PONE-D-20-06005

Elevated serum magnesium lowers calcification propensity in Memo1-deficient mice

PLOS ONE

Dear Dr. Bonny,

I hope this note finds you well. We’re all in a bit of a fog coping with the c19 pandemic. At some level, it seems odd to be concerned with esoteric scientific problems with so much more at stake. That said, I now received reviews on your submitted work from two internationally respected experts. Both found your work of substantial potential interest but raised a number of salient questions and concerns that will have to be favorably addressed before a final decision can be reached.

Reviewer #1 raises the obvious and sensible question regarding the paradoxical increase of Npt2a with elevated Fgf23 levels, when reduced transporter expression would be expected. Moreover, if Npt2a is elevated, what counterregulatory mechanism keeps serum Pi normal?

Reviewer #2 expressed greater reservations and questions whether MEMO1 deficiency causes such profound pathology that the phenotype arises secondarily. The reviewer also notes the apparent contradictory findings with Fgf23 and Npt2a, along with other significant concerns. Some suggestions for addressing these perceived deficiencies are included by both reviewers.

The journal looks forward to your response and an amended manuscript.

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"is an inventor of the T50-Test and stock-holder and employee of Calciscon AG, which commercialized the T50-Test (calcification propensity test). MB is an employee of Calciscon AG. The other authors declare that no conflict of interest exists."

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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?

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Reviewer #1: Yes

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

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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: The manuscript by Moor et al describes a potential mechanism for the reduction of calcifications in Memo-1 deficient mice. The authors’ work suggests this could be due to a role of Memo-1 in magnesium handling. Overall, the work adequately supports the hypothesis, however several clarifications should be made:

1) It is not clear why in the cKO mouse that if FGF23 is elevated, why is NPT2a increased, when a principle action of FGF23 is to down-regulate this protein? Further, why are there conflicting data with the 24-OHase mRNA levels? It appears as if this gene is properly regulated by the elevated FGF23, as it shows increased expression.

2) Would suggest that the mice be re-assessed with the Quidel Rodent specific FGF23 intact ELISA, not the Kainos human FGF23 ELISA that was used previously.

3) If NPT2a is elevated, what is the counter regulatory mechanism to keep the serum Pi normal in the cKO mice since 1,25D and PTH are normal?

4) It is clear that FGF23 and KL are genes involved in phosphate handling, and are not true aging-related genes as in progeria. References to aging-like phenotypes in the cKO mouse model should be minimized in the Introduction and a sentence should be added that these phenotypes can be explained by altered phosphate and vitamin D handling.

5) If possible, in vitro experiments would strengthen the idea that Memo-1 directly controls Trpm6-7 expression.

Reviewer #2: Mediator of cell ErbB2-driven MOtility1 (MEMO1) is a ubiquitously expressed protein with vital functions. Structurally, MEMO1 is related to iron- and zinc-binding enzymes, and exerting a Cu-II dependent oxidase activity affecting the cellular redox status and controlling the activity of a wide range of redox-regulated cellular proteins. MEMO1 affects cellular motility presumably by direct interaction with cellular motility proteins, and in addition through scaffolding of signalosomes. Thus MEMO1 exhibits profound protein moonlighting, which renders in vivo studies near impossible because or complex interaction and interdependencies of the claimed functions. Whole body MEMO1 knockout is embryo lethal, while conditional knockout cKO produces a complex phenotype of growth retardation and drastically reduced life span (mean survival about 2 months). These findings suggest that MEMO1 deficiency renders the animals profoundly sick and that it will be very difficult to decide if aspects of the phenotype are the cause or a consequence of the progressively poorer health status of the animals, again begging the question, which of the many claimed functions of MEMO1 is chief in this scenario. Studies suggested that a major function of MEMO1 is enhancing FGF signaling by stabilizing the FGFR signalosome. This broad function downstream of receptor tyrosine kinases was narrowed down when it was realized that the MEMO1 cKO phenotype was similar to the phenotype of FGF23 and klotho deficient mice. This finding put MEMO1 into FGF23-klotho phosphaturic signaling, however with one major shortcoming, namely that the overt hyperphosphatemia of FGF23 and klotho knockout mice that is meant to be instrumental for a large part of the detrimental knockout phenotypes is completely absent in MEMO1 cKO. One driving feature of FGF23 and klotho knockout morbidity is pathological calcification, which can be (partially) rescued by low phosphate diet or by reducing Vit D activity in the mice. Against this background the authors study calcification in MEMO1 cKO mice and unlike in FGF23 mice, find none. Dietary phosphate or vitamin-D restriction do not change the survival of the MEMO1 cKO mice. The authors report however, slight differences in serum calcification propensity, that they attribute to serum Mg.

The amount and quality of work presented in the manuscript make it well worth reporting even if it were purely descriptive. In the current mechanistic presentation I have great difficulties following the authors’ reasoning, especially that a slight change in serum magnesium will affect the health of the animals so profoundly.

Major points

1) It would help a lot if the authors would lay out their working hypothesis more clearly given the many purported functions of MEMO1, especially since some of the crucial disturbances in mineral handling observed in FGF23 and klotho mice are completely absent in MEMO1 cKO mice, or minor at best.

Minor points

1) Hyperphosphatemia and calcification are absent in the mice, why go on and study calcification propensity? Please explain.

2) Experimental diets; 0.65% phosphorous in the control diet is still pretty high, 0.8% definitely is. For comparison see a recent study by Babler et al. 2020 PLoS ONE (https://doi.org/10.1371/journal.pone.0228938)

3) The differences in control serum magnesium in Fig 5C and Fig 7B are almost as large like the differences between control and kKO. Please discuss.

4) Fig 4F x-rays are pretty bad quality. Since the authors used a Skyscan micro-CT, can they provide higher resolution CT scans? Perhaps these would show microcalcifications.

5) Fig 7 and the function of Mg transporters in various tissues, please discuss against a recent publication on this topic (Chubanov et al. 2016, doi.org/10.7554/eLife.20914)

6) Fig 9 does not add any information to the text, delete.

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Reviewer #1: No

Reviewer #2: Yes: Willi Jahnen-Dechent

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Elevated serum magnesium lowers calcification propensity in Memo1-deficient mice

PONE-D-20-06005R1

Dear Dr. Bonny,

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.

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Peter A. Friedman, Ph.D.

Academic Editor

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Additional Editor Comments (optional):

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: 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 #1: (No Response)

Reviewer #2: Yes

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

Reviewer #1: (No Response)

Reviewer #2: N/A

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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

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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 #1: (No Response)

Reviewer #2: Yes

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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: (No Response)

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Reviewer #1: No

Reviewer #2: Yes: Willi Jahnen-Dechent