above, radiolabeled sucrose was used in our former, in situ brain perfusion study (Cottier et al., 2018) to investigate changes in BBB paracellular permeability after CSD induction. Sucrose was applied in those and the current study as well, as a model molecule without known transcellular transporter. Therefore, it can represent the paracellular transport, and the paracellular leakage of BBB. We stated the rationale of using sucrose in the revised version of the manuscript. Reviewer #2: A) “However, I have a minor concern on the expression of NHE1 in Figure 4C and 4D. It would be in a better readable form if the authors replace these western blotting figures.” Answer: As the reviewer suggested we replaced the representative Western blots within Fig 6C and 6D (Original Fig. number is 4, new number is 6). We would like to note that after quantification of a new set of TG samples, the expression of NHE1 after KCl treatment was significantly increased, as we indicated in the graph. B) “Another concern is that the authors have given 4 supplementary figures. If the journal has an option to give these figures as main figures, it would be better to include as main figures.” Answer: Having consulted the editorial office on the policy for Supplemental Information, we have moved the former S2, S3, and S4 figures to main figures and left S1 and S5 as supplemental figures. We would like to note that our blot/gel image data with other raw data can be found in Supporting Information file. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0227463.r002
5 May 2020 Decision Letter - Eliseo A Eugenin, Editor Functional NHE1 Expression is Critical to Blood Brain Barrier Integrity and Sumatriptan Blood to Brain Uptake PONE-D-19-34794R1 Dear Dr. Largent-Milnes 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. Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. 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 enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and 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. With kind regards, Eliseo A Eugenin, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Thank you for a great re-submission and for taking the time and effort to answer all the questions and concerns Best regards Eliseo 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 Reviewer #3: 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 Reviewer #3: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #3: 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 Reviewer #3: 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 Reviewer #3: 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) Reviewer #3: This revised version of the manuscript by Liktor-Busa and colleagues has properly addressed the major concerns and comments raised in the first review round. The authors improved the western blot figure 4 and performed new experiments in order to demonstrate BBB integrity in vivo, particularly, using FITC-dextran transport in aCSF and KCl injected rats. I have not further comments and the manuscript should be published as it is. ****** 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: Yes: Botir T Sagdullaev Reviewer #3: Yes: Juan A. Orellana https://doi.org/10.1371/journal.pone.0227463.r003
Formally Accepted
15 May 2020 Acceptance Letter - Eliseo A Eugenin, Editor PONE-D-19-34794R1 Functional NHE1 Expression is Critical to Blood Brain Barrier Integrity and Sumatriptan Blood to Brain Uptake Dear Dr. Largent-Milnes: I am 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 notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, 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. For any other questions or concerns, please email plosone@plos.org. Thank you for submitting your work to PLOS ONE. With kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Eliseo A Eugenin Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0227463.r004

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .