PONE-D-19-19628

C5aR agonist enhances phagocytosis of fibrillar and non-fibrillar Aβ amyloid and preserves memory in a mouse model of Familial Alzheimer’s disease

PLOS ONE

Dear Professor Kyriakides,

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.

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Wataru Araki

Academic Editor

PLOS ONE

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2. We note that you have indicated that Avertin was used to sacrifice animals in your study. We would be grateful if you could clarify how death was confirmed in these animals, following administration of Avertin in the dose described. If animals were decapitated or other secondary methods were used to ensure death please include this information in your Methods section.

Please also state whether your ethics committee specifically approved the use of this compound.

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4. Thank you for stating the following in the Competing Interests section:

"The authors E.P. and T.K. have filed a PCT application on the use of the modified C5aR agonist EP67 in cerebral and peripheral amyloidoses (PCT/EP2018/053362)."

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Additional Editor Comments:

Both reviewers raise some methodological concerns, as described in their comments.  I recommend to make an appropriate revision to address these criticisms carefully. Especially, it is necessary to consider seriously about the validity of some immunohistochemical analyses such as synaptophisin immunostaining. In addition, please provide the method of immunohistochemistry more in detail. Also please recheck the data of Fig 3c, in which very large reductions are observed in 5XFAD, compared with WT.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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

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

Reviewer #1: Yes

Reviewer #2: Partly

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

Reviewer #1: Yes

Reviewer #2: Yes

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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: No

Reviewer #2: Yes

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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

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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: Pannayiotou et al, PLOS One

This is a relatively straightforward paper demonstrating amyloid reducing effects of a Complement C5a receptor agonist peptide. In general the paper is written clearly, and the results, as shown, appear to support the hypothesis. However, there are some methodological concerns that the authors should try to address. Also, some of the supplemental data should be in the manuscript proper. This referee was not able to access the supplemental material during the review (a frustrating experience), some of which is essential to the arguments.

Issues the authors need to consider addressing in revising the manuscript.

1. The agent used to treat the mice is a 9 amino acid peptide. They administer it orally in the drinking water for a week once each month. Most peptides do not survive transit through the GI tract. There is no demonstration that the drug gets into either plasma or the central nervous system. Do the authors have any pharmacokinetic data they can supply or at least refer to that demonstrates CNS penetration by this relatively hydrophilic peptide? Why did they not provide the agent continuously?

2. The authors claim they modified the peptide such that it activates the macrophage receptor, but not the neutrophil receptor. Are these two receptors different genes, or splice variants? How can this be achieved?

3. The sample size is specified as 6 per group. They state that one hemisphere was fixed for histological processing and the other frozen. They then indicate that the Aβ measurement was performed on the entire hemisphere (line 186). The kits they used specify the use of guanidinium buffer, which would dissolve both fibrillar and soluble Aβ, yet they claim to only measure non-fibrillar Aβ. Was there a centrifugation step?

4. The authors then indicate that they used a RIPA buffer (line 199) to perform immunoblots. Where did the other tissue come from (unless they did not follow the Aβ kit instructions). They further indicate extracting total RNA (line 241). Where did the tissue for this come from? There needs to be some further methodological detail to indicate how they measured these multiple markers from the same tissue. Or perhaps, they did not make the measurements on the same tissue but had either dissected brain regions, more mice, or perhaps reduced the numbers of mice for each measurement.

5. The authors need to find a reasonable convention for specifying the Aβ peptide and stick with it. They variably refer to it as Amyloid β, as αβ, A4, or Aβ.

6. For immunohistochemistry, the authors need to specify the number of sections they analyzed for each stain and the distribution of the sections throughout the tissue. Paraffin sections are often sampling from a limited portion of the region and not very representative overall.

7. Is there evidence that C5a increases the expression of its receptor? Typically, agonists decrease the cognate receptor. As this is the only evidence the authors offer of CNS penetration, it would be more convincing if this was a well known phenomenon.

8 Line 288 seems a bit odd. First, most anti-Aβ antibodies list the aa in the Aβ sequence. It would appear that the aa listed (672-714) are from the amyloid precursor protein sequence, not the Amyloid A4 sequence (which is the same as Aβ). Second, it is also unclear what is meant by A4 precursors

9. The reductions in NeuN staining in the 5xFAD line of 70-80% are beyond any reductions reported previously. These mice have a modest reduction in neuron number in layer V of the anterior cerebral cortex. It is important that the authors specify what regions they are imaging and the number of measurements made per mouse if these data are to be believed. Further the images in the pdf are so dark that this referee is not able to evaluate what is being stained.

10. Figure S1 and S10 should probably be in the manuscript (although this referee was not able to view them). The results presented in S10 at least are integral to the overall interpretation of the mechanism by which this agent appears to prevent amyloid induced changes in the mice.

In summary, this is a manuscript which examines a novel agent for amyloid reducing effects in a mouse model of amyloid deposition (its not really FAD without tau pathology and brain atrophy). There are some methodological issues which need to be addressed. It also should be the case that evidence that the agents does gain access to both plasma and brain should be at least referenced. Given that we have close to 500 manipulations that reduce amyloid in APP mice, it is unclear how much of an advance this is. However, the reported effect sizes are substantial and the approach is relatively novel.

