Calcium phosphate formation and deposition in ischemic neurons

Shu Q. Liu; John B. Troy; Jeremy Goldman; Roger J. Guillory

doi:10.1371/journal.pone.0317055

Peer Review History

Original SubmissionMarch 23, 2024
29 Apr 2024 Decision Letter - Alexander G Obukhov, Editor PONE-D-24-11802Calcium phosphate formation and deposition in ischemic neuronsPLOS ONE Dear Dr. Liu, 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. Additionally, please provide the uncropped images of all Western blots in supplemental figures. Please also show the molecular weights of all products on all Western blots. Please submit your revised manuscript by Jun 13 2024 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. 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Please include your full ethics statement in the ‘Methods’ section of your manuscript file. In your statement, please include the full name of the IRB or ethics committee who approved or waived your study, as well as whether or not you obtained informed written or verbal consent. If consent was waived for your study, please include this information in your statement as well. [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 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: 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: Review of Manuscript Number: PONE-D-24-11802 The manuscript entitled “Calcium phosphate formation and deposition in ischemic neurons”, by Shi Q. Liu, John B Troy, Jeremy B Goldman and Roger J. Guillory, describes a potential mechanism for the formation of calcium phosphate precipitates in cortical neurons post-ischemia. This manuscript proposes a compelling mechanism where several annexins form complexes with CaP, in a cytoskeletal dependent manner. However, the argument the author makes would be strengthened by providing more evidence to support and clarify the logic of their proposed mechanism. Some key assumptions require further justification and /or clarification. Concerns: 1) In the current literature intraneuronal CaP precipitates are considered a post-ischemic protective step of calcium homeostasis. How do the authors reconcile this view with their conclusion that preventing CaP deposition during ischemia can be protective and a potential treatment strategy, particularly while the plasma membrane integrity is still compromised (see Figure 13 C)? 2) Using a biochemistry approach the authors conclude that annexins translocate from the cell membrane to the cytoskeleton. However, they also conclude that when the translocation occurs the plasma membrane integrity is compromised. A leaky plasma membrane is the first step versus irreversible cell death and disassembly of the cytoskeleton. Indeed, recently a paper has shown that ischemic cortical neurons display a dramatic increase in their F-actin content in the somatodendritic region, and that this response is inversely correlated to plasma membrane leakage. Can the authors support their conclusion by using an intracellular in situ approach to monitor the interaction between Annexins and the cellular cytoskeleton post-ischemia? For example PLA? 3) In the manuscript is not explained why when the annexins are added to the healthy mouse brains, this resulted in cerebral CaP deposition and Anx binding to the cytoskeleton. Why in these healthy brains the exogenously added Anx wouldn’t just remain bound to the plasma membrane? Why this manipulation would even cause infarction? 4) In Figure 6 the green actin signal is expected to have the same intensity and distribution in all images, but it doesn’t. Why? 5) Figure 11 is the most convincing piece of evidence that annexins are connected to calcium precipitates and infarct size. However, these findings are counterintuitive based on the current knowledge that knocking down annexins would result in plasma membrane damage and excessive calcium influx/levels. 6) The authors should make sure that all citated papers are correctly referenced. For example Liu 2019 cited on page 28 does not have any mention of CaP depositions. Conclusion: Overall, this manuscript represents an informative new set of experiments that provides insights in the connection between Annexins and CaP precipitates. However, by addressing the limitations outlined above, the authors could strengthen the overall impact of their work. Reviewer #2: This study aims to establish that CaP deposition occurs early after an ischemic stroke and that it participates in the associated injury. The authors propose a mechanism after which specific annexins are crucial for CaP deposition via binding to ß actin filaments. First, the study tries to establish the presence of CaP deposits after ischemia in a mouse model sytem. Then the expression and subcelluar distribution of annexins is investigated by Western blot followed by an analysis of binding of annexins to ß actin. The alteration in distribution of actin and annexins was investigated by immunofluorescence after stroke induction. Furthermore, actin and annexin dependent CaP deposition was investigated. As a behavioral measure, grip strength was investigated and compared under the different conditions for the lesioned and intact site. Finally, the specific effect of particular annexins was investigated by blocking annexin expression by siRNAs. The experiments follow a logical outline and are conclusive. However, the data presented do not convince in all aspects: -the pictures in Figure 3A which shall illustrate an increase in alkaline phosphatase activity are not convincing as the increased staining is hard to distinguish from the background. - in Fig 6, the alteration in distribution of actin and annexins is illustrated by single pictures (representative?) but not quantified. - it remains unclear whether the proposed CaP deposits form in intact cells or only after disruption of the cell membrane, as indicated in Fig 13. If Ca2+ entry and CaP deposition are a consequence of cell membrane damage, prevention of CaP deposition for treatment as proposed in the discussion would be too late. The individual data points underlying the summary statistics are not available. ****** 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: Barbara Calabrese 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.] 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.0317055.r001
Revision 1
19 Sep 2024 Author Response Response to the Reviewers The authors would like to thank the reviewers for providing thoughtful and informative comments to the manuscript. The manuscript has been revised in accordance to the reviewers’ comments. Please note that all revisions are marked in blue in the Word document. The comments are addressed here. Response to Reviewer 1 The manuscript entitled “Calcium phosphate formation and deposition in ischemic neurons”, by Shi Q. Liu, John B Troy, Jeremy B Goldman and Roger J. Guillory, describes a potential mechanism for the formation of calcium phosphate precipitates in cortical neurons post-ischemia. This manuscript proposes a compelling mechanism where several annexins form complexes with CaP, in a cytoskeletal dependent manner. However, the argument the author makes would be strengthened by providing more evidence to support and clarify the logic of their proposed mechanism. Some key assumptions require further justification and /or clarification. Concerns: 1) In the current literature intraneuronal CaP precipitates are considered a post-ischemic protective step of calcium homeostasis. How do the authors reconcile this view with their conclusion that preventing CaP deposition during ischemia can be protective and a potential treatment strategy, particularly while the plasma membrane integrity is still compromised (see Figure 13 C)? Response: Intracellular CaP precipitation may represent a protective mechanism against Ca2+ overload and its cytotoxicity in ischemic neurons [Ramonet D, Pugliese M, Rodríguez MJ, de Yebra L, Andrade C, Adroer R, Ribalta T, Mascort J, Mahy N. Calcium precipitation in acute and chronic brain diseases. J Physiol Paris. 96(3-4):307-312, 2002.]. However, a consensus from a larger number of previous investigations is that the presence of intracellular CaP nanoparticles can cause cell injury and death. For instance, the presence of CaP nanoparticles in human vascular smooth muscle cells can cause cell death [Ewence AE, Bootman M, Roderick HL, Skepper JN, McCarthy G, Epple M, Neumann M, Shanahan CM, Proudfoot D. Calcium phosphate crystals induce cell death in human vascular smooth muscle cells: a potential mechanism in atherosclerotic plaque destabilization. Circ Res. 103(5):e28-34, 2008]. Other studies support this observation [Epple M. Review of potential health risks associated with nanoscopic calcium phosphate. Acta Biomaterialia 77, 1-14, 2018; Z.L. Shi, X. Huang, Y.R. Cai, R.K. Tang, D.S. Yang. Size effect of hydroxyapatite nanoparticles on proliferation and apoptosis of osteoblast-like cells. Acta Biomater., 5 (2009), pp. 338-345; Z. Xu, C. Liu, J. Wei, J. Sun. Effects of four types of hydroxyapatite nanoparticles with different nanocrystal morphologies and sizes on apoptosis in rat osteoblasts. J. Appl. Toxicol., 32 (2012), pp. 429-435; H. Turkez, M.I. Yousef, E. Sonmez, B. Togar, F. Bakan, P. Sozio, A.D. Stefano. Evaluation of cytotoxic, oxidative stress and genotoxic responses of hydroxyapatite nanoparticles on human blood cells. J. Appl. Toxicol., 34 (2014), 373-379; E. Sonmez, I. Cacciatore, F. Bakan, H. Turkez, Y.I. Mohtar, B. Togar, A.D. Stefano. Toxicity assessment of hydroxyapatite nanoparticles in rat liver cell model in vitro. Hum. Exp. Toxicol., 35 (2016), 1073-1083; Y. Jin, X.L. Liu, H.F. Liu, S.Z. Chen, C.Y. Gao, K. Ge, C.M. Zhang, J.C. Zhang. Oxidative stress-induced apoptosis of osteoblastic MC3T3-E1 cells by hydroxyapatite nanoparticles through lysosomal and mitochondrial pathways. RSC Adv., 7 (2017), 13010-13018; Y. Dautova, D. Kozlova, J.N. Skepper, M. Epple, M.D. Bootman, D. Proudfoot. Fetuin-A and albumin alter cytotoxic effects of calcium phosphate nanoparticles on human vascular smooth muscle cells. PLoS One, 9 (2014), e97565]. Based on these observations, it is possible that the impact of CaP formation on cell survivability may depend on the level of free CaP nanoparticles and the degree of CaP deposition to intracellular structures. The presence of a low density of free CaP nanoparticles without significant deposition to intracellular structures may be protective against Ca2+ overload and cytotoxicity; whereas the deposition of a high level of CaP nanoparticles to intracellular structures, especially those critical to cell signaling and integrity, may elicit a harmful impact on cell survivability. β actin filaments support many neuronal functions such as signal transduction, maintenance of cell integrity, regulation of synaptic plasticity, and control of axonal branching and regeneration. In particular, the dynamic assembly and disassembly processes of β actin is essential to neuronal survival [Patrinostro X, Roy P, Lindsay A, Perrin BJ. Essential nucleotide- and protein-dependent functions of Actb/β-actin. PNAS 115 (31) 7973-7978, 2018]. Exposure to phalloidin, a substance that stabilizes actin filaments and prevents the dynamic actin processes, can cause cell death [Cooper, J. A. Effects of cytochalasin and phalloidin on actin. J. Cell. Biol. 105, 1473–1478, 1987]. CaP deposition on β actin filaments, as observed in this investigation, can interfere with these dynamic actin processes, thereby contributing to neuronal injury and death. The points above are addressed in the revised manuscript (page 19 – 20 of the Word document). 2) Using a biochemistry approach the authors conclude that annexins translocate from the cell membrane to the cytoskeleton. However, they also conclude that when the translocation occurs the plasma membrane integrity is compromised. A leaky plasma membrane is the first step versus irreversible cell death and disassembly of the cytoskeleton. Indeed, recently a paper has shown that ischemic cortical neurons display a dramatic increase in their F-actin content in the somatodendritic region, and that this response is inversely correlated to plasma membrane leakage. Can the authors support their conclusion by using an intracellular in situ approach to monitor the interaction between Annexins and the cellular cytoskeleton post-ischemia? For example PLA? Response: In this investigation, an in situ approach was used to demonstrate the interaction between annexins and β actin filaments (Fig. 8 in the manuscript). Freshly prepared healthy mouse brain specimens, instead of ischemic brain specimens, were used for this test. The reason is that annexin – β actin filament interaction occurs in ischemia, making it difficult to distinguish between the newly induced annexin – β actin filament interaction and the ischemia-induced annexin – β actin filament interaction. In this test, individual annexins were added to brain sections in the presence of Ca2+ and PO43-, in the presence or absence of an annexin, in the presence or absence of monomeric β actin (capable of binding to annexins to block annexin – β actin filament interaction), and in the presence or absence of phalloidin. The outcomes can be used to evaluate whether annexins bind to β actin filaments to cause CaP deposition to β actin filaments as well as whether the phalloidin-induced stabilization of β actin filaments interferes with annexin binding to the β actin filaments and CaP deposition. Please note that the total level of β actin was not changed from sham control to ischemic stroke as evaluated by immunoblot analysis in this investigation (Figure 4 in the manuscript). 3) In the manuscript is not explained why when the annexins are added to the healthy mouse brains, this resulted in cerebral CaP deposition and Anx binding to the cytoskeleton. Why in these healthy brains the exogenously added Anx wouldn’t just remain bound to the plasma membrane? Why this manipulation would even cause infarction? Response: The relative level of annexin binding to the cell membrane or β actin filaments may depend on the quantity of exogenous annexins delivered to cells. At a large quantity, the annexin binding sites on the inner face of the cell membrane may all be bound with annexins, and the excessive exogenous free annexins may bind to β actin filaments. Free annexins within the cell can attract calcium ions, which in turn attract phosphate ions to form CaP nanostructures on one side of a disk-like annexin molecule. The other side of the annexin molecule can bind to β actin filaments, resulting in annexin/CaP complex deposition onto the β actin filaments or β actin filament calcification. This process contributes to neuronal death and brain infarction. These points are addressed in the revised manuscript (page 20 of the Word document). 4) In Figure 6 the green actin signal is expected to have the same intensity and distribution in all images, but it doesn’t. Why? Response: This could be a result of variations in experimental preparations and locations for specimen collection among different mice. 5) Figure 11 is the most convincing piece of evidence that annexins are connected to calcium precipitates and infarct size. However, these findings are counterintuitive based on the current knowledge that knocking down annexins would result in plasma membrane damage and excessive calcium influx/levels. Response: It is possible that knocking down annexins could result in plasma membrane damage and excessive calcium influx. Even though additional injury may be induced by annexin siRNA delivery, silencing of the annexin genes resulted in a significant alleviation of CaP deposition and brain infarction, supporting the role of annexins in ischemic neuron calcification and infarction. 6) The authors should make sure that all citated papers are correctly referenced. For example Liu 2019 cited on page 28 does not have any mention of CaP depositions. Response: This error has been corrected. Conclusion: Overall, this manuscript represents an informative new set of experiments that provides insights in the connection between Annexins and CaP precipitates. However, by addressing the limitations outlined above, the authors could strengthen the overall impact of their work. Response: Thanks. The comments have been addressed to our best knowledge. Response to Reviewer 2 This study aims to establish that CaP deposition occurs early after an ischemic stroke and that it participates in the associated injury. The authors propose a mechanism after which specific annexins are crucial for CaP deposition via binding to ß actin filaments. First, the study tries to establish the presence of CaP deposits after ischemia in a mouse model system. Then the expression and subcelluar distribution of annexins is investigated by Western blot followed by an analysis of binding of annexins to ß actin. The alteration in distribution of actin and annexins was investigated by immunofluorescence after stroke induction. Furthermore, actin and annexin dependent CaP deposition was investigated. As a behavioral measure, grip strength was investigated and compared under the different conditions for the lesioned and intact site. Finally, the specific effect of particular annexins was investigated by blocking annexin expression by siRNAs. The experiments follow a logical outline and are conclusive. However, the data presented do not convince in all aspects: -the pictures in Figure 3A which shall illustrate an increase in alkaline phosphatase activity are not convincing as the increased staining is hard to distinguish from the background. Response: Although changes in alkaline phosphatase activity are not apparent enough to be recognized by naked eyes, optical measurements have shown significant changes from the images shown in the manuscript. - in Fig 6, the alteration in distribution of actin and annexins is illustrated by single pictures (representative?) but not quantified. Response: Yes, the images are representative, but all samples for each group showed similar distributions in annexins. The outcomes were not quantified because it is difficult to quantify the distributions of annexins and β actin filaments in neurons by an imaging approach. - it remains unclear whether the proposed CaP deposits form in intact cells or only after disruption of the cell membrane, as indicated in Fig 13. If Ca2+ entry and CaP deposition are a consequence of cell membrane damage, prevention of CaP deposition for treatment as proposed in the discussion would be too late. Response: Multiple factors contribute to cell death. Cell membrane damage is one of these factors, and β actin filament calcification may represent another cell death-inducing factor. Preventing annexin-induced β actin filament calcification may partially alleviate neuronal death. This point is addressed in the revised manuscript (page 19 of the revised manuscript). The individual data points underlying the summary statistics are not available. Response: All raw data are now available in the Supplemental Information section. Attachments Attachment Submitted filename: 2024 Response to Reviewers PlosOne.docx https://doi.org/10.1371/journal.pone.0317055.r002
10 Oct 2024 Decision Letter - Alexander G Obukhov, Editor PONE-D-24-11802R1Calcium phosphate formation and deposition in ischemic neuronsPLOS ONE Dear Dr. Liu, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that your manuscript has merit, but it still requires additional revisions to meet PLOS ONE’s publication criteria. Specifically, Reviewer 2 raised several serious concerns. Therefore, we invite you to submit a revised version of the manuscript that addresses the concerns of Reviewer 2. Please submit your revised manuscript by Nov 24 2024 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: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Alexander G Obukhov, Ph.D. Academic Editor PLOS ONE [Note: HTML markup is below. Please do not edit.] 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 #2: (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: (No Response) Reviewer #2: Partly ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: (No Response) Reviewer #2: 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: (No Response) Reviewer #2: 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: (No Response) Reviewer #2: 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 #2: My concerns were not sufficiently addressed by the response of the authors. - Selecting areas for quantification in Fig 3 appears very arbitrary given the high background. - I do not see, why quantification for Fig 6 distribution of actin and annexins poses a major problem. - Prevention of CaP deposition for treatment as proposed in the discussion would be too late as the cell membranes are already damaged. The response to this criticism does not settle the point. - The primary data were now supplied as supplement. ****** 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 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.] 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.0317055.r003
Revision 2
25 Nov 2024 Author Response Response to Reviewer 2 The authors would like to thank the reviewer for providing insightful comments. The manuscript has been revised based on the reviewer’s concerns. All changes are marked in blue in the revised manuscript. The reviewer’s concerns are addressed here. Reviewer’s concern: My concerns were not sufficiently addressed by the response of the authors. Response: Sorry that the authors did not address fully the reviewer’s concerns. In this revision, these concerns have been carefully addressed and substantial changes have been made accordingly. Reviewer’s concern: - Selecting areas for quantification in Fig 3 appears very arbitrary given the high background. Response: Figure 3A, B and associated data have been removed from the revised manuscript. Results from the remaining p-nitrophenyl phosphate (pNPP) test would be sufficient to support the observation that the activity of alkaline phosphatase was increased in the cortex in response to ischemic stroke. Reviewer’s concern: - I do not see, why quantification for Fig 6 distribution of actin and annexins poses a major problem. Response: The distribution of annexins in reference to that of the β actin filaments has been measured and presented in Fig. 6B. Reviewer’s concern: - Prevention of CaP deposition for treatment as proposed in the discussion would be too late as the cell membranes are already damaged. The response to this criticism does not settle the point. Response: The authors are in agreement with the reviewer. The statement on “prevention of CaP deposition for the treatment of myocardial infarction” has been removed from the Abstract and Discussion sections. The revised manuscript focusses primarily on the molecular mechanisms of calcium phosphate formation and deposition in ischemic neurons. Reviewer’s concern: - The primary data were now supplied as supplement. Response: Thanks. Attachments Attachment Submitted filename: 2024 Response to Reviewer 2.pdf https://doi.org/10.1371/journal.pone.0317055.r004
20 Dec 2024 Decision Letter - Alexander G Obukhov, Editor Calcium phosphate formation and deposition in ischemic neurons PONE-D-24-11802R2 Dear Dr. Liu, 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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, 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, Alexander G Obukhov, Ph.D. Academic Editor PLOS ONE 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 #2: 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 #2: (No Response) ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #2: (No Response) ****** 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 #2: (No Response) ****** 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 #2: (No Response) ****** 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 #2: (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 #2: No ******** https://doi.org/10.1371/journal.pone.0317055.r005
Formally Accepted
7 Jan 2025 Acceptance Letter - Alexander G Obukhov, Editor PONE-D-24-11802R2 PLOS ONE Dear Dr. Liu, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, 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 customercare@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 Dr. Alexander G Obukhov Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0317055.r006

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