Reviewer #2: In this study, Panayiotou et al., authors demonstrated that the reduction of amyloid-β (Aβ) accumulation in the brains and improvement of cognitive impairment in a model mouse of AD (5XFAD) by the intermittent administration of a modified C5a receptor agonist EP67. Authors also suggested that this therapeutic effects of EP67 are exerted by the preservation of synaptic integrity following the promotion of Aβ clearance by microglia and infiltrated myeloid cells such as monocytes/macrophages but not astrocytes and neutrophils.

This manuscript suggests very important information and evidences for the development of a novel immunotherapeutic strategy against AD: the modulation of brain immunity through the regulation of compliment pathways, especially using a modified C5a receptor agonist, would be the attractive strategy for treating AD.

Text is well written, and discussions are quite fair and reasonable. However, the problem is the poor quality of immunohistochemistry. Except for figure 1a, immunohistochemical data including supplemental figures should be replaced to those of more good quality. Alternatively, it would be possible that authors delete the immunohistochemical analysis in this manuscript as the future study.

For example, staining of Synaptophysin and beta tublin III should be laminar but not dots nor like postsynaptic. In the NeuN staining, the nuclei of neurons are too big. GFAP and F4/80 signals should be detected over a wide area even in wild type mice, Iba1 and CD68 staining should indicate microglia but not neuron like structure. DAPI staining in figure S10 also shows too big nuclei.

Minor comment;

Please define and unify the short form of amyloid-β to ‘αβ’ or ‘Aβ’.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-19-19628R1

C5aR agonist enhances phagocytosis of fibrillar and non-fibrillar Aβ amyloid and preserves memory in a mouse model of Familial Alzheimer’s disease

PLOS ONE

Dear Professor Kyriakides,

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.

In revising your manuscript, please respond to all the comments raised by the editor, which are described below.

We would appreciate receiving your revised manuscript by Dec 05 2019 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,

Wataru Araki

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

In the revised manuscript, the authors addressed most of the comments raised by the reviewers. However, their responses to several comments are not sufficient; especially it is necessary to clarify the following points.

1. Regarding the issue of whether EP67 can get into either the plasma or the brain, the authors need to add more explanations in the discussion part.

2. Regarding the comment No2 of Reviewer#1, the response should be added somewhere in the manuscript.

3. Methods of immunoblotting (page 11, 211-216) are not described clearly. This part should be corrected in such a way that other researchers can reproduce the experiments.

4. The subtitles “Fibrillar Abeta quantification” and “Non-fibrillar Abeta quantification” are not appropriate. The former means the quantification of Thioflavin S-positive Abeta plaques and the latter means the quantification of GuHCl-soluble Abeta40 and Abeta42 by ELISA. As the expressions “Fibrillar Abeta quantification” and “Non-fibrillar Abeta quantification” can be misleading, these should be rephrased throughout in the manuscript.

5. Fig 6B: The immunoblotting data of NeuN appears unnatural. As NeuN is a nuclear protein and is not easily extracted by RIPA buffer, the data does not appear to reflect the actual situation in the brain. It is recommended to delete this data and to leave only the immunohistochemistry data.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #2: All comments have been addressed

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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: Yes

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

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 #2: Yes

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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: 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 #2: This manuscript has been addresed all my comments and revised properly. Statistical analysis been performed appropriately. Authors has made all data underlying the findings in their manuscript fully available, and the manuscript presented in an intelligible fashion and written in standard English.

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Reviewer #2: Yes: Kazuyuki Takata

[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.]

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PONE-D-19-19628R2

C5aR agonist enhances phagocytosis of fibrillar and non-fibrillar Aβ amyloid and preserves memory in a mouse model of Familial Alzheimer’s disease

PLOS ONE

Dear Professor Kyriakides,

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.

Please revise carefully according to the editor's comments, including the previous ones.

We would appreciate receiving your revised manuscript by Dec 14 2019 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,

Wataru Araki

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

In the revised manuscript, the authors’ responses are not complete.

The editor noted some points need to be clarified, as follows.

The responses to comment No3 are not complete.

Please correct the following points.

L189 “Fibrillar Abeta quantification”

L195 “soluble Abeta quantification” This means GuHCl-soluble.

L323 “non-fibrillar peptides”

L197-198 “manufacturer’s instructions” This part should be described more in details.

L373-375 “neuronal loss”

Synaptophysin loss indicates synaptic loss, not neuronal loss.

This part should be changed to “synaptic and neuronal loss”.

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[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.]

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C5aR agonist enhances phagocytosis of fibrillar and non-fibrillar Aβ amyloid and preserves memory in a mouse model of Familial Alzheimer’s disease

PONE-D-19-19628R3

Dear Dr. Kyriakides,

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

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

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With kind regards,

Wataru Araki

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The authors have made an appropriate revision.

Reviewers' comments: